SUNDAY

SUNDAY, DECEMBER 4- Late Abstracts 1

Membrane Trafficking

1970 A novel SNX3-dependent Retromer pathway is required for Wnt secretion. I. McGough1, M. Harterink2, F. Port3, M. Lorenowicz2, R. Korswagen2, P. Cullen1; 1Biochemistry, University of Bristol, Bristol, England, 2Hubrecht Institute, Utrecht, Holland, 3Institute of Molecular Life Sciences, University of Zurich

Wnt proteins play a central role in development. Wnt secretion is mediated by the Wnt-binding protein Wntless (Wls), which transports Wnt from the Golgi to the cell surface for release. Wnt secretion requires recycling of Wls through a retromer-dependent endosome-to-Golgi trafficking pathway, but the mechanism remains poorly understood. Using C. elegans and Drosophila genetics, we established that Wls recycling requires an evolutionarily conserved, ‘non-classical’ retromer pathway, that functions independently of the classical retromer sorting nexins. We found sorting nexin-3, SNX3, has an evolutionarily conserved function in Wls recycling and Wnt secretion. SNX3 interacts directly with the cargo-selective sub-complex of retromer. Live cell imaging reveals WLS exits SNX3 labelled endosomes through vesicular carriers rather than classic retromer decorated tubular profiles. These results establish SNX3 as part of an alternative retromer pathway that functionally separates retrograde transport of Wls from other retromer cargo.

1971 The lectin ArtinM binds to and is internalized by hematopoietic cell lines. P. A. Buranello1, V. M. Mazucato1, M. R. Pinto1, M. C. Barreira1, M. C. Jamur1, C. Oliver1; 1Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, Brazil

The D-mannose binding lectin ArtinM, extracted from Artocarpus integrifolia, has an important role in the defense against pathogens. Artin M is imunnomodulatory and activates immune cells, such as mast cells, neutrophils and macrophages. It is known that ArtinM can induce cell death in various hematopoietic cell lines. The aim of the present study is to further characterize the effects of Artin M on hematopoetic cell lines. The human hematopoietic cell lines NB4, K562 and U937 were used. Flow cytometric analysis showed that NB4 cells bind 2.4 times less ArtinM than K562 and U937 cells. By scanning electron microscopy, it was possible to observe that ArtinM activated the various cell lines. After 5 minutes of incubation, the K562 cells already presented ruffles on their surface. Only after 15 minutes of incubation was this activation observed in NB4 and U937 cells. However, the ruffling was less intense. After one hour of incubation in presence of ArtinM, all of the cell lines exhibited extensive ruffling. This ruffling was most evident in the K562 cells. Artin M internalization was investigated by fluorescence microscopy using biotinylated Artin M followed by streptavidin-FITC. After 15 minutes of incubation the K562 and U937 cells had already internalized the lectin, but internalized lectin was seen only after 30 minutes in the NB4 cells. One hour after incubation, ArtinM had accumulated in cytoplasmic vesicles in all of the cell lines. This internalization was independent of clathrin and caveolin. In addition, the cytoplasmic vesicles containing ArtinM did not co- localize with CD63, a lysosomal marker. Our results show that ArtinM activates and is internalized by hematopoietic cells and that the ArtinM then accumulates in cytoplasmic vesicles. The binding, activation and internalization of ArtinM may all contribute to the ArtinM induced cell death seen in hematopoietic cells.

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1972 Proteomic Characterization of the Binding Partners of Dopamine Transporter (DAT) and Functional DAT Mutants. S. M. Moore1,2, A. Rao2, A. Sorkin1, C. C. Wu1; 1Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA, 2Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO

Dopamine transporter (DAT) is a 12-transmembrane domain, integral membrane protein that acts to terminate the synaptic transmission of the neurotransmitter dopamine. DAT is normally expressed primarily on the plasma membrane of the cell and is internalized through clathrin- mediated endocytosis. It has been shown that with N-terminal deletion, DAT undergoes increased internalization through augmented levels of endocytosis. Conversely, a C-terminal deletion variant of DAT has been shown to reside primarily in the endoplasmic reticulum, with unknown endocytic effects. The main objective of this project is to utilize mass spectrometry profiling methods to analyze the binding partners of these different DAT constructs in order to increase the understanding of DAT trafficking and endocytosis. Initial studies in mice expressing a full-length hemagglutinin (HA)-tagged DAT construct combined immunoprecipitation (IP) using an HA antibody with profiling mass spectrometry. These studies resulted in the identification of a variety of potential DAT binding partners. Some of the identified proteins were previously known to be DAT binding partners, others were known to be involved in endocytosis, and still others were known to be involved in synaptic transmission. These mouse studies inspired further proteomic studies in cell lines containing the aforementioned DAT constructs, due to the identification of endocytic proteins in the mouse studies along with the difference in endocytic patterns in the DAT construct containing cell lines. Full length (FL), N-terminally deleted (ΔN), and C-terminally deleted (ΔC) DAT constructs, dually tagged with YFP and HA, were stably expressed in porcine aortic endothelial (PAE) cells. These constructs then underwent IP using either HA or GFP antibodies. After IP the samples were digested with trypsin and analyzed on either an LTQ linear ion trap mass spectrometer, for profiling experiments, or a TSQ Vantage triple quadrupole mass spectrometer, for quantitative experiments. In these cell studies, several proteins were identified that were present in all three samples while others were expressed differentially between the samples when compared to the parental PAE cell line. Proteins present in all samples included ubiquitin, calmodulin, and alpha-tubulin. Differentially expressed proteins included clathrin heavy chain, Rho-GEF 12, and AP2 alpha-1 subunit, among a variety of others. This differential expression indicates that the endocytic changes that occur with these deletion mutations are accompanied by changes in the proteins that associate with DAT.

1973 Cell adhesion and survival requires integrin rapid recycling. N. Waxmonsky1, S. Conner1; 1University of Minnesota, Minneapolis, MN

Integrins are transmembrane heterodimers composed of an α and a β subunit that mediate attachment between the cell and its environment, which is a requirement for cell survival in many cell types. Cells maintain adhesion by trafficking integrins to the cell surface through endosomal sorting pathways; however, the molecular mechanisms governing this process are undefined. We hypothesized that integrin recycling through the endosomal compartment is important for maintenance of cell adhesion. In order to test this, we depleted HeLa cells of factors with known roles in long- and short-loop recycling. Depletion of a long-loop recycling factor, EHD1 (Eps15 Homology Domain), did not lead to loss of adhesion. However, depletion of factors that have established roles at the early endosome, AAK1 (Adaptor-Associated Kinase 1) and EHD3, led to cell rounding and an eventual loss of cell adhesion. Defects in integrin delivery to the cell surface disrupts formation of focal adhesion complexes. We investigated SUNDAY whether there were differences in number of focal adhesion complexes in cells with short-loop recycling defects. To do so, we visualized complexes using a vinculin antibody and found a decrease in number of focal adhesion complexes in AAK1 and EHD3 depleted cells. To observe focal adhesion formation in cells with recycling defects, we used a TIRF live cell imaging approach to visualize the dynamics of β3 integrin-GFP positive focal complex formation. The experiment revealed a reduction in the rate of focal adhesion complex formation in AAK1 and EHD3 depleted cells. Commensurate with this observation, we found that β3 integrin total levels were also reduced in AAK1 and EHD3, but not EHD1, depleted cells. This raised the possibility that the observed reduction might result from a biosynthetic defect or a mis-sorting toward the degradative pathway. To distinguish between these possibilities, we disrupted the degradative pathway by depleting Tsg101 (Tumor susceptibility gene 101), a factor with a role in the ESCRT complex. In doing so, we found partial restoration of β3 integrin total levels and cell attachment upon depletion of AAK1/Tsg101 and EHD3/Tsg101. Depletion of factors involved in short-loop recycling resulted in a loss of adhesion, a reduction in focal adhesion formation rate and mis- sorting to the degradative pathway. This experimental evidence indicates that cell adhesion requires integrin short-loop recycling.

1974 CFTR anion channel modulates expression of human transmembrane mucin MUC3 through the PDZ protein GOPC. T. Pelaseyed1, G. C. Hansson1; 1Department of Medical Biochemistry, Institute of Biomedicine, University of Gothenburg, Göteborg, Sweden

The transmembrane mucins in the enterocyte are type 1 transmembrane proteins with long and rigid mucin domains, rich in proline, threonine and serine residues that carry numerous O- glycans. Three of these mucins, MUC3, MUC12 and MUC17 are unique in harboring C-terminal class I PDZ motifs, making them suitable ligands for PDZ proteins. A screening of 123 different human PDZ domains for binding to MUC3 identified a strong interaction with the PDZ protein GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein). This interaction was mediated by the C-terminal PDZ motif of MUC3, binding to the single GOPC PDZ domain. GOPC is also a binding partner for cystic fibrosis transmembrane conductance regulator (CFTR) that directs CFTR for degradation. Overexpression of GOPC downregulated the total levels of MUC3, an effect that was reversed by introducing CFTR. The results suggest that CFTR and MUC3 compete for binding to GOPC, which in turn can regulate levels of these two proteins. For the first time a direct coupling between mucins and the CFTR channel is demonstrated, a finding that will shed further light on the still poorly understood relationship between cystic fibrosis and the mucus phenotype of this disease.

1975 For “The Perfect Antagonists” the winners are: MyoIC and MyoIE, for their role in “Actin shaping for vesicle exocytosis at the immune synapse.” J. M. DIAZ MUNOZ1,2, M. I. Yuseff1, D. Lankar1, P. Pierobon1, M. Rosemblatt3, A. M. Lennon- Dumenil1; 1INSERM U932, Immunity and cancer, Institut Curie, Paris, France, 2Genetica Molecular y Microbiologia, P. Universidad Catolica de Chile, Santiago de Chile, Chile, 3Fundacion Ciencia para la Vida, Santiago de Chile, Chile

Engagement of the B Cell Receptor (BCR) by surface-tethered antigens (Ag) leads to formation of a synapse that promotes Ag uptake and presentation onto MHCII molecules. Here, we highlight the membrane trafficking events and associated molecular mechanisms required for efficient Ag extraction and processing at the B cell synapse. We show that MHCII-containing lysosomes are recruited at the synapse and locally undergo exocytosis, a process that relies on SUNDAY the SNARE protein Vamp-7. Lysosome secretion allows the extracellular release of proteases, whose activities promote the extraction of the immobilized Ag. We further show that local re- organization of cortical actin by type I Myosins, which link the cortex to the plasma membrane, is required for lysosome exocytosis and Ag extraction. Remarkably, while the short-tail Myosin IC is recruited at the synapse and facilitates vesicle secretion and Ag uptake, the long-tail Myosin IE negatively regulates both processes. These results suggest that the two class I Myosins play antagonistic roles in the local reorganization of cortical actin for vesicle secretion and Ag uptake. The B cell synapse therefore emerges as a highly specialized site where tightly regulated exocytic and endocytic events take place thanks to the local shaping of the membrane-cytoskeleton interface by class I Myosins

1976 Determination of the Subcellular, Surface, and Extracellular Localization of Hsp70s in Mammalian Cells. R. Medina*1, M. Rashedan*1, N. Nikolaidis1; 1Biological Science, California State University, Fullerton, Fullerton, CA 70-kD Heat shock proteins (Hsp70s) are a family of molecular chaperones that play essential roles in stress response by promoting protein homeostasis. Members of this family are primarily localized in different subcellular compartments, including the cytosol, the endoplasmic reticulum, and the mitochondria. Apart from their primary location in specific parts of the cell, different Hsp70s have also been found in other places within the cell, at the cellular membrane, and at the extracellular milieu. Although a few stresses and pathophysiological conditions, like cancer, have been related with the re-localization of Hsp70s within the cell, their translocation to the membrane, and their secretion from viable cells, the majority of these conditions remain unknown. Additionally, the signaling mechanisms involved in the trafficking of intracellular Hsp70s remain elusive. To this end we studied the intra-, membrane- and extra-cellular localization of four members of the Hsp70 family. Specifically, we determined the re-localization of HSPA1A and HSPA8, primarily localized in the cytosol, HSPA5, primarily localized in the endoplasmic reticulum, and HSPA9, primarily localized in the mitochondria. Human embryonic cells treated with different stressors, including heat and ethanol, or untreated were subjected to sub-cellular fractionation and the presence of Hsp70s was determined by Western. The amount of Hsp70s in the different fractions was quantified and normalized for equal loads using fraction- specific antibodies, e.g., beta-actin for the cytosolic fraction. These experiments revealed that under normal growth conditions all four proteins were present in the nuclear, cytosolic, mitochondrial, and membrane fractions, as well as in the extracellular medium. In heat-shocked cells, although all proteins were still present in all fractions their amounts in each fraction were significantly different from the untreated cells. These results strongly suggest that after stress the different Hsp70s are being re-localized within the cell, are anchored at the cellular membrane, and are secreted from the cell. Additional experiments using pharmacological manipulation of intracellular trafficking pathways are currently being performed to identify the molecular mechanism used by Hsp70s to achieve their translocation and subsequent secretion. This study is the first step towards elucidating the biological importance of the relocalization and membrane occurrence of these chaperones.

*equal contribution

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1977 Fab1/PIKfyve Is Required in Multiple Organs, and is the Major Pathway for Production of PI(3,5)P2 and PI5P in Mammals. S. N. Zolov1, D. Bridges1, Y. Zhang2, R. Verma1, W-W. Lee1, G. M. Lenk3, K. Converso-Baran4, R. Albin5, A. Saltiel1,6, M. Meisler3, M. Russell4, L. S. Weisman2; 1Life Sciences Institute, University of Michigan, Ann Arbor, MI, 2Department of Cell and Developmental Biology and Life Sciences Institute, University of Michigan, Ann Arbor, MI, 3Department of Human Genetics, University of Michigan, Ann Arbor, MI, 4Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI, 5Department of Neurology, University of Michigan, Ann Arbor, MI, 6Departments of Internal Medicine, University of Michigan, Ann Arbor, MI

Mutations that cause defects in the levels of the signaling lipids PI(3,5)P2 and PI5P lead to profound neurodegeneration in mice. Moreover mutations in human FIG4 predicted to lower PI(3,5)P2 and PI5P levels underlie some cases of Charcot-Marie-Tooth syndrome and Amyotrophic Lateral Sclerosis. In mammals, PI(3,5)P2 is generated by a protein complex that includes the lipid kinase Fab1/PIKfyve, the scaffolding protein Vac14, and the lipid phosphatase Fig4. Fibroblasts cultured from Vac14-/- mutant mice have a 50% reduction in the levels of PI(3,5)P2 and PI5P, which leaves open the question of whether there is a second pathway to generate these lipids. To address this question we characterize a Fab1 gene-trap mouse (Fab1β-geo/β-geo). The homozygous Fab1β-geo/β-geo mouse is a hypomorph that expresses 10% of the normal levels of Fab1 and produces 50% of the normal levels of PI(3,5)P2. When we used shRNA silencing in Fab1β-geo/β-geo fibroblasts to knock-down the remaining Fab1, we observed a loss of detectable PI(3,5)P2 and an 85% reduction in PI5P. These fibroblasts also have a substantial increase in the amount of PI3P, the for Fab1. Thus, similar to yeast, conversion of PI3P to PI(3,5)P2 in mammals requires the Fab1 pathway. Surprisingly, much of the PI5P pool also requires the Fab1 pathway. Similar to Vac14-/- and Fig4-/- mice, the Fab1 hypomorphic mouse exhibits neurodegeneration in the brain and in peripheral sensory neurons. However, these mice also have profound defects in the heart, lung, kidney, thymus, spleen and intestine. Importantly, we examined the heart in Vac14-/- and Fig4-/- mouse mutants and found β-geo/β-geo similar defects to those observed in the Fab1 mouse. Thus PI(3,5)P2 and possibly PI5P have major roles in multiple organs.

1978 Characterization of Trak1, a novel protein implicated in hypertonia and epilepsy. C. A. Lee1, L. Li1, L-S. Chin1; 1Pharmacology, Emory University, Atlanta, GA

Hypertonia—a pathophysiological condition characterized by postural abnormalities, jerky movements, and tremor—is associated with a number of neurological disorders, including cerebral palsy, dystonia, Parkinson's disease, stroke, and epilepsy. The pathogenic mechanisms that trigger hypertonia remain elusive. Interestingly, a homozygous truncation mutation in the trafficking protein, kinesin-binding 1 (Trak1) protein causes a recessively transmitted form of hypertonia in mice. Moreover, recent genome wide high-density SNP-based linkage analysis has linked variants in Trak1 to childhood absence epilepsy in humans. Despite the critical importance of Trak1 in maintaining neuronal function, the mechanism and sites of Trak1 action remain unclear and the pathogenic mechanism by which Trak1 mutation causes hypertonia is unknown. We have generated a highly specific anti-Trak1 antibody and shown that endogenous Trak1 protein is expressed in multiple tissues, including the brain. Immunofluorescence confocal microscopic studies show that endogenous Trak1 protein is localized in axons, dendrites, and cell bodies of mouse cortical neurons, supporting a functional role of Trak1 in neuronal physiology. We found that endogenous Trak1 protein is associated with both early endosomes and mitochondria in cortical neurons. Our data suggest that Trak1 SUNDAY protein may play a critical role in maintaining neuronal homeostasis by regulating endosomal trafficking and mitochondrial transport. Further characterization of the mechanisms of Trak1 action and its regulation will provide a better understanding of the role of Trak1 in hypertonia and epilepsy.

1979 Post-Fusion Actin Coating of Secretory Vesicles is required for Active Content Extrusion in Alveolar Type II Cells. P. Miklavc1, E. Hecht1, N. Hobi2, O. H. Wittekindt1, T. Haller2, P. Dietl1, E. Felder1, C. Kranz1, M. Frick1; 1University of Ulm, Ulm, Germany, 2Innsbruck Medical University, Innsbruck, Austria

Exocytosis of secretory vesicles is a fundamental cellular process allowing regulated secretion of vesicle contents in the extracellular space. Growing evidence suggests that regulation of fusion pore dilation during the post-fusion stage of exocytosis plays an important role in release of secretory vesicle contents. However, dependent on the nature of the cargo additional mechanisms might be essential to facilitate effective release. We have recently described in alveolar type II cells that surfactant-storing secretory vesicles (lamellar bodies) are coated with actin following fusion with the plasma membrane. Surfactant, a lipoprotein-like substance, does not readily diffuse out of fused lamellar bodies following opening and dilation of the fusion pore. Using fluorescence microscopy, atomic force microscopy and biochemical assays we present evidence that actin coating and subsequent compression of the actin coat is essential to facilitate surfactant secretion. Simultaneous imaging of the vesicle membrane and the actin coat revealed that contraction of the actin coat compresses the vesicle following fusion. This leads to active extrusion of vesicle contents. Preventing actin coating of fused LBs with latrunculin A inhibits surfactant secretion almost completely. Subsequent compression of the actin coat is modulated by myosin II. In summary our data suggest that fusion pore opening and dilation is not sufficient for “passive” release of bulky vesicle cargo in alveolar type II cells and that active extrusion mechanisms are required.

1980 Synaptotagmin-7 - the link between fusion-activated Ca2+-entry and regulation of fusion pore expansion? N. Sharma1, P. Miklavc1, K. Thompson1, O. H. Wittekindt1, E. Felder1, P. Dietl1, M. Frick1; 1Institute of General Physiology, University of Ulm, Ulm, Germany

Ca2+ is the key element in regulated exocytosis controlling multiple steps during the exocytic pre- and post-fusion stages. We have recently described a “fusion-activated” Ca2+-entry (FACE) via vesicular P2X4 receptors during the post-fusion stage of lamellar body exocytosis in alveolar type II (ATII) cells. FACE regulates fusion pore expansion and vesicle content release during the post-fusion phase of exocytosis (PNAS, 2011 Aug 30;108(35):14503-8). Yet, the question remained how this locally restricted Ca2+ signal is translated into mechanical force promoting fusion pore expansion. In this study we tested whether synaptotagmins could form a molecular link between FACE and fusion pore expansion. Members of the synaptotagmin family that localize to secretory vesicles have been found to promote fusion pore expansion via Ca2+ binding to C2 domains. Using RT-PCR and western-blotting we identified synaptotagmin-7 as the Ca2+-binding isoform predominantly expressed in primary ATII cells. Immunofluorescence confirmed for the first time that synaptotagmin-7 is localised on lamellar bodies. We next performed functional studies over-expressing wt synaptotagmin-7 or mutants abolishing Ca2+- binding to either the C2A, C2B or C2A and C2B domains. We measured diffusion rates of fluorescent dyes through the fusion pore to directly assess dynamic changes in fusion pore diameters. Our results show that overexpression of neither wt nor mutant synaptotagmin-7 SUNDAY affects fusion pore expansion in the absence of FACE. However, when fusions were followed by FACE fusion pore expansion was modulated by over-expression of wt or mutant synaptotagmin- 7. In summary these data confirm that FACE constitutes an “autoregulatory” mechanism, modulating the post-fusion fate of individual vesicles. In this model synaptotagmin-7 constitutes the Ca2+ sensor for FACE providing the link between FACE and fusion pore expansion in lamellar body exocytosis.

1981 Analysis of GGA null mice demonstrates a non-redundant role for mammalian GGA2 during development. B. Doray1, J. Govero1, S. Kornfeld1; 1Washington University Sch Med, St Louis, MO

The mammalian GGAs (Golgi-localized, gamma-ear containing, ADP-ribosylation factor- binding proteins) comprise three homologous proteins that function as monomeric clathrin adaptors. Numerous studies using cultured mammalian cells have shown that the three GGAs function in the transport of cargo proteins between the trans-Golgi network and endosomes. However, the in vivo role(s) of these adaptor proteins and their possible functional redundancy has not been analyzed. To address this issue, gene ablation studies were performed in mice using insertional mutagenesis. Our data demonstrate that loss of GGA1 or GGA3 alone is well tolerated whereas the absence of GGA2 results in embryonic lethality (C57BL/6J and 129/Ola mixed parentage), or neonatal lethality (C57BL/6NJ background). Out of 169 pups from Gga2 heterozygous crosses in the C57BL/6NJ genetic background, a single Gga2 -/- mouse escaped lethality, remains alive and is fertile when crossed with a Gga2 +/- male. This result indicates that GGA2 mediates some vital function during embryogenesis and the neonatal period that cannot be compensated for by GGA1 and/or GGA3 but beyond this period, GGA2 may be dispensable. This result is also consistent with our expression studies showing that the level of GGA2 is highest in the brain during embryonic development and early life but decreases substantially two weeks after birth. In contrast, brain expression of GGA1 and GGA3 remains consistently high from late embryogenesis through the adult stage. The fact that all three GGAs are expressed at comparable levels in the brain during embryonic development excludes the possibility that GGA2 is the sole GGA present during this phase, at least in the brain. The combined loss of GGA1 and GGA3 also results in a high incidence of neonatal mortality (58% mortality within the first 3 weeks) but in this case the expression level of GGA2 may be inadequate to compensate for the loss of the other two GGAs. We conclude that the three mammalian GGAs are essential proteins that are not fully redundant.

1982 Severing of actin filaments by cofilin is required for both disassembly and assembly of the actin patch in fission yeast. Q. Chen1, T. Pollard1,2; 1Department of Molecular, Cell and Developmental Biology, Yale University, New Haven, CT, 2Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT

We use mutant cofilins to test the proposal that cofilin severs actin filaments during endocytosis in fission yeast. We replaced the endogenous protein with a mutant cofilin that is defective in severing actin filaments. We used quantitative fluorescence microscopy to study the endocytic actin patches in these mutant cells. We tracked GFP tagged patch markers in the patches, including early endocytic adaptor proteins, activators of Arp2/3 complex and actin filaments. Consistent with the hypothesis, the actin patches disassembled far slower during their inward movement than in wild type cells. Abundant actin filaments accumulated at endocytic sites, even SUNDAY after the membrane adaptor proteins dissociated from the patches. We were surprised to find that actin patches also assembled slowly in these cofilin mutant cells. Adaptor proteins such as End4p and Pan1p accumulated and persisted at the endocytic sites more than 10 times longer than in wild type cells, followed by delayed put persistent recruitment of activators of Arp2/3 complex, including WASP and myosin I. We propose that severing by cofilin normally produces short actin filaments that diffuse out of actin patches and stimulate Arp2/3 complex in adjacent patches by serving as the mother filaments that initiate the autocatalytic branching reaction. Additional feedback mechanisms seem to prolong the early steps in the pathway until the later steps are executed.

1983 Structural and functional characterization of cargo-binding sites of the µ4-subunit of adaptor protein complex 4. B. H. Ross1, E. A. Corales1, Y. Lin1, J. H. Hurley2, J. S. Bonifacino3, P. V. Burgos1, G. A. Mardones1; 1Universidad Austral de Chile Sch Med, Valdivia, Chile, 2LMB, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD, 3CBMP, National Institute of Child Health and Human Development, NIH, Bethesda, MD

Adaptor protein (AP) complexes assist protein trafficking by playing key roles in the selection of cargo molecules to be sorted in post-Golgi compartments. The medium-sized subunit (µ1-µ4) of the four heterotetrameric AP complexes recognizes YXXØ-sequences (Ø is a bulky hydrophobic residue), which are sorting signals in transmembrane proteins. A conserved region, the µ2- , mediates recognition of YXXØ-signals. Recently we found that a non-canonical YXXØ-signal binds to a distinct µ4-binding site of the AP-4 complex. In this study we aimed to determine the functionality of both binding sites on the recognition of this non-canonical YXXØ- signal. We used site-directed mutagenesis, yeast-two hybrid (Y2H) analyses, isothermal titration calorimetry (ITC), and X-ray crystallography. Substitutions in either of both binding sites on µ4 abrogated binding to the APP-tail in Y2H experiments. Further characterization by ITC showed no binding only with the R283D substitution at the µ4-binding site, in contrast with a decrease in binding affinity with the substitution D190A at the µ2-binding site. We solved the crystal structure of the C-terminal domain of the D190A mutant of the µ4 subunit bound to the non- canonical YXXØ-signal. This structure showed no significant difference compared to that of wild-type µ4 subunit. Our mutational, biochemical and structural analyses established the role of the µ4-binding site for the non-canonical YXXØ-signal. FONDECYT 1100896

1984 Regulation of the RalGAP Complex by Akt-Catalyzed Phosphorylation. D. Leto1,2, X-W. Chen1,2, A. Burk1,2, T. Xiong1,3, G. Yu1,2, A. Saltiel1,2; 1University of Michigan, Ann Arbor, MI, 2Life Sciences Institute, University of Michigan, Ann Arbor, MI, 3Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI

In response to feeding, the anabolic hormone insulin increases glucose uptake into adipocytes by stimulating exocytosis of vesicles containing the facilitative glucose transporter, Glut4. The small vesicle-localized G protein, RalA, is rapidly and transiently activated by insulin in an Akt- dependent manner. When bound to GTP, RalA facilitates recognition of Glut4 vesicles at the plasma membrane by interacting with the exocyst, an 8-subunit tethering complex. We have previously shown that in the absence of insulin, RalA is retained in a largely inactive state by a RalGAP Complex comprised of a regulatory subunit, RGC1, and a catalytic subunit, RGC2, that contains a GAP domain with specific activity toward RalA. Activation of this GTPase by insulin requires inhibition of the RalGAP complex. Here, we show that insulin stimulates Akt-catalyzed SUNDAY phosphorylation of RGC1/2 on at least three residues, and that phosphorylation inhibits the complex without directly inactivating its catalytic activity. We have identified a novel interaction between 14-3-3 and RGC1/2. This interaction is dependent on phosphorylation of RGC2 on a residue that was previously identified as an Akt target site. 14-3-3 binding does not interrupt the interaction between RGC1 and RGC2 or the catalytic activity of the complex. Thus, 14-3-3 may regulate RGC1/2 by altering its interaction with RalA, as has been reported for other GAP complexes.

1985 The Rab GAP Msb3/Gyp3 regulates dose-dependent Rab5/Vps21 signaling. D. Nickerson1, A. Merz1; 1Biochemistry, University of Washington, Seattle, WA

Membrane transport into and out of endosomes in yeast requires the function of a trio of Rab5 orthologs: Vps21, Ypt52 and Ypt53. While loss of Vps21 causes obvious trafficking phenotypes, the roles of its paralogs Ypt52 and Ypt53 are mostly unexplored and the regulatory logic of the trio is unclear. We report that Ypt52 and Ypt53 cooperate with Vps21 to mediate biosynthetic transport of CPY and multivesicular body cargoes to the vacuole. Cells deficient in multiple Rab5 paralogs display synthetic defects in both these processes and in calcium resistance. Ypt53 functions as a calcineurin-dependent calcium stress response factor. We also find that conditions of Rab5 signaling deficiency result in significant upregulation of YPT53, suggesting that Ypt53 functions as a positive modulator of Rab5 signal in response to both environmental stresses and trafficking malfunctions. We further report that Msb3/Gyp3 functions as the principle GAP for Vps21 in vivo. Loss of Gyp3 activity results in both greater accumulation of GTP-bound Vps21 on endosomes and mislocalization of Vps21 to vacuoles, suggesting that Gyp3 serves the dual purposes of negatively modulating the degree of Rab5 signaling and spatially restricting Vps21 to endosomes.

1986 Glycosaminoglycan attachment affects the intracellular transport of the amyloid precursor protein. D. Mihov1, M. Spiess1; 1Biozentrum, University of Basel, Basel, Switzerland

Amyloid precursor protein (APP) is a type I transmembrane protein implicated in the pathophysiology of Alzheimer disease by releasing the amyloidogenic Aβ peptide. The intracellular distribution of the different secretases and the sorting and transport of APP define the extents of amyloidogenic and non-amyloidogenic processing of the protein. APP splice variants lacking exon 15 contain a glycosaminoglycan (GAG) attachment site and are modified with a single chondroitin sulfate (ChS) chain. We have previously shown that GAG attachment affects exocytic and endocytic traffic of model proteins. Therefore, we investigated the effect of GAG attachment on intracellular traffic and processing of APP. HeLa cells were transiently transfected with the neuronal-specific splice variant APP-695 or with its corresponding proteoglycan isoform APP-677, lacking exon 15. Since APP is naturally tyrosine sulfated, we used radioactive sulfate to metabolically label transfected cells and to simultaneously detect the GAG-containing and GAG-free forms of APP. About 50% of APP-677 was GAG-modified, containing ChS chains. Pulse-chase experiments showed that GAGs accelerated the biosynthetic exocytosis of the APP-677 proteoglycan in comparison with APP-695 and APP-677 lacking ChS. APP contains well characterized signals for endocytosis from the plasma membrane. While GAG-free APP was internalized at a rate of about 6% per min, GAG attachment resulted in a significant reduction of endocytosis of at least threefold. The rates of recycling, however, were not affected. Our results indicate that exon-15 splicing and subsequent SUNDAY

GAG attachment alters APP intracellular transport, potentially influencing its processing and Aβ generation.

1987 Methyl esterification of retinal proteins is essential for rod-mediated vision. J. R. Christiansen1, S. Kolandaivelu1, M. O. Bergo2, S. G. Young3, V. Ramamurthy1,4; 1Ophthalmology, West Virginia University, Morgantown, WV, 2Cancer Center Sahlgrenska, University of Gothenburg, 3eDepartments of Medicine and Human Genetics, David Geffen School of Medicine University of California, Los Angeles, 4Biochemistry, West Virginia University

Background: Proteins ending in a “CAAX” box are first prenylated at their c-terminal cysteine and then RAS-converting 1 (RCE1) cleaves the final three amino acids before isoprenylcysteine methyltransferase (ICMT) catalyzes the methyl esterification of the newly prenylated cysteine residue. Prenylation is crucial for a protein’s ability to interact with membrane domains. The contribution of the final two steps to a protein’s localization and function are variable and depends on the protein being studied. We recently demonstrated the importance of RCE1-mediated proteolysis in phototransduction. In the absence of Rce1, photoreceptors do not function and rapidly degenerate. To determine if the effects seen in the retina conditional knockout of Rce1 were related to the lack of proteolysis, lack of carboxyl methylation, or both we analyzed the retina of mice with reduced levels of Icmt. Methods: We utilized the hypomorphic Icmt fl allele as a tool to generate animals expressing various levels of Icmt in the retina. Electroretinogram (ERG) recordings were used to analyze visual function of littermate animals with a range of Icmt levels. Morphology and phototransduction protein expression profiles were investigated by immunofluorescence and immunoblotting respectively. Results: ERG recordings displayed delayed photoreceptor cell responses and reduced downstream electrical responses to light stimuli. The delayed photoreceptor cell response correlates to changes in Icmt message levels in the retina. In agreement with delayed photoreceptor cell responses, critical phototransduction protein levels were also altered. To investigate the cause of the reduced downstream neuronal responses, we are currently verifying the development and proper synaptic stratification of inner retinal neurons. Conclusion: Efficient coupling of signal transduction in photoreceptor neurons requires methyl esterification of a phototransduction protein. In comparison with Rce1 CKO, we did not observe rapid retinal degeneration or loss of visual function. At low light levels, the visual response was reduced corresponding to reductions in Icmt. In agreement with the loss of visual response phototransduction protein turnover was also increased.

1988 Turnover of amyloid precursor protein carboxy terminal fragment beta (C99) in lysosomal compartments. A. Rivera-Dictter1, H. Bustamante1, V. Muñoz1, V. Cavieres1, G. A. Mardones1, J. S. Bonifacino2, P. V. Burgos1; 1Universidad Austral de Chile Sch Med, Valdivia, Chile, 2CBMP, National Institute of Child Health and Human Development, NIH, Bethesda, MD

Proteolytic processing of the amyloid precursor protein by β-secretase generates C99, which subsequently is cleaved by γ-secretase, yielding the amyloid β peptide (Aβ). C99 contains within its cytosolic tail key signal motifs for its delivery to lysosomal compartments. Our aim was to investigate lysosomal turnover of C99 in a mechanism independent of γ-secretase cleavage. H4 neuroglioma cells stably expressing C99-EGFP-WT were analyzed in pulse-chase experiments with cycloheximide, in the absence or presence of γ-secretase inhibitors testing several conditions in order to: 1) block delivery to lysosomes and 2) disrupt lysosomal function. SUNDAY

Blocking delivery of C99 to lysosomes or disrupting lysosomal function caused an enhancement in its proteolytic processing by γ-secretase. Similar conditions but in the presence γ-secretase inhibitors showed a significant delay in C99 turnover with a strong accumulations in lysosomal compartments. Our results show that lysosomes play an important role in the turnover of C99 suggesting that dysfunction of these organelles might have key implications in Aβ production and Alzheimer. FONDECYT 1100027 and DID-UACH

1989 Ciliary targeting of sensory receptors is coordinated by Arf and Rab GTPases and their effectors. D. Deretic1, J. Wang1; 1University of New Mexico, Albuqueruqe, NM

Ciliopathies encompass a wide range of human diseases caused by dysfunctional primary cilia, however trafficking of signaling receptors to the ciliary membrane remains poorly understood. A recently described ciliary targeting complex organized by Arf4 binds at the trans-Golgi network (TGN) to the VxPx motif present in membrane proteins targeted to primary cilia, including rhodopsin. We now show that the ciliary targeting is initiated at the TGN, by the formation of a tripartite complex between rhodopsin, Arf4GTP and the Arf-GAP ASAP1, which each recognize one of the two ciliary targeting motifs of rhodopsin, VxPx and FR, respectively. Subsequently, ASAP1 recruits Rab11a and the Arf/Rab11 effector FIP3 displaces rhodopsin from ASAP1. On ciliary transport carriers, ASAP1 provides an activation platform for the Rab11a-Rabin8-Rab8 ciliary destination module. Ciliary localization of rhodopsin-GFP-VxPx expressed in epithelial cells is lost upon ablation of ASAP1 that causes formation of actin-rich periciliary membrane projections, to which ciliary cargo is redirected. Furthermore, [FR-AA]rhodopsin-GFP-VxPx that is defective in ASAP1 binding fails to engage Rab8 and translocate across the periciliary diffusion barrier. As the VxPx and FR targeting motifs are present in other sensory receptors, comparable coupling of cargo recognition with cargo destination likely confers directionality to ciliary membrane transport.

Organelles and Membrane Biology

1990 Determinants of ciliary identity in Giardia intestinalis. A. Wiedmann1, K. Hagen1, S. C. Dawson1; 1Department of Microbiology, University of California, Davis, Davis, CA

Flagella (or cilia) are essential for the function of many cell types yet little is known about how different cell types, given the same genetic information, can build flagella of different size, protein composition and function. The unicellular intestinal parasite Giardia intestinalis forms four pairs of flagella distinct in length, behavior and protein composition (1). Identifying factors that allow for the formation of these flagella could inform research on human diseases caused by defects in flagellar growth, such as polycystic kidney disease, as well as well as lead to better treatment of the disease caused by this parasite. To determine how flagellar identities are established in Giardia we are (1) developing a novel whole-genome overexpresision anti-sense screen, and (2) in a candidate approach, are investigating the role of three putative NEK kinases in ciliogenesis. Giardia is a binucleate organism and thus random mutagenesis is not a feasible approach for identifying ciliogenesis factors. We are using T7 polymerase to direct overexpression of a library of small genomic DNA fragments to allow for a whole genome screen for factors important for SUNDAY the establishment of ciliary identity in Giardia intestinalis. Our candidate approach focuses on three NEK kinases. NEK kinases were first identified for their role in mitosis but have been shown to play a role in ciliogenesis and flagellar length determination in model systems such as Tetrahymena (2,3). Similar to the Tetrahymena genome, the giardial genome encodes an unusually large number of these kinases, suggesting that some may have non-mitotic, regulatory roles. Consistent with this hypothesis, we have identified several putative NEK kinases associated with the giardial ventral disk and flagella. Most of these are predicted to be catalytically inactive and may play structural or kinase activity independent regulatory roles. Three of the kinases, GL50803_11311, _8445, and _95593, however, contain a canonical kinase . We are using morpholino knockdown as well as overexpression of kinase- inactive and constitutively active variants to assess whether these putative kinases contribute to Giardia motility and/or ciliogenesis.

References: 1. Dawson SC and House SA (2010) Methods Cell Biol. 2. Quarmby LM and Mahjoub MR (2005) J Cell Sci. 3. Wloga D, et al (2006) Mol Biol Cell.

1991 Trophic accumulation of the antidepressant sertraline/Zoloft in fungal secretory membranes. E. Perlstein1, J. Chen1, D. Korostyshevsky1; 1Lewis-Sigler Institute/Princeton University, Princeton, NJ

Many antidepressants are cationic amphipaths, which spontaneously accumulate in cellular membranes in the absence of their protein targets. However, the clinical relevance of progressive cellular membrane infiltration by antidepressants in humans is poorly understood. Here we take an “evolutionary pharmacology” approach to studying the effects of the selective- serotonin reuptake inhibitor sertraline/Zoloft on cellular membrane homeostasis in the simple eukaryote Saccharomyces cerevisiae, which lacks a serotonin transporter entirely. We biochemically characterized the association of tritiated sertraline with the membranes of the fungal secretory pathway, and in parallel performed a quantitative ultrastructural analysis of membrane quality control in untreated vs. sertraline-treated cells. These experiments have revealed that sertraline enters yeast cells and then reshapes the secretory pathway by a complex process. Internalization of the neutral species proceeds by simple diffusion amplified by lysosomotropism, but is counteracted by ATP-dependent xenobiotic efflux. At equilibrium, a small fraction (10-15%) of spontaneously reprotonated sertraline is soluble while the remainder partitions into secretory pathway membranes by both adsorption and intercalation. Asymmetric accumulation of sertraline results in concentration- and time-dependent local membrane curvature stresses, which in turn trigger an adaptive autophagy-dependent relief response. Our model appears to support the notion of a serotonin transporter-independent, “amphipath accumulation” component of antidepressant pharmacology in humans.

1992 The UBX-domain-containing protein Ubx2/ Ubxd8 regulates lipid droplet homeostasis. C-W. Wang1, S-C. Lee1; 1Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei, Taiwan

Lipid droplets (LDs) are central organelles for maintaining lipid homeostasis. However, how cells control the size and number of LDs remains largely unknown. Herein, we report that Ubx2, a protein involved in endoplasmic reticulum (ER)-associated degradation (ERAD), is crucial for LD SUNDAY maintenance. Ubx2 redistributes from ER to LDs when LDs start to form and enlarge during diauxic shift and in the stationary phase. ubx2Δ cells contain abnormal number and reduced size of LDs and their triacylglycerol (TAG) is reduced to 50% of the normal level. Deletion of either UBX or UBA domain in Ubx2 has no effect, but deletion of both causes LD phenotypes similar to that in ubx2Δ. The reduced TAG in ubx2Δ is likely due to mislocalization of Lro1, one of the required for TAG synthesis, that normally resides at the putative LD assembly sites abutted LDs. The mammalian Ubxd8 expressed in yeast complements the defect of ubx2Δ, implying a functional conservation for these UBX-domain-containing proteins in lipid homeostasis.

1993 Mitochondrial Cardiolipin and Phosphatidylethanolamine have Overlapping Functions in Mitochondrial Fusion in Saccharomyces cerevisiae. A. S. Joshi1, M. N. Thompson1, M. Hüttemann2, M. L. Greenberg1; 1Department of Biological Sciences, Wayne State University, Detroit, MI, 2Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI

The two non-bilayer forming mitochondrial phospholipids cardiolipin (CL) and phosphatidylethanolamine (PE) play crucial roles in maintaining mitochondrial morphology. We have previously shown that these phospholipids have overlapping functions and the loss of both is synthetically lethal. Recent in vitro studies have shown that CL stimulates the GTPase activity of the fusion protein Mgm1p and is required for assembly of its isoforms. Because the lack of CL does not lead to defects in the mitochondrial network, we hypothesized that PE may compensate for CL for maintenance of mitochondrial tubular morphology and fusion. To test this hypothesis, we constructed a conditional double mutant crd1psd1 containing null alleles of CRD1 (lacking CL) and PSD1 (lacking mitochondrial PE), in which the wild type CRD1 gene is present on a plasmid under control of the TET OFF promoter. Our in vivo studies demonstrate for the first time that mitochondrial fusion and maintenance of a mitochondrial tubular network require the presence of CL and mitochondrial PE. The crd1psd1 mutant in the presence of tetracycline exhibited highly fragmented mitochondria, a phenotype similar to that observed in fusion mutants. The crd1psd1 cells further exhibited loss of mtDNA and reduced membrane potential, characteristic of cells devoid of mitochondrial fusion. Deletion of FIS1 or DNM1, which are required for mitochondrial fission, restored the tubular mitochondrial morphology in crd1psd1 cells. Loss of CL and mitochondrial PE led to reduced levels of small and large isoforms of the fusion protein Mgm1p, possibly accounting for the fusion defect. Taken together, these data suggest that mitochondrial phospholipids CL and PE are required to maintain tubular mitochondrial morphology and have overlapping functions in mitochondrial fusion. Defects in mitochondrial fusion may underlie the mitochondrial morphological variation observed in Barth syndrome (BTHS) lymphoblasts and in cardiac and skeletal muscles of the mouse model of BTHS.

1994 CPSAR1, a protein involved in vesicular trafficking inside the chloroplast. N. Z. Khan1, E. Lindquist1, S. Karim1, H. Aronsson11; 1Plant and Environmental Sciences, University of Gothenburg, Gothenburg, Sweden

COPII coated vesicles are involved in the secretory pathway transporting proteins and lipids from the endoplasmic reticulum (ER) to the Golgi. On the basis of Arabidopsis genome sequences and web prediction tools some important components of COPII i.e. the Sec23-Sec24 complex, the Sec13-Sec31 complex, and the small GTPase SAR1 homologues were predicted to be chloroplast localized. It has recently been verified that CPSAR1 is involved in a vesicular SUNDAY transport system inside the chloroplast similar to the transport mechanism that exists between the ER and the Golgi. CPSAR1 has a role in thylakoid biogenesis and in vesicle budding from the inner envelope membrane for fusion with the thylakoids. As this mechanism is similar to the cytosolic vesicular transport, a putative model is proposed in which the CPSAR1 need other protein partners for the vesicular formation and transport. For this purpose we are using co- immunoprecipitation and yeast-2-hybrid for identification of protein-protein interaction involving CPSAR1. Ongoing bioinformatic studies of common motifs important for interactions and cargos of the secretory pathway gives that they also exist for chloroplast localized proteins i.e. possible protein interactors to the chloroplast vesicle traffic system. In addition, we are studying the intraplastidial role of CPSAR1 from isolated chloroplast envelopes in vitro.

1995 A Novel Function of AWP1/ZFAND6: Regulation of Pex5p Export by Interacting with Cys- monoubiquitinated Pex5p and AAA ATPase, Pex6p. N. Miyata1, K. Okumoto1,2, S. Mukai1, M. Noguchi2, Y. Fujiki1,3; 1Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka, Japan, 2Graduate School of Systems Life Sciences, Kyushu University, Fukuoka, Japan, 3JST, CREST, Tokyo, Japan

In biogenesis of peroxisome, a subcellular organelle, Pex5p is the shuttling receptor for peroxisomal matrix proteins harboring peroxisome-targeting signal 1 (PTS1). Several peroxins are involved in the Pex5p shuttling between the cytosol and peroxisomes. However, the precise mechanism underlying the PTS1 receptor shuttling remains elusive. We herein suggest that liver cytosol contains at least two distinct factors involved in the Pex5p export. We isolate one of the factors by biochemical fractionation and in vitro Pex5p export assay and identify it as AWP1/ZFAND6 (termed p40), a ubiquitin-binding NF-κB modulator. In in vitro Pex5p export assay, recombinant p40, stimulates Pex5p export, whilst anti-p40 antibody interferes with Pex5p export. p40 interacts with AAA ATPase Pex6p, but not Pex1p-Pex6p complex. p40 binds Cys- ubiquitinated form of Pex5p more preferentially than unmodified Pex5p, apparently via its A20 zinc-finger domain. RNA interference for p40 significantly affects the PTS1 protein import into peroxisomes. Furthermore, in the p40 knocked-down cells Pex5p is unstable, as in fibroblasts from patients each defective in Pex1p, Pex6p, and Pex26p, all prerequisite to the Pex5p export. Taken together, p40 is a novel of Pex6p involved in the regulation of Pex5p export in peroxisome biogenesis.

1996 Docosahexaenoic acid is required for peroxisomal elongation, a prerequisite for division of peroxisomes. A. Itoyama1, M. Honsho2, Y. Abe2, Y. Yoshida2, Y. Fujiki2,3; 1Graduate School of Systems Life Sciences, Kyushu University, Fukuoka, Japan, 2Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka, Japan, 3CREST, JST, Tokyo, Japan

Peroxisomal division is strictly regulated by several fission factors and cellular environments. In the past decade, metabolic control of peroxisomal morphogenesis is postulated but remains largely undefined. We herein identify docosahexaenoic acid (DHA, C22:6n-3) as the regulator of the peroxisomal morphogenesis. Peroxisomes are much less abundant in fibroblasts from patients defective in peroxisomal fatty-acid β-oxidation. Supplementation of DHA to such fibroblasts induces proliferation of peroxisomes up to the same level in normal fibroblasts. DHA- inducible peroxisomal proliferation is abrogated upon treatment with dynamin-like protein 1 siRNA, suggesting the DHA-induced division of peroxisomes. DHA-induced peroxisomal division is initiated by elongation of peroxisomes in a Pex11p-dependent manner. Furthermore, DHA augments hyper-oligomerization of Pex11pβ, giving rise to Pex11pβ-enriched regions on SUNDAY the elongated peroxisome membrane. Collectively, these findings suggest that DHA is responsible for the elongation step of peroxisomes, the prerequisite stage for the subsequent fission of peroxisomes.

1997 Molecular Basis for Targeting of C-tail-anchored Proteins to Peroxisomes. Y. Yagita1, Y. Fujiki2,3; 1Graduate School of Systems Life Sciences, Kyushu University, Fukuoka, Japan, 2Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka, Japan, 3CREST, JST, Tokyo, Japan

C-tail anchored (TA) proteins are a distinct class of membrane proteins that harbor a single transmembrane domain at the extreme C-terminal region and expose their N-terminal functional domains to the cytosol. Although TA proteins are found in all of subcellular membranes facing the cytosol and play pivotal roles in various biological processes, the pathways by which they are targeted to and inserted into specific organellar membranes, including peroxisomal membranes, are not fully defined. We herein show that knockdown of Pex19p, a predominantly cytosolic protein that functions as a chaperon and/or soluble receptor for newly synthesized peroxisomal membrane proteins (PMPs), eliminates the import of peroxisomal TA proteins (P- TAs) in vivo. Pex19p forms complexes with P-TAs in the cytosol. These results indicate that Pex19p is involved in the import of P-TAs as well as non-TA-type PMPs. We also show that P- TAs, which form complexes with Pex19p, are specifically targeted to peroxisomes, onto Pex3p, even under ATP-depleted condition in an import assay using semi-intact mammalian cells. The targeting of P-TAs to peroxisomes is driven by Pex19p-Pex3p interaction. Collectively, these results strongly suggest that P-TAs are, like most PMPs, targeted to peroxisomes in a Pex19p- and Pex3p-dependent manner. Thus, P-TAs share the import pathway with non-TA-type PMPs, in contrast to the TA proteins directed to the ER and mitochondrial outer membrane that do not share the import pathways with non-TA proteins.

1998 Novel Function of Peroxisome Targeting Signal Type-1 Receptor Pex5p in Pex14p Stability: Study Using a Newly Isolated Peroxisome-Deficient CHO Cell Mutant, ZPEG101. R. Natsuyama1, K. Okumoto1,2, Y. Fujiki1,3; 1Grad. Sch. of Sys. Life Sci. of Kyushu University., Fukuoka, Japan, 2Dept. of Biol., Grad. Sch. of Sci., of Kyushu University., 3JST, CREST

Pex5p is one of the peroxins required for peroxisome biogenesis and plays an important role in peroxisomal matrix protein import. Majority of peroxisomal matrix proteins possess peroxisome targeting signal type-1 (PTS1) at the C-terminus. Pex5p recognizes PTS1 in cytosol via its C- terminal region consisting of seven tetratricopeptide repeats and also interacts with peroxisome membrane peroxins Pex14p and Pex13p via its N-terminal region. Together with extensive studies in yeasts and mammals, Pex5p is now proposed as a shuttling receptor between the cytosol and peroxisomes. Here, we found a novel function of Pex5p in stabilizing Pex14p by making use of a newly isolated ZPEG101, a PEX5-deficient CHO cell mutant, showing typical mutant phenotypes with defects in import of both PTS1 and PTS2 proteins. No Pex5p was detected in ZPEG101, indicating ZPEG101 is the first pex5 CHO cell mutant completely lacking Pex5p. Interestingly, the expressed level of Pex14p was significantly decreased in ZPEG101, as compared to that in wild-type CHO-K1 and other previously isolated pex5 mutants. More detailed analyses of ZPEG101 are in progress.

SUNDAY

1999 P7BP1 E3 ubiquitin controls the quality of PTS2 receptor, Pex7p. Y. Miyauchi1, Y. Fujiki1; 1Kyushu University, Fukuoka, Japan

Peroxisomes are ubiquitous single membrane-bound organelles that contain about 100 different catalyzing various metabolic pathways such as fatty-acid ƒÀ-oxidation and etherglycerolipid synthesis. Peroxisomes are formed by growth and division of preexisting peroxisomes after posttranslational import of newly synthesized peroxisomal matrix and membrane proteins. Two distinct signals, peroxisomal targeting signal type 1 (PTS1) and PTS2, direct proteins to the peroxisomal matrix. PEX5 and PEX7 encode the cytosolic receptors for PTS1 and PTS2, respectively. Mono-Ubiquitination of Pex5p is required for its export from peroxisomal membrane to the cytosol in yeast to mammals. However, regulation of the function and transport of Pex7p remains undefined. We first investigated whether or not Pex7p is ubiquitinated and degraded by proteasomes in cells. Addition of a proteasome inhibitor, MG132 to cell culture delayed the turnover rate of Pex7p. We also attempted to isolate any potential Pex7p-binding proteins potentially involved in the degradation pathway of Pex7p. From a cell line stably expressing FLAG-Pex7p, we isolated several Pex7p-binding proteins, termed P7BPs: Pex7p binding proteins. Suppression of P7BP1 by RNAi resulted in a delay of Pex7p turnover rate, as observed in the MG132-treated cells, hence implying that P7BP1 plays a role in Pex7p ubiquitination. Furthermore, we also show that the degradation of dysfunctional Pex7p mediated by P7BP1 is crucially required for the maintenance of normal PTS2 import. Our findings may define a mechanism underlying Pex7p degradation and its importance in regulating PTS2 import.

2000 A System to Quantify the Differential Import of Peroxisomal Matrix Protein by Measuring Fluorescence Intensity. M. Noguchi1, Y. Fujiki1,2; 1Graduate School of Systems Life Sciences, Kyushu University, Fukuoka, Japan, 2CREST, JST

Fourteen distinct peroxins are identified as essential factors for peroxisome biogenesis in mammals, of which ten are involved in peroxisomal import of matrix proteins. Peroxisomal matrix protein import is regulated by various cellular factors. However, mechanisms underlying such regulations are poorly understood, partly, if not all, due to the lack of quantitative detection method with high resolution. Here, we developed a monitoring system that utilizes the differences in the stability of fluorescent reporters localized between peroxisomes and the cytosol. An FKBP12 variant, termed destruction domain (DD), is rapidly and constitutively degraded by proteasomes when expressed in mammalian cells. Degradation of DD is reversibly protected by addition of a specific synthetic ligand. In the absence of the ligand, a reporter DD- EGFP-PTS1, EGFP fused with the DD and peroxisomal targeting signal 1, is largely degraded in the cytosol prior to entering peroxisomes in wild-type cells. In contrast, in the presence of the ligand the reporter becomes stable and translocates into peroxisomes. Upon withdrawal of the ligand, the reporter in peroxisomes remains intact, whilst that in the cytosol is rapidly degraded. Thus, peroxisomal protein import can be readily quantified by simply measuring the fluorescence intensity of whole cells.

SUNDAY

2001 Molecular architecture of OPA1, the dynamin-related GTPase involved in mitochondrial fusion. A. M. Fontainhas1, J. A. Heymann1, S. Fang1, J. E. Hinshaw1; 1LCBB, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD

Mitochondria, the powerhouse of the cell, continually undergo fission and fusion. OPA1 (Optic atrophy 1) - a dynamin-related GTPase is essential for the fusion of the inner mitochondrial membrane and is involved in cristae maintenance. Once OPA1 is transported to the mitochondria, OPA1 is proteolytically processed into a long and a short form. The long form of OPA1 is anchored to the inner membrane of the mitochondria through a N-terminal transmembrane domain, while the short form of OPA1 (OPA1s) is found in the inner mitochondrial space. Both forms are required for mitochondrial membrane fusion in vivo. Over 200 mutations of OPA1 have been identified that lead to vision loss, mainly through retinal ganglion cells. Ban, et al. (2010) showed that OPA1s preferentially tubulates and clusters large, negatively charged liposomes. However, the short form alone was unable to mediate membrane fusion. It has been proposed that tubulation may be the mechanism by which fusion of mitochondrial membranes occur. To elucidate OPA1’s 3D structure and how it interacts with lipids we used cryo-electron microscopy to visualize and analyze the topological arrangement of OPA1 on these protein-lipid tubules. We show that OPA1s wraps around the perimeter of these tubes in a helical fashion. We observed that tubulation is not GTP dependent. However, GMPPCP stabilizes the chevron like pattern. OPA1s also forms an organized 2D protein lattice, indicating that specific intermolecular interactions are at play. Tubes, under all conditions, range in diameter from 50 to 150 nm with a majority of them falling in the 60-70 nm range. Understanding how OPA1’s interactions with the membrane leads to fusion of inner mitochondrial membrane will give us a better understanding of how mutations in the protein affect its function and give rise to vision loss. Reference: Ban, T., Heymann, J. A., Song, Z., Hinshaw, J. E., Chan, D. C.: OPA1 disease alleles causing dominant optic atrophy have defects in cardiolipin-stimulated GTP hydrolysis and membrane tubulation. Hum. Mol. Genet. 2010, 19: 2113-22.

2002 Probing the dynamics of single lipids in supported lipid bilayers and living cells by fluorescence correlation spectroscopy and confocal microscopy. C-F. Hsieh1, L-L. Yang1, Y-L. Lin2, C-F. Chou1; 1Institute of Physics, Academia Sinica, Taipei, Taiwan, 2National Taiwan University, Institute of Chemistry, Taiwan

Lipid raft served as a platform for recruiting signaling components of effective signal transduction involving various cellular activities, such as cell proliferation, differentiation, migration, and apoptosis in living cells. However, the single-lipid dynamics of lipid raft or non- lipid raft domain in living cells are not well elucidated.

We apply fluorescence correlation spectroscopy (FCS) to probe the mobility of single lipids. We observed the behaviors of non-raft lipids and raft lipids in supported lipid bilayers and found that the diffusion constant of raft lipids (~2.4 × 10-8 cm2/s) is much smaller than non-raft lipids (~8.1 × 10-8 cm2/s) and spans a wide range. These results suggest that the raft lipids are in a trend to aggregate in supported bilayers while non-raft lipids are not.

Confocal microscopy was used to monitor the dynamics of raft lipids induced by ligand-receptor binding in living cells. The RGD-coated quantum dots (Qdots) were recognized and bound to SUNDAY integrin to trigger the aggregation of lipid raft. The duration of lipid-raft aggregation was extended from 10 minutes (induced by ligand-coated micro-bead) to 2 hours (induced by ligand- coated Qdot), and finally the aggregate reached a diameter around 5 micrometers. In our studies, RGD-coated Qdot bound to a small number of integrin proteins may provide an effective means to observe the early events of lipid raft aggregation in a living cell.

2003 Molecular Mechanism of F-BAR Protein Pacsin2 in Caveolae Biogenesis. Y. Senju1, S. Suetsugu1; 1Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan

Protein kinase C (PKC) and casein kinase substrate in neurons 2 (Pacsin2) belongs to the EFC/F-BAR domain and deforms membrane. The N-terminal F-BAR domain of pacsin2 forms a crescent-shaped dimer with a positively charged surface. The C-terminal SH3 domain associates with dynamin2, a GTPase implicated in vesicle endocytosis. Therefore, pacsin2 is involved in membrane invaginations including caveolae (Senju et al., 2011). Previous study has demonstrated that the cells overexpressing pacsin2 showed membrane invaginations. However, the number of membrane invagination was larger in the cells overexpressing pacsin2 F-BAR domain than in the cells overexpressing pacsin2 full length, which was interpreted as autoinhibition of pacsin2 resulted from the intramolecular interaction between F-BAR domain and SH3 domain. Because pacsin2 has been identified as a PKC substrate, we investigated the autoinhibition mechanism based on the phosphorylation of pacsin2 by PKC. First, we performed an in vitro kinase assay, and confirmed that PKC actually phosphorylated pacsin2. Then, we generated two different pacsin2 mutants by substitution with either alanine or glutamic acid, and identified the phosphorylation site in pacsin2 by in vitro kinase assay. Next, we generated a phospho-specific antibody, and confirmed that endogenous pacsin2 was phosphorylated after the PKC activator PMA treatment using immunofluorescence microscopy and western blotting analysis. The number of membrane invaginations induced by phospho-mimic mutant of pacsin2 was lesser than that induced by wild-type pacsin2. After the PMA treatment, the number of membrane invagination was decreased in the cells expressing wild-type pacsin2, but there was no significant effect in the cells expressing either phospho-mimic, phospho-deficient mutant, or F-BAR domain of pacsin2. Therefore, these data suggest that phosphorylated pacsin2 prefers an open protein conformation that permits interaction with its partners such as dynamin2. The interaction of pacsin2 with dynamin2 probably caused dynmin2-induced scission of membrane invaginations, which in turn decreased the number of pacsin2-induced membrane invaginations. Further experimentation will be required to test this hypothesis and reveal the physiological role of pacsin2 in caveolae biogenesis.

2004 Identification and characterization of atypical FYVE Domains for specific interaction with phospholipids. J-E. Gil1, I-S. Kim2, B. Ku1, W. Park1, B-H. Oh1, S. Ryu2, W. Heo1; 1Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2Pohang University of Science and Technology, Korea

Interactions of lipid binding domains with intracellular lipids play a significant role in regulation of numerous signaling and cellular mechanisms. To characterize interactions of lipid binding domains with phospholipids, five classes of lipid binding domains were screened by fluorescent imaging and classified based on their subcellular localizations. Six of over one hundred kinds of lipid binding domains were localized in the plasma membrane where a number of signaling cascades are generally initiated. Interestingly, we found FYVE domain of ZFYVE27 which is SUNDAY localized in the plasma membrane unlike typical FYVE domains which are localized in the endosomes. Conventionally, it has been known that lipid binding pocket of FYVE domains has WXXD, R+HHC+XCG and RVC regions through which FYVE domains bind to PI(3)P. However, from analysis of amino acid sequences and model of 3D protein structure, we found that ZFYVE27-FYVE domain did not contain WXXD, R+HHC+XCG and RVC regions, reflecting it may have unconventional property in lipid interaction of FYVE domain. It was demonstrated that ZFYVE27-FYVE domain specifically interacts with plasma membrane phospholipids by using inducible strategies in which phospholipids are depleted by translocating phosphatase to the plasma membrane and specific inhibitor treatment. We also observed that growth factor-induced ZFYVE27 translocation was not induced when this interaction was ruined.

2005 Plin 2 and Plin 3 exhibit characteristics of functionally diverse C-terminal domains. B. Chong1,2, L. Cicchini1,2, P. Reigan3, J. McManaman1; 1OB/GYN, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, 2Graduate Program in Molecular Biology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, 3Pharmaceutical Science, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO

Perilipin family members, adipophilin (ADPH/Plin2) and TIP47 (Plin3) have the highest degree of sequence similarity within the perilipin family, with nearly 60% similarity, and are thought to be redundant lipid droplet associated proteins. Our laboratory previously demonstrated that the N-terminal half of ADPH is required for association with the CLD monolayer and this interaction protects the CLD core from lipolysis. More recent studies indicate that the C-terminal half of ADPH is a lipid membrane association domain that is required for milk lipid secretion. TIP47 was originally identified as a vessicle trafficking protein but evidence supporting this is controversial. Additionally, less is known about the unique functional domains within TIP47. In mammary epithelial cells, the two family members have distinct cellular localizations and differential expression patterns over the course of pregnancy and lactation, which suggest that the two proteins are not functionally redundant. We took a structural and biochemical approach to understanding how these two proteins are functionally distinct. ADPH and TIP47 share 43% amino acid similarity in their C-terminal domain and are hypothesized to be structural homologs. The C-terminus of TIP47 has been crystallized and is known to form a four-helix bundle motif with a centrally located hydrophobic cleft motif. We constructed a homology model for the C- terminus of ADPH, which shows that ADPH forms a putative four-helix bundle and hydrophobic cleft, similar to that of TIP47. Closer examination of these structures shows that these two proteins have noticeable differences in overall shape, with TIP47 being more compact and ADPH having a slightly more extended in structure. We expressed and purified recombinant ADPH and TIP47 C-termini from E. coli and analytical size exclusion chromatography (SEC) shows that the two proteins have different retention times, suggestive of differences in overall shape. Additionally, native PAGE comparison shows differences in migration between the two proteins and unique sensitivies to urea denaturation. Analytical ultracentrifugation sedimentation velocity and SEC suggest that ADPH forms higher order structures and TIP47 does not. We provide the first side-by-side comparison of ADPH and TIP47 and show that they are not structurally identical. Further work is needed to determine how these structures account for functional diversity.

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2006 Perilipin, Moesin and Microtubule Dependence of Lipid Droplet Clustering and Dispersion. D. Orlicky1, A. Stefanski2, B. Chong2, J. Monks2, C. Monks3, J. L. McManaman2; 1Pathology, UC Denver, Anschutz Medical Campus, Aurora, CO, 2Ob/Gyn, UC Denver, Anschutz Medical Campus, Aurora, CO, 3Intelligent Imaging Innovations, Inc., Denver, CO

Cytoplasmic lipid droplets (CLD) function in storage, trafficking and secretion of lipids as well as a depot for fatty acid signaling precursors. Perilipin 1, the primary CLD binding protein in adipocytes, is thought to contribute to CLD trafficking by directing fragmentation of CLD into smaller droplets, making the lipids more enzymatically available. To study the role of perilipins in mediating intracellular CLD trafficking we used stable ectopic expression of perilipin 1, or truncated perilipin 1, in HEK293 cells. Expression of either full-length or C-term perilipin 1 (amino acids 198-517) initiated formation of single, dense CLD clusters that localize near the microtubule organizing center (MTOC). Using live-cell imaging, we showed these clustered CLD became fragmented and moved to the periphery of the cell in response to isoproterenol or forskolin activation of adenylate cyclase- which has previously been shown to regulate PKA- mediated phosphorylation of perlipin 1. We further show that nocodozole disruption of microtubules could inhibit this process, while staurosporine blockage of PKA activity disrupted dispersion, but not the initial fragmentation of the CLD cluster. Finally, siRNA knockdown of the cortical actin-binding protein, moesin, prevented dispersed CLD from reforming dense clusters when isoproterenol was removed. These data suggest a model in which CLD clustering and dispersion are mediated by both microtubules and actin, and trafficking is controlled by PKA- phosphorylation of perilipin 1.

2007 Netrin-1-induced axon branching of cerebral cortical neurons requires DCC and lipid raft integrity. H. Matsumoto1, M. Nagashima1; 1Department of Anatomy, Faculty of Medicine, Saitama Medical University, Saitama, Japan

A multifunctional axon guidance cue netrin-1 is known to induce axon branching in developing cerebral cortical neurons of mammals. In this study we investigated whether netrin-1-induced cortical axon branching involves DCC (deleted in colorectal cancer; one of known receptors for netrin-1) and lipid rafts (cholesterol- and sphingolipid-enriched membrane microdomains), employing dissociated cortical neurons prepared from neonatal Syrian golden hamsters. Stimulation of neurons by netrin-1 caused an increase in the number of branch points along the primary axons, which is in consistent with previous reports. We then observed that this increase was attenuated by application of a neutralizing antibody against DCC to the culture. Attenuation of netrin-1-induced axon branching was also caused by disruption of lipid rafts, while association of DCC to lipid rafts was revealed by Western blot analysis. These data indicate a significant contribution of DCC to, and requirement of lipid raft integrity for, the promotion of cortical axon branching caused by netrin-1. Furthermore, the data suggest the possibility that association of DCC with lipid rafts is involved in netrin-1-induced axon branching. (Supported by Grant-in-Aid for Scientific Research (C) No. 23590225 from Japan Society for the Promotion of Science.)

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2008 HGSNAT mono-multimer formation revealed by live-cell two-photon FRET-FLIM microscopy. F. Majer1, J. Sikora1, J. Lukas1, D. Musalkova1, O. Chernyavskiy2, M. Hrebicek1; 1Inst Inherited Metabolic Disorders, Charles University, Prague 2, Czech Republic, 2Department of Biomathematics, Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i., Prague 2, Czech Republic

Human HGSNAT (Acetyl-: Alpha-Glucosaminide N-) is a membrane enzyme localized in endosomal/lysosomal microdomains catalyzing transmembrane acetylation of heparan sulfate. This synthetic modification is mandatory prerequisite for subsequent intralysosomal degradation of heparan sulfate. Mutations in HGSNAT gene cause rare but clinically serious lysosomal storage disorder - mucopolysaccharidosis IIIC (Sanfilippo C syndrome). Our aim was to find intracellular membrane protein partners of HGSNAT. We have prepared plasmids for transient expression of HGSNAT with two alternative starting ATG codons as well as plasmids coding potentially interacting protein partners of HGSNAT. To detect in vivo protein-protein interactions, we used live two-photon excitation FRET-FLIM time correlated single photon counting technique with EGFP/mCherry donor-acceptor pairs. Additionally, we also prepared fraction of enriched lysosomal membranes using density gradient ultracentrifugation and by 2D electrophoresis (Blue Native / SDS PAGE) we identified region of HGSNAT protein electrophoretic mobility and controlled protein integrity. Basic co-localizing experiments implied LAMP1, LAMP2 and Rab7 their intracellular co- occurrence with HGSNAT by resolution limited light microscopy. Nevertheless two-photon FRET-FLIM methodology revealed only HGSNAT-EGFP and HGSNAT-mCherry as potential interacting partners suggesting mono-multimer formation within the endosomal/lysosomal membrane. This study was funded by the research project MSM0021620806 from the Ministry of Education, Youth and Sports of the Czech Republic and by the Internal Grant Agency of the Ministry of Health of the Czech Republic, Project IGA MZ NS / 10342 - 3 / 2009.

2009 Mechanism underlying the expression of Na-H Exchanger isoforms 1 and 3 in SHR rat colon and ileum in hypertension. A. A. Ghefreh1, I. Khan2, A. A. Ghefreh1; 1Biochemistry, Kuwait University, Kuwait, 2Kuwait University, Kuwait

Ileum and colon play an important role in regulating water and electrolyte homeostasis mechanisms which are deregulated in hypertension. Role of colonic and ileal Na-H exchanger (NHE) isoforms-1 and -3 which regulate water and electrolyte absorption from GI-tract and kidneys is poorly investigated in hypertension. In this study we investigated a putative role of colonic and ileal NHE-1 and -3 isoforms in hypertension using a 12-week old spontaneously hypertensive rat (SHR) model. A group of SHR animals was treated with captopril (300 mg/L) ad libitum starting week 4 post birth and followed till week 12. Age matched genetic counterparts, WKY animals served as controls in this study. Further to elucidate the underlying mechanism, expression of NHERF-1 and cGKII which regulate activity of NHE was also examined. SHR animals showed a significant increase in kidney and heart hypertrophy and proteinuria, while decrease in urine output as compared to WKY controls. These changes were reversed by captopril treatment. There was significant elevation in myeloperoxidase activity in SHR ileum and colon as compared to WKY controls. These changes were reversed by captopril treatment. Expression of NHE-1, NHE-3 and NHERF-1 proteins was significantly decreased, while that of SUNDAY cGKII was increased in SHR ileum and colon as compared to WKY controls. Captopril treatment of SHR caused a partial reversal of these changes, and a significant reversal of NHERF-1. These changes do not seem to be due to any difference in the microsomal protein yield. We demonstrate evidence of mild inflammation in SHR colon and ileum due to hypertension. Our findings showed decreased expression of NHE-1, -3 and NHERF-1, which together would suppress uptake of water and NaCl from the GI-tract and hence may be a counteracting response in the development of hypertension in this model. Acknowledgments: Kuwait University Research administration (grant YM 06/2010) and College of Graduate Studies for financial support and Shared Core Facility, Health Science Center, Kuwait University for allowing to use the instrumentation.

2010 Gustatory receptors not G protein coupled receptors are required for the response to L- canavanine. Y. Lee1, M. Kang2, J. Shim3, C. Cheong2, S. Moon2, C. Montell1; 1Department of Biological Chemistry and Neuroscience, Center for Sensory Biology, The Johns Hopkins University School of Medicine, 2Department of Oral Biology, Brain Korea21 project, Yonsei University College of Dentistry, Seoul, Korea, 3Department of Phamacology, Yonsei University College of Medicine, Seoul, Korea

Insects respond to their surroundings through sensing chemical cues and then decide their behaviors. Gustatory sense is one of the most important chemical sensory systems. There are 68 gustatory receptors (GRs) encoded by 60 gustatory receptor genes. Several gustatory receptors are known to be required for sensing of sugars or bitter chemicals, such as caffeine. However, the functions of most GRs are unknown. Here we show that Gr8a and Gr66a are required for sensing insecticide, L-canavainine, which is produced by plants. Gr8a is expressed in a subset of Gr66a expressing gustatory receptor neurons (GRNs). The action potentials to L- canavanine are detected only in the Gr8a expressing neuron. The consistence of Gr8a expression pattern and electrophysiological response to L-canavanine suggests the possibility that Gr8a is the specific receptor to L-canavanine, while Gr66a is the broad receptor to bitter chemicals. In contrast, DmXR, one of the G-protein coupled receptors (GPCRs), has been reported to be required for detecting L-canavanine. However, DmXR missing flies show the avoidance to L-canavanine and action potentials to L-canavanine as wild type flies do in our results. These results imply that Gr8a and Gr66a are indispensible for detection of L- canavanine, but not DmXR.

2011 NHERF- 1 Modulates Intestinal NaPi transporter NaPi-2b expression in Apical Microvilli. H. Giral1, Y. Caldas1, L. Lanzano2, E. Gratton2, M. Levi1; 1University of Colorado Denver, Aurora, CO, 2University of California Irvine, CA

The regulation of phosphate (Pi) homeostasis is maintained by the coordinated function of the renal and intestinal phosphate transporters. Several PDZ (PSD-95/discs large/ZO-1 homologous) domain proteins, including NHERF-1, PDZK1, ShanK2, and PIST play an important role in the regulation of the renal sodium-phosphate (NaPi) co-transporters (NaPi-2a and NaPi-2c). The main mediator of intestinal sodium dependent transcellular Pi transport, NaPi-2b, also contains a PDZ-binding motif consensus in the C-terminal region. However, interactions of the transporter NaPi-2b with PDZ proteins have been not described and their potential role in regulation of the intestinal transporter is not known. For this purpose we performed studies with knock-out (KO) mice models and cell culture to determine a potential role for NHERF-1 and PDZK1 in the regulation of NaPi-2b. SUNDAY

To study the putative interaction between NaPi-2b and PDZ proteins we determined the Forster Resonance Energy Transference (FRET) by using Fluorescence Lifetime Imaging Microscopy (FLIM). OK cells, an extensively used proximal tubule model, and CaCo-2BBE cells, an enterocyte cell model, were used to perform this technique. First, expression of EGFP-NaPi-2b was confirmed in the microvilli of both cell types, and images along a single microvillus were obtained with the novel Modulation Tracking (MT) method. Cells co-expressing EGFP-NaPi-2b and mCherry-NHERF-1 were analyzed by FLIM-FRET technique revealing significant FRET between NaPi-2b and NHERF-1. Parallel studies between the pair NaPi-2b and PDZK1 proteins resulted in non occurrence of FRET. To evaluate the functional significance of these results we study NaPi-2b expression and activity in NHERF1 KO and PDZK1 KO mice models, where we found that adaptation to a low Pi diet of NaPi2b was markedly impaired in the NHERF-1 KO mice but not in the PDZK1 KO. Our results therefore suggest an important role of NHERF1 in modulation of NaPi-2b expression or stability in the microvilli of the mouse intestine. This research was supported by NIH R01 DK066029 to YC, HG, ML, EG and LL; NIH445 P41R03155 to EG and LL; and the R01 DK-080769 to BD.

2012 Cytoskeleton modulates Ca2+ sensor Stim1. C-M. Li1, M. Joensuu1, E. Jokitalo1; 1Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki, Helsinki, Finland

Stim1 is a 685 amino acids, single-pass transmembrane protein with N-terminal EF hand located in the endoplasmic reticulum (ER) lumen and C-terminal coiled-coil domains facing cell cytosol. As a sensor Stim1 plays essential role in Ca2+ signalling, Store-operated Ca2+ entry (SOCE) channel activation, and influx of Ca2+ in response to depletion of ER luminal Ca2+. Depletion of intracellular Ca2+ stores results in dissociation of Ca2+ from EF-hand, thus activating store-operated channels, and trigger translocation of STIM1 proteins to the plasma membrane [1].

Rosado and his colleagues reported in their recent study that: cytoskeleton regulates the interaction between Stim1 and the plasma membrane SOCE channel components such as Orai1 and TRPCs. In Human Embryonic Kidney (HEK-293) cells, disruption of microtubules enhanced both the activation of SOCE and the association between STIM1 and Orai1 or TRPC1 induced by thapsigargin (TG). Conversely, stabilization of microtubules or actin filaments attenuated both TG evoked activation of SOCE and the interaction between STIM1 and the channel components. However, disruption of the actin filament network did not affect TG-evoked association between STIM1 and Orai1 or TRPC1 but enhanced TG stimulated SOCE [2]. They concluded that the microtubules act as a negative regulator of SOCE.

We study the location of Stim1 in association with disruption/stabilization of microtubule/actin at immunofluorescence microscopy and immuno electron microscopy level with HEK-293 cells and hepatocarcinoma Huh-7 cells. We show that Stim1 proteins translocate and aggregate on the plasma membrane upon Calcium depletion by TG treatment.

References: Zhang SL, Yu Y, Roos J, Kozak JA, Deerinck TJ, Ellisman MH, et al. STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane. Nature 2005;437:902–5. SUNDAY

Galán C, Dionisio N, Smani T, Salido GM, Rosado JA. The cytoskeleton plays a modulatory role in the association between STIM1 and the Ca2+ channel subunits Orai1 and TRPC1. Biochem Pharmacol. 2011;82:400-10.

Nuclear Structure and Function

2013 Proteostatic control of nuclear targeting pathways by the nuclear pore complex protein, Nup42. L-K. LIU1, C-C. LIN1, D. S. Goldfarb1; 1Biology, University of Rochester, Rochester, NY

Multiple transport receptors ferry nuclear localization signal (NLS)-bearing cargos across the nuclear pore complex (NPC). The FG-domains of nucleoporins in the NPC are thought to be required in specific combinations for efficient transport receptor-mediated transports. A model of multiple independent NPC translocation routes exist for different transport receptors was presented. Based on this model, direct competition for the same translocation pathway shared by multiple transport receptors can be expected. In this study, we have found that Nup42 governs a unique stress response that differentially controls import mediated by Kap95/60 and Kap104. In vivo kinetic analyses show that Kap95/60 and Kap104 compete for limiting docking sites at Nup42. Binding to Nup42 is the rate-limiting step in import, since the import rates of Kap95/60 and Kap104 NLS-cargos are faster in nup42 DFG cells. Nup42 collaborates with the Hsp70s Ssa1/Ssa2 (but not Ssa3/Ssa4) and the co-chaperone Swa2 to differentially control nuclear targeting by Kap95/60 and Kap104. Specifically, Kap95/60 import is favored over Kap104 import when Ssa1/Ssa2 levels are high, such as in response to heat shock or nutrient depletion. In contrast, Kap104 import is favored when the free Hsp70 pool is depleted, for example, by sequestration into Huntingtin polyQ protein aggregates. Kap123-mediated import rates are unaffected by this regulatory circuit. Here we present the first evidence that two specific transport receptors, Kap95/60 and Kap104, share and compete for similar translocation route in the NPC. We also conclude that Nup42 is a Kap-specific gatekeeper that controls import rates in response to proteostatic stress.

2014 Evidence of asymmetric assembly pathway of Nuclear Pore Complex. P. Colombi1, C. Vogel1, P. Lusk1; 1Department of Cell Biology, Yale School of Medicine, New Haven, CT

In an effort to investigate regulatory mechanisms governing nuclear pore complex (NPC) assembly, we investigated alterations in the subcellular distribution of multiple nucleoporins (Nups) through the cell cycle in the budding yeast S. cerevisiae. Consistent with the idea that NPC assembly is under the control of cell cycle regulators, we observe that several specific Nups accumulate in the cytoplasm of cells arrested in S-phase but not in G1. Interestingly, these likely NPC intermediates accumulate asymmetrically between the mother and bud and appear to be strikingly biased towards the bud, often accumulating at the bud cortex. These intermediates can often be observed integrating into the daughter cell nuclear envelope during anaphase supporting the idea that there is a pathway in which Nups are specifically targeted to the bud to be incorporated in the NPC of the daughter cell. Perturbing this pathway by conditionally sequestering specific newly synthesized Nups results in a decrease in the size of daughter nuclei. Most strikingly, these daughter cells are no longer able to progress through the cell cycle, whereas the mother cells continue to divide. Together these results support the SUNDAY existence of a pathway critical for continued daughter cell proliferation linked to the assembly of NPC.

2015 Temporal Control of Nuclear Envelope Assembly by Phosphorylation of Lamin B Receptor. L-C. Tseng1, R-H. Chen1; 1Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan

The nuclear envelope of metazoans disassembles during mitosis and reforms in late anaphase after sister chromatids have well separated. The coordination of these mitotic events is important for genome stability, yet the temporal control of nuclear envelope reassembly is unknown. Although the steps of nuclear formation have been extensively studied in vitro using the reconstitution system from egg extracts, the temporal control can only be studied in vivo. Herein, we use time-lapse microscopy to investigate this process in living HeLa cells. We demonstrate that Cdk1 activity prevents premature nuclear envelope assembly and that phosphorylation of the inner nuclear membrane protein lamin B receptor (LBR) by Cdk1 contributes to the temporal control. We further identify a region in the nucleoplasmic domain of LBR that inhibits premature chromatin binding of the protein. We propose that this inhibitory effect is partly mediated by Cdk1 phosphorylation. Furthermore, we show that the reduced chromatin-binding ability of LBR together with Aurora B activity contribute to nuclear envelope breakdown. Our studies reveal for the first time a mechanism that controls the timing of nuclear envelope reassembly through modification of an integral nuclear membrane protein.

2016 Amphiregulin, an EGF family protein localized to the nuclear envelope, activates directional cell migration via histone H3K9 methylation. M. Hieda1, Y. Yokoyama1, Y. Nishioka1, H. Tanaka1, S. Higashiyama2, N. Matsuura1, S. Matsuura1; 1Graduate School of Medicine, Osaka University, Suita, Japan, 2Proteo Medicine Research Center, Ehime University, Toon, Japan

EGF family members are expressed on the cell surface in pro-form, comprising an extracellular EGF-like domain, a transmembrane segment, and a short cytoplasmic tail. The EGF-like domain is sufficient to activate the EGF receptor, whereas the function of the cytoplasmic tail remains unknown. Previously we showed that in the case of one EGF family member, amphiregulin (AREG), shedding stimuli result in release of soluble EGFR ligand and migration of the pro-form (proAREG) to the nuclear envelope (NE) and induction of global histone H3K9methylation. Here we found that NE-localized proAREG activates cell migration. This activation was dependent on the cytoplasmic tail of proARG but independent of the extracellular growth factor domain. The activation of cell migration did not require EGFR function, but did occur concomitantly with a slight increase in a specific histone modification, histone H3 lysine9 methylation (H3K9me3). Expression of the H3K9 methyltransferase SUV39H1 activated cell migration; inversely, knockdown of SUV39H1 or chemical inhibition by chaetocin reduced cell migration. Chaetocin also suppressed the cell migration activated by NE-localized proAREG. Moreover, impairment of H3K9me3 suppressed the proper formation of cell polarity and led to a reduction in directional cell movement. Thus, NE-localized proAREG regulates cell migration via histone modification, indicating that chromatin organization influences both cell polarity and motility.

SUNDAY

2017 Samp1 is functionally associated with the LINC complex and A-type lamina networks. S. Gudise1, R. Figueroa2, V. Larsson3, E. Hallberg3; 1Department of Biosciences and Nutrition, Karolinska Institute, Sweden, 2Department of Biochemistry and Biophysics, Stockholm University, 3Department of Neurochemistry, Stockholm University, Stockholm, Sweden

The transmembrane inner nuclear membrane (INM) protein Samp1 is required for anchoring centrosomes near the nuclei. Using highresolution fluorescence microscopy we show that Samp1 is distributed in a distinct and characteristic pattern in the nuclear envelope (NE), where it partially colocalizes with the LINC complex protein Sun1. By studying the localization of Samp1 deletion mutants and fusion proteins, we conclude that the cysteine-rich N-terminal half of Samp1 is nucleoplasmically exposed and is responsible for targeting to the INM. It contains four conserved CxxC motifs with the potential to form zinc fingers. The distribution of cysteine-toalanine substitution mutants, designed to prevent zinc finger formation, showed that NE localization of Samp1 depends on intact CxxC motifs. Overexpression of Samp1 zinc finger mutants produced an abnormal dominant phenotype characterized by disrupted organization of a selective subset NE proteins, including emerin, Sun1, endogenous Samp1 and, in some cases, lamin A/C, but not lamin B, Sun2 or nucleoporins. Silencing of Samp1 expression showed that emerin depends on Samp1 for its correct localization in the NE. Our results demonstrate that Samp1 is functionally associated with the LINC complex protein Sun1 and proteins of the A-type lamina network.

2018 NE localization of the LINC complex requires multiple elements of SUN and KASH proteins. Y. G. Turgay1, R. Ungricht2, A. Rothballer2, A. Kiss2, G. Csucs2, P. Horvath2, U. Kutay2; 1Institute of Biochemistry, ETH Zurich, Zurich, Switzerland, 2ETH Zurich, Zurich, Switzerland

Inner nuclear membrane (INM) proteins are co-translationally inserted into the endoplasmic reticulum membrane (ER) and need to cross the nuclear pore complex (NPC) to reach their final destination. Originally, targeting of INM proteins to the INM was solely explained by a “diffusion- retention” model. However, recent studies on yeast INM proteins revealed the existence of classical nuclear localization signals (cNLS) that mediate the active transport of the proteins across the NPC. Whether active transport mechanisms also play a role in the targeting of INM proteins in mammalian cells is still unclear. We have used human SUN2 to study INM targeting and identified three different elements, which collectively mediate NE targeting. The N-terminal nucleoplasmic domain of SUN2 comprises a cNLS and a Golgi retrieval signal. The cNLS was shown to constitute a functional binding site for the heterodimeric transport receptor importin α/β. A nearby arginine cluster was found to serve as binding platform for the coatomer complex, which mediates retrieval of SUN2 from the Golgi to the ER and ensures efficient INM targeting. The conserved SUN domain at the C terminus represents the third targeting element we have identified in our study. Together, our study shows that multiple elements in SUN2 contribute to NE targeting and that these elements are not limited to cytoplasmic or transmembrane domains.

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2019 LINC complexes mediate the positioning of cone photoreceptor nuclei in mouse retina. D. Razafsky1, S. Zang1, D. Hodzic1; 1Ophthalmology and Visual Sciences, Washington University School of Medicine, St Louis, MO

LINC complexes form through the interaction between inner nuclear membrane Sun proteins (Sun 1 and Sun 2) and outer nuclear membrane Nesprins (Nesprin 1, 2, 3 and 4). The Suns and Nesprins interact within the perinuclear space thereby connecting the interior of the nucleus to the cytoplasm. In C.elegans and D. melanogaster, mutation of Sun and Nesprin orthologs prevent nuclear anchorage and/or migration within cells or syncitia. Mouse models deficient for both Sun1 and Sun2 expression display abnormal migration of cortical neurons and thereby phenocopies human lissencephaly phenotypes associated with mutations in Lis1 and Dcx. Our goal is to identify the role of the LINC complexes in mammalian tissue development and homeostasis. To this end, retinal development provides an excellent model for two reasons: 1) different types of nucleokinetic events such as interkinetic nuclear migration and postmitotic neuronal migration can be studied and 2) several waves of neuronal migration culminate in the development of a stratified tissue whereby nuclei of various cell types are anchored specific spatial positions. Here, we characterized the expression of LINC complex components during mouse retinal development and present a new versatile transgenic mouse model allowing for the conditional expression of a dominant negative EGFP-KASH2 protein. Using this model, we disrupted LINC complexes within the photoreceptor layer of mouse retina and report the ectopic positioning of cone photoreceptor nuclei. These studies show, for the first time, the relevance of LINC complexes in the positioning of cell nuclei in a mammalian CNS tissue.

2020 Molecular Basis of the SUN-KASH interaction. B. A. Sosa1, A. Rothballer2, U. Kutay2, T. U. Schwartz1; 1Biology, MIT, Cambridge, MA, 2ETH Zurich, Switzerland

The position of the nucleus within the eukaryotic cell is defined. To achieve specific anchorage and movement nucleoskeleton and cytoskeleton are mechanically connected across the double- layered membrane of the nuclear envelope. These bridges, also called LINC-complexes, are formed by KASH-peptide containing proteins spanning the outer nuclear membrane tightly bound to SUN-domain proteins spanning the inner nuclear membrane. We present the crystal structure of the SUN domain of human Sun2 in complex with the KASH peptides of Nesprin-1 and -2, respectively. The unexpected structure reveals several key features of this novel interaction that help explain how it can work as the mechanical tether it is proposed to be.

2021 Building SUN-KASH Complexes at the Nuclear Envelope. A. Rothballer1, B. A. Sosa2, T. U. Schwartz2, U. Kutay1; 1Institute of Biochemistry, ETH Zurich, Zurich, Switzerland, 2Department of Biology, Massachusetts Institute of Technology, Cambridge, MA

Physical connections between the nuclear envelope and the cytoskeleton are an essential feature of eukaryotic cells. They are required for diverse biological processes such as nuclear anchorage and migration, or homologous chromosome pairing. Nucleo-cytoskeletal connections are built by SUN and KASH domain proteins, transmembrane proteins of the inner and the outer nuclear membrane, respectively. A key element lies in the perinuclear space where the conserved SUN and KASH domains form a complex. Thereby, SUN-KASH protein assemblies bridge the entire nuclear envelope to connect nuclear structures with the cytoskeleton. SUNDAY

We set out to identify the determinants of SUN-KASH interaction and to understand complex formation in molecular detail. Using pulldown experiments, we were able to reconstitute SUN- KASH binding in vitro. We analyzed luminal constructs of human SUN1 and SUN2 expressed either in the ER of Hela cells or purified from E. coli. Both the conserved SUN domains and the preceding coiled-coil regions were required for interaction. Replacing the endogenous coiled- coils of SUN1 or SUN2 by an unrelated coiled-coil domain allowed efficient KASH binding. This indicates that oligomerization of SUN domains by coiled-coils is a key determinant of SUN- KASH interaction.

The crystal structure of the complex between the SUN domain of human SUN2 and the Nesprin-2 KASH domain (see poster presented by Brian Sosa and Thomas Schwartz) revealed essential features of SUN-KASH interaction in molecular detail. Using mutational analyses of both binding partners, we confirmed the key determinants in in vitro binding assays. Furthermore, we could show that the disruption of SUN-KASH interaction affects nuclear envelope targeting of both SUN and KASH proteins.

2022 NSrp70 is a novel nuclear speckle-related protein and modulates alternative pre-mRNA splicing in vivo. Y-D. Kim1, J-Y. Lee1, K-M. Oh1; 1Life Science, Gwangju Inst Science/Technol, Gwangju, Korea

Nuclear speckle is known to serve as the storing place of mRNA splicing regulators. We report here the identification and characterization of a novel speckle protein, referred to as NSrp70, based on its sub-cellular location and apparent molecular weight. This protein was firstly identified as the NSrp70, as the of the NIH Mammalian Gene Collection, while no function has been assigned yet. NSrp70 was co-localized and physically interacted with both SC35 and ASF/SF2 in speckles. NSrp66 has putative RNA recognition motif, RE/RD rich domain, and two coiled-coil domains, suggesting role in RNA processing. Accordingly, using CD44, Tra2b1 and Fas constructs as splicing reporter minigenes, we found that NSrp70 modulated alternative splice site selection in vivo. The C-terminal ten amino acids (aa) (531- 540) including 536RD537 were identified as the novel nuclear localization signal, and the region spanning 290 to 471 aa was critical for the speckle localization and the binding to SC35 and ASF/SF2. The N-terminal region (107-161) was essential for the pre-mRNA splicing activity. Finally, we found that knockout of NSrp70 gene in mice produced no progeny including fetal embryos. Collectively, we demonstrate that the NSrp70 is a novel splicing regulator and essentially required earlier during embryonic development.

2023 Quality control and stress response in mammalian ribosomal biogenesis. M. Wang1,2, D. Pestov2; 1UMDNJ-GSBS, Stratford, NJ, 2Cell Biology, UMDNJ-SOM, Stratford, NJ

Ribosome is the subcellular component that translates mRNA to proteins. This complex molecular machine is composed of two subunits, and contains four different ribosomal RNAs (rRNAs) and 79 ribosomal proteins (RPs). The assembly of eukaryotic ribosomes is a multi-step process involving numerous of proteins and RNA factors. Compromised ribosome integrity contributes to premature aging syndromes, cancer and other diseases. Surveillance mechanisms act throughout the entire ribosome synthesis pathway to eliminate defective intermediates and thereby promote structural and functional integrity of the final ribosomal particles. However, ribosome biogenesis is very sensitive to various disturbances of cellular SUNDAY metabolism. Multiple studies have shown that “nucleolar stress” caused by disruption of ribosome biogenesis activates the p53 pathway. However, the critical component(s) in ribosome synthesis machinery that trigger the p53 response are unknown. We propose that nucleophosmin (NPM), a molecular chaperone with predominant nucleolar localization, is able to transmit stress signal via binding to ribosomal proteins or other factors involved in ribosome synthesis. Although NPM is thought to be required for ribosome biogenesis, its down-regulation in NIH3T3 cells, unlike other ribosome synthesis factors, does not trigger cell growth arrest. Our immuno-precipitation data show that certain amount of NPM could be pulled down by ribosomal proteins RPL5 and RPL11. This suggests that instead of participating in pre-ribosome processing directly, NPM may associate and stabilize free RPs in the nucleolus, thereby preventing binding of Mdm2 to RPs. We also found that expressing a mutant form of NPM with two amino acids substitutions in its oligomerization domain triggered cell growth arrest but did not affect pre-rRNA processing. Immunostaining also indicates that the mutant NPM becomes distributed over the whole nucleus. We are currently testing the ability of NPM (both wild-type and mutant forms) to bind RP in the in vitro system. Our data support the idea that under normal growth condition, free ribosomal proteins (RPL5 or RPL11) are sequestered by nucleolar NPM and thus do not interact with Mdm2, an inhibitor of p53. Upon stress, delocalization of NPM may liberate ribosomal protein and make them available for binding to Mdm2 in the nucleoplasm which eventually leads to stabilization p53.

2024 Nucleologenesis in Giardia lamblia by light, electron and atomic force microscopy Nucleolog. L. F. Jiménez-García1, R. Lara-Martínez2, I. de la Mora-de la Mora2, H. Reyes-Vivas3, G. López- Velázquez4, M. Segura-Valdez2; 1Cell Biology, UNAM, México City, Mexico, 2Cell Biology, UNAM, Mexico, 3Instituto Nacional de Pediatría, 4Instituto Nacional de Pediatría, Mexico

The nucleolus is the site of most ribosome biogenesis in eukaryotes. During cell division, the nucleolar elements are redistributed from prophase and they reorganize during telophase by a process called nucleologenesis. There are several studies on the nucleologenesis in animal and plant cells Nucleologenesis has been studied in animal and plant cells, but less work is known in protest cells. The smallest nucleolus was described recently in Giardia lamblia. Here we show by light, electron and atomic force microscopy, that nucleolar elements are present during cell division in G. lamblia. Samples of trophozoites growing onto glass coverslips were processed for standard transmission electron microscopy and silver staining for NOR both for light and electron microscopy. Atomic force microscopy was conducted as described using semithin sections intended for electron microscopy. During cell division, silver staining was observed at the internal periphery of each cell nuclei. Atomic force microscopy revealed some nuclear peripheral material. We conclude that nucleolar elements are present throughout cell division of G. lamblia (This work is supported by UNAM DGAPA PAPIIT IN227810).

2025 Mitotic Lamin disassembly is triggered by a lipid-mediated mitotic Lipin signaling pathway. M. Mall1, T. Walter1, M. Gorjánácz1, J. Ellenberg1, I. W. Mattaj1; 1European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

Disassembly of the lamina, an intermediate filament meshwork that underlies the nuclear envelope, is a key step in early mitosis. While the activity of several kinases including cyclin- dependent kinase 1 (CDK1) and protein kinase C (PKC) have been reported to trigger this process, the relative contribution of these kinases to lamina disassembly remains unclear. SUNDAY

We have established an automated image-processing pipeline that allows us to quantitatively monitor lamina disassembly in live HeLa cells. Using kinase inhibitors, we could show that both CDK1 and PKCs significantly contribute to efficient Lamin B1 disassembly in HeLa cells. Similarly, overexpression of Lamin B1 reporters with mutated CDK1 or PKC consensus phosphorylation sites also impeded Lamin B1 disassembly. Since the role of PKC-dependent pathways during mitosis and their effects on lamina disassembly are poorly understood, we focused on elucidating this process in more detail. Using RNAi we found that specific PKC isoenzymes that require diacylglycerol (DAG) for their activation trigger rate-limiting steps during mitotic Lamin B1 disassembly in our in vivo assay.

Interestingly, we recently showed that the enzyme Lipin, which produces DAG by dephosphorylation of phosphatidate, is essential for Lamin disassembly in the nematode C. elegans (Gorjánácz and Mattaj, 2009). This suggested that Lipin might act upstream of DAG- dependent PKCs. To investigate this possibility we used RNAi to deplete the three redundant human Lipin enzymes and monitored Lamin B1 disassembly. Strikingly, Lipin depletion inhibited Lamin B1 disassembly to a similar extent as did PKC depletion. Furthermore, the effect of Lipin RNAi could be rescued by the addition of a DAG analogue.

This supports a model where Lipins function in a lipid-mediated manner to activate a PKC- dependent pathway of mitotic Lamin disassembly.

Gorjánácz, M., and Mattaj, I.W. (2009). Lipin is required for efficient breakdown of the nuclear envelope in Caenorhabditis elegans. J Cell Sci 122, 1963-1969.

Signal Transduction and Signaling Networks I

2026 Negative regulation of Notch1 signaling by Serum- and glucocorticoid-inducible kinase 1. J-H. Yoon1, E-J. Ann1, J-S. Ahn1, H-S. Park1; 1Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju, Korea

Notch is a transmembrane protein that acts as a transcriptional factor in the Notch signaling pathway for cell survival, cell death, and cell differentiation. However, little is known about mechanisms and regulators that are responsible for attenuating the Notch signaling pathway and protein stability. Here, we report that SGK1 remarkably reduced the protein stability of Notch1 through Fbw7. The protein level and transcriptional activity of the Notch1 intracellular domain were higher in SGK1 null cells than in SGK1 wild-type cells. Notch1-IC was able to form a trimeric complex with Fbw7 and SGK1, thereby SGK1 enhanced the protein degradation of Notch1-IC via a Fbw7-dependent proteasomal pathway. Furthermore, activated SGK1 phosphorylated Fbw7 at Ser227, an effect inducing Notch1-IC protein degradation and ubiquitination. Moreover, accumulated dexamethasone-induced SGK1 facilitated the degradation of Notch1-IC through phosphorylation of Fbw7. Altogether our results suggest that SGK1 inhibits the Notch1 signaling pathway via phosphorylation of Fbw7.

SUNDAY

2027 Secretory Protein Expression Under Microgravity Conditions. M. Mednieks1, I. Khan2, A. Khatri3, J. Perrier1, A. Hand4; 1Oral Health and Diagnostic Sciences, University of Connecticut Health Center, Farmington, CT, 2University of Connecticut School of Dental Medicine, Farmington, CT, 3Wesleyan University, Middletown, CT, 4Departments of Craniofacial Sciences and Cell Biology, University of Connecticut Health Center, Farmington, CT

A major goal of the space shuttle biological experiments is to identify the physiological subsystems that require in-flight monitoring and post-landing testing to evaluate spaceflight crew status and to devise countermeasures for longer duration missions. Previous studies have shown that exposure of rodents to microgravity results in changes in the regulation of protein secretion. To test the hypothesis that signaling via cyclic AMP-dependent pathways is affected by microgravity the expression of several salivary proteins (both regulated by cyclic AMP and not) was determined. Methods: Adult female C57Bl/6J mice housed in Animal Enclosure Modules (AEMs) were flown on the space shuttles Discovery and Atlantis. Findings from flight animals were compared with those from ground control animals housed in AEMs and from vivarium-housed controls. Tissue samples were collected within 5 hours of landing, and glands were fixed and processed for ultrastructure and frozen for biochemistry. Specific proteins, including the PKA type II regulatory subunit (RII), were identified by immunocytochemistry and by electrophoresis and Western Blotting (WB), digitized and quantified using NIH ImageJ. Results: The ultrastructure of serous and mucous secretory cells, as well as ducts, was similar in flight and control animals. Quantitative analysis of immunogold labeled thin sections of parotid acinar cells of flight animals showed a decrease (p < 0.01) in the expression of RII, amylase, and proline-rich proteins (PRPs), whereas parotid secretory protein (PSP) expression was unchanged. In the sublingual gland the expressionof PSP in serous demilune cells and mucin (Muc-19) in mucous acinar cells was increased (p < 0.001), while RII was slightly increased in demilune cells (p = 0.23). Demilune cell and parotid protein (DCPP) was increased in intercalated ducts of the parotid (p < 0.001), whereas a slight decrease occurred in the sublingual gland (p = 0.27). Electrophoretic banding patterns were not markedly altered in flight parotid or submandibular glands compared to controls. Analysis by WB, however, showed that in the submandibular gland expression of IgA (p < 0.05) was decreased while there was an increase in RII (p < 0.02). Recent data from the last flight of the space shuttle Discovery show that protein expression in flight samples begins to return to normal by day 5 after landing and approaches control values by day 7. Conclusions: The expression of several parotid, submandibular and sublingual gland secretory proteins is altered by travel in space. These data also indicate that the responses of the different salivary glands to microgravity are cell, tissue, and organ specific. As these secretory proteins are present in saliva, these findings can be used to establish this easily collected biofluid as a diagnostic tool to assess physiological responses of astronauts as well as for clinical applications on Earth. Support: NASA grant NNX09AP13G

2028 A-kinase Anchoring Proteins: Mediators in Neonatal Rat Schwann Cell Proliferation. A. L. Asirvatham1, R. Stahl2, C. M. Schworer2, E. Shoemaker1, D. J. Carey2; 1Biology, Misericordia University, Dallas, PA, 2Weis Center for Research, Geisinger Clinic, Danville, PA

A-kinase Anchoring Proteins: Mediators in Neonatal Rat Schwann Cell Proliferation Schwann cell proliferation in the peripheral nervous system is mediated by the heregulin/neuregulin family of growth factors that are secreted by neurons. Previous studies have revealed that culture of neonatal rat Schwann cells exhibit a synergistic response in growth when treated with both heregulin and forskolin, in comparison to cultures incubated with SUNDAY either heregulin or forskolin. The molecular mechanisms that mediate the proliferation of Schwann cells in response to heregulin and forskolin are not well known. This study was undertaken to identify the role of the cyclic AMP protein kinase A signaling pathway in Schwann cells that are cultured with heregulin and forskolin. Studies conducted previously have shown that the A-Kinase anchoring proteins of the cAMP/PKA pathway play an important role in Schwann cell division. Incubation of neonatal rat Schwann cells with SiRNA oligos synthesized against AKAP95 and AKAP150 resulted in a reduction in protein levels of both AKAPs accompanied by a decrease in cell proliferation. In addition to a decrease in AKAP protein levels, Schwann cells cultured with the SiRNA oligos against AKAP95 and AKAP150, exhibited a reduction in protein levels of cyclin D3 and PKB/Akt respectively. To investigate if the loss of AKAPs along with cyclin D3 occurred at the level of synthesis or degradation, neonatal rat Schwann cells were cultured with the protein synthesis inhibitor cycloheximide and the proteasome inhibitor lactacystin. Immunoblot analysis of Schwann cells incubated with cycloheximide and SiRNA oligos or cycloheximide alone showed a reduction in both AKAP95 and cyclin D3 levels. Schwann cells cultured with lactacystin and SiRNA oligos showed a marked reduction in AKAP95 and cyclin D3 levels in comparison to cultures incubated with lactacystin only. These preliminary observations suggests that the loss of AKAP95 along with the cyclin D3 proteins does not occur at the synthesis or degradation levels and instead may be mediated by a different mechanism. To examine if AKAP150 associates with PKB/Akt to stimulate Schwann cell proliferation, cells were incubated with modified N2 media or modified N2 media with heregulin and forskolin. Using immunofluorescent staining techniques, preliminary results demonstrate that Schwann cells treated with heregulin and forskolin exhibit distinct colocalization patterns for AKAP150 and PKB/Akt. These results suggest that in the presence of forskolin and heregulin, AKAP150 may associate with PKB/Akt to mediate cell proliferation.

2029 Turn motif phosphorylation negatively regulates activation-loop phosphorylation in Akt. D. Hiraoka1, E. Okumura1, T. Kishimoto1; 1Tokyo Institute of Technology, Yokohama, Kanagawa, Japan

Akt, also known as PKB, plays a central role in various signaling pathways that regulate cellular processes such as metabolism, proliferation and survival. Under stimulation, phosphorylation of the activation loop (A-loop) and hydrophobic motif (HM) of Akt by the kinase, PDK1, and the mammalian target of rapamycin complex 2 (mTORC2), respectively, results in its activation. A well-conserved threonine in the turn motif (TM) is also constitutively phosphorylated by mTORC2 and contributes to the stability of Akt. However, the relationship between TM phosphorylation and the phosphorylation of Akt on the HM and A-loop has not been sufficiently evaluated. Using starfish oocytes as a model system, our study provides the first evidence that TM phosphorylation plays a negative role in A-loop phosphorylation. In this system, the maturation-inducing hormone, 1-methyladenine, stimulates Akt to induce meiotic reinitiation through the activation of cyclin B-Cdc2. The phosphorylation status of Akt was monitored via introduction of exogenous human Akt in starfish oocytes. Injection of an anti-starfish TOR antibody inhibited TM and HM phosphorylation, suggesting that phosphorylation of these sites depends on TOR, as reported in mammalian cells. Precise analyses of single or double alanine substitution mutants at each of three phosphorylation residues revealed that TM phosphorylation renders Akt susceptible to dephosphorylation on the A-loop. When A-loop phosphatase was inhibited by okadaic acid, TM phosphorylation still reduced A-loop phosphorylation, suggesting that the effect is caused at least partially through reduction of sensitivity to PDK1. By contrast, HM phosphorylation enhanced A-loop phosphorylation and achieved full activation of Akt via a mechanism at least partially independent of TM SUNDAY phosphorylation. These observations provide new insight into the mechanism controlling Akt phosphorylation in the cell.

2030 MK-STYX Reduces eIF2α Phosphorylation. J. E. Barr1, S. D. Hinton1; 1Biology, College of William and Mary, Williamsburg, VA

The pseudophosphatase MK-STYX [MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding protein] has been implicated in the stress response pathway. MK-STYX interacts with Ras-GTPase activating protein SH3 domain binding protein-1 (G3BP-1), and inhibits stress granule assembly. Stress granules, cytoplasmic storage sites for mRNA, form as a protective mechanism against stress caused by UV irradiation, hypoxia, and heat shock. In addition, the overexpression of G3BP-1, when dephosphorylated induces stress granule assembly. Initially, we hypothesized that MK-STYX inhibition of stress granules was G3BP-1 phosphorylation-dependent. However, data with G3BP-1 phosphomimetic and non- phosphorylatable mutants suggest differently so we turned our focus to the eukaryotic initiation factor 2 alpha (eIF2). eIF2 initiates translation by forming a ternary complex with methionine bound to tRNA and GTP. Phosphorylation of eIF2 arrests translation and results in stress granule formation, whereas dephosphorylation promotes polysomes. To determine if MK-STYX inhibits stress granule assembly via upstream interactions, we investigated its affects on eIF2 phosphorylation. HeLa cells were transfected with pMT2-FLAG-MK-STYX-FLAG or pMT2 expression vectors, and heat shocked for an hour to induce stress. Cells were lysed and immunoblots for phosphorylation of eIF2 were performed. We show that the presence of MK- STYX reduced the phosphorylation of eIF2. Furthermore, MK-STYX also reduced the eIF2 phosphorylation in heat shocked cells, which showed a drastic increase in phosphorlyation in the absence of MK-STYX. These data are significant because they provide more insight into how MK-STYX inhibits stress granule assembly.

2031 VEGF modulates Col I expression in human adipose derived stem cells through Akt dependent mechanisms. C-J. Li1, V. Madhu1, A. Dighe1, G. Balian1,2, Q. Cui1,3; 1Orthopaedic Research Labs, University of Virginia, Charlottesville, VA, 2Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA, 3Orthopaedic Surgery, University of Virginia, Charlottesville, VA

Objective: We earlier demonstrated that in vivo osteogenesis induced by mouse bone marrow derived osteoprogenitor cells expressing bone morphogenetic protein – 6 (BMP-6) or Lim Mineralization Protein - 1 (LMP-1) was enhanced significantly by co-expressing vascular endothelial growth factor (VEGF) gene. The facts that increase in osteogenesis did not completely correlate with VEGF-induced angiogenesis and that LMP-1 is downstream activator of BMP-6 signaling pathway clearly indicated interaction between VEGF and other signaling pathways. In order to understand mechanisms of the interaction and to determine if similar interactions occurred in human mesenchymal stem cells we determined in vitro osteogenesis of human adipose derived stem cells (hADSCs) in presence of purified VEGF and BMP-6 proteins.

Results: The results of calcium deposition showed no significant difference between control, BMP-6, VEGF or BMP-6+VEGF group in hADSC at day 14th. However, significant increase in expression of Col I α2 gene was observed in VEGF group. At day 14, a synergistic increase in Col I α2 expression was observed in VEGF+BMP-6 group. Addition of Akt1/2/3 inhibitor SUNDAY suppressed Akt and Smad1/5 phosphorylation, calcium deposition and Col I α2 mRNA expression in hADSCs. Most importantly, blockage of Akt activation significantly inhibited the synergistic enhancement of Col I α2 mRNA expression, which was induced by BMP-6 and VEGF combined treatment.

Conclusion and Discussion: Our data showed a striking observation that VEGF and BMP-6 synergistically enhanced Col I α2 gene expression in hADSCs. Furthermore, biochemical blockage of Akt activation significantly inhibited the enhancing effects of Col I α2 mRNA expression by BMP-6 and VEGF combined treatment. It is reported that PI3-kinase catalytic isoform is coupled to VEGFR2 and regulates the bulk of Akt activity. Moreover, dominant-negative Akt inhibited Smad5 phosphorylation. Our data and these reported observations together establish a novel axis of control of Col I expression as VEGF-VEGFR2-PI3K-Akt-Smad1/5-Col I in hADSCs. However, Akt-dependent BMP-6 modulation of this axis remains to be investigated in near future.

2032 A novel gene encoding a candidate CK2 substrate involves in cancer cell proliferation. H. Yang1, S. Liu1, H. Zheng1, H. Jiang1, Y. Qu1, Y. Li1; 1Harbin Institute of Technology, Harbin, China

CK2, a ubiquitous protein serine/threonine kinase, is essential for the decision of cell survival and cell death. In the present study, we identified a novel gene, Homo Sapiens Chromosome 1 ORF109 which protein product might be a candidate CK2 substrate. The C1ORF109 protein presented in the phosphorylated form in vivo, and could be phosphorylized by the protein kinase CK2 in vitro. Truncated constructs and site direct mutagenesis analysis revealed that Ser104, Ser 134, and Ser182 were phosphorylated sites. Both TATA box and CAAT box within the crucial region of c1orf109 promoter are required for maximal transcription of the c1orf109 gene. The C1ORF109 protein was mainly located in the nucleus and cytoplasm. Upregulated expression of c1orf109 was detected in multiple breast cancer cells. Exogenous expression of C1ORF109 in breast cancer Hs578T cells promoted colony formation and cell proliferation along with increased level of PCNA and cyclinD1. Meanwhile, knockdown of c1orf109 by siRNA in breast cancer MDA-MB-231 cells resulted in inhibition of cell proliferation. Taken together, our findings suggest that C1ORF109 may be the down stream target of protein kinase CK2 and involved in the regulation of cancer cell proliferation. Key words: promoter, transcription, CK2 kinase, c1orf109, proliferation

2033 Quantitative detection of phosphorylation of STAT3 and p38 by Western blotting. K. Söderquist1, S. Grimsby1, M. Winkvist1; 1GE Healthcare, Uppsala, Sweden

Introduction Western blotting is a well established technique used to study proteins from a wide variety of sources. The technique is used throughout life sciences from basic research to medical diagnostic applications. Western blotting is at best considered as semi-quantitative and hence limited to studies involving large protein differences. However development of detection reagents as well as imagers has open up for quantitative applications when high sensitivity and broad dynamic range is needed.

Here we demonstrate how the Western blotting technique can be used to determine phosphorylation levels of various proteins in whole cell lysates upon stimulation with various well known inducers. SUNDAY

Methods HeLa cells were stimulated with INF-β. Phosphorylation level of STAT3 was determined by Western blotting performed according to standard procedure and protocol supplied with the Amersham™ ECL™ Prime reagent using optimized blocking solution and antibodies concentrations. 293T cells where stimulated with TGF-β. Phosphorylation level of p38 was determined by Western blotting performed according to standard procedure and protocol supplied with the Amersham ECL Prime reagent using optimized blocking solution and antibodies concentrations. Detection reagent was added to the membrane and the chemiluminescent signal was captured using CCD cameras.

Results The result demonstrates that phosphorylation of STAT3 upon stimulation with INF-β can be quantitatively determined by Western blotting. In addition, the result demonstrates that time dependent phosphorylation of p38 upon stimulation with TGF-β can be quantitatively determined by Western blotting.

Conclusions The Western blotting technique can be used for accurate quantitative analysis of phosphorylation level of various proteins, such as p38 and STAT3, by combining the use of a detection reagent, providing high sensitivity and broad dynamic range, with a CCD camera.

2034 A distinctive salt bridge arrangement in RSK P-loop enables discovery of isoform- selective RSK1/2 inhibitors that protect from myocardial infarction injury. V. O. Paavilainen1, I. Serafimova2, M. S. Cohen3, M. Spreafico2, J-I. Abe4, M. Jacobson2, J. Taunton2; 1Cellular and Molecular Pharmacology, UC-San Francisco, San Francisco, CA, 2UC- San Francisco, 3Oregon Health and Science University, Portland, OR, 4University of Rochester, Rochester, NY p90 ribosomal protein S6 kinases (RSK) are serine/threonine kinases under the MAPK/ERK signaling pathway, which have been implicated in a variety of cellular functions such as cell proliferation and survival. Their expression is upregulated in primary breast and prostate cancers, and mutations in RSK2 gene lead to Coffin-Lowry syndrome, an X-linked form of mental retardation. We sought to discover potent RSK1 inhibitors with high selectivity over RSK2. These inhibitors utilized diverse electrophilic warheads that covalently target a non- conserved cysteine in the RSK active site and led to the identification of an irreversible RSK inhibitor, fmk, with ~20-fold selectivity difference between the highly identical RSK1 and RSK2 isoforms both in vitro and in vivo. Detailed biophysical and structural experiments showed that this selectivity arises from differences in the reversible binding affinity mediated by subtle differences in the structure and dynamics of the kinase P-loop region due to a specific salt bridge arrangement around the P-loop region. In a mouse model of myocardial infarction (MI), fmk treatment resulted in a dramatic improvement in survival and heart function, possibly by inhibition of NHE1 phosphorylation. Future studies will aim to dissect the roles of different RSK isoforms during MI injury.

SUNDAY

2035 Inhibition of Notch1 signaling by APP intracellular domain. E-J. Ann1, J-H. Yoon1, J-S. Ahn1, H-S. Park1; 1Chonnam National University, Gwangju, Korea

The Notch1 receptor is a crucial controller of cell fate decisions, and is also a key regulator of cell growth and differentiation in a variety of contexts. In this study, we have demonstrated that the APP intracellular domain (AICD) attenuates Notch1 signaling by accelerated degradation of the Notch1 intracellular domain (Notch1-IC) and RBP-Jk, through different degradation pathways. AICD suppresses Notch1 transcriptional activity by the dissociation of the Notch1-IC– RBP-Jk complex after processing by gamma-secretase. Notch1-IC is capable of forming a trimeric complex with Fbw7 and AICD, and AICD enhances the protein degradation of Notch1- IC through an Fbw7-dependent proteasomal pathway. AICD downregulates the levels of RBP- Jk protein through the lysosomal pathway. AICD-mediated degradation is involved in the preferential degradation of non-phosphorylated RBP-Jk. Collectively, our results demonstrate that AICD functions as a negative regulator in Notch1 signaling through the promotion of Notch1-IC and RBP-Jk protein degradation.

2036 Isoprenylcysteine methylation is required for growth and endocytosis in Dictyostelium discoideum. K. J. McQuade1, J. Bollan1, K. Bailey1, J. Fritz1; 1Colorado Mesa University, Grand Junction, CO

Many members of the Ras superfamily of small monomeric GTPases and other membrane- associated proteins ending in carboxy-terminal –CAAX motifs are proteolytically processed and reversibly methylated on isoprenylated terminal cysteine residues. Methylation is thought to regulate localization and activity of these proteins. Ras GTPases are often overactive in cancer cells and inhibitors of isoprenylcysteine methyltransferase are being tested as potential chemotherapies, but the cellular effects of these inhibitors are unclear. We are characterizing the effects of loss of isoprenylcysteine methyltransferase activity in the social amoeba, Dictyostelium discodeum. Amoebae lacking methyltransferase activity grow slowly on bacterial lawns and do not grow in shaking or adherent axenic culture. Methyltransferase-deficient cells also migrate slowly towards folate, have reduced rates of endocytosis and are sensitive to osmotic shock. The mechanisms underlying these defects are being investigated.

2037 Understanding the cellular function of poly(ADP-ribose) and its importance in cancer. K. Krukenberg1, T. Mitchison2; 1Systems Biology, Harvard Medical School, Boston, MA, 2Systems Biology, Harvard Medical School

Poly(ADP-ribose) is a unique but poorly understood post-translational modification. It has been implicated in multiple cellular processes including DNA damage, cell death, inflammation, protein stability and cell division. In the clinic, inhibition of poly(ADP-ribose) formation is showing promise as a cancer therapeutic. Poly(ADP-ribose) polymerases (PARPs) catalyze the addition of ADP-ribose onto acceptor proteins using NAD+ as a substrate. Though the importance of PARPs and poly(ADP-ribose) is clear, their biological functions are not well understood. We are working on developing tools for characterizing the function of PARPs and poly(ADP-ribose) and are using these tools to further identify and define the biological roles of these intriguing and essential molecules. Here we describe an assay for quantifying the levels of poly(ADP-ribose) in cells that has increased sensitivity as compared to previous approaches. Using this assay, we quantified the extent to which poly(ADP-ribose) levels vary throughout the cell cycle. We have also found that different cancer cell types have different amounts of poly(ADP-ribose) and these SUNDAY levels appear to be largely attributable to PARP1 activity. Current efforts are focused on better understanding the molecular basis for the differences in polymer levels. Not only will this provide insight into the biological function of poly(ADP-ribose) but poly(ADP-ribose) levels may be useful as an indicator of which patients will respond best to PARP inhibitors or other treatments.

2038 Monitoring Post-Translational Modifications Using Antibody-Based Real-Time PCR. R. Bruinsma1, K. G. Huwiler1, B. D. Marks1, M. Shannon2, D. Ruff2, B. Schweitzer3; 1Life Technologies, Madison, WI, 2Life Technologies, Foster City, CA, 3Life Technologies, Carlsbad, CA

Protein post-translational modifications (PTM) are covalent modifications to specific amino acids within a protein. PTMs often occur via reversible enzymatically catalyzed reactions and numerous types of PTMs exist, including phosphorylation, acetylation, methylation, and ubiquitination. The importance of PTMs resides in their ability to influence the function, stability, and localization of the modified protein within cells. These changes in protein properties due to PTMs are critical to the regulation of cellular signaling networks and the deregulation of protein PTMs is associated with many diseases, including cancer. Two proteins that are recognized to be important in cancer biology and that undergo PTMs are p53 and AKT. p53 is central to maintaining the integrity of the genome and is found to be mutated in ~50% of all cancers. Phosphorylation of p53 at Ser15 has been shown to result in p53 protein stabilization, which can result in transcriptional activation. The PI3K/AKT pathway is critical for normal cell growth and survival, but persistent uncontrolled activation of this pathway is associated with cancer. Full kinase activity of AKT and downstream cellular signaling involves phosphorylation of Ser473 and Thr308. We report the application of a technique that partners the specificity of antibody detection with the signal amplification of real-time PCR (RT-PCR) to monitor changes in PTMs. Specifically, we were able to quantitatively monitor changes in p53 Ser15 phosporylation and AKT Ser473 phosphorylation from very small cellular samples. To our knowledge, this is the first demonstration of monitoring PTMs for p53 and AKT via RT-PCR. This is a powerful method for monitoring PTMs due to the high specificity, high sensitivity, and small sample requirement.

2039 Association of Connexin 36 with synaptic scaffolding proteins in the retina. A. Vila1, C. Whitaker1, J. O'Brien1; 1Opthalmology and Vision, University of Texas Health Science Center, Houston, TX

Our visual world contains an incredible range of light intensities. This necessitates adaption at many levels of the visual system to produce useful vision. In the retina, electrical synapses composed of gap junctions display extensive plasticity associated with light adaptation. Recent studies have found that NMDA-type glutamate receptors regulate coupling among AII amacrine cells and that NMDA receptors are localized near Cx36 gap junctions on AII amacrine cells. These studies indicate that a localized Ca2+ signal derived from NMDA receptor activation drives Cam kinase II-dependent phosphorylation of Cx36 to produce activity-dependent enhancement of coupling. To maintain the integrity of this signaling mechanism, we hypothesized that scaffolding proteins may assemble Cx36 gap junctions and NMDA receptors into a complex analogous to the post-synaptic density of conventional synapses. To explore this hypothesis, we examined the association of Cx36 with traditional synaptic scaffold proteins Synapse- Associated Protein 97 (SAP97), SAP102, and Post-Synaptic Density 95 (PSD95) by immunofluorescence in rabbit retina and 3-D image reconstruction. We further examined the association of these proteins by co-immunoprecipitation and western blot techniques. Antibodies to SAP97 and SAP102 gave punctate labeling throughout the inner synaptic layer of SUNDAY the retina, in which AII amacrine cells make their gap junctions. Antibodies to PSD95 did not label the inner synaptic layer, but did label the outer synaptic layer, where photoreceptor synapses are located. In the inner synaptic layer, approximately 50% of the Cx36 gap junctions were co-localized with either SAP97 or SAP102, and 15% co-localized with both. SAP102 co- localized with about twice as many Cx36 gap junctions as did SAP97. Furthermore, of the SAP102 puncta in the inner synaptic layer, fully one third were associated with Cx36 gap junctions. In the outer synaptic layer, some PSD95 immunoreactivity was closely associated with Cx36. Our data show that synaptic scaffolding proteins are associated not only with conventional synapses, but also with electrical synapses. These scaffolding proteins may facilitate assembly of non-synaptic neurotransmitter receptors, such as the NMDA receptors on AII amacrine cells, with Cx36 gap junctions. They may alternatively, or in addition, facilitate the association of conventional synapses and gap junctions to form mixed synapses.

2040 A structural biology approach to predict proteolytic sites of accessory gland proteins in D. Melanogaster. C. A. Del Carpio1, M-T. Yamamoto1; 1Drosophila Genetic Resource Center (DGRC), Kyoto Institute of Technology, Kyoto, Japan

Extra and intra-cellular protein processing and degradation accounts for one of the main biochemical processes involved in series of signal transduction and regulation pathways. Moreover, genome-wide studies of higher organisms have unveiled that a large proportion of encoded proteins are enzymes oriented to the cleavage of other proteins. Thus predicting the cleavage sites of substrate proteins and the enzyme involved is highly required in functional genome analyses including the elucidation of intra and extra-cellular mechanisms at the origin of cellular malfunction and diseases. Sequence based inference of proteolytic cleavage sites in polypeptides frequently leads to several sites that are located in the core of the molecule or are sterically hindered and hardly accessible to the cleaving enzyme. Catering to the need of including 3D structural factors in the assessment of cleavage sites in polypeptides and reduce the number of candidates output at the primary structure level, we propose a bio-computational strategy to firstly evaluate substrate-enzyme affinity and subsequently infer putative scissible bonds. Affinity is evaluated by docking the 3D structures of the substrate and the enzyme, and molecular dynamics (MD) simulation is performed to assess the formation of the transition state (TS) complex triggering the proteolytic reaction. The cleavage sites are then inferred by tracing the main atomic interactions throughout the MD simulation leading to formation of the TS species. The methodology involves protein structure prediction, as well as protein docking to assess substrate-enzyme interaction. As a case study we applied the methodology to the prediction of cleavage sites for Acp36DE, a D. Melanogaster seminal fluid protein that plays an important role in sperm storage and competition in multiply mated females. The results show that among several candidate regions amenable to protease attack, the region LEU514- ASP517, located at a disordered region between two alpha helices shows geometrical complementarity and energetic affinity for an astacin like protease. The TS species derived through the MD simulation shows the metal (Zn) in the astacin pulling the LYS515.O, and the contraction of the distance between the SER516.N and GLU101.OE2 in astacin suggests the scissible peptide bond. Repeating the calculations with serine proteases reported to be expressed at copulation shows a higher affinity region for the hydrophobic region that includes amino acids SER21-GLU22-SER23-PHE24 the cleavage bond being GLU22-SER23. Cleavage of Acp36DE at LYS515-SER516 generates fragments of Acp36DE of molecular weights that compare well with reported experimental findings

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2041 Evolutionary analysis of the phosphorylation signaling reveals functionally conserved motifs. H. Yoshizaki1, S. Okuda2; 1Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, Japan, 2Department of Bioinformatics, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, Japan

(Background) Protein kinases regulate various cellular processes by phosphorylating specific substrates. Phosphoproteomic studies recently have shown that tens of thousands of sites are phosphorylated. In addition, in silico systemes biological analyses have revealed protein phosphorylations dynamically regulate cellular networks. These studies provide powerful tools for studying intra-cellular signal transduction pathways. High throughput proteomic techniques provide a massive amount of protein information but we can not directly retrieve protein-protein interactions organizing cellular processes. Toward understanding the complicate intracellular signaling networks, we performed a comparative analysis about evolutionally conserved phosphorylation motifs. (Methods and Results) First, we retrieved the known phosphorylated protein sequences from the PhoshoSitePlus database (http://www.phosphosite.org/) and determined phosphorylation motifs by clustering them. As a result, we acquired 186 original phosphorylation motifs from 434 clusters obtained by the MCL clustering method. Subsequently, we assigned kinase information to a motif based on the known kinase-subustrate combinations stored in the PhosphoSitePlus database and predicted a probable kinase phosphorylating the motif. Furthermore, we evaluated the evolutionary conservation of the phosphorylation motifs by a comparative sequence analysis from yeast to human. We found that most of the motifs conserved along the general evolutionary scenario, but some motifs showed the evolutionary patterns different from them. Additional protein functional analysis showed that these phosphorylation sites were involved in a specific protein function. Our comparative evolutionary analysis of phosphorylation motifs is useful methods to highlight protein groups with specific functions from complicated signal transduction pathways.

2042 Network topologies evolve during neutrophil polarization. C-J. Ku*1, Y. Wang*1, O. Weiner2, S. J. Altschuler1, L. F. Wu1; 1Dept of Pharmacology, Green Ctr for Systems Biology, Simmons Cancer Ctr, University of Texas Southwestern Med Ctr, Dallas, TX, 2Cardiovascular Research Institute and Department of Biochemistry, University of California, San Francisco, San Francisco, CA

*These authors contributed equally to the work.

Much attention has focused on how complex cellular behaviors can emerge from static networks. Here, we found that the rapid response of neutrophils to stimulus arises from a time- varying series of distinct signaling network topologies. Not only do the signaling topologies change over time, but the topology also depends on the phenotype. Surprisingly, the set of persistent links revealed an underlying simplicity of the network structure: a linear cascade controlling signaling activities, and a feed-forward network in the opposite direction controlling signaling polarity. Our work provides a clear example that simple circuits orchestrating cellular behaviors may lay hidden beneath the complexity of comprehensive “everything-connects-to- everything” networks obtained by combining links from different times and phenotypes.

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2043 The Drosophila Transcription Factor Protein Interactome. D. Y. Rhee1, B. Zhai1, C. Wong1, C. Beekman1, S. Gygi1, R. Obar1, S. Artavanis-Tsakonas1; 1Department of Cell Biology, Harvard Medical School, Boston, MA

From the earliest embryo to the adult, the spatiotemporal expression of genes is essential for normal development and physiology. At the basis of this is the regulation of transcription via transcription factors (TFs), proteins that physically bind DNA to activate or suppress gene expression. As the target of signaling pathways, TFs represent a crucial point of regulation relating to the vast majority of cellular processes and as a rule function through interactions with other proteins. Consequently, the characterization of protein-protein interactions involving TFs is essential for understanding how they function to regulate gene expression and, in turn, the biology of the cell. Towards this end, we have generated a co-AP/MS-based interaction map encompassing more than 500 from the total of 750 TFs estimated, by various criteria, to represent the entire gamut of TFs in Drosophila melanogaster.

Cell Migration and Motility

2044 Macrophage Strength: Quantifying Phagocytic Forces and Kinetics. N. Sosale1, T. Rouhi2, P. Rodriguez1, R. Lipowsky2, D. Discher1; 1Biophysical Engineering Lab, University of Pennsylvania, Philadelphia, PA, 2Max Planck Institute of Colloids and Interfaces, Potsdam, Germany

Macrophages act as immunological gatekeepers at the interface of tissue, blood, and lymph as these cells take up antigens from the extracellular environment and then present them to the immune system stimulating lymphocytes. We have studied the force and kinetics of phagocytosis by human macrophages. To this aim, we studied phagocytosis of opsonized red blood cells. The force calculation is based on blood cell's shape deformation and elastic properties. Our analysis shows that the range of the resultant force imposed by Macrophages is up to 100 pN and is imparted within 10 minutes after initial contact.

2045 Integrin Linked Kinase Modulates Phagocytosis through Rac1 Activation in Epidermal Keratinocytes. S. Sayedyahossein1,2, L. Nini1, L. Dagnino1,2; 1Physiology and Pharmacology, The University of Western Ontario, London, ON, Canada, 2Children’s Health Research Institute, London, ON, Canada

Integrin linked kinase (ILK) is an adaptor protein that regulates numerous biological processes, such as cell survival and migration. ILK is also essential for keratinocyte interactions with the extracellular matrix and development of polarity, but the exact role of ILK in other epidermal functions, such as melanosome phagocytosis, is not clear. We examined alterations in phagocytic capacity in ILK-deficient epidermal keratinocytes, using 0.5-µm latex microspheres. Inactivation of the Ilk gene resulted in severe impairment of microsphere uptake. Further, stimulation of the keratinocyte growth factor (KGF) receptor or PAR-2 failed to increase phagocytosis in these cells. Activation of ERK in response to KGF stimulation is unaltered in ILK-deficient keratinocytes, indicating that the KGF receptor likely signals normally in the absence of ILK. In contrast, ILK deficiency was associated with impaired Rac1 activation in response to KGF. The latter was accompanied by reduced formation of membrane ruffles and SUNDAY abnormal F-actin dynamics. Thus, ILK is essential for normal keratinocyte phagocytosis in response to KGF. Supported by the Canadian Institutes of Health Research (CIHR).

2046 Myristolated Alanine-Rich C-Kinase Substrate (MARCKS) is an Important Regulator of Equine Neutrophil Migration and Adhesion. M. K. Sheats1, K. L. Carren2, E. M. Hefner3, E. J. Sung2, K. B. Adler4, S. L. Jones2; 1Molecular Biological Sciences, North Carolina State University, Holly Springs, NC, 2Clinical Sciences, North Carolina State University, 3Animal Science, North Carolina State University, 4Molecular Biological Sciences, North Carolina State University

Neutrophil infiltration is a prominent feature in a number of pathologic conditions including recurrent airway obstruction and ischemic reperfusion injury. Cell signaling components involved in neutrophil migration represent targets for novel anti-inflammatory therapies. This study was conducted to test the hypothesis that the 32 kD protein, Myristolated Alanine-Rich C- Kinase Substrate (MARCKS) is involved in equine neutrophil migration and adhesion. In other species (i.e. human) MARCKS phosphorylation and dephosphorylation causes the protein to cycle between the cell membrane and cytosol, respectively. To investigate MARCKS phosphorylation in horses, neutrophils were isolated from whole blood using Ficoll gradient centrifugation and stimulated with platelet activating factor (PAF), leukotriene B4 (LTB4) or phorbol myristate acetate (PMA). Western blot was performed using specific phospho-MARCKS and total MARCKS primary antibodies. These results determined that MARCKS phosphorylation in equine neutrophils is maximal 30 seconds following stimulation with 100 nM PAF or LTB4 and that dephosphorylation occurs within 3 minutes. MARCKS interacts with the resting neutrophil cell membrane through the combined efforts of its myristolated N-terminus and basic amino acid effector domain; potentially regulating interactions between PIP2 and actin binding proteins. To investigate the requirement for MARCKS in equine neutrophil chemotaxis, isolated neutrophils were pre-treated with MANS (a cell permeant peptide identical to the N-terminal 24 amino acids of MARCKS), RNS (a scrambled sequence control peptide) or vehicle control prior to migration toward known neutrophil chemoattractants (LTB4 or PAF). Pre-treatment of equine neutrophils with the MANS peptide significantly inhibited LTB4 and PAF stimulated migration while the control RNS peptide had no effect. Beta-integrin upregulation and adhesion is an essential process to neutrophil chemotaxis. To investigate the requirement for MARCKS in equine neutrophil adhesion, isolated neutrophils were pretreated with MANS, RNS or vehicle control and stimulated to adhere to Immulon 2 plates coated with 5% FBS (a β-integrin dependent substrate) in PBS with 10 ng/ml PMA or vehicle control. Pre-treatment of equine neutrophils with the MANS peptide significantly inhibited PMA induced adhesion; while the control RNS peptide had no effect. Pre-treatment of equine neutrophils with a cell permeant peptide identical to the N-terminus of MARCKS significantly inhibited equine neutrophil PMA induced adhesion and LTB4 and PAF induced migration. These results indicate that MARCKS is an important regulator of equine neutrophil migration and adhesion and MARCKS regulation of neutrophil chemotaxis has been conserved across evolutionary lines.

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2047 The Scar/WAVE complex is necessary for proper regulation of traction stresses during amoeboid motility. E. E. Bastounis1, R. Meili2, B. Alonso Latorre2, J-C. del Alamo2, J. Lasheras2, R. Firtel2; 1Bioengineering, University of California, San Diego, San Diego, CA, 2University of California, San Diego

Chemotaxis, or directed cell migration, is involved in a broad spectrum of biological phenomena, ranging from the metastatic spreading of cancer to the active migration of neutrophils during wound healing or in response to bacterial infection. Chemotaxis requires a tightly regulated, spatiotemporal coordination of underlying biochemical processes. SCAR/WAVE-mediated dendritic F-actin polymerization at the cell’s leading edge plays a key role in cell migration. Our study identifies a mechanical and biochemical role for the SCAR/WAVE complex in modulating the traction stresses that drive cell movement. We demonstrate that the traction stresses of wild-type Dictyostelium cells or cells lacking the SCAR/WAVE complex protein PIR121 (pirA-) or SCAR (scrA-) exert stresses of different strength that correlate with their levels of F-actin. By processing the time records of the cell length and the strain energy exerted by the cells on their substrate, we show that wild-type cells migrate by repeating a specific set of mechanical steps (motility cycle), whereby the cell length (L) and the strain energy exerted by the cells on their substrate (Us) vary periodically. Our analysis also revealed that scrA- cells exhibit an altered motility cycle with a longer period (T=160s) and a lower migration velocity (V =6μm/min) compared to those of wild-type cells (T=80s, V =13μm/min). In marked contrast to these strains, pirA- cells, although they have a higher F-actin content, migrate as slowly as scrA- cells but their migration occurs in a seemingly random manner in that they lack the periodic changes in traction stresses that are observed for the other two strains. Finally, by quantifying the level of F-actin in the leading edge in combination with the traction stresses, we demonstrated that the level of leading edge, SCAR/WAVE complex-mediated F-actin polymerizations is critical for the level and spatiotemporal control of the traction stresses, cell-substrate interactions, and the motility cycle.

2048 Self-Generated EGF Gradients Guide Epithelial Cell Migration. D. Irimia1, A. J. Aranyosi1, B. Kulemann1, S. Thayer1, M. Toner1, O. Illiopoulos1, C. Scherber1; 1BioMEMS Resource Center, Massachusetts General Hospital/Harvard Medical School, Boston, MA

Migrating epithelial cells can contribute to wound healing processes, or can trigger lethal complications during cancer through invasion and distant metastasis. During their migration, cells must be able to orient accurately and it is generally assumed that gradients of chemokines or growth factors are required to guide epithelial cells towards their target location. Unexpectedly, we uncovered a novel strategy for epithelial cell orientation during migration that does not require pre-existent chemical gradients. Using microscale engineering techniques, we demonstrate that the strategy is dependent on the competition between epidermal growth factor (EGF) uptake by the cells and the restricted diffusion of EGF from surrounding microenvironment. Both normal and malignant epithelial cells are capable of EGF uptake and can use this strategy when placed in confined environment. The strategy has some surprising consequences for epithelial cells, like the ability to efficiently disperse through channels, or to navigate along the shortest path to exit from various microscopic mazes. Inhibition of EGF- receptor but not the inhibition of chemokine receptors, reduces the ability of the cells to orient, without altering their velocity of migration. Better understanding of the epithelial cells guidance strategy by EGF uptake-dependent self-generated gradients could lead to approaches for SUNDAY restricting the migration of malignant cells to delay local invasion and distant metastases, or enhance the migration of normal cells to promote wound healing.

2049 Three dimensional traction forces exerted by migrating amoeboid cells.* R. Meili1, B. Alvarez-Gonzalez2, J. C. del Alamo2, R. A. Firtel1, J. C. Lasheras2; 1Division of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, 2MAE Department, University of California, San Diego

Cell migration is essential for many physiological processes in multicellular organisms including embryonic development, wound healing and cancer metastasis. Amoeboid motility requires the spatiotemporal coordination of forces generated by biochemical processes. This results in directional motility by repeated protrusion of the front and retraction of the rear driven by actin polymerization and actomyosin contraction. Mechanically, movement requires the modulation of the cell’s interactions with the environment, either with surrounding cells or with extracellular matrix. For quantitative analysis, these interactions have so far typically been characterized as tangential traction stresses imposed by the cell on a flat substrate. We have expanded the method for measuring traction stresses to determine the stresses exerted perpendicular to the substrate in addition to the in-plane stresses. We find that the deformation perpendicular to the substrate is significant and that calculating the stresses using one of the existing two-dimensional traction cytometry methods misses an important aspect of cellular mechanics. We used our new method to better understand the mechanical role of individual cytoskeletal components by comparing traction stress maps and dynamics of wild type cells moving over flat substrates with measurements of Dictyostelium cells with mutations in cytoskeletal proteins. We focused our study on mutant cell lines with crosslinking defects, such as myosin II-null cells and cortexilin-null cells. We find that cells from all cell lines studied push downward on the substrate near the center of the cell and pull up near the periphery. The magnitude of the perpendicular forces is comparable to the magnitude of the tangential forces exerted on the substrate; therefore the perpendicular component is expected to play a significant role in the cell behavior and cannot be neglected. Our initial findings show that the effects of the crosslinking mutations on the parallel forces do not track with the effects on the perpendicular forces. For example, myosin II- null cells show a significant reduction of the front to back organization of the parallel traction forces while the push pull distribution of forces remains unaffected. Our observations suggest that the generation of the stresses perpendicular and tangential to the substrate is possibly controlled independently. *Work supported by NIH grant 1RO1 GM084227.

2050 Profilin-1: a biomarker for breast cancer malignancy? Z. Ding1, R. Bhargava2, N. Lakshman3, M. Petroll4, P. Roy5; 1Bioengineering, University of Pittsburgh, Pittsburgh, PA, 2University of Pittsburgh, Pittsburgh, PA, 3University of Texas Southwestern Medical Center Dallas, Dallas, TX, 4Ophthalmology, University of Texas Southwestern Medical Center Dallas, Dallas, TX, 5Bioengineering and Pathology, University of Pittsburgh, Pittsburgh, PA

This study unexpectedly reveals that stable loss of expression of profilin1 (Pfn1), an actin- binding protein that has been traditionally shown to be required for cell proliferation and migration, in fact promotes tumor growth and overall distant metastasis of breast cancer SUNDAY xenografts in mice. Comparative studies in spontaneous vs experimental metastasis models suggest that loss of Pfn1 expression likely promotes metastasis-promoting events at the primary tumor site. ECM invasion (an important step for tumor metastasis) of breast cancer cells is increased upon loss of Pfn1 expression. Experiments involving selective perturbation of signaling pathway downstream of PI3-Kinase (PI3K) further revealed that hyper-invasiveness of Pfn1-depleted cells is mediated by a PI3K-PI(3,4)P2-lamellipodin signaling axis. Finally, as a clinical correlate of these in vitro and in vivo findings, analyses of Pfn1 expression in primary tumors revealed that Pfn1 downregulation occurs in all molecular subtypes of breast cancer, somewhat more prominently in estrogen receptor-negative tumors. Furthermore, tumors involving significant lymph node infiltration and/or distant metastasis present the most dramatic downregulation in Pfn1 expression. These findings imply that Pfn1 could serve as a new molecular marker for breast cancer malignancy.

2051 Reestablishing the polarity of Dictyostelium cells to understand the organization of the cytoskeleton and signaling complexes during chemotaxis. D. Jowhar1, G. Wright1, C. Janetopoulos1,2; 1Biological Sciences, Vanderbilt University, Nashville, TN, 2Cell and Developmental Biology, Vanderbilt University, Nashville, TN

Cells migrating in a chemical gradient typically have a polarized morphology, where they display a distinct front and back. Cells can also have different localized sensitivities to chemoattractants depending on their degree and type of polarity. To better understand these phenomena, we used the social amoeba Dictyostelium discoideum that expressed GFP-tagged proteins known to localize to distinct regions of the cell during cell migration. Dictyostelium cells were lured into a specialized microfluidic device developed in our laboratory which confined the cells in narrow 3-dimensional Polydimethylsiloxane (PDMS) channels. The chemoattractant gradient in this experimental platform could be reversed so that the rear of the cell was exposed to a higher concentration of chemoattractant than the front of the cell. Cells in the channels would freeze, become unpolarized, and were then capable of forming a new front at the former rear of the cell. We have observed the temporal and spatial loss and gain of localization of various signaling and cytoskeletal molecules at both the rear and front of the cell as polarity is broken down and re-established. We monitored the localization of PHcrac (a biosensor for PI(3,4)P2 and PI(3,4,5)P3), RBD (a biosensor for Ras activity), PTEN- GFP (a phosphatase which elevates levels of PI(4,5)P2), LimE (a marker for F-actin), and TACC-GFP (a microtubule centrosome and plus-end marker.) The signaling markers showed a re-distribution to opposite sides of the cell during the reversals, as did LimE. We also monitored the microtubules and centrosomes and tracked their redistribution during polarity re-establishment using TACC. Further experiments were performed in the absence of an actin cytoskeleton and in a variety of signaling mutants. These experiments have helped elucidate the molecular mechanisms that contribute to the establishment and maintenance of cell polarity.

2052 Alteration of α6 integrin expression and chemotactic motility of microglia upon ADP stimulation. S-H. Lee1, C. Y. Chung2; 1Pharmacology, Vanderbilt University Medical Center, Nashville, TN, 2Vanderbilt University Medical Center, Nashville, TN

Microglia are the immune effector cells that are rapidly activated in response to even minor pathological changes in the central nervous system. It has been demonstrated that microglia SUNDAY attach well to fibronectin and vitronectin, but only weakly to laminin, and that laminin exerts a dominant anti-adhesive effect on microglial adhesion. Microglial activation is reported to be accompanied by the alteration of integrin expression. Inflammatory cytokines increased expression of α4β1, α5β1, and Mac-1 integrins on microglia but changes of integrin expression upon ADP (a chemoattractant for microglia) stimulation remain unknown. We, in this study, investigated if ADP can induce the alteration of integrin species on cell surface, leading to changes in chemotactic ability on different ECM. FACS scans and surface biotinylation assays showed that ADP stimulation induced a significant increase in the expression of α6 integrin, but not α5 on the surface of microglia cells. Examination of microglia motility on different ECM revealed that cells have greater motility on laminin than fibronectin upon ADP stimulation, presumably due to the increased expression of α6 integrin. The alteration of integrin-mediated adhesion may regulate the extent of microglial infiltration into the site of damage by controlling their chemotactic ability.

2053 Directing cell migration with nano-ridges/grooves. X. Sun1, M. Driscoll2, J. Fourkas1, W. Losert2; 1Chemistry and Biochemistry, University of Maryland, College Park, MD, 2Physics, University of Maryland, College Park, MD

We evaluated the contact guidance of nano-ridges/grooves on the amoeba Dictyostelium discoideum, a model system for studying cell migration. Nano-ridges were fabricated with Multiphoton Absorption Polymerization (MAP). These nano-topographies, as well as complementary nano-grooves, were replicated with high fidelity from an improved composite polydimethylsiloxane (PDMS) mold. By analyzing the cellular velocity, orientation, boundary curvature and shape dynamics, we found that the ridges and grooves exert bidirectional guidance on migrating cells. More cells migrate in the direction of the nano-ridges/grooves than perpendicular to them. We also found that cells aligning in the direction of the nano- ridges/grooves are more elongated and that cells migrating in the direction of the nano- ridges/grooves migrate faster. Contact guidance is only achieved with topotraphies of proper pitch. Nano-ridges/grooves with a pitch smaller than 0.2 μm or greater than 10 μm do not exhibit effective guidance. This study may offer insights into fabricating complex 3D scaffolds for tissue engineering with MAP.

2054 The N-formylpeptide Receptor FPR1 Is Functionally Expressed in Lens Epithelial Cells and is Critical for Lens Maintenance in Mice. J-L. Gao1, J. Tuo1, E. H. Schneider1, D. Despres1, M. Lizak1, A. Maminishkis1, C. C. Chan1, P. M. Murphy1; 1National Institutes of Health, Bethesda, MD

Most mouse knockouts for inflammatory chemoattractants and their receptors lack spontaneous phenotypes. We considered whether an aging study might identify slowly developing phenotypes in such mice. Here we show that mice lacking Fpr1 gene (Fpr1-/- mice) have severe cataracts at an early age. FPR1 is known to mediate pro-inflammatory chemotactic responses by phagocytic leukocytes to N-formylpeptides produced by bacteria or mitochondria and plays a role in host defense against certain bacteria. Fpr1-/- mice developed small eyes starting at 3 month of age because of lens degeneration and rupture. Histopathological analysis did not show any infection or inflammation in these eyes, but showed abnormal lens cell migration. Consistent with this, we detected FPR1 mRNA in both mouse and human lens epithelial cells, and FPR1 proteins on primary human fetal lens cells with the level compatible to that detected on human neutrophils. Stimulation of lens epithelial cells with FPR1 ligands induced intracellular SUNDAY calcium release and actin polymerization. Taken together, the data implicate FPR1 in mediating lens cell migration, a critical event for lens development and maintenance.

2055 PRL-3 promotes migration and invasion by up-regulating MMP-7 in human colorectal cancer cells. Y-M. Han1, S-K. Lee2, D-S. Sin2, Y-R. Ha2, J. Kim2, C-W. Lee3; 1University of Science and Technology, Daejeon, Korea, 2Laboratory of Chemical Biology and Genomics, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea, 3Bio-Evaluation Center, Korea Research Institute of Bioscience and Biotechnology, Chungbuk, Korea

PRL-3, a member of a subgroup of protein tyrosine phosphatases (PTPs) that can stimulate the degradation of the extracellular matrix, is over-expressed in metastatic colorectal cancer relative to primary tumors. To determine whether PRL-3-induced enhancement of migration and invasion is dependent on the expression of matrix metalloproteases (MMPs), PRL-3 was expressed in DLD-1 human colorectal cancer cells. The motility, migration and invasion characteristics of the cells were examined and metastasis to the lung was confirmed in a nude mouse using PRL-3-overexpressing DLD-1 cells [DLD-1 (PRL-3)]. Migration and invasion of the cells were inhibited by phosphatase and farnesyl inhibitors. Expression of MMPs was enhanced 3- to 10-fold in comparison to control cells, and migration and invasion were partially inhibited by siRNA knockdown of MMP-2, -13, or -14. Importantly, siRNA knockdown of MMP-7 completely inhibited the migration and invasion of DLD-1 (PRL-3) cells, while overexpression of MMP-7 increased migration. The expression of MMP-7 was also down- regulated by phosphatase and farnesyl transferase inhibitors. It was found that PRL-3 induced MMP-7 through oncogenic pathways including PI3K/AKT and ERK, and that there is a relationship between the expression of PRL-3 and MMP-7 in human tumor cell lines. The expression of MMP-13 and MMP-14 was very sensitive to the inhibition of farnesyl transferase; however, the migration and invasion of DLD-1 (PRL-3) cells did not strongly depend on the expression of MMP-13 or -14. These results suggest that the migration and invasion of PRL-3- expressing colorectal cancer cells depends primarily on the expression of MMP-7.

2056 Serine phosphorylation in paxillin is important for focal adhesion formation during cell adhesion onto collagen type I. T. Kwak1, J. W. Lee1; 1Department of Pharmacy, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, Korea

Integrin-mediated adhesion to extracellular matrix proteins is dynamically regulated during morphological change and cell migration. Upon cell adhesion, protein-protein interaction at focal adhesions consisting of FAK, talin, paxillin, and others, plays major roles in regulation of cell morphogenesis and migration. Although phosphorylation at tyrosines of paxillin has been well known to be critically involved in adhesion-mediated signaling, little is known about the significance of paxillin phosphorylation at serine 85 in cell migration. Here in this study, we provide evidences that paxillin phosphorylation at serine 85 occurred during HeLa cell adhesion onto collagen I, being concomitant with FAK and talin phosphorylations. However, unphosphorylatable mutant S85A paxillin impaired cell spreading, focal adhesion turnover, and migration toward collagen I but not to serum components. Further, the physical interaction between paxillin and talin was through the C-terminal tail of talin leading to focal adhesions at the cell boundary, but S85A paxillin did not bind talin and caused random focal adhesion formation. Together, these observations suggest that paxillin phosphorylation at Serine 85 depending on cell adhesion is important for interaction with talin and for cell morphological and SUNDAY migratory ability. [This work was supported by NRF by senior researchers program (Leap research, 2011-0001160) and Global Frontier Project grant (NRF-M1AXA002-2010-0029778), and a grant of the Korean Health Technology R&D Project (A100727), MHWFA, Korea to JW Lee]. Key words: focal adhesion, paxillin, talin, morphology, migration

2057 Nck Is Required for Focal Adhesion Maturation and Polarized Cell Motility. S. P. Chaki1, R. Barhoumi2, R. Yog1, M. E. Berginski3, S. M. Gomez3, G. M. Rivera1; 1Veterinary Pathobiology, Texas A&M University, College Station, TX, 2Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, 3University of North Carolina at Chapel Hill, NC

The establishment of cell polarity is essential for directional migration of endothelial cells and the organization and function of endothelial layers. The SH2/SH3 domain-containing adaptors Nck (Nck1 and Nck2) link signaling by tyrosine phosphorylation with effectors that mediate remodeling of the actin cytoskeleton. We previously determined an important role for Nck adaptors in endothelial cell migration, invasion, and in vitro morphogenesis. To understand the mechanisms by which Nck promotes these processes, we used differential interference contrast (DIC) and total internal reflection fluorescence (TIRF) microscopy to compare membrane and adhesion dynamics of human umbilical vein endothelial cells (HUVEC) with or without perturbation of Nck signaling. Expression of specific short hairpin RNAs induced > 90% and 50% reduction in Nck1 and Nck2 levels, respectively. Double knockdown cells (Nck1 and Nck2) were rescued by expression of a siRNA-resistant Nck2 cDNA. Kymographs derived from time- lapse DIC images showed a significant (p<0.001) increase in membrane protrusion/retraction velocity with simultaneous decrease in protrusion/retraction persistence in Nck-knockdown vs. control/Nck-rescued cells. Importantly, Nck-depleted cells formed simultaneous protrusions in multiple directions and failed to establish a front-rear axis of polarity. In contrast, a clear front- rear axis of polarity was almost always discernible in control and Nck-rescued cells regardless of changes in the directionality of movement. The transient nature of the protrusions formed by Nck-depleted cells prompted us to examine the dynamics of focal adhesions in cells expressing fluorescently-labeled paxillin. Time-lapse TIRF imaging showed that control and Nck-rescued cells formed small focal complexes immediately behind the edge of newly forming protrusions. Focal complexes matured into focal adhesions in synchrony with protrusion extension, as evidenced by an increase in adhesion size and fluorescence intensity. Similarly, focal complexes readily formed at the edge of the emerging, multiple protrusions of Nck-depleted cells. However, in contrast to the maturation observed in control and Nck-rescued cells, focal complexes of Nck-knockdown cells rapidly disassembled concurrently with protrusion retraction. Thus, multiple and transient cycles of protrusion/retraction were associated with increased adhesion turnover in Nck-depleted but not control/Nck-rescued cells. These results demonstrate the requirement of Nck in the coordination of protrusion formation with the assembly and subsequent maturation of focal adhesions at the cell’s edge during polarized cell movement. Experiments to assess the activation status of the Rho GTPases in cells with or without perturbation of Nck signaling are underway.

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2058 The F-BAR domain protein PACSIN2 associates to Rac1 and regulates cell spreading and migration. B-J. de Kreuk1, M. Nethe1,2, M. Fernandez-Borja1, E. Anthony1, P. Hensbergen3, A. Deelder3, M. Plomann4, P. Hordijk1; 1Molecular Cell Biology, Sanquin Research, University of Amsterdam, Amsterdam, Netherlands, 2University of Virginia, Charlottesville, VA, 3Leiden University Medical Center, Leiden, Netherlands,, 4Institute for Biochemistry 2, University of Cologne, Germany

The Rac1 GTPase controls cytoskeletal dynamics and is a key regulator of cell spreading and migration via signaling through effector proteins such as the PAK kinases and the Scar/WAVE proteins. We previously identified a series of regulatory proteins that associate with Rac1 through its hypervariable C-terminal domain. These include the Rac1 activator β-Pix, which recruits Rac1 to focal adhesions, and the membrane adapter Caveolin1, which regulates the poly-ubiquitylation and degradation of activated Rac1. Here, we show that Rac1 associates with the F-BAR domain protein PACSIN2, an inducer of membrane tubulation and a regulator of endocytosis. We show that loss of PACSIN2 increases Rac1-GTP loading and promotes cell spreading and –migration. Conversely, expression of PACSIN2 reduces Rac1-GTP levels in a fashion which is dependent on the PACSIN2-Rac1 interaction, on the membrane-tubulating capacity of PACSIN2, and on Dynamin. In addition, we show that GEF-mediated Rac1-GTP loading is unaffected when PACSIN2 is expressed. These results demonstrate that PACSIN2 is an important regulator of the small GTPase Rac1 and that PACSIN2 limits Rac1-GTP signaling by promoting Rac1 inactivation.

2059 Control over acquisition of cell motility and cell migration by the RNA binding protein Dead end. M. Goudarzi1, T. U. Banisch1, E. Raz1; 1Cell Biology, ZMBE, Muenster, Germany

Specification of zebrafish primordial germ cells (PGCs) depends on maternally inherited determinants. These factors, collectively termed the germ-plasm direct cells to enter the germline fate and to acquire proper cell behavior. An important such maternally-provided germ- plasm component in zebrafish is encoded by the dead end (dnd) gene. The Dnd protein contains an RNA recognition motif and is specifically expressed in PGCs, where it is localized to the perinuclear granules. Knockdown of dead end results in severe defects in cell behavior (e.g. the acquisition of motility), cell fate maintenance and survival. We could previously show that Dnd functions by protecting specific mRNAs (e.g. tdrd7, nanos1 and hub) from micro RNA–mediated translational inhibition and degradation in the germ cells, a process by which the same RNAs are repressed and degraded in somatic cells. To determine the molecular basis of the dramatic changes in PGC properties upon loss of Dnd, we identified RNAs regulated by miRNAs and the Dnd protein. The functional role the proteins encoded by these mRNAs play in controlling cell shape and behavior will be discussed.

2060 Nonmuscle Myosin II Isoforms regulate microtubule dynamics in three-dimensional cell migration through the distinct MLC phosphorylations. S. Komatsu1, M. Ikebe2; 1Microbiology and Physiological Systems, UMass Medical School, Worcester, MA, 2UMass Medical School, Worcester, MA

Cell migration is an extremely complex process which is controlled by the rearrangement of cytoskeletal systems. Most studies of cell migration have been focused on controlling the actomyosin and microtubule networks, and studied a migrating cell on two-dimensional (2D) SUNDAY surfaces such as plastic or glass. However, cells under physiological environment interact with extracellular matrices on all surfaces, not just on the basal surface. This raises an idea that cells showing rounded spindle shape in 3D matrices migrate with the different mechanism from flat- shaped cells in 2D surface. To address this question, we use a 3D migration in collagen gel as an in vitro model system for identifying the mechanisms that regulate cell migration under physiological conditions. First, we observed distribution of nonmuscle myosin IIA (NMIIA) and IIB (NMIIB) isoforms in 3D migrating cells by using NMII isoforms specific antibodies and fluorescent protein-fusion NMII isoforms, mCherry-NMIIA and GFP-NMIIB. Although fluorescent signals of the NMIIA and the NMIIB were observed at both anterior and posterior regions in the 3D migrating cells, the detailed distribution of both isoforms were different from each other. The strong signals of NMIIA were particularly detected at the tips of cell extensions, while the signals of NMIIB were accumulated at the cortex around the spindle-shaped cell body. Inhibition of either actomyosin or microtubule networks by blebbistatin or nocodazole resulted in a decrease in 3D cell migration. Isoform-specific knockdowns by recombinant adenovirus siRNA for NMIIA and NMIIB reveal that NMII isoforms are respectively involved in the regulation of the microtubule dynamics in the 3D migrating cells. We also studied the localization of myosin II isoforms phosphorylated at functionally distinct sites of myosin light chain (MLC) in the 3D migrating cells. Interestingly, our results showed that NMIIA phosphorylated at Ser19 localized at the anterior tip of cell protrusion, while NMIIB phosphorylated at Ser1 localized at posterior region of the protrusion. On the other hand, di-phosphorylated myosin II at Thr18 and Ser19 colocalized with cortical actin structure. Our results suggest that the functionally distinct MLC phosphorylation plays an important role in the function of NMII isoforms in 3D cell migration. Further studies are in progress to elucidate the role of the functionally distinct MLC phosphorylation in the 3D cell migration.

2061 Wound healing: The combination use of regenerative agents and bioavailable antioxidant constituents of vitamin E and vitamin D in a model in vitro system. A. Bettica1, N. Christensen2, T. Ragasha2; 1Biology, Manhattanville College, Purchase, NY, 2Manhattanville College, Purchase, NY

Wound healing models have fostered a greater understanding of the cell types involved and the many complex series of events from the site of trauma through the phases of inflammation, proliferation, and remodeling. Many in vitro studies have concentrated on the proliferation stages to elucidate agents that may aid migrating fibroblasts in producing growth factors, laying down fibers and ground substance, and attracting epithelial cells to the site. Fat-soluble vitamins such as vitamin D3 and vitamin E are potent antioxidant and anti-inflammatory agents and have been shown to be effective in ameliorating dental and surgical wounds, burns, and scars. To determine the optimal effectiveness of these properties, appropriate formulations of the isomeric constituents must be tested. The combination of these vitamin species may show additive effects and enhanced efficacy under in vitro wound healing conditions. In dental wound healing, liners and sealers containing varying amounts of calcium hydroxide can trigger a cascade of events which include increased cell proliferation, growth factor synthesis, and cell differentiation. Skin cells, keratinocytes, and fibroblasts are susceptible to a range of UVA and UVB wavelengths that cause cellular and DNA damage, including the formation of thymine dimers. Avobenzone and zinc oxide (ZnO) provide true broad-spectrum protection against UVA wavelengths above 360 nm and are the only two sunscreen active ingredients approved in the US. Studies suggest that proper formulation strategies are necessary for superior attenuation of UVA wavelengths and may be achievable at 3% avobenzone or 5% ZnO. An in vitro wound healing model was standardized to test the combination of vitamin D3 and vitamin E isomers for SUNDAY additive effectiveness with proven or suggested regenerative agents. WS1 human skin fibroblasts are seeded at a density of 105 cells/well on a 6-well culture plate. Using a 4mm sterile biopsy punch, three wound areas are created in each well at confluency. WS1 cell wounds are exposed to 10-fold serial dilutions of calcium hydroxide, avobenzene, or ZnO in appropriate solvents for 24 hours in control wells, or before cells are exposed to UVA. Effective concentrations are selected from standard dose response curves for the various regenerative reagents and are used in combination with the vitamin D3 and E isomers. During the test periods, wound areas are digitally recorded beginning at day 0, for every 12 hours over a four day period. Measurements of the radius (rw - radius of the minimal diameter of the wound), the wound area (Aw) are taken using Motic 2.0 software and compared to day 0 (r0 and A0). Triplicate data sets are statistically analyzed using a one-way ANOVA with Tukey’s post hoc tests. Effective concentrations of calcium/ D3 and cholcalciferol alone are seen at 10-4 mg/ml. Vitamin E isomers did improve cell proliferation and overall wound healing. The most effective doses were 10-7 mg/ml for alpha-tocopherol and 10-8 mg/ml for delta-tocotrienol, showing complete wound healing between 48 and 60 hours. It is suggested that agents with greater antioxidant activities may shorten healing times, reduce the incidence of wound infection and, in the most serious wound cases, lower the mortality rate in burn patients.

2062 Arp 2/3 Plays a Crucial Role in Focal Adhesion Initiation at the Leading Edge. R. J. Vasquez1, K. Sayegh2, J. Stricker3, Y. Beckham3, M. Gardel3; 1Department of Pediatrics, Section of Hematology, Oncology and Stem Cell Transplantation, University of Chicago, Chicago, IL, 2University of Chicago, Chicago, IL, 3Department of Physics, University of Chicago, Chicago, IL

The lamellipodia is a dense, Arp-2/3 mediated actin network at the leading edge of migrating cells that generates forces sufficient to generate sheet-like protrusions of the cell edge. In addition to this force generating role, the initiation of focal adhesions also occurs within the lamellipodia. However, the role of lamellipodial actin in focal adhesion initiation is not well understood. We used a recently identified inhibitor of the ARP 2/3 complex, CK 869, to investigate the role of the lamellipodia in leading edge protrusion and focal adhesion formation in cell motility and adhesion in two cell types- MCF10A cells (a human breast epithelial cell line) and U20S cells (a human osteosarcoma cell line). We find that treatment with CK 869 disrupts or inhibits the formation of an organized lamellipod, as demonstrated by phalloidin staining and immunofluoresce with cortactin, a protein enriched in the lamellipod. Motility assays of single cells and scratch assays with MCF-10A cells demonstrate that treatment with CK-869 reduced the number of motile cells and reduced the motility rate, and in the case of the scratch assay, greatly increased the time to closure of the cell sheet. Despite these effects on cell migration, protrusions of the cell edge persist, suggesting that ARP 2/3 mediated actin assembly is not the only mechanism through which to generate sheet-like membrane protrusions. We hypothesized that these membrane protrusions might differ from lamellipodial protrusions in their ability to form new focal adhesions. Immuno-staining of the focal adhesion proteins paxillin and vinculin demonstrated that the ARP 2/3 inhibited cells had fewer but larger peripheral adhesions and fewer central focal adhesions. Live cell imaging with GFP-paxillin in U2OS cells revealed that the protrusions formed in the presence of the ARP-inhibitor revealed reduced focal adhesion assembly. Collectively, these results indicate that ARP 2/3 is not necessary for sheet-like protrusions of the cell edge but plays a crucial role in focal adhesion formation.

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2063 Quantitative Analysis of Protein Binding Dynamics in Live Cells with Photoswitchable Fluorescent Tags. M. Rubashkin1, M. J. Paszek1, C. C. Dufort1, P. Kumar2, T. Wittmann2, V. M. Weaver1,3; 1Surgery and Center for Bioengineering and Tissue Regeneration, University of California at San Francisco, San Francisco, CA, 2Cell and Tissue Biology, University of California at San Francisco, San Francisco, CA, 3Anatomy, Bioengineering and Therapeutic Sciences, Eli and Edythe Broad Center of Regeneration Med., University of California at San Francisco, San Francisco, CA

The kinetics of individual proteins can dictate focal adhesion dynamics and cellular behaviors such as polarity, motility and migration. We developed a new quantitative fluorescence microscopy technique, RAPS (Regression After Photo-Switching), to study the binding state and kinetics of proteins in live cells. In brief, RAPS involves tagging a protein with a photoswitchable fluorescent protein, such as mEOS2. Initially, an image is taken of the protein in the unswitched green state, 488nm, to identify focal adhesions of interest. Then, a digital micro-mirror device concurrently illuminates all adhesions of interest at the activation wavelength, 405nm, converting a subpopulation of the protein to a stable red fluorescent state. The cell is imaged in the converted state, 568nm, to measure the dynamics of the protein. Lastly, kinetic modeling analysis is implemented to determine the Koff and mobile fraction of the protein. In comparison to existing numerical fluorescence photobleaching techniques, RAPS reduces the time and intensity of light needed for activation, enables imaging in a secondary wavelength to increase contrast and minimize noise, permits diffusion kinetics to be ignored in analysis, and facilitates simultaneous investigation of multiple cell structures within a single cell. In order to verify that RAPS can be used to quantify changes in protein binding kinetics, we studied focal adhesion proteins in MCF10a breast cancer epithelial cells. In the study of talin, we observed a Koff rate of 0.024 ± 0.007 (1/s) and a bound protein fraction of 63 ± 12%. Furthermore, we detected that the mobile fraction of talin significantly increased after inducing expression of the glycoprotein Mucin1, a cancer oncogene associated with changes in cell motility.

Development and Morphogenesis I

2064 Expression of new growth factor of the VEGF family in sea urchin development. Y. O. Kipryushina1,2, K. V. Yakovlev1, N. A. Odintsova1,2; 1A.V. Zhirmunsky Institute of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok, Russia, 2Far Eastern Federal University, Vladivostok, Russia

Growth factors regulate many processes, including cell proliferation, differentiation, migration and apoptosis, and play an important role in embryonic development. Among them vascular endothelial growth factors (VEGF), of the VEGF/PDGF superfamily, are important signaling proteins involved in induction of angiogenesis and playing a central role in the regulation of vasculogenesis in mammals. In invertebrates, such as insects and nematodes, VEGF/PDGF superfamily members guide cell migration and cell differentiation during development. The aim of our study is to investigate the role of VEGFs during development of the sea urchin Strongylocentrotus intermedius. Three Sp-Vegf genes have been predicted in the genome of a closely related sea urchin S. purpuratus. One of them, Sp-Vegf3, has been reported to play an essential role in sea urchin development (Duloquin et al., 2007). In this study, we investigated the expression of a new gene of the VEGF family, Si-Vegf2, and expression of the Si- Pdgfr/vegfrL gene in sea urchin development by RT-PCR. We demonstrated a high similarity of SUNDAY the central part of the new gene, which we found in the sea urchin S. intermedius, with that of the predicted gene Sp-Vegf2 in S. purpuratus. The Si-Vegf2 was found to be expressed from the zygote stage, whereas Si-Pdgfr/vegfrL expression began later at the blastula stage. Our data have shown that a nonspecific inhibitor of heparine-binding GF receptors, suramin, results in significant disturbances in embryonic development: archenteron formation is suppressed, and spicule growth is inhibited. An addition of human PDGF-AB to embryonic culture leads to partial recovery of archenteron growth. These results match the findings on another sea urchin species (Katow and Aizu, 2002). In summary, we have discovered the expression of a new VEGF family member in sea urchin development and shown that temporal expression profiles of the Si-Vegf2 and Si-Pdgfr/vegfrL are not similar in sea urchin development, but signaling through VEGF/PDGF pathway is essential for embryonic development. (This work was supported by Presidium of the FEB RAS (grants 09-I-P22-04, 09-II-SB-06-001) and Program at the Far Eastern Federal University (grant 11 G34.31.0010)).

2065 How “acellular” embryos gastrulate: Investigation of cytoplasm movements during gastrulation in developing Drosophila melanogaster embryos. B. He1, O. Polyakov2, E. Wieschaus1,3; 1Department of Molecular Biology, Princeton University, Princeton, NJ, 2Department of Physics, Princeton University, Princeton, NJ, 3Howard Hughes Medical Institute

During Drosophila gastrulation, the mesoderm precursor cells are internalized from the ventral surface of the embryo and form a ventral furrow. The invagination of these cells is accompanied by apical constriction, a process driven by cycles of pulsed contraction and stabilization of the actin-myosin network underneath the apical membrane. Previous studies have shown that in response to individual constriction pulses, the ventral cells elongate along their apical-basal axis and their nuclei move basally in an incremental manner. However, it is unclear how contractile forces constrained to the vicinity of the apical surface drives cell shape change along the entire cell length. We recently identified a cellularization mutant in which the periplasm of the syncytial blastoderm is never partitioned into individual cells. Intriguingly, these “acellular” embryos still gastrulate, and exhibit initial gastrulation movements largely reminiscent of those in normal cellular embryos. In particular, a contractile actin-myosin network forms on the ventral cortex and drives apical constriction, which leads to formation of a pseudo-ventral-furrow. The observation suggests that the cytoplasm can respond to cellular forces and undergo morphogenetic movements even in the absence of cell membrane. To test this, we investigated the micromechanical property of the cytoplasm and its response to constriction pulses by tracking injected fluorescent polystyrene microspheres. Constriction pulses were identified by simultaneously imaging a GFP-tagged membrane marker or myosin subunit. We found that the periplasm in both cellular and acellular embryos was highly viscous (~3 orders magnitude higher than water) but on the other hand did not show measurable elasticity over developmentally relevant timescales. In the cellular embryos, upon gastrulation, beads within the ventral cells moved basally in an incremental manner that largely correlated with pulsed constriction. Interestingly, beads initially localized to the apical region above the nucleus were never observed to flow past the nucleus upon apical constriction. Furthermore, the distance between beads localized in the same cell remained correlated during cell lengthening and did not change abruptly over time, as if the cytoplasm was a gel that underwent gradual deformation under force. Strikingly, this behavior does not appear to rely on the presence of cell membrane, as in the acellular embryos the beads within the ventral periplasm display similar global movements as in the cellular embryos. Based on these observations we propose that during Drosophila ventral furrow formation the cytoplasm responds to cellular forces in a more “gel-like”, rather than a “liquid-like”, manner. This “gel-like” property would allow for an SUNDAY instantaneous transmittance of apically generated force throughout the entire tissue to drive cell shape change and their corresponding invagination. Our study also implicates that cellular components that determine the viscoelastic property of the cytoplasm are potential targets of regulation during tissue morphogenesis.

2066 Probing tension and dynamics in actomyosin mediated cell shape change. C. D. Higgins1, U. S. Tulu2, L. Gao3, E. Betzig3, D. P. Kiehart2, B. Goldstein1; 1Biology, UNC Chapel Hill, Chapel Hill, NC, 2Biology, Duke University, Durham, NC, 3Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA

Apical constriction is a cell shape change critical for morphogenesis in diverse organisms. Apical constriction requires the activity of a dynamic, contractile actomyosin meshwork that is enriched at the apical cortex of the cell. In the 26-cell C. elegans embryo, two cells apically constrict and internalize. We have found previously that contractility in the apical actomyosin meshwork of these cells is initially uncoupled from apical constriction, and embryos deficient for RacGEF ced-5 and classical cadherin hmr-1 display normal myosin dynamics that fail to drive apical constriction. These findings suggest a model where the contractile actomyosin meshwork must become coupled to apical cell-cell contacts to drive cell shape change. Here, we use particle image velocimetry (PIV) to map the movements of the actomyosin meshwork and cell- cell contacts over time. An unbiased PIV algorithm confirms our manual measurements indicating that actomyosin contractility precedes cell shape change. Furthermore, we estimate the tensile properties of the meshwork by cortical laser ablation and show that contractile forces are established prior to cell shape change. We also show that tension in the apical meshwork is higher in apically constricting cells than neighboring cells. To examine actomyosin dynamics and cell shape change with improved axial resolution, we have also taken advantage of Bessel beam structured plane illumination microscopy. Together, our results suggest that both apical actomyosin contractility and coupling to cell-cell contacts are required to facilitate cell shape change.

2067 Imaging Assays for High-throughput Quantification of Synaptic Formation and Neurite Dynamics in Human iPSC-derived Neurons. J. A. Hesley1, L. Chase2, M. Strathman2, O. Sirenko1, P. Tavormina1; 1Molecular Devices, LLC, Sunnyvale, CA, 2Cellular Dynamics International, Inc., Madison, WI

Central nervous system (CNS) disorders affect millions of people worldwide. Considerable effort has been made in an attempt to develop relevant in vitro human model systems for studying degenerative (i.e. Alzheimer’s and Parkinson’s disease) and genetic (i.e. Huntington's disease and muscular dystrophy) disorders as well as neurotoxicity. Induced pluripotent stem cells (iPSCs) of human origin provide an ideal model for scientists both within the academic and pharmaceutical settings. By pairing these cells with an automated high content imaging system, endpoint and live-cell assays can be used to determine whether pharmaceutical drug candidates or environmental contaminants exert neurotrophic, neuroprotective, or neurotoxic effects. High content imaging of neurons allows scientists to both characterize and measure changes in neuronal networks such as neurite number, length, and branching, as well as to quantitate synaptogenesis. iPSC-derived iCell® Neurons were treated with neurite modulating compounds at varying doses and images were acquired over time using the ImageXpress® Micro High Content System. These experiments were done using time-lapse imaging of live cells with transmitted light or fluorescence as well as using immunofluorescence staining of fixed cells with the neuronal SUNDAY marker class III beta tubulin (TuJ1). Images of the neural networks were analyzed with MetaXpress® Software to identify numerous aspects of the cells and their outgrowths. The data shows over a doubling of neurite outgrowths over a 12 hour period without stimulation and a clear response to drug doses as well. In addition, emergence of GABAergic and glutamatergic synaptic phenotypes was seen by tracking the number of vGAT+ and vGLUT2+ puncta within a 1-3 week post-thaw timecourse. These results demonstrate that human iPSC-derived neurons paired with high content imaging techniques provide valuable information for assessing the effects of compounds on neuronal networks and can be applied to various academic and pharmaceutical applications.

2068 Growth Hormone Regulates Survival of Primary Cultures of Embryonic Neural Precursor Cells. C. Regalado Santiago1, M. L. Lopez Meraz2, E. Juarez Aguilar3; 1Doctorado en Ciencias Biomedicas, Instituto de Ciencias de la Salud Universidad Veracruzana, Xalapa, Mexico, 2Centro de Investigaciones Cerebrales Universidad Veracruzana, Xalapa, Mexico, 3Departamendo de Biomedicina, Instituto de Ciencias de la Salud Universidad Veracruzana, Xalapa, Mexico

During central nervous system development, cellular proliferation and cellular survival play a critical role. It has been suggested that these biological processes are regulated in an orchestrated way by numerous neurotrophic factors. In addition to the well known effects of growth hormone (GH) on growth and metabolism regulation, there is a growing body of evidence that suggest a central role of this hormone in the development and function of the brain. The presence of the GH and its receptor has been widely reported in different regions of the brain. In spite of these findings, the biological role of GH in the CNS is not completely understood. In order to analyze the effect of GH during CNS development, we performed studies on primary cultures of embryonic (~E14) neural precursor cells from striatum. In presence of the epidermal growth factor (EGF), this population gives rise to neurospheres of undifferentiated cells that can generate neurons, oligodendrocytes and glia. In a first approach, we showed that GH was unable to stimulate the formation of neurospheres in absence of EGF. Secondly, we incubated primary cultures of striatum with different concentrations of GH (1, 10, 50, 100, 500 and 1000 ng/ml) in presence of EGF and determined the total number of neurospheres produced. The results showed a typical “bell shaped” curve with an increase since 1 ng/ml of GH and a maximal response at 10 ng/ml of hormone. In order to discriminate whether the increase in the number of neurospheres stimulated by GH was due to a mitogenic effect, primary cultures of striatum were incubated with different doses of GH and the analogue of thymidine BrdU (10 µM), during 24 hours. Counting of BrdU positive cells showed no difference between control and cultures treated with different doses of GH (10 ng/ml - 100 ng/ml), while this hormone inhibited BrdU incorporation at doses of 500 ng/ml and 1000 ng/ml. Interestingly, GH stimulated a proliferative effect on primary cultures of striatum in absence of EGF. Next, we evaluated if GH has a neuroprotective action on the primary cultures using an apoptosis assay (TUNEL). Results showed a decrease in the apoptosis rate on cultures treated with GH at 10 ng/ml. Altogether, these results suggest that GH could be considered a survival factor rather than a mitogenic factor during embryonic development of the CNS at the E14 stage.

SUNDAY

2069 Endothelium effects on the differentiation of iPSCs to insulin-producing cells. D. Talavera1, S. Kurtovic1, D. C. Dafoe1; 1Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA

Formation of fully differentiated insulin-producing beta cells depends of the interaction between endothelial cells (ECs) and pre-patterned endodermal cells in vivo. It has been described that endothelial-derived bone morphogenetic proteins (BMPs) play an important role in this differentiation process. However, such EC signals are not fully characterized. Therefore, our objective is to study the effects of ECs in the differentiation of induced pluripotent stem cells (iPSCs) to insulin-producing cells and the role of EC-BMPs in this process. To accomplish this objective, we generated human embryoid bodies (EBs) from iPSCs. Different groups of EBs were cultured in a three-dimensional system of collagen-laminin gels that allowed EB-EC interaction for longer periods. After four weeks, the differentiated cells from EBs were analyzed by immunocytochemistry (ICC), immunohistochemistry (IHC), qRT-PCR, and Western blot (WB) looking for the expression of specific beta-cell, progenitor-cell, and islet-cell markers. To study the role of BMP-2/-4 and BMPR1A in the differentiation process, expression of BMP-2/-4, and BMPR1A as well as co-expression of C-peptide and phosphorylated Smad1/5/8 (pSmad1/5/8) were analyzed in all EB groups. Our results showed a significant increase in the expression of specific beta-cell markers (insulin, PDX-1, GLUT2, Nkx6.1, Kir6.2, SUR1, GKS, PC1/3, and PC2), progenitor-cell markers (Isl-1, Ngn3, Pax4, Pax6, Nkx2.2, Nkx6.1, and Ptf1a), and islet- cell markers (GCG and STT) in those EBs co-cultured with ECs in comparison to EBs cultured alone. Islet-like structures composed of cells that co-expressed C-peptide and PDX-1 or C- peptide and pSmad1/5/8 were identified exclusively in co-cultured EBs. In addition, higher expression of BMP-2, BMP-4, and BMPR1A was found in these EBs in contrast to EBs cultured alone. Our results indicate that ECs promote the differentiation of iPSCs to insulin-producing cells and that the BMP pathway activation plays a key role in this process.

2070 Myogenic progenitors regulating skeletal muscle homeostasis through Bmpr1a signaling. P. Huang1, T. J. Schulz2, A. Beauvais1, Y-H. Tseng2, E. Gussoni1; 1Children's Hospital Boston, Harvard Medical School, Boston, MA, 2Joslin Diabetes Center, Harvard Medical School, Boston, MA

Skeletal muscle maintenance and regeneration require finely coordinated interaction of multiple cell types. The molecular mechanism regulating this coordination is not clear. Bmp-signaling is suggested to balance proliferation and differentiation of myogenic cells in vitro. Here, we ablated expression of Bmpr1a in Myf5+ and MyoD+ progenitors in vivo (Myf5+/cre:Bmpr1af/f and MyoD+/cre:Bmpr1af/f ). Mutant Myf5+/cre:Bmpr1af/f mice were born runted and remained significantly smaller in size throughout life. Ablation of Bmpr1a resulted in increased fat infiltration and smaller regenerated myofibers in acutely injured Myf5+/cre:Bmpr1af/f and MyoD+/cre:Bmpr1af/f muscles. Interstitial myogenic cells (IMCs, Sca-1posCD45neg CD31posPDGFRaneg) isolated from mutant mice proliferated more slowly as compared to wild type cells. Following culture and differentiation in vitro, Sca-1posCD45neg cells from wildtype mice gave rise to myogenic and adipogenic progenitors, whereas Sca-1posCD45neg cells from mutant mice generated, nearly exclusively, adipocytes. Ablation of Bmpr1a expression in Bmpr1a f/f Sca-1posCD45neg progenitors also resulted in adipocyte differentiation, as opposed to myogenic cells and adipocyte when Bmpr1a is expressed. Altogether, these results show that Bmpr1a signaling is necessary for the commitment and proliferation of interstitial progenitors towards the myogenic lineage, and its presence might regulate intramuscular adipogenesis. SUNDAY

2071 Human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by AMP-activated protein kinase. E-K. Kim1, S. Lim1, J-M. Park1, J. Seo1, J. Kim2, K. Kim3, S. Ryu3, P-G. Suh1; 1School of Nano- Bioscience & Chemical Engineering, UNIST, Ulsan, Korea, 2Physiology, Pusan National University, Busan, Korea, 3Division of Molecular and Life Sciences, POSTECH, Pohang, Korea

AMP-activated protein kinase (AMPK) is an energy-sensing kinase that has recently been shown to regulate the differentiation of preadipocytes and osteoblasts. However, the role of AMPK in stem cell differentiation is largely unknown. Using in vitro culture models, the present study demonstrates that AMPK is a critical regulatory factor for osteogenic differentiation. We observed that expression and phosphorylation of AMPK were increased during osteogenesis in human adipose tissue–derived mesenchymal stem cells (hAMSC). To elucidate the role of AMPK in osteogenic differentiation, we investigated the effect of AMPK inhibition or knockdown on mineralization of hAMSC. Compound C, an AMPK inhibitor, reduced mineralized matrix deposition and suppressed the expression of osteoblast-specific genes, including alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN). Knockdown of AMPK by shRNA-lentivirus infection also reduced osteogenesis. In addition, inhibition or knockdown of AMPK during osteogenesis inhibited ERK phosphorylation, which is required for osteogenesis. Interestingly, inhibition of AMPK induced adipogenic differentiation of hAMSC, even in osteogenic induction medium (OIM). These results provide a potential mechanism involving AMPK activation in osteogenic differentiation of hAMSC and suggest that commitment of hAMSC to osteogenic or adipogenic lineage is governed by activation or inhibition of AMPK, respectively.

2072 Thymic nurse cells express signature markers consistent with epithelial progenitor phenotype. R. V. Chilukuri1, V. K. Patel1, M. D. Samms2, J. C. Guyden1; 1Biology, The City College of New York, New York, NY, 2The City College of New York, The City College of New York, New York, NY

Thymic nurse cells (TNCs) represent a subset of thymic cortical epithelial cell with the unique ability to entose other cell types. Our earlier studies demonstrated that TNCs bind and internalize immature thymocytes bearing the αβ TCRloCD4+CD8+ phenotype. Subsequent studies showed that the binding and internalization of thymocytes by TNCs is mediated by a TNC-specific antigen that is recognized by the monoclonal antibody, pH91. Internalized thymocyte subset received life or death signals during the process of MHC restriction, resulting in survival and release of some thymocytes or the induction of apoposis in the thymocyte subset that remained trapped within specialized TNC compartments. In this study, we examined the ontogenesis of the TNC-specific antigen. Using confocal microscopic analyses, the onset expression of the pH91-specific antigen occurs as early as E11.5 stage of development and persists throughout embryological development as well as postnatally in mice. Further analyses of PH91+ cells show the co-expression of K5, K8, Foxn1 and P63. At E17.5 stage of development, we observe the formation of the multicellular, lympho-epithelial complexes that exhibit the pH91+K8+K5Foxn1+P63+ phenotype. These markers have been implicated in the identification of thymic epithelial progenitors elsewhere. Interestingly, in a previous report, we showed pH91+K5+K8 TNCs to be strategically localized within the cortico-medullary junction of the thymus, thus possibly allowing for differentiation of K8+cells to proceed toward cortex and K5+cells to the medulla. By E18.5 stage of development, the pH91+K8+K5+Foxn1+P63+ TNCs express elevated levels of MHC-class II antigens on their cell surfaces. In addition, confocal SUNDAY analyses show that in post-natal mice 26% of pH91+ TNCs also express K8 and K5, whereas 76% of the K8+K5+ cells in the thymus are TNCs. Taken together our data suggest that pH91+K8+K5+Foxn1+P63+ TNCs have thymic epithelial progenitor potential.

2073 The Role of Endogenous FGF-2 in heterogeneity and pluripotency of human embryonic stem cells. K. Matulka1, L. Eiselleova1, M. Kunova1, A. Hampl2,3, Y-M. Sun1, P. Dvorak1,3; 1Department of Biology, Masaryk University, Brno, Czech Republic, 2Department of Histology, Masaryk University, Brno, Czech Republic, 3st. Anne´s University Hospital, International Clinical Research Center, Brno, Czech Republic

Exogenous fibroblast growth factor-2 (FGF-2) is one of crucial supplements in media for human embryonic stem (ES) cells. In our systematic work, we investigate its roles in plating efficiency and survival rate under stressed conditions. We show that exogenous FGF-2 enhances the expression of high molecular mass (HMM) isoforms of FGF-2, which have been implicated in promoting proliferation and survival in most cell types. Stably over expression of HMM FGF-2 isoforms has been shown to link to malignant phenotypes. We found that all isoforms of FGF-2 are expressed in undifferentiated human ES cells, whereas the low molecular mass FGF-2 retains its expression when cells undergo differentiation. The withdrawal of exogenous FGF-2 results in cell differentiation depending upon the size of colonies. Our data suggest that exogenous FGF-2 is involved in the early establishment of heterogeneous population. Furthermore, FGF-2 knockdown human ES cells using shRNA are predisposed to differentiate, suggesting that FGF2 plays a pivotal role in pluripotency. We hypothesise that FGF-2 signalling maintains the undifferentiated growth of hESCs. This action is enhanced by exogenous FGF-2. Both, extrinsic and intrinsic, activities may synergize to regulate transition between self-renewal and differentiation of hESCs.

2074 Mutant frequency increases with the length of cultivation in human embryonic stem cells. M. Kruta1, L. Balek1, R. Hejnova1, M. Seneklova1, A. Hampl2, P. Dvorak1, V. Rotrekl1; 1Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic, 2Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic

Human embryonic stem cells (hESCs) derived from blastocyst of early embryo are capable of differentiation into all three germ layers and can produce all cell types of human body. Although hESCs immortality and their differenciation potential make them promising candidates to become powerful tool of regenerative medicine, their long-term propagation in vitro induces accumulation of genetic changes as demonstrated by karyotyping, SNP analysis or analysis of copy number variation. These changes might render the cells useless in regenerative medicine. We have shown one of the mechanisms contributing to these changes, Our data demonstrate that decreased efficiency of base excision repair (BER) in late passage hESCs contributes to the genome instability. Decrease of apurinic/apyrimidinic endonuclease APE1, a key enzyme of BER, with increasing time in culture leads to changes in DNA repair dynamics as well as to changes in DNA damage signaling. To dissect the role of such changes in DNA repair and damage signaling during prolonged hESC cultivation we have designed specific mutation assay. It is a method based on mutagenesis of hypoxanthine-phosphoribosyltransferase (HPRT) gene. Despite the current thinking based on computer models, that time in culture does not increase the mutagenic pressure, we have shown significantly increased both spontaneous and IR- induced frequency of mutants in late passage hESCs in comparison with early passage. SUNDAY

However the higher doses of IR (above 1Gy) induce similar effect in early and late passage, suggesting that the most dramatic effect happens under non stressing conditions. Taken together, our results suggest that the changes in the APE1 level during long-term cultivation of hESCs play an important role in their response to acquired DNA damage and that the long-term propagation increases the mutagenic burden and that these events present a chain of events from DNA repair failure all the way to genome instability, presented as increased mutation burden.

Supported by Ministry of Education, Youth and Sports the Czech Republic (LC06077) and by the MZ CR (grant NS10237-3)

2075 Primary Cilia of Proliferating Cells in Rodent Dorsal Root Ganglia. M. V. Coronel1, D. Hao1, W. Huang1, H. D. Schwark1; 1Biology, University of North Texas, Denton, TX

Primary cilia are non-motile, microtubule based organelles that arise from the membrane of most vertebrate cells. Mutations in primary cilia genes can lead to severe developmental abnormalities. In the nervous system these include hydrocephaly, neural tube patterning defects and sensory deficits that may include anosmia, hearing loss, retinal degeneration and impaired somatic sensory inputs. Many of the developmental defects seen in primary cilia mutants have been attributed to impaired sonic hedgehog signaling. The mitogen sonic hedgehog is essential in development of neural crest-derived structures such as the dorsal root ganglia (DRG): disruption of sonic hedgehog signaling results in failure of primary sensory neurons to develop. This essential developmental role played by primary cilia may continue into adulthood. Each sensory neuron in the DRG is tightly ensheathed by surrounding satellite glial cells. These cells arise around E9.5 (neurons) and E11 (glia) from multipotent progenitors derived from the neural crest. Neurons, and perhaps satellite glial cells, continue to be added to the postnatal DRG, but the origin of these new cells is unknown. It seems likely that primary cilia play a role in postnatal proliferation and maintenance of DRG homeostasis for the following reasons: primary cilia play an active role during the cell cycle; proliferation of stem cell niches is aberrant in primary cilia mutants; peripheral nerve injury induces sonic hedgehog expression in the DRG; and patients with abnormal primary cilia have sensory deficits. We have begun our investigation into the role of primary cilia in the developing and adult rodent DRG by determining the incidence and lengths of the cilia. In the rat DRG, primary cilia decrease in length from P3 (2.6 um) to P14 (2.1 um) to adult (1.9 um). A similar trend of decreasing cilium length with development is seen in proliferating satellite glial cells labeled by antibodies to the cell cycle marker Ki67. At P1 the average cilium length is 1.4 um, and at P7 the average length is 1.2 um. The percentage of ciliated proliferating cells also decreased across these ages, from 35.5% to 28.5%. Finally, we also identified primary cilia in cells expressing the transcription factor Sox2, which is thought to maintain certain cell types in an undifferentiated state. Cilia characteristics in this small sample of cells were not quantified. The decreasing lengths and incidence of primary cilia with development may underlie a decreased sensitivity to sonic hedgehog signaling.

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2076 HNF-4α control of pancreatic endocrine differentiation from pluripotent stem cells. E. C. Moorefield1, X. Guan1, M. E. Furth1, C. E. Bishop1; 1Wake Forest University, Winston- Salem, NC

The HNF4A gene codes for hepatocyte nuclear factor (HNF)-4α, which has an important role in pancreatic development and maintenance of β-cell function. Haplo-insufficiency of this transcription factor causes a form of monogenic type 2 diabetes (T2D), maturity-onset diabetes of the young 1 (MODY1). Recently, the advancement of embryonic stem (ES) cell and induced pluripotent stem (iPS) cell technology makes study of the genetic basis of the disease achievable. Pluripotent stem cells can be differentiated into insulin producing β-cells using step- wise protocols that recapitulate signaling pathways necessary for endocrine pancreas development in vivo. Growth factors and small molecules are applied in a step-wise fashion to replicate the stages in β-cell development which include endoderm formation, pancreas specification, endocrine specification and β-cell maturation. By applying this protocol to normal human ES cells we have shown that HNF-4α is expressed at high levels during pancreas specification and continues to be expressed at lower levels in throughout the differentiation process. To investigate the effect of HNF-4α deficiency on pancreatic β-cell development and function we have examined two systems. First, we have generated iPS cells from MODY1 patient fibroblasts using a polycistronic human lentivirus carrying Oct4, Sox2, Klf4 and c-myc. In addition, we have created dox-inducible HNF-4α knockdown hES cells. The stemness of both systems has been confirmed by analysis of a panel of pluripotency markers. However, the MODY1 iPS showed a large clone to clone variation in differentiation capability toward insulin- producing cells. On the contrary, we have confirmed that HNF-4α expression can be efficiently knocked down throughout in vitro differentiation to endocrine pancreas by shRNA delivery. Thus, the HNF-4α KD hES system will allow us to elucidate the impact of genetic variation on the development and function of pancreatic β-cells which may lead to identification of novel therapeutic targets for treatment of T2D.

2077 RNAi screen of predicted E3 ubiquitin that may mark MEX-3 for degradation in the Caenorhabditis elegans embryo. A. Lin1, K. Reichard1, A. Desai2, P. Heng2, K. Schwartz2, N. Huang1; 1Biology, Colorado College, Colorado Springs, CO, 2Colorado College, Colorado Springs, CO

Early embryonic development in C. elegans depends on precise timing of division and differentiation, which requires precise regulation of maternally supplied cell fate determinants. MEX-3, a maternally supplied RNA binding protein, is present throughout the oocyte, 1-cell, and 2-cell embryo. By the 4-cell stage, MEX-3 becomes depleted in the posterior blastomeres. By binding to the 3’ UTR of pal-1 mRNA, MEX-3 prevents translation of PAL-1 in anterior blastomeres. PAL-1 is a homeodomain transcription factor that promotes posterior development, including muscle development. In mex-3 mutants, PAL-1 is expressed throughout the early embryo, leading to muscle excess and embryonic lethality. After the 4-cell stage of embryogenesis, MEX-3 is rapidly broken down. The E3 ubiquitin ligase component of the ubiquitin-proteosome system determines protein specificity of ubiquitination, which leads to degradation. There are numerous predicted E3 ubiquitin ligases in the C. elegans genome.

The goal of this project is to use RNA inhibition (RNAi) to knock out predicted E3 ubiquitin ligases, and determine which one(s) mark MEX-3 for degradation in the C. elegans embryo. Genes encoding predicted E3 ubiquitin ligases that were previously shown to be maternally expressed and/or that produced embryonic lethality in previous RNAi experiments were chosen SUNDAY as candidate genes to knock out. We first determined if knockout of the candidate genes resulted in embryonic lethality higher than 20%. Then, fluorescence microscopy was used to determine whether RNAi effected the degradation of a GFP::MEX-3 fusion protein. If an E3 ubiquitin ligase contributing to MEX-3 degradation was successfully knocked out, GFP::MEX-3 would remain present throughout the embryo. RNAi was induced by feeding young adults with bacteria expressing dsRNA, or by directly injecting the dsRNA. To date, numerous predicted HECT-domain, U-box, and monomeric RING finger E3 ubiquitin ligases have been knocked out by RNAi. Depletion of five of these genes resulted in high levels of embryonic lethality, with two resulting in abnormal patterns of GFP::MEX-3 expression. However, mis-expression of MEX-3 does not appear to be the primary defect in these RNAi embryos. The screen is being continued with additional predicted E3 ubiquitin ligases.

2078 Osteoblast response to a nanostructured titanium surface. M. M. Beloti1, R. B. Kato1, A. Nanci2, P. T. de Oliveira1, A. L. Rosa1; 1School of Dentistry of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil, 2Faculté de Médecine Dentaire, Université de Montréal, Canada

Titanium (Ti) surface nanotopography obtained by chemical deoxidation and controlled reoxidation affects early events of osteoblast phenotype development in cultures derived from rat calvaria. The aim of our study was to evaluate the effect of the nanostructured Ti surface on the responses of human osteoblastic cells. Machined Ti discs, 12 mm in diameter, were chemically treated with H2SO4/H2O2 for 2 hours. Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured under osteogenic condition until subconfluence. First passage cells were cultured (2104 cells/disc) on nanostructured and machined Ti discs for periods of up to 21 days. For cell attachment assay, adherent cells were counted at 24 hours and data were expressed as a percentage of the initial cell number. For proliferation, cells were counted at 1 and 7 days and data were expressed as doubling time in hours. At day 7, cell viability was evaluated using trypan blue and expressed as a percentage of viable cells. At days 1, 3, and 7, cell morphology was evaluated with phalloidin and DAPI; alkaline phosphatase (ALP) labeling was performed using a primary mouse anti-human ALP antibody. Extracellular matrix mineralization was detected by Alizarin red staining at 21 days and data were expressed as a percentage of the total disc area. Quantitative data were compared by Mann-Whitney test (n=3). There were no statistically significant differences between nanostructured and machined Ti in terms of cell attachment (34.7±6.4 and 40.3±10.5; p=0.70), proliferation (50.4±2.7 and 48.1±2.1; p=0.28), and viability (92.1±2.8 and 93.1±1; p=0.83). At day 1, cells were spread and exhibited an elongated polygonal shape on machined Ti whereas on nanostructured Ti they were mostly stellate-shaped. At day 7, both Ti surfaces exhibited areas of cell multilayering and cells were predominantly elongated. ALP positive cells were observed at all time points with a similar labeling pattern on both Ti surfaces. It was noticed five times more extracellular matrix mineralization (2.7±0.2 and 15.2±4.6; p=0.01) on nanostructured compared with machined Ti. In conclusion, our results indicate that nanotopography induces a significant increase in the late key event of in vitro osteogenesis, i.e.: extracellular matrix mineralization and this surface modification may, therefore, be considered a useful strategy to accelerate/enhance the process of Ti implant osseointegration.

Acknowledgements: FAPESP (Grant # 2010/18395-3, 2010/19280-5)

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2079 Effects of hedgehog signaling on human differentiated osteoblasts. F. S. Oliveira1, G. B. Junqueira1, M. M. Beloti1, A. L. Rosa1; 1School of Dentistry of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil

Hedgehog (Hh) signaling is involved in many developmental processes including osteoblast differentiation of mesenchymal stem cells. Here, we aimed at determining the effect of Hh signaling modulation on phenotype expression of differentiated osteoblasts. Cells harvested from human dental alveolar bone were cultured in osteogenic media under four conditions: non- supplemented (control), supplemented with the Hh agonist purmorphamine (2 M), supplemented with the Hh antagonist KAAD-cyclopamine (100 nM), and supplemented with both purmorphamine and KAAD-cyclopamine. Hh signaling was evaluated by gene expression of Gli1 at day 7. Osteoblast phenotype expression was evaluated by activity and gene expression of alkaline phosphatase (ALP) at day 7, and extracellular matrix mineralization at day 21. Data (n=3) were compared by Kruskall-Wallis test followed by Fisher’s test when appropriated. Hh pathway was stimulated by purmorphamine as revealed by an increase of almost four times in the expression of Gli1 compared to control (p=0.001). This increase was only partially reversed by KAAD-cyclopamine (p=0.05) that by itself did not present any effect when compared to control (p>0.05). Despite this, ALP activity (p>0.05), ALP gene expression (p>0.05) and matrix mineralization (p>0.05) were not modified by any culture condition. These results suggest that the modulation of Hh signaling does not affect the phenotypic expression of human differentiated osteoblasts.

Acknowledgments: FAPESP and CNPq

2080 Bavachinin A modulates the differentiation of hADMSCs on adipogenesis and osteogenesis. H-J. Jang1,2, S. Lim1, J-M. Kim1,2, S. Ryu2, P-G. Suh1; 1School of Nano-Bioscience and Chemical Engineering, UNIST, Ulsan Metropolitan, Korea, 2Division of Molecular and Life Science, POSTECH, POHANG, Korea

Human mesenchymal stem cells are an attractive model not only to investigate molecular mechanisms of cell growth and differentiation under normal and pathological conditions but also to use cell-based therapies for regenerative medicine. Therefore, it is important to understand how their differentiation is regulated. We screened the molecules which could regulate adipogenesis using natural compound library. As a screening result, we found that bavachinin A enhanced adipogenesis of human adipose-derived mesenchymal stem cells(hADMSCs). Bavachinin A also increased the mRNA level of adipocyte markers, such as CCAAT-enhancer- binding proteins α, peroxisome proliferator-activated receptor γ(PPAR-γ) and adipocyte fatty acid–binding protein during adipocyte differentiation. Furthermore, PPARγ activity was increased by treatment of bavachinin A. To evaluate the effect of bavachinin A in vivo, we used implanted de novo adipose tissue formation system. It showed that intraperitoneal administration of bavachinin A in mice, implanted with mass of 3t3-f442A cells, increased de novo adipogenesis and weight of inborn adipose tissues. Whereas bavachinin A inhibited osteogenesis of hADMSCs. Our results suggest that bavachinin A is a potent activator of adipogenesis and a inhibitor of osteogenesis via activation of PPAR-γ.

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2081 Directed Stem Cell Differentiation Using PEG/α-CD-derived Biomaterials. A. Singh1, J. Zhan1, J. Elisseeff1,2; 1BME, Johns Hopkins University, Baltimore, MD, 2Ophthalmology, Wilmer Eye Institute, Baltimore, MD

Adult stem cells (ASCs), e.g., mesenchymal and adipose-derived (MSCs and ADSCs) hold great promise in tissue engineering because of their proliferative capacity and ability to differentiate into several phenotypes, and ultimately built new tissues. However, to enable translation of stem cell therapies, there is a significant need to generate materials that can be used to study fundamentals in biology and modulate or enhance differentiation. Poly(ethylene glycol) (PEG)-based hydrogels are frequently utilized to encapsulate cells in a 3D environment. Unfortunately, PEG based biomaterials lack functionality to incorporate multiple chemical and biological moieties as signals for stem cells to probe behavior and guide development. Conventional methods to modify PEG hydrogels include copolymerization and chain extension, which leads to changes in mechanical properties of the material. Therefore, we developed an advanced multifunctional hydrogel by combining PEG- and α-Cyclodextrin (α-CD). α-CD is a six membered oligosaccharide ring, which threads onto the PEG chains. [1] The hydroxyl groups of α-CD provide necessary functional sites to introduce various moieties, such as hydrophobic - groups (CH3), hydrophilic groups (PO4 ) and integrin-binding peptide RGD by chemical modifications. This simple strategy provides us a unique ability to independently control chemical functionality, mechanical and bioadhesive properties of the hydrogels to probe stem cell behavior. ASCs were either seeded onto or encapsulated in UV-photopolymerizable PEG-α-CD hydrogels with varying stiffness (0.5 kPa to 30 kPa), adhesive peptide concentrations (0% to 10% w/v) and different functional groups. In an initial experiment, the enhanced chondrogenic differentiation of gMSCs in 10% PEGDA/1% α-CD hydrogel was evident from increased gene expression for Col II and Aggrecan, and Safranin O staining for GAG deposition. At the similar mechanical properties of the hydrogel, introducing a hydrophobic group (CH3) in α-CD enhanced adipogenic - differentiation, while a hydrophilic group (PO4 ) increased osteogenic differentiation of hADSCs. We also found that cell spreading and morphology are dependent on both stiffness and concentrations of integrin-binding peptide. On the stiffer substrate, hMSCs adopted elongated morphology at all concentrations of RGD. Interestingly, on the softer substrate hMSCs adopted elongated morphology at higher concentrations of RGD. RT-PCR experiments showed that softer matrix upregulates gene-expression markers for adipogenesis at both lower and higher cell-binding peptide concentrations, while moderately stiffer substrate with lower cell-binding peptide concentration upregulates markers for myogenesis. The stiffer substrate enhances both chondrogenesis and osteogenesis at higher concentrations of integrin-binding peptide. This indicates that both substrate-stiffness[2] and concentration of cell-binding peptide sites dictate stem cell differentiation. In summary, this biomaterial enables us to probe stem cell behavior, such as proliferation and lineage specific differentiation by independently controlling the functionality, mechanical and bioadhesive properties of the material. References: 1. A. Harada, J. Li, M. Kamachi, Nature, 1992, 356, 325-327. 2. A.J. Engler, S. Sen, H.L. Sweeny, D.E. Discher, Cell, 2006, 126(4), 677-689.

Acknowledgements: Maryland Stem Cell (TEDCO) fellowship, Dr. O. Manuel Uy, and Cindy Berlinicke, and JHE group.

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Cell Biology of the Neuron

2082 PINK1 and Parkin Target Miro for Phosphorylation and Degradation to Arrest Mitochondrial Motility. X. Wang1, D. Winter2, G. Ashrafi2, J. Schlehe3, Y. Wong4, D. Selkoe3, S. Rice4, J. Steen2, M. LaVoie3, T. Schwarz3; 1Stanford University/Harvard Medical School, Boston, MA, 2Children's Hospital Boston, MA, 3Harvard Medical School, Boston, MA, 4Northwestern University, Evanston, IL

Cells keep their energy balance and avoid oxidative stress by regulating mitochondrial movement, distribution, and clearance. We report here that two Parkinson's disease proteins, the Ser/Thr-kinase PINK1 and ubiquitin-ligase Parkin, participate in this regulation by arresting mitochondrial movement. PINK1 phosphorylates Miro, a component of the primary motor/adaptor complex that anchors kinesin to the mitochondrial surface. The phosphorylation of Miro activates proteasomal degradation of Miro in a Parkin-dependent manner. Removal of Miro from the mitochondrion also detaches kinesin from its surface. By preventing mitochondrial movement, the PINK1/Parkin pathway may quarantine damaged mitochondria prior to their clearance. PINK1 has been shown to act upstream of Parkin but the mechanism corresponding to this relationship has not been known. We propose that PINK1 phosphorylation of substrates is necessary for the subsequent action of Parkin and the proteasome.

2083 Essential Role of a Conserved Homophilic Interaction Domain of SYD-2/Liprin-alpha Protein in Presynaptic Assembly in C.elegans. H. Taru1; 1Creative Research Institution, Hokkaido University, Sapporo, Japan

Background Synapses are asymmetric structures that are specialized for neuronal signal transduction. A unique set of proteins is present at the presynaptic active zone, which is a core structure essential for neurotransmitter release. C. elegans SYD-2, a Liprin-α family protein, is localized at the center of presynaptic active zones and acts together with a GAP protein SYD-1 to promote active zone organization in various types of neurons. Liprin-α proteins are scaffolding proteins and composed of coiled-coil rich regions in the N-terminal halves, which mediate interactions with adapter proteins at the presynaptic active zone, and three SAM domains in the C-termini, which bind proteins such as LAR receptor tyrosine phosphatase. Objective To address the molecular mechanism by which SYD-2 activity in presynaptic assembly is regulated, core functional domains of SYD-2 were dissected out by transgenic structure-function analyses and characterized biochemically. Methods A series of SYD-2 deletion constructs were transgenically expressed in C.elegans, and were analyzed for their abilities to rescue presynaptic assembly defects in HSN neuron in loss-of- function mutants of syd-2 or syd-1. Recovery of presynaptic assembly was evaluated by restored egg-laying behavior and localization of fluorescent tagged presynaptic protein reporters such as synaptobrevin::YFP. Biochemical property of recombinant SYD-2 coiled-coil region was analyzed by pull-down, crosslinking and gel-filtration assay. Clustering of human Liprin-α was analyzed by Blue Native-PAGE and live-cell imaging of GFP in cultured cells. Results Transgenic structure-function studies of SYD-2 revealed that the N-terminal half of SYD-2 is minimally required for rescuing presynaptic defects in syd-2 mutants. A highly conserved short SUNDAY coiled-coil segment named Liprin Homology 1 (LH1) domain is both necessary and sufficient to suppress presynaptic defects in syd-1 mutants. Biochemical analyses revealed that the LH1 domains of SYD-2 and Liprin-α form dimer in vitro. The role of SYD-2 LH1 domain in presynaptic assembly can be partially complemented by artificial dimerization. In human Liprin-α deletion of the LH1 domain impaired proper clustering and/or complex formation in cells. Conclusions These findings suggest a model by which the self-assembly of SYD-2/Liprin-α proteins mediated by the evolutionally conserved coiled-coil LH1 domain is one of the key steps to the accumulation of presynaptic components at nascent synaptic junctions.

2084 Quantum-dot based presynaptic probe reveals dynamic modulation of synaptic vesicle trafficking behaviors by synaptic activity. S. Lee1, S. Chang1; 1Biomedical science, SNU, Seoul, Korea

Presynaptic contribution during synaptic plasticity is a matter of debate. A small nature of presynaptic bouton and a lack of suitable technique, however, hamper detailed analysis of synaptic vesicle (SV) behaviors at synapse. Here, we created a quantum dot-based presynaptic probe and characterized dynamic behaviors of individual SVs in CA3-CA1 synapse of cultured neurons. As previously reported, SVs show multiple exchanges between neighboring boutons. Actin disruption induces a dramatic decrease in diffusive behaviors of SVs at synapse while microtubule disruption only reduce extrasynaptic mobility. Chemical LTP induces NMDA receptor-dependent increase in synaptic mobility of SVs and in inter-boutonal traffickings of SVs between neighboring boutons, causing a bias to incorporation of SVs into synapses. NMDA induced-LTD decreased diffusion coefficient of SVs at synapse. Together, our results suggest that a dynamic modulation of SV diffusive behaviors could be the one of key mechanisms of regulating presynaptic efficacy during presynaptically expressed long-term synaptic plasticity.

2085 Characterization of the DOPA transporter in an in vitro cellular model of dopaminergic neurons. E. Moura1,2, E. Magalhães Ribeiro1, E. Silva3,4, M. P. Serrão1, J. Guimarães5, M. A. Vieira Coelho1,2; 1Pharmacology and Therapeutics, Faculty of Medicine University of Porto, Porto, Portugal, 2Neuropharmacology, Institute for Molecular and Cell Biology, Porto, Portugal, 3Laboratório de Biologia Celular e Molecular, Faculty of Medicine University of Porto, Porto, Portugal, 4Departamento de Stress in Animals, Instituto de Biologia Celular e Molecular, Porto, Portugal, Portugal, 5Neurology Department, Faculty of Medicine University of Porto & Hospital de São João

L-3,4-dihydroxyphenylalanine (DOPA) is the immediate product of the rate-limiting step in catecholamine biosynthesis and the precursor of all the endogenous catecholamines Treatment with DOPA remains to date the most effective treatment of the slowness of movement, increased muscle tone, and tremor that are typical of Parkinson’s disease (PD). The human neuroblastoma cell line SH-SY5Y has been widely used as a cellular model of dopaminergic neurons for PD research. In this study we investigated the transporter involved in DOPA uptake in SH-SY5Y cells. DOPA levels in cells were evaluated by high performance liquid chromatography with electrochemical detection. Results are presented as arithmetic mean ± standard error mean. SH-SY5Y cells take up DOPA in a time dependent (linear until 6 minutes) and concentration dependent (2.5-2500 µM) manner. Non-linear analysis of the saturation curves revealed for DOPA a KM (in µM) of 570±97 and a Vmax (in nmol/mg protein/6 min) of 611±34. The uptake of SUNDAY

DOPA (2.5 µM) was reduced by the inhibitor of the L-type amino acid transporters 2- aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH, 0.1-1000 µM) (IC50 = 47 ± 2 nM; Emax = 24 ± 10 % control uptake) and neutral amino acids (1mM), but no by the inhibitor of the A-type amino acid transporters N-(methylamino)-isobutyric acid (MeAIB, 0.1-1000 µM) nor by the acidic and basic amino acids (1mM). DOPA uptake (2.5 µM) was unaltered by lowering the pH from 7.4 to 6.2. In the absence of Na+ there was a 20% reduction in the Vmax values for DOPA uptake. Accumulation of DOPA in SH-SY5Y cells was largely inhibited by the L-isomers of the small and large neutral amino acids (alanine, serine, threonine, cysteine, leucine, isoleucine, phenylalanine, methionine, and tyrosine), histidine, tryptophan, valine, asparagine and glutamine. Whereas the amino acids glycine, proline and the basic amino acid arginine also produced an inhibition of DOPA uptake, albeit minor, the basic amino acids lysine and cystine, and acidic amino acids aspartate and glutamate did not inhibit the uptake of L-DOPA. In conclusion, DOPA uptake in SH-SY5Y cells was sensitive to inhibition by BCH, but not to MeAIB, and was more sensitive to inhibition by neutral than to basic and acidic amino acids. Although most of DOPA was entering the cells in a Na+-independent manner, a minor component of DOPA uptake (25%) was found to require extracellular Na+. In general, these findings support the view that DOPA may be transported by systems B0 (Na+-dependent) and L (Na+-independent). The fraction of DOPA that is handled through system L is through a high- affinity (KM values in the micromolar range) and pH-insensitive transport, which are characteristic of the LAT1.

2086 Deciphering the molecular interaction between sodium channel Nav1.8 and ankyrin G. F. Castets1,2, A. Montersino1,2, M-P. Fache1,2, B. Dargent1,2; 1INSERM U641, Marseille, France, 2Aix-Marseille University, Marseille, France

In the central nervous system (CNS), the accumulation of the voltage-gated sodium channels, Nav1 (Nav1-2 and Nav1.6) at the axon initial segment (AIS) results from a direct interaction between the ankyrin-binding motif of Nav1 and ankyrin G. This interaction is regulated in a phospho-dependent fashion by protein caseine kinase 2 (CK2). The peripheral channel Nav1.8 is predominantly expressed in dorsal root ganglion neurons. However, Nav1.8 is ectopically expressed in Purkinje cells in EAE animal model and in human multiple sclerosis. Interestingly, this channel possesses a motif close to the conserved ankyrin-binding motif found in the Nav1 expressed in CNS. This study was aimed at deciphering the molecular interaction between Nav1.8 and ankyrin G. We observed by GST pull-downs that Nav1.8 associates with endogenous ankyrin G from rat brain membranes. Using surface plasmon resonance, we found that Nav1.8 constitutively interacts with ankyrin G 190 kDa in contrast to Nav1.2, which requires the phosphorylation of CK2 to bind to ankyrin G 190 kDa. Furthermore when the ankyrin-binding motif of Nav1.8 was fused to the somatodendritic channel Kv2.1, the resulting chimeric protein was not only clustered at the AIS of transfected primary hippocampal neurons but also acted as a dominant negative displacing the endogenous Nav1 from AIS of hippocampal neurons. Altogether, these results indicate that Nav1.8 carries all the information required for clustering at the AIS and that its accumulation is not regulated by protein kinase CK2, unlike CNS Nav1. Our work shows a new kind of interaction between sodium channel and the scaffold protein ankyrin G, which is likely to contribute to the pathological aspects of multiple sclerosis.

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2087 N-cadherin induces synapse formation in heterologous cultures of brainstem cholinergic neurons and CHO cells. R. Flannery1, J. L. Brusés2; 1Department of Anatomy and Cell Biology, University of Kansas School of Medicine, Kansas City, KS, 2Department of Anatomy and Cell Biology & Department of Psychiatry and Behavioral Sciences, University of Kansas School of Medicine, Kansas City, KS

N-cadherin is a cell adhesion molecule that contributes to synaptic differentiation, synaptic vesicle recycling, and synaptic plasticity. It promotes the contact between pre and postsynaptic membranes and acts co-operatively with synaptogenic proteins to induce synapse development. Much of what is known about the regulation of synapse formation and function by N-cadherin has been elucidated from experiments with glutamatergic synapses. Here we have used heterologous cell cultures with primary cholinergic neurons and transfected Chinese Hamster Ovary (CHO) cells to determine whether N- cadherin is sufficient to initiate formation of functional cholinergic synapses. Central cholinergic neurons were cultured from brainstem nuclei explants of transgenic mice expressing EGFP under the control of choline acetyltransferase (ChAT) transcriptional regulatory elements, and cultured with CHO cells (which do not express endogenous N-cadherin) transfected with N-cadherin or a fluorescent protein (FP) as control. CHO cells were co-transfected with nicotinic acetylcholine receptor (nAChR) subunits alpha 3 and beta 4 forming functional AChRs that are activated by acetylcholine. Immunostaining for synapsin I and SV2 demonstrated that synaptic vesicle proteins accumulate at contact sites between cholinergic axons and N-cadherin expressing CHO cells. There was a ~3-fold increase in the total area of SV2 immuno- labeling and a ~2-fold increase in the amount of synapsin I immuno-puncta accumulation per axon length on N- cadherin expressing CHO cells as compared to FP expressing CHO cells. N-cadherin expression in CHO cells in contact with axons extending from cholinergic neurons resulted in the appearance of excitatory postsynaptic potentials (EPSPs), as measured by whole cell recordings of the CHO transfected cells. While 67% of N-cadherin transfected CHO cells displayed synaptic events (6 out of 9 cells) only 11% of control cells (1 out of 9 cells) showed activity. The mean EPSP amplitude for heterologous synapses was 66.7 +/- 13.0 pA (227.7 +/- 36.7 pA after normalizing current density). While the mean decay time constant for heterologous cholinergic synapses was 10.1 +/- 1.0 msec, events in one cell had a biphasic decay with time constants tau1 = 42.4 +/- 10.4 msec and tau2 = 5.2 +/- 0.6 msec, suggesting the presence of immature synaptic release sites. Miniature EPSPs were not detected in control (n=7) or N- cadherin transfected CHO cells (n=12), which we attribute to a low release probability intrinsic to brainstem cholinergic neurons. These results indicate that expression of N-cadherin in non- neuronal cells is sufficient to initiate formation of functional synapses with brainstem cholinergic neurons; however, complete development or maturation of release sites may require expression of additional synaptogenic proteins.

2088 Fast-scanning AFM analysis of agonist-induced height transition of the NMDA receptor. Y. Suzuki1, T. A. Goetze2, D. Stroebel3, S. H. Yoshimura1, P. Paoletti3, R. M. Henderson2, K. Takeyasu1, J. M. Edwardson2; 1Laboratory of Plasma Membrane and Nuclear Signaling, Graduate School of Biostudies, Kyoto University, Kyoto, Japan, 2Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom, 3Institut de Biologie de l'Ecole Normale Supérieure (IBENS), Ecole Normale Supérieure, Paris, France

NMDA receptors (NMDARs) are widely expressed in the central nervous system, and play a major role in excitatory synaptic transmission. Activation of NMDARs requires the binding of two SUNDAY agonists, glutamate and glycine (or D-serine). Here, we used fast-scanning atomic force microscopy (AFM) to analyze agonist-induced structural changes in the extracellular domain of the heterotetrameric GluN1/GluN2A receptor integrated into lipid bilayers. In the absence of agonist, the frequency distribution of heights of the receptors above the bilayer surface had two peaks, at 3.5 nm and 8.5 nm. The taller particles were specifically recognized by an anti-GluN1 antibody that recognizes the S2 segment of the agonist-binding domain, suggesting that these particles represent the extracellular region of the receptor. Receptor activation was triggered by UV photolysis of caged glutamate. Release of glutamate into a glycine-containing buffer resulted in a significant structural change, with the height of the receptor decreasing from 8.5 nm to 7.2 nm. This height shift did not occur in the absence of glycine or in the presence of the selective NMDAR antagonist, D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5). Hence, the height of the extracellular domain of the receptor is reduced upon agonist activation. Such a conformational change could affect interactions that NMDARs engage with synaptic partners.

2089 Potential of neuronal progenitor stem cell therapy for Alzheimer's disease. D. Raman1, M. Choudhary1, J. P. Cleary1, R. R. Punzalan1, C. V. Sharma1, M. V. Sharma1, R. Balmiki2, N. Chopra2, D. K. Lahiri2, J. P. Sharma3; 1Stem Cell Biology, Celprogen Inc, San Pedro, CA, 2Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, IN, 3Celprogen Inc, San Pedro, CA

Alzheimer's disease (AD) is a progressive neurodegenerative disorder which impairs the memory and intellectual abilities of the affected individuals.The basal forebrain cholinergic system is the population of neurons most affected by the neurodegenerative process. Depositions of beta-amyloid or Abeta (Abeta) protein, neurofibrillary tangles and neuronal loss are the neuropathological hallmarks of AD. The goal of our labs is to unveil the mechanism of neurodegeneration and devise ways to prevent /reverse neurodegeneration by various approaches in primary brain cultures and AD animal models. For example, we have recently shown that rivastigmine can preserve or enhance neuronal and synaptic terminal markers in degenerating primary embryonic cerebrocortical cultures (Bailey et al, PLOS One, 2011). Further, we demonstrated neuroprotective and neurorescue effects of a novel polymeric nanoparticle in neuronal cultures and AD animal model (Ray et al- JAD-2011). Similarly the role of specific micro-RNA species is being studied (Long & Lahiri-CMC-2011). From the pharmacological -chemical perspective to si RNA approach, here we report of testing a novel cell replacement therapy based on the hypothesis that cell replacement therapy would provide cure by compensating the lost neuronal systems. Neuronal progenitor stem cells (NPSCs) obtained from adult human post mortem brains from transplant rejects were grafted into the intact brain of mice (10) with matching controls (10), which was then followed by their incorporation into the host parenchyma and differentiation into functional neurons. In the lesioned mouse brains (SCID), NPSCs exhibited a significant targeted migration towards the damaged regions of the brain, where they engrafted, proliferated and matured into functional neurons. These NPSCs were intravenously administered and migrated into brain damaged areas and induce functional recovery. These results in animal models of AD suggest that transplanted NPSCs survive, migrate, and differentiate into cholinergic neurons, astrocytes, and oligodendrocytes with possible amelioration of the learning/memory deficits. In addition to replenish lost or damaged cells, NPSCs most likely stimulate endogenous neural precursors, enhance structural neuroplasticity, and down regulate proinflammatorycytokines and neuronal apoptotic death. Further, the NPSCs could be genetically modified to express growth factors into the brain. An attempt at mobilizing this endogenous pool of resident neuronal progenitor stem cell-like cells would provide another attractive approach for the treatment of AD. This paved the way towards exploring the possible cell based therapy of neurogenesis SUNDAY which would offer an alternative approach for the treatment of AD.

2090 DSCAM Associates with UNC5C in Netrin-1-Mediated Growth Cone Collapse. A. Purohit1, C. Qu1, W. Li2, K-L. Guan3, G. Liu1; 1Department of Biological Sciences, University of Toledo, Toledo, OH, 2University of Michigan Health Systems, University of Michigan, Ann Arbor, MI, 3Department of Pharmacology, University of California San Diego, La Jolla, CA

In the developing nervous system, neuronal growth cones explore the extracellular environment for guidance cues, which can attract or repel axons and guide them along specific trajectories towards their targets. Netrin-1, a bifunctional guidance cue, binds to receptors deleted in colorectal cancer (DCC) and Down syndrome cell adhesion molecule (DSCAM) mediating axon attraction, and uncoordinated-5 (UNC5) mediating axon repulsion. Here, we show that DSCAM interacts with UNC5C and netrin-1 stimulation increases this interaction in primary cortical neurons and postnatal cerebellar granule cells. Netrin-1 induces mouse cerebellum external granule layer (EGL) cell growth cone collapse and knocking-down DSCAM or UNC5C, by specific shRNAs or blocking their signaling by over-expressing mutants lacking the intracellular domains, eliminates netrin-1-induced growth cone collapse. Simultaneous knockdown of DSCAM and UNC5C blocks netrin-1 induced growth cone collapse in EGL cells as well. Netrin- 1 increases the tyrosine phosphorylation of endogenous DSCAM, UNC5C, FAK, Fyn, and PAK1, and promotes the formation of a protein-protein interaction complex of DSCAM with these signaling molecules in primary postnatal cerebellar neurons. PP2, a Src family kinase inhibitor, efficiently inhibits the tyrosine phosphorylation of these molecules and netrin-1-induced mouse EGL cell growth cone collapse. These data present the first evidence that DSCAM coordinates with UNC5C in netrin-1 repulsion.

2091 p75 neurotrophin receptor mediates ephrin-B3 signaling for axonal growth inhibition. N. Uesugi1, T. Yamashita1; 1Osaka University, Suita, Japan p75 neurotrophin receptor, a member of the TNF (tumor necrosis factor) receptor family, is known as a key molecule for axonal growth inhibition in the central nervous system (CNS). Axon elongation in the CNS is inhibited by molecules expressed in oligodendrocytes, e.g. MAG, Nogo, OMgp. These inhibitory molecules bind to p75 receptor indirectly, activate RhoA, known as a cytoskeletal regulator, thus suppressing axonal elongation. It has been reported that ephrin-B3, a member of the ephrin family, is also expressed in oligodendrocytes in the CNS and inhibits axonal elongation in vitro. Here we report that p75 neurotrophin receptor mediates the signal of ephrin-B3 for axonal growth inhibition. The axonal growth of cortical neurons was inhibited on ephrin-B3 coated dishes. This inhibitory effect of ephrin-B3 was reversed by treatment with Pep5, an inhibitory peptide of the signal transduction of p75 receptor. Furthermore, knockdown of p75 receptor by siRNA also silenced the inhibitory effect of ephrin- B3. Our results reveal a novel signaling cascade of ephrin-B3-induced axonal growth inhibition via p75 receptor. Moreover, in this study, we assessed if regeneration of optic nerve was induced by Pep5 application after crush injury.

SUNDAY

2092 Maintenance of Dendritic Spine Morphology by PAR1b through Regulation of Microtubule Growth. K. Hayashi1, A. Suzuki1, C. C. Hoogenraad2, S. Ohno1; 1Molecular and Cellullar Biology, Yokohama City University Graduate School of Medical Science, Yokohama, Japan, 2Cell Biology, Faculty of Science, Utrecht University, The Netherlands

Dendritic spines are postsynaptic structures that receive excitatory synaptic input from presynaptic terminals. Actin and its regulatory proteins play a central role in morphogenesis of dendritic spines. In addition, recent studies have revealed that microtubules are indispensable for the maintenance of mature dendritic spine morphology by stochastically invading dendritic spines and regulating dendritic localization of p140Cap, which is required for actin reorganization. However, the regulatory mechanisms of microtubule dynamics remains poorly understood. PAR1b, a cell polarity-regulating serine/threonine protein kinase, is thought to regulate microtubule dynamics by inhibiting microtubule binding of microtubule-associated proteins. Results from the present study demonstrated that PAR1b participates in the maintenance of mature dendritic spine morphology. Immunofluorescent analysis revealed PAR1b localization in the dendrites, which was concentrated in dendritic spines of mature neurons. PAR1b knockdown cells exhibited decreased mushroom-like dendritic spines, as well as increased filopodia-like dendritic protrusions, with no effect on the number of protrusions. Live imaging of microtubule plus-end tracking proteins directly revealed decreases in distance and duration of microtubule growth following PAR1b knockdown in a neuroblastoma cell line and in dendrites of hippocampal neurons. In addition, reduced accumulation of GFP-p140Cap in dendritic protrusions was confirmed in PAR1b knockdown neurons. In conclusion, the present results suggested a novel function for PAR1b in the maintenance of mature dendritic spine morphology by regulating microtubule growth and the accumulation of p140Cap in dendritic spines.

2093 PDZ-RhoGEF ubiquitination by Cullin3-KLHL20 controls neurotrophin-induced neurite outgrowth. M. Y. Lin1,2, Y. M. Lin1,3, T. C. Kao1,3, H. H. Chuang1, R. H. Chen1,3; 1Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, 2Institute of Molecular Medicine, College of Medicien, National Taiwan University, Taipei, Taiwan, 3Institute of Biochemical Science, National Taiwan University, Taipei, Taiwan

The induction of neurite outgrowth and arborization is critical for developmental and regenerative processes. Here we report that the BTB-kelch protein KLHL20 promoted neurite outgrowth and arborization in hippocampal and cortical neurons through its interaction with Cullin3 to form a ubiquitin ligase complex. This complex targeted PDZ-RhoGEF, a protein abundantly expressed in brain, for ubiquitin-dependent proteolysis, thereby restricting RhoA activity and facilitating growth cone spreading and neurite outgrowth. Importantly, targeting PDZ-RhoGEF to KLHL20 required PDZ-RhoGEF phosphorylation by p38 MAPK. In response to p38-activating neurotrophins, such as brain-derived neurotrophic factor and neurotrophin-3, KLHL20-mediated PDZ-RhoGEF destruction was potentiated, leading to neurotrophin-induced neurite outgrowth. Our study identified a ubiquitin-dependent pathway that targets PDZ- RhoGEF destruction to facilitate neurite outgrowth, and indicates a key role of this pathway in neurotrophin-induced neuronal morphogenesis.

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2094 c-Abl inhibition decreases the levels of Amyloid beta peptide in plasma of Alzheimer transgenic mice. L. D. Estrada1, D. chamorro1, N. C. inestrosa2, A. R. alvarez1; 1Lab. Señalizacion celular, Depto Biología Celular y Molecular, P. University of Catolica De Chile, Santiago, Chile, 2Centro de Envejecimiento y Regeneración, P. Universidad Catolica de Chile, Santiago, Chile

Introduction: AD is a progressive degenerative disorder characterized by memory loss, confusion and a variety of cognitive disabilities. The major neuropathological features of AD are the presence of amyloid plaques and neurofibrillary tangles. The main component of amyloid plaques is the amyloid-beta peptide (Aβ), which is a 39-42 amino acid peptide part of a much larger transmembrane protein; the amyloid precursor protein (APP). Accumulation of toxic Aβ- oligomers in the brain of the AD patients appears to be central in the pathogenesis of AD. Our group previously report a dramatic clearance of amyloid plaques in old AD transgenic mice treated with the c-Abl kinase inhibitor; STI571. Objective: The aim of this work was to investigated the effect of STI571 on peripheral blood and evaluate the cellular mechanisms involved in this clearance. Material and Methods: APPsw/PSEN1ΔE9 mice (6-months) were treated with imatinib (STI571, Novartis) or vehicle every other day for 14-days by intraperitoneal injection. Plasma fractions were isolated using a Ficoll gradient and amyloid beta was measured using an anti-A ELISA. Brains were removed and fixed in paraformaldehide for histological analysis. In addition, primary hippocampal neurons and HT22-hippocampal cells lines expressing APP-GFP were treated with STI571 and APP localization and processing was evaluated. Results: The inhibition of c-abl by peripheral administration of the inhibitor STI571 decreased both, cerebral and plasmatic levels of A-oligomers in APPswe/PSEN1ΔE9 transgenic mice. In addition, hippocampal neurons and HT22-APP(swe) cells treated with STI571 show differences in the distribution of APP and decreased levels of A in comparison with control cells. Conclusion: It has been shown that c-Abl participates in endosomal regulation and since APP amyloidogenic proteolytic cleavage occurs in endosomal compartments, we believe that this processing may be regulated by inhibiting c-abl by STI571, thus decreasing A production. Besides, the fact that the administration of STI571, a drug that poorly cross the blood–brain barrier, reduced accumulation of Aβ in both peripherical blood and the brain, suggest a peripheral clearance of A. The findings of these experiments support a therapeutic value for STI571 and related compounds in Alzheimer's disease. Fondecyt-Postoctorado-3110052/Fondecyt-1080221

2095 Septin-dependent microtubule control during mammalian neurite outgrowth. N. Ageta-Ishihara1, M. Kinoshita1; 1Graduate School of Science, Nagoya University, Nagoya, Japan

The outgrowth, arborization, connection and functional organization of neurites (axons and dendrites) are prerequisites for the development of the nervous system. Previous studies revealed the implications of mammalian septins in multiple steps in neural development including neuritogenesis and synaptogenesis. However, the mechanism of action remains unclear due to the functional redundancy and pleiotropic roles of this cytoskeletal/scaffold protein family. Here we focus on the role of septins in neurite outgrowth of mouse cerebral cortical neurons. Quantitative morphometric analysis demonstrated that shRNA-mediated depletion of the pivotal subunit SEPT7 inhibited the outgrowth of both axons and dendrites in SUNDAY vitro, which was recapitulated in vivo. Septin-depleted neurons contained more acetylated α- tubulin, indicating hyperstabilized microtubules are responsible for the phenotype at least in part. Tubacin, a small molecule inhibitor of the major tubulin deacetylase HDAC6 gave a phenocopy of septin depletion, i.e., neurite outgrowth and hyperacetylated α-tubulin. Biochemical analyses indicated that septins, HDAC6 and acetylated α-tubulin can physically interact and that septin depletion can reduce the interaction between HDAC6 and acetylated α- tubulin. These and other data indicate that septins serve as a scaffold that facilitates the interaction between HDAC6 and acetylated α/β-tubulin heterodimer, which ensures microtubule remodeling for neurite outgrowth by optimizing the acetylation level of α-tubulin.

2096 Neuroproteomic Analysis of Homer2 Knockout and Wildtype Mice Reveals Novel Gene Products Contributing to Alcohol Aversion Behavior. S. P. Goulding1,2, N. Shulman3, N. W. Bateman2, M. J. MacCoss3, K. K. Szumlinski4, C. C. Wu2; 1Neuroscience Program, University of Colorado Anschutz Medical Campus, Aurora, CO, 2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 3Department of Genome Sciences, University of Washington, Seattle, WA, 4Department of Psychology and the Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA

In excitatory synapses of the mammalian central nervous system, the Homer family of proteins mediates excitatory signal transduction and receptor plasticity. The product of three genes, Homer1/2/3, this family shares domain features that include a conserved amino-terminal Ena/Vasp Homology 1 (EVH1) domain and two leucine zippers that form the hydrophobic core of a coiled-coil motif unique to the carboxyl-terminal of long, but not short, Homer isoforms. EVH1 containing Homers bind proline-rich sequences found in numerous postsynaptic glutamatergic signaling proteins such as group 1 metabotropic glutamate receptors (mGluRs) and the N-methyl D-aspartate (NMDA) receptor adaptor protein Shank. Coiled-coil, or long Homer isoforms, oligomerize into scaffolds and regulate signaling through interactions with cytoskeletal constituents like Drebrin, an actin-binding protein. Consistent with structural observations, Homer2 knockout mice present with numerous molecular, neurochemical and behavioral phenotypes in response to acute and chronic alcohol administration. Since ethanol alters glutamate neurotransmission in parts of the brain implicated in alcohol addiction, such as the hippocampus, and Homers regulate excitatory neural signaling through interactions with binding partners in the postsynaptic density (PSD), we set out to investigate alcohol avoiding behavior of the Homer2 knockout mouse at the neuroproteomic level. To better understand molecular mechanisms contributing to alcohol aversion, we studied differentially expressed peptides in the hippocampus of wildtype and Homer2 knockout mice using label-free mass spectrometry. By combining liquid chromatography-tandem mass spectrometry (LC-MS/MS) and a suite of software tools capable of detecting chromatographic features of differential peptide peak areas between samples, we demonstrate that Homer2 deletion alters global gene products between wildtype and knockout mice. While Homer2 deletion is necessary and sufficient for alcohol aversion in mice, these data suggest that differentially abundant gene products also contribute to the alcohol aversive phenotype and provide a readout of functional protein effectors of the Homer2 protein.

SUNDAY

2097 Seizure susceptibility in mice lacking somatostatin type 3 receptors. S. K. Evans1, S. Lewis1, J. L. Fuchs1; 1Biological Sciences, University of North Texas, Denton, TX

The role of primary cilia as essential mediators of cell proliferation in the developing brain is well established, but the function of cilia in mature, postmitotic neurons is just beginning to be investigated. The somatostatin type 3 receptor (Sst3R) is concentrated mainly on neuronal primary cilia in most brain regions. We found that this receptor was typically absent in cilia of neural progenitors and immature neurons, but was present in cilia of more mature neurons in postnatal mice. Given the generally homeostatic, inhibitory, anti-mitotic effects of the hormone somatostatin, we hypothesized that Sst3R on neuronal cilia serves to protect neurons against excitotoxicity, thus enhancing neuron survival in the face of stressful conditions. We compared seizure-related behaviors in C57BL/6 wildtype versus SstR3 knockout mice, in response to intranasal kainic acid (KA) administered under isoflurane anesthesia. Mice were observed periodically for 24 hours. The mutant mice showed behavioral symptoms of seizures at level 4-5 on a standard seizure scale, where 0 is no symptoms and 5 is the most severe. In contrast, wildtypes scored only 0-1, even with up to two additional doses of KA (0.3 mg total per mouse). After a 3-day post-KA survival period, the brains of control and KA-treated mice were immunostained for GFAP to visualize fibrous astrocytes and adenylate cyclase type 3 to visualize primary cilia. KA-treated mutants showed evident astrogliosis, a hallmark of epilepsy, whereas KA-treated wildtypes showed only mild astrogliosis. We examined cilia in the hilus of the dentate gyrus in all four groups of mice and found that KA administration was associated with reductions in the numerical density of immunostained cilia in the mutants. These observations support the hypothesis that ciliary Sst3R has a neuroprotective role in mature, postmitotic neurons.

2098 SNX26, a GAP for Cdc42, Interacts with PSD-95 and Regulates Dendritic Spine Formation in Neurons. Y. Kim1, S. Chang1; 1Department of Physiology and Biomedical Sciences, Seoul National University College of Medicine, Seoul, Korea

Dendritic spines are tiny protrusions composed of highly specialized actin microfilaments that receive the majority of excitatory glutamatergic synaptic inputs in the CNS. SNX26 (Sorting nexin 26) is a brain-enriched Rho GTPase-activating protein known to be localized in spines, though little is known about its neuronal function. Here, we report that SNX26 interacts with PSD-95, a well-known glutamatergic postsynaptic scaffolding protein and regulates spine formation in cultured hippocampal neurons. SNX26/TCGAP is present in dendritic spines where it colocalizes with PSD-95. We found that C-terminal domain of SNX26 interacts with SH3 domain of PSD-95. Overexpression of SNX26 decreased the number of dendritic spines while it increased the number of filopodial form of protrusions. RhoGAP domain is responsible for its effect on spines to filopodia transition. Accordingly R350I, a GAP defective mutant form of SNX26/TCGAP, has no effect on spines to filopodia transition. We further found that expression of SNX26 largely decreases the F-actin contents in COS-7 cells as well as in neurons, suggesting that SNX26 as a RhoGAP destabilizes F-actin, which may mediate spines to filopodia transition in neurons. Evidently, knocking-down endogenous expression of SNX26 by shRNA affects the number and morphology of dendritic spines. Taken together, our results indicate that SNX26 acts as a GAP for Cdc42, interacts with PSD-95 in dendritic spines, and regulates spine formation. Our results also raise the possibility that coordinated interaction of SUNDAY

Cdc42 with SNX26 and other GEF proteins during dendritic spinogenesis in developing neurons may also play a pivotal role during synaptic plasticity in mature neurons. MONDAY

MONDAY, DECEMBER 5- Late Abstracts 2

Science Education

2099 Industry Engagement in STEM Education. S. P. Denker1, L. Lindburg2, G. Maderis3; 1BayBio Institute, South San Francisco, CA, 2BayBio Institute, 3BayBio

Bio-Community: Building the Next Generation of Life Science Professionals is the BayBio Institute′s Science Education program aimed at bringing quality science, technology, engineering and math (STEM) educational experiences to students of diverse socio-economic and ethnic backgrounds. It is a hands-on program designed to connect students and educators to life science professionals, and to bring these professionals as volunteers into middle/high school and community college classrooms. Members of the Bio-Community are Northern California life science companies and educators committed to improving STEM education. The program and network provide a mechanism to establish the critical partnerships between industry, community based organizations and schools necessary to improve STEM education, teacher impact and student motivation. The objectives of Bio-Community are to 1) enhance STEM education in the region′s middle, high schools, and community colleges, 2) engage life science employees and companies in STEM-related activities and 3) increase student interest in STEM subjects, STEM-related majors, and awareness of the variety of careers available within the life science industry. The program was developed and launched in Fall, 2010 and has reached nearly 900 students thus far.

2100 Case-based learning to understand Cell Communication. S. W. Gorsich1; 1Biology, Central Michigan University, Mount Pleasant, MI

Cell Communication is an advanced biology course. The goal of this course is for students to learn various cell communication mechanisms, become familiar with critically evaluating the primary literature, and to improve critical thinking skills. Thirteen students were enrolled in the class and they were divided into five groups with each group having a lap top computer. During the first week of the course an introduction to how cells communicate was given in a more traditional lecture style. In addition during this first week I introduced the Osternsky family to the class. Beginning in the second week we began a 3-4 day cycle of 1) case presentation, 2) discussion of cell communication topic, and 3) discussion/presentation of primary literature. During the case presentation a member of the Osternsky family is presented, which often centers on a medical concern. As the case unfolds students are provided leading information for them to think about and make predictions at each stage. At the end of the case students will diagnose what’s wrong with the family member and then link it to a cell communication problem. For instance in week one the class diagnosed E. Osternsky as having diabetes. They then found a primary paper connecting G-protein coupled receptors to diabetes. During the next class I provide an interactive lecture introducing the specific cell communication pathway the students previously identified (e.g. G-protein coupled receptors). On the final day of the cycle we critically discuss the paper. Everyone in the class is expected to read the entire paper, but each group has one figure they present. To assure students are reading the paper before class, individual and group quizzes are given over the main take home points of the paper and the communication pathway being discussed. The three-part cycle is then repeated with a different Osternsky family member. Some outcomes that have resulted include: 1) students came more prepared to class, 2) students became proficient in critically evaluating and presenting the MONDAY primary literature, 3) improved attendance, 4) retention of material improved, and 5) at a qualitative level student engagement, critical thinking, curiosity, and enthusiasm were high, 6) improved confidence, and 7) student’s became invested in the individual family members. Within this course additional learning strategies were used. They include: multiple quizzing, research ethic case studies, and oral midterm and final exams. With the success of this course I plan to implement a similar strategy in my larger classes.

2101 Go Figure: Comparing Graphics in Biology Textbooks. K. Chiem1, N. K. Dave1, R. Ghasemian1, E. J. Ortiz1, D. R. Perusse1, J. T. Sandu1, I. M. Tabel1, B. A. Velasco1, T. Vu1, M. L. Casem1, S. E. Walker1; 1Biological Science, California State University, Fullerton, Fullerton, CA

The quality of a biology textbook can immensely affect a student’s ability to learn, comprehend and integrate concepts. Figures, drawings or photographs, are often used in textbooks to convey large and complex concepts that would be otherwise difficult to follow. We explored the quality and quantity of figures in majors, non-majors, and mixed biology textbooks. A rubric was developed to measure the quality of figures. Figures were evaluated for three different topics typically taught in a general biology course: meiosis, alternation of generations, and endosymbiosis. We hypothesized that major’s textbooks would provide greater quality and quantity of figures than non-majors/mixed textbooks. On average the majors textbooks scored higher in all quality categories, except for figure location and number of figures when compared to non-majors. Major’s textbooks tend to include entire complex processes in one large figure versus a non-majors text that might break down a complicated, lengthy concept (e.g. meiosis) into smaller more easily understandable pieces.

2102 An interdisciplinary team approach in undergraduate biological instruction and research. V. C. Ware1, N. G. Simon1; 1Biological Sciences, Lehigh University, Bethlehem, PA

Lehigh University has an undergraduate science education award from the Howard Hughes Medical Institute (HHMI) that prepares students to integrate perspectives from multiple disciplines to address complex questions in the biosciences. Lehigh is also a member of the HHMI-sponsored Science Education Alliance (SEA), where students and faculty nationwide use multidisciplinary approaches in mycobacteriophage research. A curricular model around the theme of “biosystems dynamics” begins in the first semester and provides continuing access to courses and research opportunities that emphasize multidisciplinary approaches. Key programmatic elements are a “Bioscience in the 21st Century” survey course and the “Biosystems Dynamics Summer Institute (BDSI),” a 10-week, research-intensive experience for interdisciplinary teams of undergraduates, graduate students, and faculty. The survey course introduces students to diverse perspectives on bioscience issues through lectures from faculty spanning science, engineering, and other disciplines to imbue a broad, trans-disciplinary perspective as a core scientific philosophy. The BDSI has supported 23 research teams, spanning projects in cell and molecular biology, bioengineering, and neuroscience with faculty from mathematics, electrical engineering, chemistry, mechanical engineering, chemical engineering, computer science and biology. Student team members (which have included some SEA alumni) are drawn from different disciplines to reinforce a multidisciplinary conceptualization to problem-solving. Participation in BDSI has had several positive effects: publications and presentations that include student authors; grant proposals to support continued collaboration; undergraduate participation in research laboratories by most students after the BDSI experience; and several graduates of the BDSI program altered their post- MONDAY graduate plans to include graduate programs that offered interdisciplinary opportunities or job opportunities in a discipline outside of their major. BDSI support has generated a “ripple effect” that significantly expanded collaborative, multidisciplinary research efforts on campus. As the HHMI-supported program has evolved over the project period, we see changes in institutional culture in teaching and research strategies designed to address complex problems in the life sciences more broadly.

2103 Undergraduate research experiences based on genomic, proteomic and gene expression libraries from Tetrahymena: launching students into cellular research on ciliated protists. L. A. Hufnagel1; 1Cell Mol Biology, University of Rhode Island, Kingston, RI

To encourage undergraduate students to consider research on the cell and molecular biology of ciliated protists in their future career plans, I have established in my laboratory an informal educational program that combines training in 1) laboratory methods for Tetrahymena thermophila, 2) in silico gene sequence analysis and 3) immunocytochemistry. Each student who joins my laboratory is encouraged to choose a human disease of interest, do background reading to establish the current understanding of molecular and cellular events underlying the disease, and identify a protein of particular importance in the disease process. The student next learns to use BLAST-searching to identify possible T. thermophila orthologues of the relevant protein, as well as orthologues in other organisms, from ciliates to humans, using portals such as TGD-Wiki and Pubmed. The orthologues identified in this way have often not yet been functionally characterized, so a variety of other on-line-accessible tools (available at NCBI, EBI and elsewhere) are then used, to further analyze the functional properties of the Tetrahymena orthologues, and their evolutionary relationships with orthologues in other protists, invertebrates and chordates. In preparation for immunocytochemical studies to characterize the spatial and temporal expression of the identified proteins, sequence alignment tools are used to compare sequences of immunogens used in preparation of commercially available antibodies with the sequences of the tetrahymena orthologues. To determine whether the predicted protein may actually be expressed, the student learns to use the expression data available at TGED, which provides temporal information that also helps to predict when during the cell cycle, starvation and mating the cells will most likely immunolabel. Using this approach, my undergraduate students are 1) characterizing the cell surface distribution of a Tetrahymena ribosomal protein, phosphoprotein P0, identified as a possible target for vaccine development in apicomplexan parasites; 2) investigating the Tetrahymena orthologue of JAK2, a protein mutated in individuals with polycythemia vera (a rare blood disorder), and 3) analyzing the Tetrahymena orthologue of human FANCM, a protein implicated in Fanconi anemia and cancer.

2104 Converting a senior-level cell biology course to a fully active learning format. D. J. Matthes1; 1Genetics, Cell Biology & Development, University of Minnesota, Minneapolis, MN

While there have been efforts nationwide to convert traditional introductory college biology courses to an active learning format, there have been few reports of shifting high-enrollment senior-level cell biology courses to incorporate such innovative instructional strategies. Here I report such a course conversion, where a modified Team Based Learning approach has been adopted for cell biology courses with approximately 100 students meeting in a SCALE-UP style active learning classroom at a large public university. Particular strategies (cell biology concept inventories, learning readiness quizzes, team challenges, and a team project to design nanoparticle-based cell-targeting therapies) will be described. Learning retention as assessed MONDAY by pre- and post-test concept inventories has improved significantly with this course format compared to the same material presented by the same instructor in similar lecture format courses.

Microtubule Cytoskeleton

2105 Analysis of the expression of microtubule plus-end tracking proteins (+TIPs) during Xenopus development. E. C. Park1,2, H. Lee3, M. Kim3, J-K. Han3, G-H. Kim1,2; 1Division of Life Science, Korea Basic Science Institute, Daejeon, Korea, 2Pioneer Research Center for Protein Network Exploration, Korea Basic Science Institute, Daejeon, Korea, 3Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Korea

Microtubules are a component of the cytoskeleton and are important for maintaining cell structure and providing platforms for intracelluar transport, as well as other cellular processes. Microtubule plus-end tracking proteins (+TIPs) are a structurally and functionally diverse group of proteins. +TIPs are specifically accumulated in microtubule plus end and regulate dynamic microtubules behavior. We characterized the +TIPs, XClip-170, Xp150glued, XClasp1b, XLis1, XStim1 and XStim2, in Xenopus laevis and report the expression patterns during embryogenesis. All six Xenopus +TIP genes are maternally expressed and show similar expression patterns during Xenopus embryo development. The expressions of Xenopus +TIPs are localized in the animal hemisphere and ectoderm region at early stages. As embryos develop to later stages, the ectodermal expression of Xenopus +TIPs persists in head and neural tube structures. XClasp1b and Xp150glued,especially, shows ectopic expressions in the cranial ganglia. Remarkably, Xenopus +TIPs are also expressed in the involuting mesoderm during gastrulation. This is the first study of developmental expression patterns of +TIPs and our analysis highlights the future insight of research fields of microtubules in vertebrate development, gastrulation cell movements and neurogenesis.

2106 Nuclear gamma-tubulin associates with nucleoli and interacts with tumor suppressor protein C53. B. Horejší1, S. Vinopal1, E. Dráberová1, V. Sulimenko1, T. Sulimenko1, A. Philimonenko2, P. Hozák2, P. Dráber1; 1Dept. Biology. Cytoskeleton, Inst. Mol. Genet AS CR, Prague, Czech Republic, 2Dept. Cell Nucleus, Inst. Mol. Genet AS CR, Prague

γ-Tubulin is assumed to be a typical cytosolic protein necessary for nucleation of microtubules from microtubule organizing centers. Using immunolocalization and cell fractionation techniques in combination with siRNAi and expression of FLAG-tagged constructs, we have obtained evidence that γ-tubulin is also present in nucleoli of mammalian interphase cells of diverse cellular origins. Immunoelectron microscopy has revealed γ-tubulin localization outside fibrillar centers where transcription of ribosomal DNA takes place. γ-Tubulin was associated with nucleolar remnants after nuclear envelope breakdown and could be translocated to nucleoli during mitosis. Pretreatment of cells with leptomycin B did not affect the distribution of nuclear γ- tubulin, making it unlikely that rapid active transport via nuclear pores participates in the transport of γ-tubulin into the nucleus. This finding was confirmed by heterokaryon assay and time-lapse imaging of photoconvertible protein Dendra2 tagged to γ-tubulin. Immunoprecipitation from nuclear extracts combined with mass spectrometry revealed an association of γ-tubulin with tumor suppressor protein C53 located at multiple subcellular MONDAY compartments including nucleoli. The notion of an interaction between γ-tubulin and C53 was corroborated by pull-down and co-immunoprecipitation experiments. Overexpression of γ- tubulin antagonized the inhibitory effect of C53 on DNA damage G2/M checkpoint activation. The combined results indicate that aside from its known role in microtubule nucleation, γ-tubulin may also have nuclear-specific function(s).

2107 Cap-gly proteins at microtule plus ends: is EB1 detyrosination involved? A. Bosson1, J-M. Soleilhac1, O. Valiron1, D. Job1, A. Andrieux1, M-J. Moutin1; Grenoble Institut Des Neurosciences, Grenoble, France

Localization of CAP-Gly proteins such as CLIP170 at microtubule + ends results from their dual interaction with α-tubulin and EB1 through their C-terminal amino acids -EEY. Detyrosination of α-tubulin by tubulin-carboxypeptidase abolishes CLIP170 binding. Can detyrosination affect EB1 as well? We developed specific antibodies to discriminate tyrosinated vs detyrosinated forms of EB1 and detected only tyrosinated EB1 in fibroblasts, astrocytes, and total brain tissue. Over-expressed EB1 was not detyrosinated in cells and chimeric EB1 with the eight C-terminal amino acids of α-tubulin was only barely detyrosinated. Our results indicate that detyrosination regulates CLIPs interaction with tubulin, but not with EB1. They highlight the specificity of carboxypeptidase toward tubulin.

2108 Tubulin Acetylation Is Required for the Maintenance of Stable Microtubules Arrays During Cell Migration. Z. Xu1,2, M. Nachury2; 1Department of Dermotology, Stanford University School of Medicine, Palo Alto, CA, 2Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Palo Alto, CA

Migrating cells possess a subset of polarized stable microtubules oriented towards the leading edge. These long lived microtubules that are highly acetylated on lysine 40 site are generally considered as tracks for delivery of signaling molecular for cell migration. However, the functional significance of tubulin acetylation is still elusive in large part because the tubulin acetyltransferase remains unidentified. Recently, in the course of proteomic study, we identified a protein with distant structural similarity to the GNAT clade of acetylatransferases that is exclusively and universally conserved in ciliated organisms. Strikingly, this putative acetyltransferase specifically acetylates a-tubulin at K40 in vitro and was therefore named aTAT1. In a variety of cell lines, overexpression of aTAT1 is sufficient to induce massive a-tubulin K40 acetylation. Furthermore, a catalytically inactive mutant of aTAT1 fails to acetylate tubulin in vivo. Thus, the enzymatic activity of aTAT1 is necessary for acetylation a-tubulin at K40 in cells. Meanwhile, Depleting aTAT1 from hTERT-RPE cells abolishes MT acetylation and affects the kinetics of primary cilium assembly. Additionally, aTAT1 is required for the maintenance of stable microtubule arrays and for directional cell migration. Interestingly, we found that the median length of nocodazole-resistant MTs falls from 4 μm in control-treated cells to less than 1 μm in αTAT1-depleted cells. This result suggests that in the absence of acetylation, stable MTs become highly fragile and fragmented. We conclude that aTAT1 is the major a-tubulin K40 acetylatransferase in mammalian cells and that a-tubulin K40 aceytlation enable the assembly of stable microtubule tracks utilized in various cellular processes.

MONDAY

2109 HURP regulates chromosome congression by modulating kinesin Kif18A function. F. Ye1, L. Tan1, Q. Yang1, Y. Xia1, L-W. Deng2, M. Murata-Hori3, Y-C. Liou1; Department of Biological Sciences, National University of Singapore, Singapore, 2Department of Biochemistry, National University of Singapore, Singapore, 3Temasek Life Sciences Laboratory, Singapore

Chromosome biorientation and congression during mitosis require precise control of microtubule dynamics. The dynamics of kinetochore microtubules (K-MTs) are regulated by a variety of microtubule associated proteins (MAPs). Recently, a MAP known as HURP (hepatoma up- regulated protein) was identified. During mitosis, RanGTP releases HURP from the Importin â inhibitory complex and allows it to localize to the kinetochore fiber (K-fiber). HURP bundles and stabilizes K-fibers and promotes chromosome congression. However, the molecular mechanism underlying the role of HURP in regulating chromosome congression remains elusive. Here, we show that overexpression of the N-terminal microtubule binding domain (1-278 aa, HURP278) of HURP induces a series of mitotic defects including lagging chromosomes during the late prometaphase to metaphase stage, prolonged mitosis and increased kinetochore oscillation amplitude, mimicking the effects of Kif18A depletion. In addition, co-immunoprecipitation and BiFC assays identify Kif18A as a novel interaction partner of HURP. Furthermore, quantitative results from live cell imaging analyses illustrate that HURP regulates Kif18A localization and dynamics at the plus-end of K-MTs. Lastly, misaligned chromosomes in HURP278- overexpressing cells can be partially rescued by the overexpression of Kif18A, suggesting the specificity of Kif18A regulation by HURP. Our results demonstrate in part the regulatory mechanism for kinesin Kif18A at the K-MTs during chromosome congression and provide new insights into the mechanism of chromosome movement at the metaphase plate.

2110 MTWD40 is a Novel Microtubule Regulatory Protein Expressed in Neurons. W. Wang1, V. Lundin1, I. Millan1, M. Maloney1, M. Kapur1, N. Chen1; 1Neurology, Stanford School of Medicine, Stanford, CA

Neurons are uniquely dependent on a highly regulated microtubule (MT) cytoskeleton. Several MT-associated proteins (MAPs) are involved in maintaining MT stability and dynamics, which support essential neuronal functions such as axonal transport. Given the critical role of MAPs in regulating MTs in both healthy and diseased neurons, we sought to understand how this regulation is achieved. Here, we identify a novel protein containing a WD40 repeat domain, tentatively named microtubule-related WD40 (MTWD40), as a potential regulator of MAP- dependent MT dynamics. mRNA and protein expression analyses in mice indicate that MTWD40 is enriched in the nervous system. Protein expression is detected as early as E11 and continues throughout adulthood. At the cellular level, MTWD40 localizes as puncta along the MT network in both COS7 cells and primary cortical neurons. Interestingly, co-overexpression with MAP8 causes MTWD40 to lose its punctate pattern and instead colocalize with MAP8 uniformly along MTs. These results suggest that MTWD40 may serve an important role in regulating the neuronal cytoskeleton through an interaction with MAP8.

MONDAY

2111 Role the Microtubule (+)-End Binding protein EB1 in differentiating osteoblasts. S. Pustylnik1, N. Nabavi2, R. Harrison3; 1Department of Biological Sciences, University of Toronto, Toronto, ON, Canada, 2Department of Cell and Systems Biology, University of Toronto, 3University of Toronto, Toronto, ON, Canada

Osteogenesis is a strictly controlled developmental process in which exogenous factors, such as hormones and growth factors activate osteoblast-specific signaling proteins and transcription factors required for osteoblast differentiation. Our microarray analysis identified the microtubule (+)-End-Binding protein (EB1) as an upregulated gene in 5-day ascorbate-induced differentiation of mouse MC3T3 mouse cells compared to undifferentiated control cells. The main functions of EB1 are regulation of microtubule dynamics, cell polarity, chromosome stability and cell migration. EB1 is known to bind to the tumor suppressor APC (Adenomatosis Polyposis Coli). This interaction has led to the speculation that EB1 is a component of the canonical APC tumor suppressor pathway, which regulates the levels of β-catenin, a key Wnt signaling effector. In this study, we examined the role of EB1 in osteoblasts during differentiation to elucidate the possible involvement of EB1 in Wnt signaling. Immunoblotting of extracts from undifferentiated and differentiated osteoblasts showed that EB1 could be detected in freshly plated cells and its level increases during the first 5 days of differentiation. Osteoblasts in which EB1 was knocked down by RNA interference showed changes in cell morphology and osteoblasts specific gene expression. EB1 and β-catenin co-immunoprecipitate in osteoblasts and we see an induction in β-catenin protein levels during differentiation using immunoblotting. Interestingly, EB1 protein knockdown led to a decrease of nuclear accumulation of β-catenin by immunofluorescence. These results suggest that EB1 is important for β-catenin degradation events that lead to its nuclear accumulation and potential activation of Wnt signaling genes. Variations in cell density significantly influenced the whole differentiation process while block of adherent junctions with E-cadherin antibody resulted in decreased cell-cell adhesions and altered bone matrix protein expression. We are now using live imaging to discern the cellular location of interactions between these 2 proteins to show a definitive role for EB1 in osteoblast differentiation.

2112 Characterization of HIV Rev and Tubulin Interactions. J. L. Chang1, B. E. Dukes1, M. W. Miller1; 1Biological Sciences, Wright State University, Dayton, OH

The HIV Rev protein has the ability to bind tubulin heterodimers and depolymerize microtubules (MTs) in vitro producing bilayered rings called Rev-tubulin toroids (RTTs) (Watts et al. 2000. J. Cell Biol. 150: 349-360). These interactions may account for MT defects observed in HIV infected cells or cells that over-express Rev. Watts et al. hypothesized Rev interacts with MTs by a mechanism shared with Kinesin-13 (Kin13) proteins owing to the presence of a shared amino acid sequence. Kin13 proteins are potent MT depolymerizing agents affecting MT behavior during mitosis. To test this hypothesis, point mutations were introduced into Rev substituting amino acids shared with Kin13. In one set of experiments, the abilities of four mutant Rev proteins (A37D, R42A, E47A, and E57A) to form RTTs when mixed with colchicine-treated tubulin were compared to wild-type Rev. With the exception of RevR42A, wild-type and mutant Rev formed comparably sized RTTs at a high frequency. In contrast, mutating arginine at position 42 to alanine completely abrogated formation of RTTs. This observation suggests this amino acid is critical for Rev-tubulin interactions, consistent with our hypothesis. MONDAY

In a second set of experiments, wild-type and seven mutant Rev proteins (T34A, A37D, R39A, R42A, E47A, R50A, E57A, and the E47A/E57A double mutant E2A) fused to YFP were over- expressed in HeLa cells and the ability of these cells to grow in culture and elaborate MT cytoskeletons was measured. Previous results show over-expression of wild-type Rev slows growth in culture and alters cell cycle progression. If these defects are due to Rev-MT interactions, mutation of residues critical for these interactions should mitigate these defects. Several Rev mutants, notably A37D, E47A and the double mutant E47A/E57A (E2A), alter cell morphology and growth in culture suggesting the affected amino acids are dispensable. Curiously, over-expression of RevR42A also alters doubling times and increases the frequency of multinucleated cells. Taken together, Rev over-expression can lead to defects not directly attributable to Rev-MT interactions.

2113 Differential Interaction of Dystrophin and Utrophin with Microtubules. J. J. Belanto1,2, D. M. Henderson1, M. A. Jaeger1, J. M. Ervasti1; 1Biochemisty, Molecular Biology, and Biophysics, University of Minnesota, Minnapolis, MN, 2Molecular, Cellular, Developmental Biology, and Genetics, University of Minnesota, Minneapolis, MN

The dystrophin gene resides on the X chromosome and encodes a 427 kD cytoplasmic protein expressed in striated muscle which links the costameric actin cytoskeleton to the extracellular matrix via a sarcolemmal-associated glycoprotein complex. Mutations in dystrophin that abolish or reduce its expression lead to Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD), respectively. Patients with DMD succumb to fatal cardiac and/or respiratory failure in their late teens to early twenties while BMD patients are more mildly affected. Currently, there is no effective treatment available for DMD or BMD. Our lab previously demonstrated that dystrophin directly binds and organizes microtubules into a rectilinear lattice beneath the sarcolemma (Prins et al., J. Cell Biol. 2009). Using an in vitro cosedimentation assay, we show that dystrophin binds to microtubules with high affinity (KD=0.20µM). We also show that transgenic expression of nearly full-length dystrophin on the dystrophin-deficient mdx background restores microtubule lattice organization whereas mdx mice present with a disorganized microtubule lattice. Based on cosedimentation assays with an array of short dystrophin isoforms and truncation constructs, we conclude/suggest that microtubule binding activity maps to a region located between spectrin-like repeats 9 and 19 of the large middle rod domain. Utrophin is the fetal homolog of dystrophin that is down-regulated in skeletal muscle at birth but ubiquitously expressed at low levels in all tissues, including skeletal muscle, in adults. Because utrophin binds to nearly all of dystrophin’s previously identified molecular partners, several therapies under development for DMD aim to upregulate utrophin in skeletal muscle. We show that utrophin binds microtubules in vitro with 10-fold lower affinity (KD=2.34µM) than dystrophin and that transgenic over-expression of utrophin is not sufficient to rescue the disorganized microtubule network in mdx muscle. Our results suggest that any deficiency in microtubule trafficking or function caused by loss of dystrophin may not be restored by upregulation of utrophin. Therefore, it remains important to determine whether any specific pathologies can be attributed to the microtubule disorganization associated with loss of dystrophin protein expression. This work was supported by the NIH Training Program in Muscle Research AR007612 and NIH RO1 AR042423.

MONDAY

2114 XMAP215 uses avidity to promote microtubule growth. P. O. Widlund1, J. Howard1, A. A. Hyman1; 1Max Planck Inst Moleculcar and Cell Biolology/Genetics, Dresden, Germany

Microtubule-associated proteins adjust the dynamic properties of microtubules so that they can be used to carry out cellular functions such as chromosome segregation, vesicle transport, and cell motility. The XMAP215/Dis1 family of proteins has been shown to dramatically promote microtubule growth and their activities significantly contribute to the rapid growth rates of microtubules seen in cells. Repeats at their N-termini, called TOG domains, are important for this function. While TOG domains directly bind tubulin dimers, it is unclear how this interaction translates to polymerase activity. Understanding the functional roles of TOG domains is further complicated by the fact that the number of these domains present in the proteins of different species varies. We determined the contributions of the individual TOG domains to microtubule growth and looked at the affinities of the TOG domains for tubulin. Based on these experiments, we have developed a model where TOG domains each have low affinity for tubulin dimers, but their combination allows for high overall avidity. This makes it possible for TOG proteins to efficiently recruit tubulin dimers, but also be able to release them into the growing microtubule end.

2115 A High-Throughput and High-Content Microscopy Approach to Identify Microtubule Regulators. X. E. Studera1,2, V. Graml1,2, M. Bortfeld-Miller2, R. E. Carazo Salas1,2; 1The Wellcome Trust/Cancer Research UK Gurdon Institute, Cambridge, United Kingdom, 2Institute of Biochemistry/ETH Zurich, Zurich, Switzerland

Microtubules (MT) are key determinants of countless eukaryotic cellular features, such as cell shape, intracellular transport, organelle positioning and cell division, yet to this day a comprehensive (genomic) catalogue of microtubule regulators is lacking even for the simplest cells.

We have carried out a screen for novel microtubule regulators, using a commercial genome- wide gene-knockout (‘deletion’) collection of haploid mutant strains of the fission yeast (Schizosaccharomyces pombe) and high-throughput/high-content microscopy. So far, only 26 genes are known from previous studies to be bona fide microtubule regulators in fission yeast, although the Gene Ontology database suggests that there could be a few hundred more.

Using a high-throughput spinning disk confocal microscope, we imaged approximately 3 000 non-essential gene knockout strains expressing GFP-labelled tubulin, allowing us to inspect their intact microtubules in live cells. The images were screened visually to look for strains displaying microtubule defects. Over 500 000 images were also analyzed computationally using custom-made, automated microtubule detection algorithms to look for knockouts with statistically significantly aberrant microtubule structures.

After preliminary quality control, we have identified approximately 100 gene knockouts displaying a phenotypic defect in microtubule organization. About 40 % of the corresponding genes have homologues in other organisms and 50 % of them have been experimentally characterized but not reported to regulate microtubules, and are hence potentially novel regulators. We are in the course of carrying out further quality control and detailed strain analysis to confirm which of those preliminary hits are conserved, new microtubule regulators MONDAY and to clarify their mode of action. Here we describe the strategy of our screen and our preliminary results.

2116 Calcium-Dependent Reorganization of Microtubules in Activated Mast Cells. V. Sulimenko1, Z. Hájková1, S. Vinopal1, E. Dráberová1, P. Dráber1; 1Institute of Molecular Genetics, Prague, Czech Republic

Mast cells play a pivotal role in innate immunity, allergy and inflammation. They express plasma membrane-associated high-affinity IgE receptors (FcεRIs), the aggregation of which by multivalent antigen-IgE complexes triggers mast cell activation resulting in degranulation and release of inflammatory mediators such as histamine, proteases, lipid mediators, and cytokines. Tyrosine kinases of the Src family are crucial for triggered signaling pathways. Although FcεRI aggregation leads to reorganization of microtubules and their accumulation in the cell periphery (J. Cell Biol. 170:115,2005; J. Immunol. 176:7243,2006), the molecular mechanisms that control microtubule rearrangement after cell activation are poorly understood. Here we show that changes in cytosolic Ca2+ concentration, controlled by store-operated Ca2+ entry (SOCE), affected microtubule plus-end dynamics detected by plus-end tracking protein EB1 and led to generation of protrusions containing microtubules. Experiments with knock-down or reexpression of STIM1, the essential regulator of SOCE, confirmed the important role of STIM1 in this process. Changes in Ca2+ concentration also modulated interactions of γ-tubulin, the key player in microtubule nucleation, with γ-tubulin complex proteins (GCPs) as well as with other proteins that are substrates for tyrosine kinases of the Src family. Combined data suggest that rearrangement of microtubules in activated mast cells depends on STIM1-induced SOCE, and that Ca2+ regulates the formation of microtubule protrusions in activated mast cells.

2117 Assembly of a millimeter-sized microtubule aster in a model cytoplasm. K. Ishihara1, C. M. Field1, A. C. Groen1, P. A. Nguyen1, T. J. Mitchison1; 1Department of Systems Biology, Harvard Medical School, Boston, MA

The cytoplasms of large egg and embryonic cells are organized primarily by radial arrays of microtubules called asters. Prior to cell division, asters expand to cover the entire millimeter- sized cytoplasm in 20 minutes. Such rapid exploration of cell shape and size is thought to be crucial for positioning of centrosomes and cleavage plane determination (Wuhr et al., 2010, Curr Biol 20, 2040-45). It is unknown how microtubules (MTs) assemble at such a large distance from their presumable nucleation centers or centrosomes. We hypothesize three distinct processes that contribute to addition of MT at the aster periphery: (1) MT polymerization at plus- ends, (2) MT transport by minus-end directed motor proteins and (3) MT-stimulated MT nucleation. We used interphase Xenopus egg extract with either centrosomes or beads coated with Aurora A antibody (Tsai & Zheng, 2005, Curr Biol 15, 2156-63) to recapitulate aster growth and assembly. To quantify the MT polymerization dynamics in the interphase extract, we tracked single MT plus-ends using time lapse microscopy. MT transport was assayed within a growing aster by fluorescent speckle microscopy. We explored the consequences of non- centrosomal MT nucleation on aster growth by constructing a biophysical model of plus-end distribution. Our model suggests that MT-mediated MT nucleation, when coupled with plus-end dynamic instability, contribute to the aster expansion velocity depending on parameter values. In the depleting limits of a hypothetical MT nucleator, the model offers an explanation to how MT density is maintained at the aster periphery. Our work begins to explore the biophysical mechanisms underlying the assembly of a millimeter-sized cytoskeletal structure.

MONDAY

2118 Evaluating models of self-organisation of linear microtubule arrays by computer simulation. Y-C. Oei1,2, X. E. Studera1, R. E. Carazo Salas1, T. Duke3; 1The Gurdon Institute, University of Cambridge, Cambridge, United Kingdom, 2Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom, 3London Centre for Nanotechnology, University College London, London, United Kingdom

In a wild-type fission yeast cell in interphase, microtubules (MTs) are organized in 3-4 bundles of 2-7 dynamic, anti-parallel MTs (interphase microtubule arrays, IMAs) whose correct number and organisation are essential for longitudinal cell growth and precise nuclear centring prior to mitosis (Sawin and Tran, Yeast 2006). Disruption of MT organisation, in cells mutant for certain genes or in cells treated with MT poisons, correlates with defects in cellular growth, shape and/or function.

Within IMAs, MTs are thought to self-organise in a process that has been shown dependent on the bundling protein Ase1 and the motor protein Klp2, resulting in a configuration in which MT plus-tips point towards the cell periphery, with a central region where MTs overlap which is traditionally referred to as the microtubule organising centre (MTOC) (Janson et al, Cell 2007).

Given the small number of constituent MTs, their continuous turnover, and the implied dynamic regeneration of IMAs, it is intriguing that the bundle number remains so stable at 3-4. Also, strikingly, cells that have had their nucleus and all IMAs removed eventually manage to re- establish IMAs in a configuration that resembles the wild-type (Carazo-Salas and Nurse, NCB 2006).

Aiming to understand how IMA number is regulated, we developed a computational Langevin simulation of interphase MT organisation, and use it to explore the influence of cell shape, of the concentrations of bundle constituents, and of the properties commonly attributed to them.

2119 Communication between Flagellar Outer and Inner Dynein Arms. T. Oda1, T. Yagi1, M. Kikkawa1; 1Department of Cell Biology, University of Tokyo Graduate School of Medicine, Tokyo, Japan

The beating motion of cilia and flagella is driven by two rows of dynein arms: the outer dynein arms (ODA) and the inner dynein arms (IDA). The ODAs are required to generate the normal beat frequency and the IDAs are responsible for the amplitude of the waveform. Although structural connections between the ODA and the IDA have been observed by cryo-electron tomography of Chlamydomonas axoneme, the functional communication between the two species of the dynein arms has not been elucidated. In this study, we investigated the roles of the two intermediate chains (IC1 and IC2) of Chlamydomonas ODA. We constructed the expression vectors of IC1 and IC2 with biotinylation-tags added to the N- and the C-termini and introduced them into the Chlamydomonas strains deficient in the respective ICs. The transformants were selected with drug resistance and the recovery of the beat frequency. The ODAs containing the biotinylated ICs were extracted from the axoneme of the transformants. We solved the three dimensional structures of the biotin-streptavidin labeled ODA-microtubule complexes using cryo-electron microscopy. We located the positions of the termini of the IC1 and IC2 near the β and the γ heavy chains, respectively. It is noteworthy that the location of the N-terminus of IC2 is close to the previously observed ODA-IDA linker. The biotin-tags at the N- terminus of IC1 and the C-terminus of IC2 did not alter the swimming phenotypes of the MONDAY transformed Chlamydomonas cells, but the biotin-tag added to the N-terminus of IC2 reduced the amplitude of the beating by half while the beat frequency was not decreased. These results suggest the N-terminus of IC2 mediates the communication between the ODA and the IDA.

2120 A Gas8 Knockout Mouse Model Reveals Defects in Cilia Motility. W. R. Lewis1, E. Malarkey1, R. Pasek1, N. Berbari1, B. Yoder1; 1Cell Biology, University of Alabama-Birmingham, Birmingham, AL

Mouse Growth Arrest Specific 8 (Gas8) has been associated with microtubules and more specifically the Dynein Regulatory Complex (DRC) where it is thought to play a role in cilia and flagella motility. While previous work on Gas8 has been done utilizing in vitro knockdown approaches and in model organisms such as Trypanosoma brucei and Chlamydomonas reinhardtii, this gene has not been studied in an in vivo mammalian genetic model. We have generated a Gas8 mutant mouse and found that mutants survive gestation; however they do not survive to sexual maturity. Consistent with a role in the DRC, it appears that the Gas8 mutant mice die as a result of severe hydrocephalus. Furthermore, analysis of ependymal cilia of the lateral ventricles of the brain indicated a severe defect in motility based on fluorescent bead tracking. Intriguingly, Gas8 is also expressed in cells that lack motile cilia, suggesting it has an additional role outside of the DRC. Recently, Gas8 has been implicated in Sonic Hedgehog signaling pathway using knockdown approaches where it has been proposed to be a positive regulator of the pathway activator Smoothened. However, our analysis of the Sonic Hedgehog pathway in the Gas8 genetic mutants using neural tube and limb bud patterning as readouts for pathway activity has yet to reveal any abnormalities consistent with a hedgehog defect. Future studies of this mouse model will focus on determining the precise nature of the motile cilia defect using high speed video waveform analysis and analyzing potential novel roles for Gas8 in cells with primary cilia.

2121 Cryo-Electron Tomography of Chlamydomonas Flagella Reveals Radial Spoke Heterogeneity and Connectivity. C. F. Barber1, T. Heuser1, B. I. Carbajal-Gonzalez1, V. V. Botchkarev Jr. 1, D. Nicastro1; 1Biology Department, Rosenstiel Center, Brandeis University, Waltham, MA

Cilia and flagella are highly conserved cellular organelles critical to many cellular, tissue and developmental processes. The core structure of cilia and flagella, called the axoneme, consists of 96-nm long units that repeat along the axoneme length. Within each repeat are two or three (depending on species) radial spokes (RSs) that transiently interact with the central pair complex (Warner and Satir, 1974). Studies of the unicellular alga Chlamydomonas reinhardtii have revealed that the RSs play a critical role in transmitting regulatory signals from the central pair complex to the axonemal dyneins, ultimately controlling the activity of dynein proteins (reviewed Porter and Sale, 2000; Smith and Yang, 2004). In both Chlamydomonas and humans, failure to assemble RSs results in flagellar and ciliary paralysis (Sturgess et al., 1979; Witman et al., 1978). While several molecular components of the regulatory pathway have been identified, little is known about their specific locations or physical interactions. To shed further light on the mechanism of axonemal dynein regulation we used cryo-electron tomography and sub-tomogram averaging of Chlamydomonas axonemes to visualize the three-dimensional structure of the radial spokes in unprecedented detail.

Unlike many other species, Chlamydomonas has only two spokes per 96-nm repeat, RS1 and RS2, which were till recently believed to be structurally and functionally homogenous. Our data MONDAY reveal that while the overall structure of RS1 and RS2 is similar - including a two-pronged spoke base and direct connections to inner arm dynein tails - unique densities are seen in each spoke. The structural comparison of Chlamydomonas wild type and the mutant pf17 with head-less spokes, also revealed the morphology and boundaries of the RS head, including a direct RS1 to RS2 interaction. Finally, in place of a third radial spoke we found an uncharacterized, shorter density, the “Radial Spoke 3 Stand-in” (RS3S), which bears structurally no resemblance to RS1 and RS2, and is unaltered in the pf17 mutant. These findings demonstrate that radial spokes are heterogeneous in structure and may play functionally distinct roles in regulating the movement of cilia and flagella.

References: Porter, M.E., and Sale, W.S. (2000). The 9 + 2 axoneme anchors multiple inner arm dyneins and a network of kinases and phosphatases that control motility. J Cell Biol 151, F37-42. Smith, E.F., and Yang, P. (2004). The radial spokes and central apparatus: mechano-chemical transducers that regulate flagellar motility. Cell Motil Cytoskeleton 57, 8-17. Sturgess, J.M., Chao, J., Wong, J., Aspin, N., and Turner, J.A. (1979). Cilia with defective radial spokes: a cause of human respiratory disease. N Engl J Med 300, 53-56. Warner, F.D., and Satir, P. (1974). The structural basis of ciliary bend formation. Radial spoke positional changes accompanying microtubule sliding. J Cell Biol 63, 35-63. Witman, G.B., Plummer, J., and Sander, G. (1978). Chlamydomonas flagellar mutants lacking radial spokes and central tubules. Structure, composition, and function of specific axonemal components. J Cell Biol 76, 729-747.

2122 How flagellum controls cell length. Y. Sun1, C. Y. He1; 1Department of Biological Sciences, National University of Singapore, Singapore

Trypanosoma brucei is an extracellular parasite causing African sleeping sickness in humans and Nagana in cattle. This single-celled pathogen possesses a single flagellum attached to the cell body via flagellum adhesion proteins that link plasma membrane to flagellar membrane, and a complex flagellar attachment zone (FAZ) mostly located in the cell body. In addition to being a motility and sensory organelle, the flagellum also acts as a molecular ruler that controls cell morphogenesis. However, it remains to be understood how the length information contained in the flagellum is transmitted to the FAZ, which directly controls cellular microtubule biogenesis and organization. Here we focused on TbFla1, a surface glycoprotein required for adhesion between flagellar and cell membranes. Using immunoprecipitation of YFP tagged Fla1 and mass spectrometry, a Fla1 binding protein (Fla1BP) was identified and was localized to flagellum membrane. Procyclic T. brucei lacking Fla1BP showed no apparent growth defects, produced flagellum of normal length but shorter FAZ of relatively fixed length, uncoupling the regulation of cell length by flagellum. Our results provided a molecular basis for flagellum membrane adhesion to cell membrane and may explain how flagellum controls cell morphogenesis.

2123 A Neuronal Calcium Sensor Protein, Calaxin, is Essential for Modulation of Ciliary Beat Direction in Sea Urchin Embryos. K. Mizuno1, J. Yaguchi1, K. Shiba1, S. Yaguchi1, K. Inaba1; 1Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka, Japan

MONDAY

Calaxin is a neuronal calcium sensor (NCS)-family Ca2+-binding protein localized in cilia and flagella. Calaxin has three EF-hand Ca2+-binding motifs, and its orthologues are present in metazoan species, but not in yeast, green algae or plant. Previous research has shown that calaxin binds to outer arm dynein or β-tubulin in the presence or absence of Ca2+, respectively [Mizuno et al., Biol. Cell. 2009]. In addition, our recent studies showed that inhibiting calaxin function greatly reduced the duration of asymmetrical waveform, resulting in the suppression of turning movement during sperm chemotaxis. These results indicate that calaxin has an essential role in the control of flagellar waveform through the control of outer arm dynein activity in Ca2+-dependent manner. However, we still do not understand the whole mechanisms of calaxin function throughout the animal kingdom, especially in their cilia. Here, we tried the functional analysis of calaxin in somatic ciliary movement in the embryo of the sea urchin, Hemicentrotus pulcherrimus, because their cilia are suitable for motility analysis and it is easy to perform knockdown experiments using morpholino antisense oligo (MO). Immunostaining showed calaxin was localized to the entire region of sea urchin embryonic cilia. To study the calaxin function, we analyzed the motility of normal and calaxin-MO embryos using a system of digital-camera equipped dark field illumination. calaxin morphants swam much slower than normal embryos and showed abnormal swimming trajectories. Detailed analysis using a high- speed camera, however, revealed that cilia of calaxin morphants beat with normal frequency. On the other hand, beating direction of cilia in calaxin morphants was greatly disorganized as compared with cilia of normal embryos that show highly organized beat direction from the animal pole toward the vegetal pole. These results suggest that calaxin is required to coordinate ciliary beat direction by controlling outer arm dyneins. Furthermore, we observed that gastrulation was greatly inhibited in calaxin morphants. Although the mechanism how the gastrulation was inhibited in calaxin morphants is not clear, it is possible that calaxin is involved in the establishment of planer cell polarity and contributes to the convergent extension of embryonic cells.

2124 Loss of basal feet on Odf2 deletion perturbs polarization of basal bodies. K. Kunimoto1, Y. Yamazaki1, T. Nishida2, K. Shinohara3, H. Ishikawa4, T. Hasegawa2, T. Okanoue5, H. Hamada3, T. Noda6, A. Tamura1, S. Tsukita7, S. Tsukita1; 1Laboratory of Biological Science, Department of Biological Science, Osaka University, Suita, Japan, 2Research Center for Ultra-high Voltage Electron Microscopy, Osaka University, Ibaraki, Japan, 3Developmental Genetics Group, Department of Biological Science, Osaka University, Suita, Japan, 4Department of Biochemistry and Biophysics, UCSF, San Francisco, 5Department of Hepatology, Saiseikai Suita Hospital, 6Department of Cell Biology, Cancer Institute of Japanese Foundation for Cancer Research, Tokyo, Japan, 7Department of Cell Biology, Kyoto University, Kyoto, Japan

Synergic multiciliary beating relies on cilia generated from basal bodies, with which basal feet are regularly associated through molecular mechanisms that remain unknown. Here we show that the coordinated multiciliary action is disturbed in Odf2 mutant mice, resulting in primary ciliary dyskinesia and a characteristic coughing/sneezing-like phenotype. Odf2 mutant depleted basal feet from basal bodies to perturb the planar cell polarity (PCP) of basal bodies, as shown by ultra-high voltage electron microscopic tomography of wild and Odf2 mutant tracheas. The apical microtubular lattice, which is organized by the keystone positioning of basal feet/basal bodies, was lost in Odf2-mutant animals, irrespective of normal localization of Vangl1, the PCP core protein. These findings demonstrate that Odf2 is required for the formation of basal feet. Odf2-based basal feet play a critical role in the PCP-based arrangement of the microtubular lattice and basal bodies, thereby enabling coordinated multiciliary beating.

MONDAY

2125 Functional Analysis of a Gliding-Associated Ciliary Protein with Putative Lipase Activity. E. Betleja1, T. Birch1, R. A. Bloodgood2, J. L. Watts3, D. G. Cole1; 1Dept of Biological Sciences, University of Idaho, Moscow, ID, 2Dept of Cell Biology, University of Virginia School of Medicine, Charlottesville, VA, 3School of Molecular Biosciences, Washington State University, Pullman, WA

The unicellular green algae Chlamydomonas reinhardtii exhibits two independent forms of whole cell locomotion, both utilizing the flagellum: swimming through a liquid medium and gliding across a solid substrate. Essential for the assembly and function of these ciliary organelles is the bidirectional transport of protein particles known as intraflagellar transport (IFT). In a search for IFT-associated cargo, pull down assays of Chlamydomonas flagellar extract with the retrograde IFT motor, cytoplasmic dynein 1b/2, yielded a set of proteins, among which the most prominent were the major flagellar surface glycoprotein, FMG-1B and a flagellar- associated protein of unknown function, which we have named GAP12 (gliding-associated protein 12). This finding suggests that the retrograde IFT motor may play a role in ciliary- dependent gliding motility. In contrast to a well established role for FMG-1B which acts as a transmembrane gliding receptor that contacts substrate during gliding motility, the biological function of GAP12 is unknown. To address the functional roles of GAP12, an artificial micro RNA interference was used to reduce expression of the GAP12 gene. Western blot analysis of whole cellular extracts from RNAi transformants showed that in 14 out of 28 knockdown lines GAP12 protein level was significantly reduced. Selected GAP12-RNAi lines were assayed for gliding phenotype. Some of the knockdown strains had difficulty attaching to the glass slides and when they did attach, they would glide for only short distances of a few microns before detaching. The observation that GAP12-deficient cells are blocked at the stage of adherence suggests that GAP12 is involved in an early stage of gliding motility. Because GAP12 contains a putative lipase domain, we also looked for effects on lipid metabolism. By shifting cells into a nitrogen-deficient medium, Chlamydomonas is forced to accumulate greater levels of triglycerides in specialized storage cytosolic organelles known as lipid bodies. When GAP12- deficient strains were examined after 72 hrs of nitrogen starvation, the lipid-soluble fluorescent Bodipy dye revealed enlarged lipid bodies, which would be consistent with a possible role for GAP12 lipase activity in the degradation or hydrolysis of lipid body triglycerides. We are currently testing, at the biochemical level, if GAP12-RNAi knockdown strains accumulate specific fatty acids when starved in N-free medium. Any further analysis of GAP12 will need to consider this potential role in lipid metabolism, which may or may not be independent of gliding motility.

2126 Monte Carlo simulation of centrosomal self-centering due to pushing by microtubules in large cells. S. K. Tang1,2, B. T. Castle3,2, D. J. Odde2,3; 1Department of Mechanical Engineering, Stanford University, Stanford, CA, 2Physiology Course 2011, Woods Hole Marine Biological Laboratory, Woods Hole, MA, 3Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN

The overarching questions of our work are: what drives self-centering in cells, and if cells with different sizes use different mechanisms for centering? Finding the center in a cell is critical for many processes such as cell division. Various models for self-centering exist. Here we implement a 1D Monte Carlo model to simulate the centering of centrosome (or other microtubule-nucleating site) due to the pushing forces generated when microtubules reach the boundary of the cell. Our model consists of one centrosome that is originally positioned close to MONDAY the cell boundary. Microtubules nucleate at this site, and grow/shrink in length governed by experimentally measured dynamic instability parameters. Our model assumes that the cell boundary is rigid, and that microtubules push the whole length of the microtubule backwards, causing the nucleating site to move.

Our model shows that the growth and pushing of multiple microtubules (up to 1000 in number) in opposing directions can center the centrosome in cells smaller than 500-600 microns in diameter. However, with the experimental dynamic instability parameters, microtubules do not grow longer than 250-300 microns within ~90 min, a typical time scale for first division in Xenopus eggs (diameter ~ 1.2 mm). As microtubules do not reach the cell boundary, the pushing model alone cannot explain centering of centrosomes for cells with diameters larger than 600 microns. In addition, the centering time scale is highly sensitive to small variations in microtubule dynamics. We conclude that the pushing model is not a robust centering mechanism in large cells and that other mechanisms must play a role in driving centering in these cells.

2127 A Sas-4/tubulin module regulates the formation of centrosomal complexes and centrosome biogenesis. J. Gopalakrishnan1, Y-C. F. Chim1, A. Ha1, M. Basiri1, T. Avidor-Reiss1; 1Harvard Medical School, Boston, MA

Centrosomes are conserved organelles that are essential for cilium formation and accurate cell division, and centrosome abnormality can result in genomic instability. A centrosome consists of a pair of centrioles surrounded by a protein network of pericentriolar material that is essential for centrosome function. Pericentriolar material assembly is a tightly regulated, critical step in centrosome biogenesis that determines a centrosome’s size and capability. Here we show that a Sas-4/tubulin module regulates the formation of centrosomal complexes and centrosome biogenesis. Using a combination of genetic and biochemical approaches in Drosophila, we found that Sas 4 and tubulin regulate the formation of several cytoplasmic complexes, which include CNN, Asl and D-PLP (named S-CAP complexes) and γ-tubulin ring proteins (named S-γ complexes). The proteins of these complexes are components of the pericentriolar material and are essential for pericentriolar material assembly and for the centrosome’s capacity to nucleate microtubule asters. Interestingly, the S-CAP complex contains several microcephaly-linked proteins and disturbing this complex causes abnormal centrosome formation. The finding that microcephaly-linked proteins form a common complex may explain why mutations in any of these lead to the same disorder. Tubulin (α-and β-tubulin heterodimer) is known to be a building block of microtubules. Our data points to a new and novel function for tubulin as a regulator of centrosome complex formation, binding to Sas-4 and regulating the interaction of Sas-4 with other centrosomal proteins. By regulating centrosome complex formation, the Sas-4/tubulin module regulates centrosome biogenesis.

2128 Mechanisms of Centrosome Separation. B. R. Mardin1, F. G. Agircan1, C. Lange1, A. M. Fry2, E. Schiebel1; 1ZMBH Uni Heidelberg, Heidelberg, Germany, 2University of Leicester, United Kingdom

In human cells, duplicated centrosomes are first held together by a proteinaceous linker that extends between the proximal ends of the two mother centrioles. To facilitate centrosome separation and bipolar spindle formation at mitotic entry, this linker is disassembled in G2/M in a process known as centrosome disjunction. Centrosome disjunction is driven by the Nek2A MONDAY kinase, which phosphorylates the linker proteins, C-Nap1 and rootletin. Previously we demonstrated that the ability of Nek2A to initiate centrosome disjunction is controlled by two components of the Hippo pathway, the mammalian sterile 20-like kinase 2 (Mst2) and the scaffold protein Salvador (hSav1). Conventionally, Hippo pathway proteins are tumor suppressors that are well known for their function in tissue growth and apoptosis. By forming a separate module, hSav1 and Mst2 associate with and regulate the localized activity of Nek2A by phosphorylation. Our recent data revealed that the Mst2-Nek2A kinase module is regulated by the Polo-like kinase 1 (Plk1). Plk1 selectively binds to and phosphorylates Mst2, which controls the binding of Nek2A to the phosphatase PP1γ providing a balance between the counteracting Nek2A and PP1γ activities on the centrosome linker. Importantly, we provided the first evidence that the Mst2-Nek2A module has overlapping functions with the kinesin motor protein Eg5 in centrosome separation and bipolar spindle formation. In fact, in cells with reduced Eg5 motor activity, the hSav1-Mst1/2-Nek2A pathway becomes essential for bipolar spindle formation. Plk1 is crucial for the targeting of Eg5 to the centrosomes and this process is supported by the microtubule cytoskeleton. Taken together, our data significantly contribute to the understanding of how centrosome disjunction and spindle formation are regulated on the molecular level.

2129 Differentiating Naegleria assemble two basal bodies de novo by counting from 0 to 1 to 2. L. Fritz-Laylin1, Y. Levy2, E. Levitan, S. Chen, Z. Cande3, E. Lai4, C. Fulton4; 1University of California, San Francsico, San Francisco, CA, 2CLARIFY Scientific and Medical Communications, 3Department of Molecular and Cell Biology, University of California, Berkeley, 4Department of Biology, Brandeis University, Waltham, MA

Naegleria gruberi differentiate from amoebae to flagellates in about an hour. This phenotypic transformation requires the de novo assembly of an entire microtubule cytoskeleton, including two basal bodies (centrioles) and flagella. The formation of new basal bodies occurs about 10 minutes before the appearance of visible flagella. Here we use antibodies against tubulin, centrin, and SAS-6 to reveal that the sequence of basal body formation proceeds in two distinct steps: first one basal body forms de novo, after which a second basal body assembles adjacent to the first. This second round of basal body assembly is similar to the centriole assembly typically seen in human cells. Interestingly, when microtubule assembly is temporarily blocked using a pulse treatment of oryzalin, followed by inhibition of protein synthesis with cycloheximide, cells form a single flagellum. Because these cells successfully assemble two basal bodies but only a single flagellum, this demonstrates that there is a moment in differentiation when one basal body is competent to template flagellar assembly while the other is not. Naegleria utilizes both commonly studied modes of centriole assembly, de novo and template-associated, and forms two basal bodies in a 0 to 1 to 2 sequence rather than simultaneously.

2130 Microtubule anchoring is facilitated by the shoulder/sidearm subunits of the dynactin complex. A. G. Le1, R. L. Schneider1, L. M. McCullough2, N. J. Quintyne2; 1Florida Atlantic University, Jupiter, FL, 2Harriet L. Wilkes Honors College, Florida Atlantic University, Jupiter, FL

Dynactin is a multifunctional protein complex identified as a required cofactor for cytoplasmic dynein. Dynactin also plays a dynein-independent role in anchoring microtubules to the centrosome during interphase. Dynactin is the only component of the microtubule anchoring complex that has been shown to directly bind to microtubules. Previous studies have shown that MONDAY the shoulder/sidearm region of dynactin, consisting of p150Glued, dynamitin and p24 are integral to this anchoring process. The p150Glued subunit of dynactin contains two microtubule binding domains of different affinity. The CAP-Gly domain binds microtubules tightly, whereas the Basic domain has a much weaker affinity for the microtubule. The function of p24 remains largely uncharacterized. Using overexpression and siRNA-mediated knockdown, we have examined the contribution of two of the three shoulder/sidearm subunits to microtubule anchoring in more detail. We find that anchoring is abrogated when either of these subunits expression levels are changed. We observe both microtubule organization and regrowth patterns to vary when comparing knockdown and overexpression. Our data suggest a model where the different microtubule-binding domains of p150Glued contribute unequally to microtubule anchoring and p24 serves as reinforcement for maintaining shoulder/sidearm rigidity at the centrosome.

2131 Centrosome inactivation in Drosophila oogenesis. I. Bento1, M. Bettencourt-Dias1; 1Instituto Gulbenkian de Ciência, Oeiras, Portugal

Centrosome is the primary microtubule-organizing center (MTOC) in animal cells, whose number is highly controlled. Duplication of this structure occurs in parallel with DNA replication. During development there are tissues where centrosome number is differently regulated. The zygote formation is one of them. During oogenesis, centrioles degenerate and female meiosis relies on an acentrosomal spindle. On the other hand, during spermatogenesis, the centriole that nucleates the axoneme looses pericentriolar (PCM) proteins. After fertilization, centrioles are paternally inherited and the PCM proteins are deposited by the mother. The absence of centrioles in the egg has been hypothesized as a strategy to avoid parthenogenesis. This is reinforced by the observations that naturally occurring parthenogenic species are able to form de novo centrosomes. Using Drosophila melanogaster oogenesis, we aim to understand how centrioles disappear during oogenesis. We are characterizing Drosophila melanogaster oogenesis by immunostaining. We observed that proteins such as Bld10, Asl, SAS6 or SAS4 are present in the oocyte´s MTOC until stage 10-11 although they are absent from the meiotic spindle. We noticed that the isolated overexpression of proteins involved in centriole biogenesis does not inhibit centriole disappearance in later stages of oocyte development, suggesting those are not limiting factors. Interestingly, Spd2, a PCM protein, is the first protein that disappears from the oocyte´s MTOC. Currently we are testing if PCM maintenance is crucial to keep centriole stability. Oogenesis characterization leads to better understanding of centriole inactivation and disappearance.

2132 Regulation of the Formation of Microtubule Organizing Centers. F. Leal1,2, S. Kandel-Lewis2, E. Karsenti2, M. Bettencourt-Dias1; 1Instituto Gulbenkian De Ciência, Oeiras, Portugal, 2European Molecular Biology Laboratory, Heidelberg, Germany

Centrosomes were originally discovered in the late 19th century[1], being named after their central position in the cell, and characterized by Theodor Boveri[2]. They are composed by a pair of orthogonally oriented centrioles, microtubule barrel shaped structures organized in a nine-fold symmetry, and a surrounding electron-dense material composed of many proteins, named pericentriolar material (PCM). The centrosome is the primary microtubule-organizing centre (MTOC) in animal cells regulating cell motility and polarity in Interphase and facilitating the organization of the spindle poles during Mitosis. Also, in animals, centrioles retain the ability to migrate to the cell membrane and form basal bodies, thus templating the assembly of cilia and flagella, important structures in motility and signalling and sensory perception. MONDAY

Centrosomes undergo duplication once every cell cycle so that, like the genetic material, their number remains stable. Abnormalities in centrosome number and structure occur in many cancers and are associated with genomic instability. In 2005, two separate studies[3-4] showed the importance of SAK/Plk4, a kinase form the serine/threonine family of polo-like kinases, in centriole duplication. Depletion or overexpression of this kinase led to the depletion or overduplication of centrioles in both Drosophila and human cells. Although SAK/Plk4 has been shown to be essential for centrosome duplication and biogenesis, very little is known about its regulation, due to the lack of knowledge of interacting partners. In order to understand the regulation and activity of this important kinase we are taking advantage of the biochemical system per excellence, the Xenopus laevis cell free extract to approach the following questions: 1. How is SAK/Plk4 activity regulated? 2. How does SAK/Plk4 activity regulate centriole formation? 3. How do centrioles regulate their own biogenesis? Up to now we have seen that the predicted Xenopus laevis ortholog (XPLK4) is able to induce the formation of MTOCs which contain centriolar markers. The number and size is directly proportional to the concentration of XPLK4 used, suggesting that this kinase specifically controls centriole biogenesis. We have also seen that addition of this kinase in different stages of the cell cycle leads to different outcomes in terms of centriole biogenesis. Strikingly, meiotic extracts are not permissive for centriole biogenesis suggesting differential regulation of the kinase activity or of other participants in this process. [1] Flemming, W. (1875). Studien uber die Entwicklungsgeschichte der Najaden. Sitzungber Akad Wissensch Wien; 71: 81-147; [2] Boveri, T. (1900). Zellen-Studien: Uber die Natur der Centrosomen. Fischer: Jena; [3] Bettencourt-Dias, M. et al. Curr Biol 15, 2199-207 (2005); [4] Habedanck, R. et al. Nat Cell Biol 7, 1140-6 (2005);

2133 The temperature-sensitive ipl1-2 mutation in yeast is suppressed by mutations altering kinetochore components Duo1 and Ndc80. K. Tatchell1, L. Brou1, J. Phillips1, E. P. Boswell1, L. C. Robinson1; 1Biochemistry and Molecular Biology, Louisiana State University Health Sci Ctr, Shreveport, LA

Ipl1/Aurora B is the catalytic subunit of a complex that is required for chromosome segregation and nuclear division. Prior to anaphase, Ipl1 localizes to kinetochores, where it is required to establish proper kinetochore-microtubule associations and regulate the spindle assembly checkpoint (SAC). At anaphase, the complex migrates to the spindle, where it stabilizes the spindle and regulates cytokinesis. We have taken a genetic approach in budding yeast to investigate Ipl1/Aurora B activity by isolating and characterizing mutations that suppress the inviability of the temperature-sensitive ipl1-2 mutant at non-permissive temperatures. These suppressors include three intragenic, second site revertants in IPL1, 19 mutations in components of the Glc7 phosphatase previously reported to suppress ipl1 (GLC7, SDS22, YPI1, and SHP1), and a null mutation in TCO89, which encodes a subunit of the TORC1 complex. Two revertants contain missense mutations in kinetochore components. rev76 contains a missense mutation in DUO1, an essential component of the microtubule binding DASH/DAM1 complex. The mutant is cold sensitive and arrests in G2-M due to activation of the SAC. rev8 contains the missense mutation ndc80-K204E, which corresponds to K116 in the CH domain of human Ndc80/Hec1. In vitro biochemical studies on human Ndc80 indicate that Ndc80-K166E binds weakly to microtubules (Ciferri et al Cell 133, 427-439, 2008). In a wild-type IPL1 background, ndc80-K204E cells grow slowly and the SAC is activated, consistent with the possibility that kinetochores bind more weakly to microtubules in the ndc80-K204E mutant. The slow growth and cell cycle delay of ndc80-K204E cells are partially alleviated by the ipl1-2 MONDAY mutation. These data are consistent with the published model that the NDC80 complex makes electrostatic interactions between negatively charged regions of Ndc80 and a positively charged C-terminal domain of tubulin. Reduction in the negative charge on Ndc80 can compensate for reduced Ipl1 activity.

Cell-Matrix and Cell-Cell Interactions

2134 A discoidin domain of the Del1 protein is essential and sufficient for its deposition in the extracellular matrix. C. Hidai1, H. Kitano2, A. Mamiya1, S. Kokubun1; 1Biomedical Sciences, Nihon University School of Medicine, Itabashi-ku, Japan, 2Dental surgery, Nihon University School of Medicine, Itabashi- ku, Japan

Objective: Because the organization of the ECM directly influences tissue structure and cell behavior, determination of how ECM organization is regulated can help clarify the mechanisms of development and diseases. In the present study, we analyzed how Del1, an ECM protein, is deposited in the ECM. Del1 consists of three epidermal growth factor repeats (E1–E3) at its N- terminus and two discoidin domains (C1 and C2) at its C-terminus. The aim of this study was to investigate which domains of Del1 mediate its deposition in the ECM. Materials & Methods: We prepared cDNAs corresponding to fusion proteins of heat-stable alkaline phosphatase (AP) and Del1 deletion mutants of various lengths. For in vitro experiments, Cos7 cells were transfected with these cDNAs and the ratio of AP activity in the ECM and the medium was measured. To evaluate deposition of Dell and its fragments into the ECM in vivo, the cDNAs encoding the AP fusion proteins were cloned into a non-viral vector and injected intravenously into mice. Exogenous AP activity in serum or in liver was measured and AP staining of tissue sections was performed. Results: In vitro experiments using AP-fusion constructs of various deletion mutants of Del1 showed that fragments containing the C-terminus of C1, which has a lectin-like structure, are deposited in the ECM. Digestion of these ECM with hyaluronidase from bovine testis released Del1 from the ECM, suggesting that glycosaminoglycans are involved in the deposition of Del1. The efficiency of Del1 C1-dependent deposition varied according to the presence of other Del1 domains. A Del1 fragment containing E3 and C1 showed the highest deposition. An immunoprecipitation assay suggested that C2, which is highly homologous to C1, inhibits C1 deposition by competitive binding to E3. In vivo gene transfer experiments showed that fusion of exogenous proteins with the C1 deposition domain of Del1 dramatically altered the distribution of these proteins in mice. Conclusions: This study suggests that C1 is essential and sufficient for deposition of Del1 into the ECM in vitro and in vivo. The deposition of Del1 could be regulated intramolecularly by its E3 and C2 domains.

2135 Tissue tension controls spatially patterned invasion of mammary epithelial tissue by regulating EMT. K. Lee1,2, N. Gjorevski1, C. M. Nelson1,2; 1Chemical & Biological Engineering, Princeton University, Princeton, NJ, 2Molecular Biology, Princeton University, Princeton, NJ

Cells within tissues constantly experience physical forces and dynamically adapt to these mechanical cues by modifying their behavior and remodeling their environment. Here we characterized the mechanical stress profile of mammary epithelium and determined how tissue MONDAY mechanics regulated tissue behavior. We found that the mechanical rigidity of the microenvironment was distributed non-uniformly in three-dimensional tissues and this patterned mechanical tone played a critical role in determining sites of mammary invasion. Cells within regions of high mechanical stress were surrounded by a stiff extracellular matrix (ECM), and were also more prone to invade. Moreover, the spatial distribution of mechanical stress was significantly correlated with patterned expression of genes associated with epithelial- mesenchymal transition (EMT), which promotes the switch to a motile phenotype. Changes in tissue rigidity altered cellular behaviors including cell morphology and scattering, and the expression of EMT markers. These findings indicate that tissue mechanics plays a pivotal role in regulating invasion of mammary epithelial cells by promoting EMT.

2136 An EGF motif of Del1 induces apoptosis in vitro. H. Kitano1, A. Mamiya1, M. Komiya1, S. Kokubun2, C. Hidai2; 1Division of Oral Surgery, Nihon University School of Medicine, Tokyo, Japan, 2Department of Biomedical Sciences, Nihon University School of Medicine

Introduction: Most cells cannot survive without integrin-mediated adherence to the extracellular matrix (ECM). However, some ECM proteins induce cell death, such as CCN1, which induces apoptosis in fibroblasts via binding with integrin ?6?1 and syndecan-4. The ECM controls cell behavior by providing either pro-survival or pro-death signals to cells. Dell, an ECM protein, is known to show pro-angiogenic or anti-angiogenic activities depending on the experimental conditions. It consists of two discoidin domains (C1, C2), and three epidermal growth factor (EGF) motifs (E1, E2, E3), of which E2 is reported to contain an RGD sequence that binds to integrin receptors and supports cell survival. In the present study, we provide evidence that E3 induces apoptosis in vitro. Materials & Methods: Several cell lines were treated with recombinant Del1 or its mutants. Apoptosis of the cells was evaluated with DNA laddering, chromatin condensation, annexin V staining and TUNEL staining. The signal transduction of apoptosis was analyzed using an inhibitor of caspase. Results: Gene transfer of Del1 induced cell death in cultured cells. DNA laddering, and the results of annexin V staining and TUNEL staining were consistent with apoptosis. The experiments with Del1 mutant proteins with deletions or point mutations revealed that apoptotic activity was localized in E3 (34 amino acids). Because an inhibitor of caspase suppressed E3 and induced apoptosis, it appears that signaling by E3 is caspase dependent. Discussion: In the Del1 protein, there is a cell death-inducing domain, E3, adjacent to the RGD sequence, which supports cell viability. The presence of domains with opposing activities next to one another in the same protein could account for the seemingly contradictory characteristics of Del1 activity in various cells. Thus, it is likely that Del1functions as a balancing factor between life and death. In the present study, we also showed that the efficiency of cancer gene therapy with the cytotoxic FasL gene can be increased by the addition of the E3 domain.

2137 Exploring the syndecan-4 adhesome. M. Montesion1, C. Flynn1, J. Ye1, M. Bonnet1, M. Frigault1, R. Bellin1; 1Biology, Holy Cross College, Worcester, MA

Syndecan-4 is a transmembrane heparan sulfate proteoglycan that has been found in almost all mammalian cell types. Specifically, syndecan-4 is localized most commonly to focal adhesion complexes, and is thought to play a role in linking cells to matrix adhesion sites. In our recent work, we have demonstrated that the attachment of cells to surfaces that are engineered to MONDAY solely support syndecan-4-based connections caused the recruitment of the focal adhesion complex proteins vinculin and talin to adhesion sites. In addition, we have shown that cells adhered solely through syndecan-4 molecules are able to transmit external mechanical strain resulting in activation of the MAPK signaling pathway within the cell. Currently, we are working to build on these findings by conducting experiments to identify the full collection of proteins that are recruited to sites of cell adhesion when cells are subjected to syndecan-4 specific adhesion conditions--i.e., the syndecan-4 adhesome. To conduct these studies, fibroblast cells are grown on non-specific growth surfaces, and then treated with anti-syndecan-4 core protein antibodies coated on magnetic core microbeads to cause the generation of new adhesion complexes at sites of cell attachment to the microbeads. After subjecting cells to a reversible, membrane- permeable chemical crosslinker to stabilize protein interactions, the adhesion complexes are collected on a magnet after cell lysis, the cross-linker based connections are broken and proteomic techniques are used to probe for specific proteins of interest, or for the contents of the entire complex. Efforts such as these have been conducted with a focus on specific integrin- based adhesion conditions, and we are very interested to compare our syndecan-4 based findings to the integrin projects' results, especially in light of long term questions in the literature of the need for both syndecan-4 and integrin molecules within focal adhesion complexes.

2138 Inverse Regulation between Integrin α2β1 and Laminin Receptors. L. Dao1, C. M. Franz1; 1Center for Functional Nanostructures, Karlsruhe Institute of Technology, Karlsruhe, Germany

Integrin α2β1, a well-characterized collagen receptor, has repeatedly been reported to function as a laminin receptor, but its relative contribution to collagen and laminin binding has not been determined. In this work, we compared α2β1-mediated adhesion to bifunctional adhesion substrates consisting of alternating collagen type I and laminin stripes. CHO cells which do not express collagen–binding integrins, showed a highly polarized morphology and adhere only on collagen stripes, whereas CHO cells stably expressing α2β1 (CHO-A2) polarized strongly on collagen, indicating a preference of α2β1 for collagen over laminin. To directly compare α2β1- mediated adhesion strength to collagen and laminin, we quantitated adhesion forces using single-cell force spectroscopy (SCFS). As expected, CHO-A2 cells adhered more strongly to collagen than wildtype cells. Comparable results were obtained for α2β1-expressing (SAOS-A2) and α2β1-deficient wildtype (SAOS-WT) human osteosarcoma cells. In turn, CHO-WT and SAOS-WT cells showed significantly stronger adhesion to laminin than the corresponding α2β1- expressing cells, pointing towards a suppressing effect of α2β1 expression on laminin-binding. In agreement, RT-qPCR and western blot analysis of α2β1-expressing cells revealed a downregulation of integrin subunits α6 and β4, both components of the major laminin-binding integrin receptors α6β1 and α6β4. In conclusion, we have shown that α2β1 is an efficient laminin but not collagen receptor. Instead, α2β1 expression decreased laminin binding and α6 and β4 expression, suggesting an inverse regulation of α2β1 and laminin receptors.

2139 Prolines in βA-sheet of neural cadherin act as a switch to control the dynamics of the equilibrium between monomer and dimer. N. Vunnam1, S. Pedigo1; 1Chemistry and Biochemistry, University of Mississippi, University, MS

Neural cadherins dimerize through the formation of calcium-dependent strand-crossover structures. Dimerization of cadherins leads to cell-cell adhesion in multicellular organisms. Strand-crossover dimer forms exclusively between the first N-terminal extracellular modules (EC1) of the adhesive partners via swapping of their βA-sheets and docking of tryptophan-2 in MONDAY the hydrophobic pocket. In the apo-state wild-type cadherin is predominantly monomer, which indicates that the dimerization is energetically unfavorable in the absence of calcium. Addition of calcium favors dimer formation by creating strain in the monomer and lowering the energetic barrier between monomer and dimer. Dynamics of the monomer-dimer equilibrium is vital for plasticity of synapses. Prolines recurrently occur in proteins that form strand-crossover dimer and are believed to be the source of the strain in the monomer. N-cadherins have two proline residues in the βA-sheet. We focused our studies on the role of these two prolines in calcium- dependent dimerization. Spectroscopic, electrophoretic, and chromatopgraphic studies showed that mutations of both prolines to alanines increased the dimerization affinity by ~20-fold and relieved the requirement of calcium in dimerization. The P5A and P6A mutant formed very stable dimers that required denaturation of protein to disassemble in the apo conditions. In summary, the proline residues act as a switch to control the dynamics of the equilibrium between monomer and dimer which is crucial for the plasticity of synapses.

2140 Calmodulin-dependent CEACAM1 monomer-dimer equilibrium of in epithelial cells. P. C. Patel1, H. S. Lee1, S. D. Gray-owen1; 1Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada

Carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs) can associate with themself and/or other CEACAM members in a manner that controls cellular growth and differentiation. CEACAM1 has previously been shown to exist in both a cis- (parallel) and a trans- (antiparallel) homophilic dimer. In this study, we used epithelial cells to determine what controls the homophilic dimerization of CEACAM1 and describe the relative effect of the monomeric versus dimeric conformation on downstream effector recruitment and cellular responses that are mediated by CEACAM1. To establish a causal relationship between the oligomeric state of CEACAM1 and its biological activities, we employed mutagenic analysis to identify specific residues that are important for CEACAM1 dimerization. In addition, we provide evidence that CEACAM1 dimerization is controlled by calmodulin activation, with increasing intracellular free calcium levels promoting dissociation of the oligomeric state in a process that requires the CEACAM1 cytoplasmic domain. This stabilization of the monomeric form has implications on tyrosine phosphatase SHP-1 recruitment, CEACAM1-dependent bacterial engulfment by epithelial cells, and cellular aggregation.

2141 Cell-to-Cell Fusion and Links to Cancer Cell Migration J. E. Boutilier1,2, R. Duncan1,2; 1Microbiology and Immunology, Dalhousie University, Halifax, NS, Canada, 2Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada

Growing evidence suggests that cell-cell fusion may play a role in the initiation and/or progression of cancer. Our lab studies a unique group of reovirus-encoded cellular fusogens termed FAST (fusion-associated small transmembrane) proteins, which serve as ideal models to study the links between cancer and cellular fusion. A genetic protein-protein interaction screen identified the receptor protein tyrosine phosphatase mu (PTPµ) as a binding partner of the p14 FAST protein. PTPµ is a member of the Ig superfamily and is inhibitory to cellular migration via its involvement in regulating adherens junction formation and stability. In glioblastoma cells, migration and dispersal is regulated by the proteolytic cleavage and down- regulation of PTPµ. Moreover, while normal prostate cancer cells express PTPµ, invasive prostate cancer cells do not. Co-immunoprecipitation and truncation studies indicated that p14 interacts with the membrane-proximal portion of the cytoplasmic tail of PTPµ, which includes the active phosphatase domain. Expression of a catalytically null PTPµ protein in HT1080 MONDAY fibrosarcoma cells, which lack endogenous PTPµ, increased p14-induced cell-cell fusion 1.7 fold. Similarly, siRNA knockdown of endogenous PTPµ in A549 lung adenocarcinoma cells increased syncytiogenesis 2.5 fold. These results indicated that PTPµ, in addition to being inhibitory to cellular migration, is also inhibitory to cellular fusion. Interestingly, there was temporal regulation of p14-PTPµ interactions. Using several fusion-dead p14 constructs and a kinetic analysis of co-immunoprecipitation, we determined that p14 only interacts with PTPµ after cell-cell fusion occurs. Syncytium formation induced by p14 requires dynamic regulation of adherens junctions, with cadherin engagement being required to promote initial cell-cell association and membrane fusion, however, adherens junctions need to be disrupted to allow microfusion pores to expand to the macropores needed for syncytiogenesis. We therefore hypothesize that the stabilization of adherens junctions by PTPµ is inhibitory to pore expansion during cellular fusion, and that cell-cell fusion triggers interactions between p14 and PTPµ that may allow p14 to downregulate PTPµ activity to promote syncytium formation. These studies reveal an interesting convergence of the processes that regulate cell migration during cancer progression and cell-cell fusion on cellular pathways that regulate cell-cell adhesion and membrane-cytoskeletal dynamics.

2142 Establishment of a cell-based assay system to screen compounds modulating tight junction barrier. A. Watari1, M. Kondoh1, K. Yagi1; 1Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan

Objective The intestinal epithelial cells are a first physical barrier to prevent entry of foreign antigens, bacteria, toxins and allergens from the external environment. Tight junction (TJ) in the epithelial cells mainly maintains the barrier function. Disruption of the intestinal epithelial TJ barrier results in increase in intestinal paracellular permeability, and induces the paracellular permeation of toxic luminal substances, which leads to inflammatory bowel disease. In this study, we tried to find compounds modulating permeability in intestinal epithelial cell sheets from various food additives, which are recognized as safe, by using claudin expression monitoring system which based on cell stably expressing claudin promoter reporter gene. Methods The cell stably expressing luciferase gene under control of 500 bp claudin-4 promoter region has been prepared. To screen compound modulating reporter activity, validated 88 food additives were tested using the claudin-4 reporter system. After adding food additives to claudin-4 reporter cell for 48 hours, luciferase activities were measured. Effect of food additives on claudin-4 expression was verified by RT-PCR and western blotting analysis. Effect on barrier function of Caco-2 cells, human colorectal adenocarcinoma cells, was measured by trans- epithelial electrical resistance assay. Results Potassium carbonate decreased the claudin-4 reporter activity, whereas thiabendazol, carotene and curcumin induced it. In accordance with the alteration of reporter activity, potassium carbonate decreased claudin-4 mRNA and protein expression. Thiabendazol, carotene and curcumin increased the expression. Furthermore, these compounds modulated barrier functions of Caco-2 monolayer correlating with the reporter activity. These results show that we successfully identified novel intestinal epithelial barrier-modulating compounds using the claudin-4 reporter system. Conclusion The cell-based assay system using claudin-4 promoter reporter gene is very useful for identifying compounds modulating permeability in intestinal epithelial cell barrier, and the MONDAY compounds identified in this system could apply to an absorption enhancer in intestine and drug for inflammatory bowel disease.

2143 ILDR1 and ILDR2 recruit Tricellulin to tricellular tight junctions. T. Higashi1, M. Furuse1; 1Cell Biology, Grad Sch of Med, Kobe University, Kobe, Japan

Tricellular tight junctions (tTJs) are specialized structures to obliterate the narrow extracellular space at tricellular contacts, where the corners of three epithelial cells meet. LSR is a tTJ- associated transmembrane protein containing a single Ig-like domain and recruits Tricellulin to tTJs to maintain a sufficient paracellular barrier function. LSR has two paralogs in mammalian genomes, ILDR1 and ILDR2. Here, we examined whether ILDR1 and ILDR2 have the same functions as LSR. Immunofluorescence microscopy of frozen sections of mouse tissues revealed that endogenous ILDR1 was localized at tTJs in several epithelial cell types including the colon and kidney. ILDR2 was localized at the tTJs of the retinal pigment epithelium and perineurium. Furthermore, we found that LSR, but not ILDR1 and ILDR2, was expressed in mouse EpH4 mammary epithelial cells. It has been reported that Tricellulin is diffusely distributed throughout the lateral membrane in the LSR-knockdown EpH4 cells and the exogenous expression of shRNA-resistant LSR rescues the tTJ-localization of Tricellulin. When ILDR1 or ILDR2 was exogenously expressed in the LSR-knockdown EpH4 cells, they were localized at the tricellular contacts as well as the lateral membrane. In these cells, Tricellulin was recruited to the tTJs. Taking these observations together, we conclude that LSR, ILDR1 and ILDR2 share the common characteristic of tTJ-localization and Tricellulin-recruiting ability.

2144 Influence of fibroblasts on the synchronization of cardiomyocyte beating and community effect. T. Kaneko1, F. Nomura1, K. Yasuda1; 1Department of Biomedical Information, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan

In the normal heart, cardiomyocytes are the dominant cell type in view of volume, and more than half of the cells consist of noncardiomyocytes, mainly fibroblasts. To elucidate the role of fibroblasts on the synchronization of cardiomyocyte beating, we have made cardiomyocyte- fibroblast network model at the single-cell level in an agarose microchamber cultivation system. The synchronization phenomenon of two cardiomyocyte networks connected by fibroblasts showed (1) propagation velocity of electrophysiological signals decreased a magnitude depending on the increasing number of fibroblasts, not the lengths of fibroblasts; (2) fluctuation of interbeat intervals of the synchronized two cardiomyocyte network connected by fibroblasts did not always decreased, and was opposite from homogeneous cardiomyocyte networks; and (3) the synchronized cardiomyocytes connected by fibroblasts sometimes loses their synchronized condition and recovered to synchronized condition, in which the length of asynchronized period was shorter less than 30 beats and was independent to their cultivation time, whereas the length of synchronized period increased according to cultivation time. The results indicated that fibroblasts can connect cardiomyocytes electrically but do not significantly enhance and contribute to beating interval stability and synchronization. This might also mean that an increase in the number of fibroblasts in heart tissue reduces the cardiomyocyte "community effect", which enhances synchronization and stability of their beating rhythms.

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2145 Determining the Molecular Link Between Adherens Junctions and Tight Junctions. J. L. Maiers1, X. Peng1, A. S. Fanning2, K. A. DeMali1; 1Biochemistry, University of Iowa, Iowa City, IA, 2University of North Carolina - Chapel Hill, NC

Cell-cell adhesion is critical to the development and maintenance of multicellular organisms. Adhesion between epithelial cells is mediated by three types of junctions: tight junctions, adherens junctions and the desmosomes. This study will focus on the adherens junctions and tight junctions because these junctions are linked spatially and functionally, with the formation of tight junctions dependent on the assembly of adherens junctions. Numerous pieces of data suggest that the assembly of tight junctions is linked to the assembly of adherens junctions. However, the molecular mechanism by which these junctions are coupled is not known. α- Catenin, a cytoplasmic component of the adherens junction, has been implicated in the coupling process as cells lacking α-catenin exhibit impaired tight junction formation and abnormal localization of tight junction proteins. Further support of this notion comes from data showing that α-catenin binds directly to the tight junction protein ZO-1. Based on these observations, I tested whether α-catenin is required for linking tight junctions and adherens junctions, and if it serves this purpose through regulated binding to ZO-1. To test this possibility, I generated a powerful knockdown/addback system for studying this interaction in epithelial cells. Using this system, I found that deletion of the alpha catenin C-terminus containing the putative binding site for ZO-1 delayed tight junction assembly and impaired paracellular resistance, while leaving adherens junction assembly and function intact. These data indicate that the α-catenin C- terminus is required for proper tight junction assembly and function. To determine if the effects of α-catenin on tight junctions require direct binding of ZO-1, I mapped the ZO-1 binding site on α-catenin and generated a full length α-catenin point mutant unable to bind ZO-1. This mutant is being introduced into epithelial cells and tight junction assembly and integrity are being analyzed. Collectively, these data are consistent with the notion that ZO-1 binding to α-catenin is required for coupling the assembly of tight junctions to adherens junctions.

2146 Gap Junction Assembly: Roles for the Formation Plaque and Regulation by the C- Terminus of Connexin43. R. G. Johnson1, J. K. Reynhout2, E. M. TenBroek1,3, B. J. Quade1,4, T. Yasumura5, K. V. Davidson5, J. D. Sheridan1, J. I. Nagy6, J. E. Rash5,7; 1Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 2Biology, Bethel University, St. Paul, MN, 3Current address: Therapy, Research and Development, Medtronic, Inc., Fridley, MN, 4Current address: Brigham and Women’s Hospital, Boston, MA, 5Biomedical Sciences, Colorado State University, Fort Collins, CO, 6Physiology, University of Manitoba, Winnipeg, MB, 7Program in Molecular, Cellular and Integrative Neurosciences, Colorado State University, Fort Collins, CO

To expand our understanding of gap junction site designation and initial assembly, we examined the molecular constituents, ultrastructural features, and effects of Cx43 truncation on the development of “formation plaques” (FPs). FPs as apparent sites of gap junction development were monitored in experimentally disaggregated and reaggregated hepatoma cells or in HeLa or N2A cells transfected with full-length Cx43 or a C-terminal truncation of Cx43 (at residue 257). In freeze-fracture replicas, filipin (a probe for membrane cholesterol) labeled membrane areas surrounding FPs but not the FPs or gap junctions. Freeze-fracture replica immunolabeling (FRIL) methods detected Cx43 in dispersed 10-nm membrane particles that characterize FPs, providing further evidence that FP membranes are distinct membrane “domains”, and that the 10-nm particles are gap junction precursors (i.e., connexin hemichannels) engaged in assembly. Analysis of FPs revealed a series of defined steps in assembly, including: a) pairing of FP MONDAY membranes and partial narrowing of the extracellular space, b) accumulation of 10-nm particles within FPs, c) linkage of individual 10-nm particles, forming conical membrane attachments, d) aggregation of 10-nm particles within FPs, with further reduction in the separation between FP membranes and e) lateral fusion of connexon aggregates. These observations further advance current models of gap junction initiation and assembly. With longer cell reaggregation times, we found a significant increase in the packing density of particles in aggregates. With different regulatory sites residing in the C-terminus of Cx43, we tested whether the M257 truncation mutants had altered FP development and/or reduced assembly. FPs continued to develop in cells with truncated Cx43, although the density of 10-nm particles within developing gap junction aggregates failed to achieve control levels. Negative regulation of assembly, as measured by intercellular dye transfer, was evaluated by following the dramatic inhibition of gap junction assembly when protein kinase C (PKC) was activated with TPA treatment. This inhibition failed to occur in the M257 cells. The studies reported here demonstrate the importance of the C- terminus of Cx43 in gap junction assembly and regulation. By confocal immunofluorescence microscopy, we found that N-cadherin was associated with Cx36 in neuronal gap junctions of rodent brain. Overall, these studies validate a model for gap junction assembly that can now be evaluated with respect to the roles played by various connexin-interacting proteins, such as N- and E-cadherin, nectins, β-catenins, ZO-1, and AF6 [aka afadin] (work in progress). Supported by NIH GM-46277 (RGJ and JDS), NS-44395 (JER), and CIHR (JIN).

2147 The Role of Connexin43 in the Development of Skeletal Muscle using Cell and Animal Models of Oculodentodigital Dysplasia. P. A. Merrifield1, S. C. Qing1, P. Vecchio1, G. I. Fishman2, D. W. Laird1; 1Anatomy & Cell Biology, University of Western Ontario, London, ON, Canada, 2Leon H. Charney Division of Cardiology, New York University School of Medicine, New York, NY

Skeletal muscle development involves the differentiation of myoblasts into myotubes; likely involving connexins (Cx) and the gap junctional exchange of secondary messengers and metabolites. Some patients with oculodentodigital dysplasia (ODDD), a rare primarily autosomal dominant disease caused by mutations in the gene encoding Cx43, become less ambulatory with aging and exhibit symptoms that may reflect defects in skeletal muscle development, maintenance, and repair. In this study, the role of Cx43 in skeletal muscle development was examined in differentiation-competent L6 myoblasts and two ODDD-linked Cx43-mutant mouse lines. Undifferentiated L6 myoblasts exhibited high levels of Cx43-based gap junctions which were inhibited by the co-expression of I130T and G60S mutants. Upon myotube formation, Cx43 was rapidly down-regulated and gap junctions were lost but myotubes continued to enlarge. Cx43-mutant mice heterozygous for the I130T mutation exhibited similar body weight, tibial length and muscle fiber size as littermate controls. While mutant mice heterozygous for the G60S mutation also had similar tibial length their overall size and muscle fibre diameter were significantly smaller. Overall, Cx43 regulation may be important for initial myoblast differentiation but not linked to myotube growth and while our mutant mouse studies suggest that some ODDD patients are predicted to have smaller muscle fibres, this appears to be dependent on the site of the Cx43 mutation or other unknown etiologies (Supported by grants from the CIHR to DWL and NSERC to PAM).

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2148 C-terminal interactions of Cx35 that regulate gap junction coupling. Y. Wang1, C. K. Mitchell1, A. Espejo2, M. T. Bedford2, J. O'Brien1; 1Department of Ophthalmology & Visual Science, UTHSC Houston, Houston, TX, 2Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Houston, TX

Connexin 35/36 gap junctions in central nervous system neurons display a large degree of plasticity. In retinal photoreceptors and AII amacrine cells, the plasticity in coupling strength is directly correlated to the phosphorylation states of two regulatory residues (Ser110 and Ser276, using numbering for Cx35). Coupling among retinal AII amacrine cells is reduced by dopamine D1 receptor activation through activation of PKA, downstream activation of protein phosphatase 2A (PP2A) and dephosphorylation of Cx36. This regulation is replicated with either Cx35 or Cx36 expressed in HeLa cells. In an earlier study (Ouyang at al. 2005, Mol Brain Res 135:1-11) we found that removal of the last 7 amino acids of the Cx35 C-terminus (Ser298ter) inverted the effect of PKA activity on coupling. One hypothesis that can account for this phenomenon is that the C-terminus of Cx35/36 is essential for an interaction with regulatory proteins, and that PP2A is part of a complex that does not associate with the mutant connexin. To examine this hypothesis, we performed a protein microarray study and peptide pulldown experiments to examine interactions with the Cx35 C-terminus. We also examined regulation of coupling in a variety of C-terminal mutants expressed in HeLa cells. A 22 amino acid peptide corresponding to the tip of the Cx35 C-terminus bound to several different PDZ domains, including domains from MUPP1, NHERF2, and PDZK1. Phosphorylation of the C-terminus at Ser298 revealed binding to 3 different isoforms of 14-3-3 proteins. Truncation of the C-terminus at Ser298 eliminated all of the interactions. These binding interactions were confirmed by pulldown experiments with recombinant PDZ and 14-3-3 proteins. In transfected HeLa cells, mutation of serine 298 to alanine, which prevents S298 phosphorylation and should prevent 14-3-3 binding, also inverted regulation of coupling by PKA activity. In contrast, Cx35 with a C-terminal YFP tag, which should prevent PDZ binding, showed wild-type regulation by PKA activity. The results suggest that phosphorylation of Ser298 and recruitment of 14-3-3 proteins is involved in the regulation of Cx35 coupling. 14-3-3 binding may facilitate assembly of a signaling complex containing PP2A or it may alter the sensitivity of Cx35 to dephosphorylation by PP2A.

2149 Matrix Stiffness Dictates Cytoskeletal Organization and Nuclear Morphology in 2D and 3D. F. Rehfeldt1,2, T. Harada2, A. E. Brown2, A. Zajac2, S. Cai2, A. Zemel3, D. E. Discher2; 13rd Inst. Physics - Biophysics, University Goettingen, Goettingen, Germany, 2Biophysical Engineering Lab, University of Pennsylvania, Philadelphia, PA, 3Hebrew University Jerusalem, Israel

Physical aspects of the microenvironment such as matrix elasticity E can influence morphology, mechanics, and even the lineage choices of stem cells. An E-series of two types of hydrogels are ligand-functionalized here with collagen and used to elucidate wide-ranging cell, cytoskeletal, and nuclear responses to E in both two and three dimensional matrix geometries. Cross-linked hyaluronan-based hydrogels show that, within just hours of initial plating, cell adhesion, shape, and cytoskeletal order depend non-linearly on E over a broad range of physiologically relevant elasticities. Force generation by non-muscle myosin IIs is required for the various cell responses. These include nuclear shape changes that can also be achieved in part by siRNA-knockdown of a key nuclear envelope gene, lamin-A/C, which is implicated in differentiation of soft tissues such as fat and stiff tissues such as bone. The results lend support to an important matrix-myosin-nucleus pathway.

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2150 Characterisation of tissue microenvironment by the direct culture of mesenchymal stem cells on tissue sections. W. Shen1, W. Y. Tong2, Y. Zhao2, C. F. Chan3, P. Chu1, W. K. Yeung2, Y. W. Lam1; 1City University of Hong Kong, Hong Kong, 2Department of Orthopaedics and Traumatology, The University of Hong Kong, Hong Kong, 3Department of Pediatircs & Adolescent Medicine, The University of Hong Kong, Hong Kong

One ultimate goal of tissue engineering is to construct functional tissues and organs from isolated cells and fabricated materials. To achieve this, it is important to understand how extracellular environments affect and control cell behaviours. Although many studies have characterised the contributions of physical and biochemical factors in extracellular environments to cell physiology, these works often fail to mimic the complexity of the native tissue microenvironment. We have developed a novel experimental system to evaluate the interactions of cells and extracellular microenvironment. We discovered that cryosections of Bovine Achilles tendon display intricate textural details on the surface, and hypothesised that these surfaces represent the internal tissue environments to which endogenous cells are exposed. By manipulating the orientation of sectioning, we could create section surfaces with highly different ultrastructures. Since these sections were carved from the same piece of tendon, their biochemical compositions were identical. To assess how cells responded to these surfaces, we directly cultured mammalian cancer cell lines and human mesenchymal stem cells (MSC), on these sections. Morphology, adhesion and proliferation of cells were highly dependent on the tissue architecture they were exposed to. In particular, cells seeded on the longitudinal sections (LS) of tendon, but not cross sections (CS), adopted a highly elongated and aligned morphology. Time-lapse microscopy revealed that MSC on both LS and CS initially projected filopodia to all directions, but only cells on LS spread along one orientation. Remarkably, MSC cultured on LS, but not CS, expressed protein biomarkers characteristic of tenocytes, suggesting that LS contained biological cues that instructed MSC to commit to the tenogenic lineage. To delineate the nature of this signal, we prepared PDMS replicas using tendon sections as the mould. The resulting replicas, which faithfully copied the physical shape of tendon sections, but not the biochemical composition, promoted a small degree of cell elongation, but failed to induce MSC differentiation into tenocyte-like cells. This suggests that although the physical structure of tissue microenvironment may be enough to modulate cell shape and morphology, specific biochemical molecules, when presented in a correct orientation, are required to drive stem cell differentiation. This study demonstrated how biophysical and biochemical information in the extracellular microenvironment intertwined to form a unique cell type specific niche that influences the fate of cells within a tissue. Direct culture of cells on sectioned tissues is a simple and useful model system for the study of this process.

Regulation and Organization of the Genome

2151 Clusterin as a target gene of HIF-1alpha in prostate cancer cells. E. Shin1, S. Park1, J. Park1, D. Lee1, G. Roh1, H. Kim1, S. Kang1, G. Cho1, W. Choi1; 1Department of Anatomy and Neurobiology, BK21 Biomedical Center, Gyeongsang National University, JINJU, Korea

Clusterin (CLU) is a stress-response glycoprotein that increases expression by diverse apoptotic triggers in several human cancers such as prostate cancers. Previous studies showed that CLU expression was up-regulated in patho-physiological condition such as oxidative stress. MONDAY

HIF-1alpha (hypoxia inducible factor-1alpha) is a major regulator in oxygen homeostasis and transcriptional factor mediating the cellular response to hypoxia. In this study we evaluated whether CLU expression is regulated by HIF-1alpha in human prostate cancer cells exposed to Cobalt chloride (CoCl2). Interestingly, we observed that both CLU and HIF-1alpha were up- regulated in mRNA and protein level. Also overexpression of HIF-1alpha up-regulates CLU expression in prostate cancer cells. Therefore, to evaluate the transcriptional regulation of CLU promoter by HIF-1alpha, we searched hypoxia response element (HRE) of Human CLU promoter sequence. Electrophoretic mobility shift assay (EMSA) and Chromatin Immunoprecipitation (ChIP) analyses demonstrated that HIF-1alpha binds the three putative HRE sites in CLU promoter. Reporter gene assay showed that three HRE sites were functionally active. Our results suggest that the transcription factor HIF-1alpha specifically binds to three HRE sites of CLU promotor and directly regulates the transcription of the human CLU gene in prostate cancer cells exposed to hypoxia.

2152 Phosphorylation of p53 by p38α regulates the expression of Gadd45β in cardiomyocytes. Y-A. Kim1,2, M-Y. Kim1, H. Yu1,3, S. Mishra1, J-H. Lee2, K. Choi2, J-H. Kim2, Y-S. Jung1,2; 1College of Pharmacy, Ajou University, Suwon, Korea, 2Brain Korea 21 for Molecular Science and Technology, Ajou University, Suwon, Korea, 3Brain Korea 21 for Medical Sciences, School of Medicine, Ajou University, Suwon, Korea

Both growth arrest and DNA damage-inducible 45β (Gadd45β) and p53 plays a critical mediatory role in cardiomyocyte apoptosis under ischemia/hypoxia. However, little is known about how Gadd45β expression is regulated under ischemia/hypoxia. In this study, we hypothesized that Gadd45β is a direct target of p53 under ischemia/hypoxia in cardiomyocytes. Using actinomycin D, it was found that the expression of Gadd45β during hypoxia is regulated at the transcriptional level in rat heart-derived H9c2 cells. Silencing p53 gene abrogated the induction of Gadd45β promoter luciferase reporter (Gadd45β-Luc) activity triggered by hypoxia, whereas overexpression of p53 enhanced activity of Gadd45β-Luc. Moreover, chromatin immunoprecipitation assay revealed that binding of p53 to Gadd45β promoter region during hypoxia, and direct binding between them was confirmed by surface plasmon resonance imaging. We also observed the necessary role of p53 in inducing Gadd45β expressions in H9c2 cells under hypoxia and in a rat myocardium of ischemia model. Further studies showed that p38α was activated and responsible for the p53-dependent expressions of Gadd45β under hypoxia. We revealed that p38α induced the distinct temporal profiles of phosphorylation at Ser15 or Ser20 of p53, and mutation of either site on p53 was sufficient to block the hypoxia- induced Gadd45β-Luc activity and Gadd45β expressions. Finally, p53-induced apoptosis during hypoxia was inhibited by Gadd45β gene silencing. These results provide first evidence that Gadd45β is a direct transcriptional target of p53 under ischemia/hypoxia and that the hypoxia- p38α-p53-Gadd45β signaling module serves as an essential regulator of cardiomyocyte apoptosis. This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0012659).

2153 Tramtrack69 Regulation of Notch Target Genes in Drosophila melanogaster. D. Eastman1, E. Hildebrand2; 1Biology, Connecticut College, New London, CT, 2Connecticut College, New London, CT

The Notch pathway is critical for determining many different cell types in developing animals. In Drosophila, the main targets of Notch signaling are the Enhancer of split (E(spl)) genes. MONDAY

Although all of these genes are activated by Notch, they have distinct expression patterns in developing embryos and larval imaginal discs. We are interested in determining the factors that are responsible for this differential expression. We previously identified sequences within the promoters of these genes that are conserved in at least 12 Drosophila species: some of these are unique to individual genes and some are found upstream of a sub-set of the E(spl) genes. One of these sequences is a putative site for the known transcriptional regulator, Tramtrack69 (TTK69). We show that over- expression of TTK69 in eye discs down-regulates HLHmgamma expression. In addition, we have found that TTK69 inhibits activated Notch (ICN) induced E(spl) HLHmgamma expression in Drosophila tissue culture cells and that this affect is dependent on the presence of the BTB/POZ repressor domain at the TTK69 N-terminus. We are currently characterizing the interaction of TTK69 with the Notch activation complex and determining whether it inhibits Notch activation of other E(spl) genes.

2154 The transcriptional activation of melanocortin 2 receptor accessory protein by PPAR[gamma]2 in adipocyte. S. KIM1, S. Kwak1, Y-J. Kim1; 1Department of Biology, Kyung Hee University, Seoul, Korea

Adrenocorticotropic hormone (ACTH) in rodents decreases body weight as well as the secretion of leptin. MC2R and Mrap are known as receptors of ACTH in adipocyte. PPARγ2 also plays roles in the transcriptional regulation in adipocyte such as adipogenesis and fatty acid beta- oxidation. Nuclear receptors activated by PPARγ2 bind to target genes for their transcriptional activation. In the present study we investigated that the Mrap transcript is regulated by PPARγ2. After the differentiation of adipocyte, the expression pattern of Mrap was similar to that of PPARγ2. In the presence of a PPAR agonist, the level of Mrap mRNA was highly increased. In addition, we identified putative PPRE site in the Mrap promoter by reporter assay, chromatin immunoprecipitation assay and the mutagenesis study. These results suggest that PPARγ2 is required for the transcriptional activity of Mrap during adipogenesis, which could be contributed to understand the molecular mechanism of lipolysis in adipocytes.

2155 Role of TopBP1 partner WDR18 in DNA damage checkpoint. J. Willis1, D. DeStephanis1, V. Gowda1, S. Yan1; 1Department of Biology, University of North Carolina at Charlotte, Charlotte, NC

DNA damage checkpoint has significant implications for biological and pathological processes such as cancer and aging. Genomes of all living organisms are exposed to a variety of threats, but the genome’s DNA damage checkpoint functions as a surveillance mechanism, monitoring damage in a genome and directing cellular responses to such damage. If failing to activate DNA damage checkpoint when necessary, the result is unrepaired damage that leads to genomic instability and tumor formation. The main barriers to understanding this process are in identifying how checkpoints sense DNA damage and how the proteins relay the damage signal. Specifically, it is not known how the activated sensor kinase ATR (ATM and Rad3-related) phosphorylates its downstream effector protein Chk1 (Checkpoint kinase 1). In addition to the critical roles in DNA replication initiation and DNA replication stress response, TopBP1 (DNA topoisomerase II β binding protein 1) plays an important role in DNA damage checkpoint signaling through its C-terminus region. However, the underlying mechanism is not known. We hypothesize that a TopBP1-interacting protein contributes to its role in DNA damage checkpoint signaling. The objective of this study is to reveal the molecular mechanism of how TopBP1 C- terminus contributes to DNA damage checkpoint signaling. Xenopus egg extracts, a reliable cell-free biochemical system, has been used for a wide variety of studies in cell cycle and MONDAY checkpoint activation. Our data have shown that WDR18 (WD40 repeat-containing protein 18), a TopBP1-interacting protein, interacts with TopBP1 C-terminus, and their interaction is required for ATR activation of Chk1 in the response to double-stranded breaks. Furthermore, immunodepletion of endogenous WDR18 from Xenopus egg extract also compromises DNA damage checkpoint signaling. Taken together, WDR18 plays an essential role in DNA damage checkpoint signaling via interacting with TopBP1 C-terminus.

This work was supported, in part, by funds provided by The University of North Carolina at Charlotte and Wachovia foundation fund for faculty excellence.

2156 A genetic screen to identify interactions between cdc13-1and yku80 mutant alleles on yeast telomeres. L. Sanchez1, G. Guillaume1, S. Flores1, C. J. Hengartner1, L. R. Vega1; 1Biology, Barry University, Miami Shores, FL

Telomeres are the physical ends of eukaryotic chromosomes that function to protect DNA ends from degradation and from end to end fusion. Telomeres consist of stretches of repeated C/G- rich DNA ending with 3’ single stranded G-rich overhangs. The maintenance and function of telomeres are facilitated by the enzyme telomerase and by accessory proteins such as Ku and Cdc13p. Cdc13p is an essential, G-strand binding protein that functions in telomere protection and in telomerase recruitment. cdc13-1 is a temperature sensitive allele of CDC13, that is defective for telomere end protection. Ku is a non-essential heterodimer composed of Ku70p and Ku80p. Ku plays multiple roles in DNA metabolism including: non-homologous end joining, recombination and end protection. Ku also interacts with TLC1, the RNA template of the telomerase enzyme and has recently been shown to exhibit end-binding activity. This study examines the effect of mutations in yKU80 on cdc13-1 strains. We will also determine genetic interactions between yku80 mutants and Pif1p, a helicase that inhibits telomerase activity. We previously showed that cdc13-1 strains deleted for PIF1 display hyper-elongated telomeres and increased temperature resistance. Telomere elongation in cdc13-1, pif1∆ strains partially depends on the Ku-TLC1 interaction. Using a genetic library of yku80 mutations generated in A. Bertuch’s laboratory, we have introduced the yku80 alleles into the cdc13-1 background. The goals of these experiments are to determine the effects on viability and telomere end protection of the various yku80 mutant alleles in cdc13-1 strains. We hope to identify a cdc13-1, yku80 double mutant that mimics the phenotypes of cdc13-1, pif1∆ strains. Supported by NIH-NIGMS MBRS RISE Grant: 2R25 GM059244-10, Barry University and NIH- NIGMS/NCI MBRS SCORE grant, 5SC 2CA 138567 to LRV

2157 Function of Daxx at centromeres and pericentromeres. V. Morozov1, E. Gavrilova2,3, A. Ishov1; 1Anatomy & Cell biology, University of Florida, Gainesville, FL, 2Institute of Cytology Russian Academy of Sciences, St. Petersburg, Russia, 3Faculty of Biology and Soil Sciences, St. Petersburg State University, St. Petersburg, Russia

Heterochromatin architecture is essential for the proper orchestration of nuclear processes, while transcription from this part of genome is required for its own maintenance. Here we present first evidence that depletion of protein Daxx affects transcription of human heterochromatin, reducing accumulation of centromeric (CEN) RNA in normal conditions and pericentromeric (periCEN) RNA after heat shock (HS) application. Searching for the mechanism of Daxx-dependent regulation of heterochromatin transcription, we found that depletion of Daxx MONDAY decreases incorporation of transcription-associated histone H3 variant, H3.3, into both CEN and periCEN. In normal conditions, Daxx is mostly accumulated at ND10/PML nuclear bodies, with minor association with CEN/periCEN in subpopulation of cells. HS changes this balance forcing very robust accumulation of Daxx on CEN/periCEN. Surprisingly, this transient redistribution of Daxx does not further elevate incorporation of H3.3 that remained steady during HS and recovery. Instead, depletion of Daxx leads to HS-induced changes in the balance of epigenetic modifications at heterochromatin, most dramatically elevating levels of H3K4Me2 at periCEN. We propose dualistic function of Daxx-containing complexes at CEN/periCEN: 1) regulation of H3.3 loading in normal conditions, and 2) protection of epigenetic status upon stress application, thus collectively guarding epigenetic identity of heterochromatin and genome integrity.

2158 Structural Requirements of the JIL-1 Tandem Kinase for Histone H3S10 Phosphorylation. Y. Li1, W. Cai1, C. Wang1, X. Bao1, H. Deng1, J. Girton1, J. Johansen1, K. M. Johansen1; 1Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, IA

The JIL-1 tandem kinase in Drosophila localizes specifically to euchromatic interband regions of polytene chromosomes and is the predominant kinase controlling the phosphorylation state of histone H3S10 at interphase. JIL-1 can be divided into four main domains including a NH2- terminal domain (NTD), two kinase domains (KDI and KDII), and a COOH-terminal domain (CTD). Our laboratory has previously determined that the CTD-domain of JIL-1 is sufficient for proper chromatin localization and for rescue of JIL- 1 null mutant chromosome morphology. Interestingly a JIL-1 construct without the CTD-domain has kinase activity for histone H3S10 despite that it does not localize properly. However, the potential contributions from the NTD was not determined. Thus, in order to further characterize the structural requirements of the JIL-1 kinase for H3S10 phosphorylation, we generated a series of mutated JIL-1 constructs and expressed them in a JIL-1 null mutant background. Our results show that a JIL-1 construct without the NTD localizes properly to chromatin and rescues mutant polytene chromosome morphology. However, in immunocytochemistry and immunoblot analyses with H3S10ph antibody no histone H3S10 phosphorylation could be detected, strongly suggesting that the NTD domain is required for H3S10 kinase activity. Furthermore, we mutated either or both kinase domains to render them "kinase dead" and assessed their ability to phosphorylate H3S10. The results indicate that both kinase domains are required for kinase activity. In addition, using a LacI/lacO-repeat tethering system we provide evidence that only JIL-1 constructs with H3S10 phosphorylation activity have the capacity to induce a change in higher order chromatin structure from a condensed heterochromatin-like band state to a more open euchromatic interband state. These findings provide direct evidence that the epigenetic histone tail modification of H3S10 phosphorylation at interphase can function as a causative regulator of chromatin structure. Supported by NIH grant GM62916.

2159 Epigenetic Regulation of MARCO in Tolerized Macrophages. J. Jing1, B. O’Connor2, B. Pederson3, E. Davidson3, I. V. Yang3, D. Schwartz3; 1University of Colorado Denver, Denver, CO, 2National Jewish Health, 3University of Colorado Denver

Macrophages play a key role in host defense against microbes through phagocytosis. MARCO (macrophage receptor with collagenous structure) is one of the scavenger receptors on the cell surface of macrophages that mediates the opsonin-independent phagocytosis. The goal of our study was to study the role of epigenetic regulation of MARCO in lipopolysaccharide (LPS) or MONDAY lipotechoic acid (LTA)-induced tolerance. We first demonstrated that phagocytosis was increased in mouse bone marrow derived macrophage (MBMM) repeated stimulation by LPS or LTA. The expression of MARCO, but not other scavenger receptors, was also elevated, suggesting that MARCO plays a key role in the phagocytosis of tolerized macrophages. We comprehensively evaluated genome-wide DNA methylation by CHARM (comprehensive high- throughput arrays for relative methylation) and profiled the mRNA expression in tolerized macrophages. Although there were no methylation changes associated with MARCO in tolerized compared to nontolerized macrophages, several transport related genes (SNX30, SNX24, Kif21b, Kif5, Kif6 and Kif9) have significant methylation changes and at least two fold mRNA expression change, indicating these genes may be involved in regulating the transport of MARCO to cells surface. We are in the process of confirming these findings by examining expression of MARCO on the cell surface following siRNA-mediated inhibition of these candidate genes. In addition, we are exploring whether H3K4me3 and H3K9me2 histone modifications contribute to increased expression of MARCO in tolerized macrophages.

2160 Molecular Basis for Interaction of let-7 microRNAs with Lin28. Y. Nam1, C. Chen1, R. Gregory1, J. Chou1, P. Sliz1; 1Harvard Medical School, Boston, MA

MicroRNAs are small non-coding RNA molecules that regulate gene expression. Members of the let-7 microRNA family control many cell fate determination genes to influence pluripotency, differentiation, and transformation. Lin28 is a specific, post-transcriptional inhibitor of let-7 biogenesis. Moreover, Lin28 is a potent cell reprogramming factor, abnormally expressed in various cancers, and linked to various developmental traits in worms and mammals. Growing evidence suggests that Lin28 also binds certain mRNAs to regulate translation independent of let-7.

A major question regarding Lin28 activity is how it recognizes its RNA substrates, and also how it inhibits microRNA processing enzymes-Drosha and Dicer-while also recruiting a modification enzyme, TUTase, to target let-7 precursors to degradation. We report crystal structures of Lin28 in complex with pre-element (preE) portions, or "terminal loop regions", of let-7d, let-7-f1, and let-7g precursors. The two folded domains of Lin28 recognize two distinct regions of the RNA; the terminal regions outside of these domains are dispensable for inhibition of let-7 in vivo. We also show by NMR spectroscopy that the linker connecting the two folded domains is flexible, to accommodate Lin28 binding to diverse let-7 family members. Mutagenesis studies of protein and RNA components identified mutations in the binding interface that affect complex formation in vitro, Dicer activity in vitro, and processing of let-7 microRNAs in cultured cells.

Our work underscores the importance of preEs in microRNA regulation, and we present a preferred preE sequence for binding to Lin28. Our model also provides a mechanistic explanation for the inhibitory effect of Lin28 on miRNA processing by Dicer; it further suggests that induced folding in the CCHCx2:GGAG part of the complex directly influences downstream factor(s) important for let-7 regulation.

MONDAY

2161 The karyopherin Sal3 is required for nuclear import of the core RNA interference pathway protein Rdp1. J. Park1, S. I. Freitag2, P. Young2, T. C. Hobman1; 1Cell Biology, University of Alberta, Edmonton, AB, Canada, 2Biology, Queen's University, Kingston, ON, Canada

RNA-dependent RNA polymerase activity is required for RNA interference (RNAi) in many lower eukaryotes including the fission yeast S. pombe. Together with Ago1 and Dcr1, the RNA- dependent RNA polymerase Rdp1 mediates small RNA-dependent transcriptional- and posttranscriptional gene-silencing. Whereas the bulk of Rdp1 is localized to the nucleus, Ago1 and Dcr1 are primarily cytoplasmic. This may reflect the fact that Rdp1 is required early in the RNAi pathway to generate double strand RNA from transcripts that originate from centromeric loci. The relatively large size of Rdp1 (139.4 kD) precludes passive diffusion of the enzyme into the nucleus suggesting that karyopherin-dependent transport is involved in nuclear targeting of this enzyme. In the present study, we report that the karyopherin Sal3 is required for nuclear import of Rdp1 in S. pombe. Loss of nuclear Rdp1 is associated with substantially reduced transcriptional gene-silencing, and surprisingly, post-transcriptional gene silencing which may occur in the cytoplasm, was also significantly affected. Together, these results identify Sal3 as a modulator of RNAi-dependent transcriptional gene silencing as well as a potential link between nuclear import and post-transcriptional gene-silencing.

This work is supported by Natural Science and Engineering Research Council of Canada (NSERC) #rgpin 183820.

Cell Polarity

2162 Siah regulation of Par3 controls neuronal cell adhesion during germinal zone exit. D. Solecki1, N. Trivedi1, J. Famulski2; 1St. Jude Children's Res Hosp, Memphis, TN, 2University of Alberta, Edmonton, AB, Canada

The brain’s circuitry is established by directed migration of neurons during development. Despite tremendous advances in our understanding of the substrates, guidance mechanisms and cytoskeletal elements required for neuronal migrations, remarkably little is known concerning the cell intrinsic machinery that initiates a neuron’s exit from their germinal zone (GZ) niche. We found that Seven in Absentia (Siah) is the first ubiquitin ligase to target Par3 for degradation and Siah-mediated proteosomal degradation of Par3 regulates the initiation of radial migration as cerebellar granule neurons (CGNs) leave the external germinal layer (EGL). Both Par3 gain of function and Seven in Absentia loss of function spur precocious radial migration. Time-lapse imaging using a novel probe to measure neuronal cell contacts in live CGNs reveals that Par3 promotes adhesive interactions needed for germinal zone exit by recruiting the JAM-C epithelial tight junction adhesion molecule to the neuronal cell surface. Our findings suggest a model in which post-translational modification of PAR complex function is a key mechanism regulating the exit of neuronal progenitors or immature neurons from a germinal zone niche. Moreover, these results identify an unexpected parallel between epithelial junction formation and the cell-cell interactions that occur during histogenesis in the developing vertebrate brain.

MONDAY

2163 Cognitive deficits associated with CNS myelin dysfunction. A. Gow1, K. Meharas1; 1Genetics, Wayne State University, Detroit, MI

Cognitive dysfunction is typically considered a secondary symptom associated with mental illness. Studies suggest that mental illnesses themselves are the cause of cognitive dysfunction, manifested as impairments in attention, memory, learning and executive function and lead to behavioral disorders. The pathophysiological processes that underlie cognitive dysfunction associated with behavioral changes have been debated for decades. Most studies focus on disrupted neural development or aberrant neurochemistry, however, an abundance of anecdotal clinical data and genetic evidence support a role for myelin and myelin-related genes in the etiology of cognitive dysfunction and behavioral disorders. In the current study, we have generated a myelin mutant mouse in which expression of the tight junction protein, claudin 11, has been ablated in oligodendrocytes. The function of this protein in contributing to the electrical resistance of CNS myelin has been elucidated in molecular and mathematical detail. Most importantly, the phenotype of these knockout mice arises from changes to the passive membrane properties of small diameter myelinated fibers and not from degenerative processes. Accordingly, we are able to interpret behavioral changes in these mutants in terms of cognitive dysfunction associated with slower conduction velocity. Our analyses of behavioral abnormalities resulting from slowed conduction will be presented.

2164 Cortical clustering of cell polarity regulators. J. Dodgson1, A. Chessel1, F. Vaggi2, S. Cox3, E. Rosten3, M. Yamamoto4, M. Geymonat1, Y. Dong1, J. Ahringer1, D. Albrecht5, M. Sato4, A. Csikasz-Nagy2, R. Carazo Salas1; 1Gurdon Institute, University of Cambridge, Cambridge, United Kingdom, 2The Microsoft Research- University of Trento Centre for Computational Systems Biology, Trento, Italy, 3Randall Division of Cell and Molecular Biophysics, Kings College London, United Kingdom, 4Department of Biophysics and Biochemistry, University of Tokyo, Japan, 5Institute of Biochemistry, ETH Zurich, Zurich, Switzerland

Cell polarity is a fundamental part of cell physiology. Despite the identification of many polarity- regulating factors in diverse organisms, the microscopic structure and dynamics of the polarity machinery itself has been difficult to elucidate. We imaged frontally the cortical areas where the polarity machinery accumulates in cells of the fission yeast Schizosaccharomyces pombe, using time-lapse and super-resolution (OMX structured-illumination and live-cell STORM) microscopy. Analysis of several key polarity factors reveals that they localize to discrete cortical clusters ('nodes'), resolvable to 50nm in diameter. Different polarity factors display different node distributions and dynamics at the cell cortex. Strikingly, we find that many factors thought to regulate polarity by interacting physically at the cortex – such as the factors Tea1p and Tea3p - do not necessarily co-localize there, suggesting unexpected layers of regulation. Artificial dimerization of Tea1p and Tea3p leads to mislocalization of these factors and cell polarity defects, suggesting that the segregation of polarity factors into adjacent but separate nodes may be crucial to their function. Similar nodes are found in Saccharomyces cerevisiae and Caenorhabditis elegans cells, suggesting conservation of this structural arrangement.

MONDAY

2165 Rapid cycles of phosphorylation and dephosphorylation on threonine 567 control ezrin localization and function in microvillus formation. R. Viswanatha1, P. Ohouo1, M. Smolka1, A. Bretscher1; 1Cornell University, Ithaca, NY

Ezrin is a member of a group of conserved plasma membrane-actin cytoskeletal cross-linkers (called ERM proteins) that is negatively regulated by an intramolecular interaction between the N- and C-termini. Ezrin is activated by threonine-567 phosphorylation near the C-terminus. In placental Jeg-3 cells at steady-state, we found that 50% of cellular ezrin is phosphorylated at threonine 567, and this phosphorylated pool is completely turned over within just 5 minutes. The N-terminus of ezrin is known to mediate its localization to the plasma membrane. Wild-type ezrin in Jeg-3 cells, however, is enriched on the microvillus membrane and virtually undetectable on other regions of the plasma membrane. Upon a brief treatment with a phosphatase inhibitor, by expressing a T567E phosphomimetic form of ezrin, or by expressing other constitutively open forms of ezrin, we find that this distribution is altered: ezrin “spills out” of the microvillar membrane and occupies other regions of the plasma membrane. To determine whether changing these phospho-dynamics by stabilizing the phosphorylated form of ezrin would affect ezrin function, we performed siRNA-rescue experiments. Ezrin depletion in Jeg-3 cell resulted in the loss of microvilli, and only wild-type, but neither T567A nor T567E ezrin restored them. Taken together, these observations hint at the presence of constant, local kinase activity toward ezrin countering ubiquitous phosphatase activity, and this balance is essential for ezrin function. To determine how phosphorylation dynamics are regulated in Jeg-3 cells, we conducted a proteomic screen to identify kinases associated with ezrin. We identified several candidate kinases previously shown to phosphorylate the C-termini of ERMs in other systems. We are in the process of examining the distribution of these kinases in Jeg-3 cells and their contribution to ezrin phospho-dynamics.

2166 Characterization of Banderuola, a novel regulator of asymmetric cell division in Drosophila melanogaster. F. Mauri1, J. L. Mummery-Widmer1, M. Yamazaki1,2, J. A. Knoblich1; 1Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria, 2The Global COE program, Akita University School of Medicine, Akita, Japan

Asymmetric cell division is a process whereby a progenitor cell can generate daughters with a different fate. The underlying molecular mechanisms have been widely studied in the Drosophila Sensory Organ Precursor (SOP) cells, that generate the 4 different cell types forming the external sensory organs (bristles) through 2 rounds of asymmetric cell division. This is achieved through the partitioning of the protein Numb, that represses Notch signaling in the daughter cell where it is segregated. To gain a better insight of this process, we used tissue specific RNAi to score the effects of the knockdown of genes on the morphology of the bristles. We have screened a library of 22,247 transgenic Drosophila RNAi lines, and identified 130 genes potentially involved in asymmetric cell division. To identify the genes involved in the asymmetric localization of Numb, we developed a live time-lapse cell-imaging assay based on a GFP reporter. Among the 130 candidates screened and not previously thought to play a role in asymmetric cell division, there were three genes whose knockdown reproducibly affected Numb localization. We have confirmed the observed phenotypes by independent secondary RNAi lines and have started investigating the function of one of the identified genes, which encodes a previously uncharacterized protein. Loss-of-function studies suggest that the protein, which we named Banderuola, specifically acts in establishing an axis of cell polarity for asymmetric cell division. MONDAY

Co-Immunoprecipitation experiments show that Banderuola binds to Discs-large (Dlg), a membrane-associated guanylate kinase acting in many cell-polarity events. Our study has identified Banderuola, a new component of the machinery of asymmetric cell division. As its binding partner Dlg acts in several polarity events, including synaptogenesis and tumor formation, our studies may be relevant for a variety of biological processes.

2167 Secretory vesicles deliver Cdc42p to sites of polarized growth in S. cerevisiae. S. A. Dighe1, K. G. Kozminski1,2; 1Biology, University of Virginia, Charlottesville, VA, 2Cell Biology, University of Virginia, Charlottesville, VA

Establishment and maintenance of cell polarity in many eukaryotes depends upon activation of the Rho GTPase Cdc42p. In the budding yeast S. cerevisiae, Cdc42p is essential for establishing and maintaining an axis of polarized growth that supports bud formation. In G1 of the cell cycle, Cdc42p asymmetrically localizes on the plasma membrane (PM), at the presumptive bud site, in an actin-independent manner. Maintenance of this axis of growth during bud formation, in S and G2, requires the actin-dependent localization of Cdc42p at the bud tip. How Cdc42p is maintained at the bud tip is unclear, leading to divergent models of Cdc42p and cell polarization (Wedlich-Soldner et al. 2003. Science 299:1231-1235; Layton et al. 2011. Current Biology 21:184-194). One model suggests that Cdc42p is delivered to the bud tip by secretory vesicles, replenishing Cdc42p that is lost at the bud tip through diffusion in the plane of the PM or by endocytosis. Consistent with this model, we show Cdc42p associated with secretory vesicles both in vivo and in vitro. By immunogold labeling EM thin sections of whole cells, both wild-type and secretory mutants, we localized Cdc42p to vesicles 80-100nm in diameter, the signature size of secretory vesicles in S. cerevisiae, in addition to the PM and ER. We also show that Cdc42p co-fractionates with secretory vesicles and the secretory vesicle markers Sec4p, Bgl2p, Osh4p, and invertase. These results indicate that secretory vesicles can serve as a vehicle for the delivery of Cdc42p to sites of polarized growth. Supported by NSF 0723342.

2168 Ral GTPases regulate biogenesis of epithelial polarity. C. Hazelett1, C. Yeaman1; 1Anatomy & Cell Biology, University of Iowa, Iowa City, IA

Tight junctions (TJs) are indispensible structures to epithelial cells, and are responsible for regulation of paracellular diffusion and maintenance of cellular polarity that is crucial to human health. A detailed mechanistic understanding of how TJs form, however, is incomplete. TJ proteins assemble at the interface between apical and lateral membrane domains, but factors that target vesicles to these sites during TJ formation have not been identified. TJ maturation is associated with establishment of polarity, and in vivo epithelial cells form tubes with distinct lumenal and basal domains. Furthermore, polarized epithelial cells generate a primary cilium on the apical surface. The Exocyst is required for ciliogenesis and cystogenesis, and under control of Ral GTPases, has been shown to direct vesicle trafficking to basolateral membranes. We investigated the roles of Ral GTPases in TJ assembly and ciliogenesis, and also in the more complex process of cystogenesis. We show that RalA and RalB serve opposing functions during the former two processes, and both are indispensible for the latter. Silencing expression of RalA or RalB altered TJ gate function in opposite ways during initial establishment of TJs in MDCK cells; RalA knockdown abrogated the trans-epithelial resistance (TER) overshoot while RalB knockdown greatly exaggerated it. However, TJ gate function, as assayed by transcellular lipid diffusion, was unaffected by either knockdown. Immunofluorescence and detergent solubility studies revealed Ral-specific differences in TJ composition. Levels of some TJ components MONDAY were reduced in RalA knockdown cells when compared to controls, but were increased in TJs of RalB knockdown cells. Additional data showed that both Ral GTPases exert effects on TJ formation through binding to the Exocyst. Furthermore, RalB was necessary for endocytosis of the junctional protein E-cadherin. RalA knockdown also decreased ciliogenesis, while RalB knockdown led to an increase of ciliated cells. Finally, formation of a central lumen during cystogenesis in three-dimensional Matrigel cultures was dependent on expression of both GTPases. Our working model is that RalA facilitates trafficking of TJ and ciliary constituents to the plasma membrane via the Exocyst, while RalB regulates endocytosis and/or recycling of these components. This and future work will significantly increase our understanding of how these GTPases function together, and in collaboration with the Exocyst, to regulate epithelial polarization.

2169 Out of the loop: The role of Bem1 in actin-independent polarization in S. cerevisiae. S. E. Smith1,2, R. Li1,2; 1Stowers Institute for Medical Research, Kansas City, MO, 2Dept of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS

Cell polarization is vital for a range of important processes across the spectrum of life, from cell migration in human development to polarized cell division in budding yeast, but the mechanisms of polarization are still not well understood. A key player is the small GTPase Cdc42, the “master regulator” of polarity. In budding yeast, polarization of Cdc42 requires neither spatial cues nor cytoskeletal structures, but occurs via a dynamic, diffusion-based process. One model consists of a positive feedback loop in which a small, stochastic accumulation of active Cdc42- GTP recruits the adaptor molecule Bem1, which then recruits the activator of Cdc42, Cdc24, resulting in an increased local active pool and further recruitment. In this work, we disrupted individual steps in the proposed feedback loop by abrogating the ability of Bem1 to interact with either Cdc42 or Cdc24. We then investigated the effect of the disruption on diffusion-based polarization. As expected, we found that abrogation of the Bem1-Cdc24 interaction resulted in a complete loss of polarization. However, we were surprised to find that Cdc42 polarization was still robust when the Cdc42-Bem1 interaction was disrupted, even though Bem1 did not localize to the polar cap. This finding invalidates the current feedback loop model, and suggests that Bem1 may have a location-independent role in activating Cdc24.

2170 Does ciliary autotomy in the kidney contribute to PKD? C. Yeaman1, N. Ma1; 1Anatomy & Cell Biology, University of Iowa, Iowa City, IA

Primary cilia are active signaling centers present on most differentiated cell types. Cells may lose their cilia either by resorption into the cell, or shedding (autotomy) from the cell surface. Autotomy is an ancient process, but its physiological significance in mammals is unclear. We have shown that diverse agents trigger ciliary shedding in renal epithelial cells both in vitro and in vivo. This occurs by a distinct mechanism than that described for ciliary resorption, as inhibitors of resorption fail to block autotomy and intact cilia are recovered in the media of cultured cells and in the urine of mice following treatment with autotomy-inducing agents. Shedding of primary cilia promotes dramatic changes to epithelial cells, including compositional and functional changes to tight junctions, induction of partial epithelial-to-mesenchymal transition and dysregulated cell cycle control. A full-length cilium is not required to trigger autotomy-induced epithelial remodeling. It occurs in cells that express stunted pro-cilia, such as non-differentiated epithelial cells and cells lacking essential ciliogenesis components (e.g. IFT88). We hypothesize that autotomy liberates the maternal centriole from its tethered state at the apical plasma membrane, thereby facilitating its differentiation into a signal-generating MONDAY centrosome. In renal epithelial cells, ciliary autotomy is dependent on the catalytic activity of Nek8 and the Exocyst vesicle tethering complex. jck mice, which develop autosomal-recessive polycystic kidney disease and harbor a mutation in the NEK8 gene, exhibit substantially elevated levels of ciliary shedding in vivo. Therefore, we propose that ciliary autotomy is a physiologic phenomenon in the kidney, and that dysregulated autotomy may contribute to PKD by altering trans-epithelial fluid transport, tissue morphogenesis and growth regulation.

Cancer Cell Biology I

2171 Evaluating the effect of South African herbal extracts on breast cancer cells. M. Choene1, L. Motadi1; 1Genetics, University of the Witwatersrand, Johannesburg, South Africa

South African plants have been used for generations by traditional healers due to their anti- bacterial, anti-viral, anti-fungal and anti-amoebic properties. Now with the emergence of natural product research in the science fraternity, a lot of these indigenous plants have been showing great potential as an alternative cancer treatment to orthodox treatments. For our research, our aim was to investigate the anti-breast cancer activities of three South African plant extracts: Kedrostis Foetidissima, Euphorbia Mauritanica and Elytropappus Rhinocerotis. We looked at the effect of these extracts on breast cancer cells since it is one of the leading cancers in females in South Africa. The herbal extracts were screened for their cytotoxicity in two breast cancer cell lines MCF-7 and YMB-1 by means of an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide) assay. MTT is a simple colorimetric assay used to measure cell cytotoxicity, proliferation or viability. The cells were treated with varying concentrations (10, 30, 50 and 100µg/ml) of the extracts, then the MTT assay was conducted to determine the IC50 (concentration of extract that causes 50% cell death) of these herbal extracts on the cell lines. We found that Elytropappus Rhinocerotis had no significant effect on both cancer cell lines, Kedrostis Foetidissima exhibited cytotoxicity on both cell lines with 48% cytotoxicity at 100µg/ml (IC50 > 100µg/ml) on MCF-7 cells and 40% cytotoxicity at 100µg/ml (IC50 > 100µg/ml) on YMB- 1 cells. Euphorbia Mauritanica showed 53% cytotoxicity at 100µg/ml and 37% cytotoxicity at 50 µg/ml (50µg/ml 100 µg/ml) on MCF-7 but had no significant effect on YMB-1. For those extracts which exhibited cytotoxicity on the breast cancer cells, we then aimed to investigate if the cells were dying due to apoptosis and not necrosis. We then employed the use of a flow cytometer using Annexin V, which can detect cells that are alive, undergoing apoptosis and necrosis (Annexin V has a strong calcium dependent affinity to phosphatidylserine residues, which are normally hidden in the cytoplasmic plasma membrane but translocate to the cell surface during apoptosis, acting as a probe to detect apoptosis). It was then observed that the herbal extracts inducing death via apoptosis and not necrosis. We can conclude that the herbal extracts K. Foetidissima and E. Mauritanica exhibit some level of cytotoxicity on breast cancer cells. It is also promising that they cause cells to undergo apoptosis showing great potential in their use in breast cancer natural product research and drug design sector.

2172 Traditional herb in cancer. L. R. Motadi1, A. Thafeni1, M. Choene1; 1School of Molecular Biology, University of the Witwatersrand, Johannesburg, South Africa

Euphorbia mauritanica and Kedrostis hirtella extracts can induce anti-proliferative activities in cancer cells. MONDAY

In recent times a significant proportion of the drugs used to treat cancer are either natural products or compounds discovered based on the study of natural products and their interaction with cellular targets. While the use of synthetic combinatorial libraries has enhanced the pace of drug discovery, the extraordinary structural diversity intrinsic to natural products assures that they will continue to find utility in defining novel cellular targets for therapeutic intervention, and also as drug candidates and structural leads for chemical optimization. With our increased understanding of the molecular mechanisms underlying cancer progression and cell death, this work aims to target E. mauritanica and K. hirtella as new therapeutic targets for treatment in cancer in particular Breast and Lung cancer. Accordingly, the study was aimed to investigate possible molecular mechanisms that are associated with the potential anti-carcinogenic property of this agrofield weed. MCF-7 and A549 cells were exposed to different concentrations (0-100 mg/ml) of the crude methanolic extract of E. mauritanica and K. hirtella to evaluate their growth inhibitory and apoptosis inducing effects. The extract elicited a dose- and time-dependent inhibition of cell proliferation, followed by a concomitant decrease in cell viability. The observed cytotoxicity was linked to the induction of apoptosis as determined by Flow cytometer Annexin V features known to be associated with the advent of apoptosis. Real time quantitative RT-PCR analyses of Bax, Bcl-2, RBBP6 and p53 exhibited aberrant expression profiles of these genes under various treatment conditions. Taken together, the data suggest that the crude methanolic extract of E. mauritanica and K. hirtella contains bioactive compounds that may be beneficial in the treatment of malignant growths. However, the mechanisms behind this activity still need to be researched.

2173 A natural small molecule YCG185 inhibits angiogenesis both in vitro and in vivo. Y. Kim1, H. Jung1, H. Kwon1; 1Chemical Genomics National Research Laboratory, TRCP, Department of Biotechnology, College of Life Science & Biotechnology, Yonsei University, Seoul, Korea

Angiogenesis, a formation of new blood vessels from pre-existing ones, plays a critical role in normal and pathological phenotypes including solid tumor growth and metastasis. Accordingly, the development of new anti-angiogenic agents has been considered as an efficient strategy for the treatment of cancer and other human diseases related with angiogenesis. To identify such small molecules, 300 crude extracts of natural plants were subjected to cell-based screening toward the proliferation of human umbilical vein endothelial cells (HUVECs). As the result, the active principle of YCG185 was identified as a new anti-angiogenic agent. YCG185 inhibited the proliferation of HUVECs at IC50 of 6.6 μg/mL without showing any toxicity to the cells. YCG185 significantly suppressed in vitro angiogenesis such as VEGF-induced tube formation and chemoinvasion. Moreover, the compound inhibits chorioallantoic membrane in vivo angiogenesis at non-toxic doses. In addition, YCG185 decreased the expression levels of hypoxia inducible factor-1a and its target gene, VEGF, in a dose-dependent manner. Taken together, these results demonstrate that YCG185 could be a novel natural small molecule targeting angiogenesis.

2174 Genetic Modification of Breast Cancer Gene Targets Using ZFN Technology Reveal Differential Responses to Drug Sensitivity. G. L. Davis1, C. Corman1, L. Daley1, G. Pegg1, H. Zakeri1, Z. Zhang1, G. Wemhoff1; 1Sigma Aldrich, St. Louis, MO

Breast cancer tumorigenesis is a complex disease where multiple signaling pathways participate in cell proliferation and invasion. The heterogeneity of this disease presents a MONDAY challenge in developing therapuetic treatments because patients respond to therapies with varying degrees of sensitvity 1,2. For example, previous studies have demonstrated that activation of the MEK/MAPK pathway, loss of cell-cell adhesion, and enahnced epithelial-to- mesenchymal transition confer resistance to the breast cancer therapeutic, gefitinib 2,3,4.

With the advent of zinc finger nuclease (ZFN) technology, it is possible to generate cancer relevant mutations in one or more endogenous genes. Zinc finger nucleases have been optimized to target specific genes where they induce a double-strand break adjacent to their binding site. Double strand-breaks are either repaired by non-homologous end joining (NHEJ) or homology dependent repair (HDR). As a result, cell lines harbor insertions, deletions, or integrations within the targeted gene of interest.

We have utilized zinc finger nuclease technology to target and disrupt endogenous breast cancer relevant genes, SYK, ESR2, BCR, and APC, in the near normal mammary epithelial line, MCF10a. These gene targets play a pivotal role in cellular proliferation, migration and cell adhesion and may thereby play a contributing role in gefitinib sensitivity 5,6,7,8,9,10. In this study, we aim to investigate how loss of SYK, ESR2, BCR, and APC gene function, in ZFN engineered cell lines, affect sensitivity towards the tyrosine kinasae inhibitor, gefitinib.

2175 Gene Specific Regulation of NFκB-Dependent Anti-Apoptotic Genes in Metastatic Prostate Cancer Cells by Bortezomib. S. Manna1, B. Singha1, T-P. Chang1, S. Sanacora1, I. Vancurova1; 1Biological Sciences, St. John's University, Queens, NY

Prostate cancer is the third most common cause of death from cancer in men of all ages. One of the critical factors in progression to the metastatic prostate cancer is the increased activity of the transcription factor NFκB, which induces synthesis of anti-apoptotic genes, such as Bcl-2, cIAP- 1 and cIAP-2, thus promoting inhibition of apoptosis and resistance to chemotherapy. Bortezomib is a potent 26S proteasome inhibitor that has been used for the treatment of multiple myeloma and mantle cell lymphoma, and has shown promising results in several other types of cancer, including the metastatic prostate cancer. However, the molecular mechanisms of bortezomib function in prostate cancer cells are not fully understood. In this study, we investigated the mechanism of bortezomib function in the metastatic prostate cancer PC-3 cells. Our data demonstrate that in PC-3 cells, bortezomib induces translocation of the NFκB inhibitory protein, IκBα, from the cytoplasm to the nucleus, thus inhibiting the constitutive NFκB activity in these cells. However, our results indicate that the regulation of NFκB activity by the bortezomib-induced nuclear IκBα is gene specific. While the expression of NFκB-dependent anti-apoptotic genes cIAP-1 and cIAP-2 is inhibited by bortezomib, expression of Bcl-2 is not suppressed. Differences in the transcriptional regulation by the bortezomib-induced nuclear IκBα might hold the key for development of more effective therapies for the advanced forms of prostate cancer and other cancers characterized by the constitutive activation of NFκB and resistance to chemotherapy. Funded by: NIH grants GM079581 and AI085497 to I.V.

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2176 The neuroprotective efficacies and anticancer properties of various botanical extracts, Rue chalpanesis and Hibiscus sabdariffa, against neurotoxic-induced Parkinson's disease and pancreatic adenocarcinoma in vitro. T. Zeleke1, H. Tsegaye1, A. Bettica1; 1Biology, Manhattanville College, Purchase, NY

Polyphenolic antioxidants have demonstrated anticarcinogenic activities by inhibiting or delaying the oxidation process caused by free radicals. Many extracts, containing anthocyanins, flavonoids, or phenolic compounds, have been shown to be strong antioxidants and possible neuroprotective agents. Common neurodegenerative disorders, such as Parkinson's disease (PD), may show neuronal cell loss as an accumulation of reactive oxygen species (ROS) due to increased oxidative stress. Roselle extract from the tropical plant Hibiscus sabdariffa contains primarily anthocyanins and protocatechuic acid, which can exhibit antioxidant, anti-inflammatory and antitumorigenic properties. Rue extract, from the tropical plant Ruta chalepensis, may also protect cells from oxidative stress-induced free radicals. The planetary structures of acridone, a major constituent in the Rue extract, can be inserted between a double stranded DNA and interfere with cellular machinery, contributing to its antitumor properties. For plant extractions, dried plant samples of R. chalapensis are centrifuged, vortexed, and agitated in the cold for 24 hours. H. sabdariffa (10g) is boiled in water, followed by filtration. To determine inhibition of growth rate in pancreatic adenocarcinoma cell lines, PANC-1 cells are seeded at a concentration of 6x104 cells/well in 96-well plates. After seeding and adherence to the well plate, all cells are allowed one to two population doubling times (PDT) before and after treatments. Single drug 24 hour treatments for dose response curves in ten fold dilutions in water determine effective IC50 values for Ruta and Hibiscus extracts. To determine the neuroprotective efficacies of these extracts with relationship to an oxidative stress-induced in vitro PD model, pretreatments of SK-N-SH neuroblastoma cells with these extracts against the neurotoxic insults, MPP+ and 6-OHDA were undertaken. The cells are pretreated in 96-well plates to formulate dose-response curves, with each of the serially-diluted extracts added for 24 hours before a potent dose of MPP+, a mitochondrial complex I inhibitor, or 6-OHDA, a potential ROS generator. The cells are exposed to each insult first for 24 hours for posttreatment studies, followed by the extracts for 24 hours. Cotreatments with each extract and the neurotoxic insults were performed to elucidate possible preconditioning or gene activation versus actual mechanisms of action. Cell viability/ proliferation of triplicate data sets are assessed either through the MTT mitochondrial (for PANC-1 cells) or Neutral Red lysosomal assay (for neuroblastoma cells). All data are statistically analyzed using a one-way ANOVA with Tukey’s post hoc tests. Preliminary dose responses for R. chalepensis and H. sabdariffa extracts show effective doses of 10-4-10-6 g/ml for SK-N-SH cells. The neuroprotective efficacies of the botanical extracts against the neurotoxin-induced PD model may indicate their successful use in the prevention or treatment of Parkinson’s disease. Effective IC50 values may lead to possible chemopreventive strategies for certain cancer cell types as well.

2177 Gefitinib resistance of cancer cells correlated with TM4SF5-mediated epithelialmesenchymal transition. M. Lee1, J. W. Lee1,2; 1Department of Pharmacy, Reseach Inst of Pharmaceutical Sciences, Cell Dynamics Res Ctr, College of P, Seoul National University, Seoul, Korea, 2Cancer Research Institute, College of Medicine, Seoul National University, Seoul, Korea

Although cancers can be initially treated with the epidermal growth factor receptor (EGFR) inhibitor, gefitinib, continued gefitinib therapy does not benefit the survival of patients due to acquired resistance through EGFR mutations, c-Met amplification, or epithelial-mesenchymal MONDAY transition (EMT). It is of further interest to determine whether mesenchymal-like, but not epithelial-like, cancer cells can become resistant to gefitinib by bypassing EGFR signaling and acquiring alternative routes of proliferative and survival signaling. Here we examined whether gefitinib resistance of cancer cells can be caused by transmembrane 4 L six family member 5 (TM4SF5), which has been shown to induce EMT via cytosolic p27Kip1 stabilization. Gefitinib- resistant cells exhibited higher and/or sustained TM4SF5 expression, cytosolic p27Kip1 stabilization, and mesenchymal phenotypes, compared with gefitinib-sensitive cells. Conversion of gefitinib-sensitive to -resistant cells by introduction of the T790M EGFR mutation caused enhanced and sustained expression of TM4SF5, phosphorylation of p27Kip1 Ser10 (responsible for cytosolic location), loss of E-cadherin from cell-cell contacts,and gefitinib- resistant EGFR and survival signaling activities. Additionally, TM4SF5 overexpression lessened the sensitivity of NSCLC cells to gefitinib. Suppression of TM4SF5 or p27Kip1 in gefitinib- resistant cells via the T790M EGFR mutation or TM4SF5 expression rendered them gefitinib- sensitive, displaying more epithelial-like and less mesenchymal-like characteristics. Together, these results indicate that TM4SF5-mediated EMT may have an important function in the gefitinib resistance of cancer cells. [This work was supported by NRF by senior researchers program (Leap research, 2011-0001160) and Global Frontier Project grant (NRF-M1AXA002- 2010-0029778), and a grant of the Korean Health Technology R&D Project (A100727), MHWFA, Korea to JW Lee].

Key words: epithelial-mesenchymal transition; drug resistance; EGFR; lung cancer; tetraspanin

2178 Bioavailability Enhancing Strategy for Oral Administration of Doxorubicin. Y. ZHAO1, M. L. Forrest1; 1Pharmaceutical Chemistry, the University of Kansas, Lawrence, KS

Doxorubicin (DOX) has been used as an anticancer drug to treat a variety of cancers. Due to the first pass effect, DOX has been only administrated intravenously. Recent studies have shown that isoflavonoid, i.e. quercetin, can improve oral bioavailability of doxorubicin by blocking the activity of p-glycoprotein. In addition, quercetin is absorbed primarily by gut lymph. Our hypothesis is that oral administrated doxorubicin-quercetin prodrug (DQ) will improve oral bioavailability and potentially enhance the absorption of DOX in gut. We have successfully synthesized DQ. The IC50 of DOX, quercetin and DQ in 4T1.2 cells were 5.7 µM, 49.2 µM and 14.04 µM, respectively. Both free DOX and DQ were orally fed to Sprague Dawley female rats. Pharmacokinetic study showed that 1h after drug administration, only DQ, but not free DOX, gave an active form of DOX in the blood serum. This study showed that the doxorubicin- quercetin prodrug strategy can be used to improve oral bioavailability of DOX.

2179 Induction of Intracellular Superoxide in Chronic Myelogenous Leukemia Cells Leads to an Antioxidant Response Downstream of NFE2L2. L. F. Gemta1, F. Alvarez-Calderon1,2, K. Hansen1, J. DeGregori1,3; 1Biochemistry and Molecular Genetics, University of Colorado - Anschutz Medical Campus, Aurora, CO, 2Medical Scientist Training Program, University of Colorado - Anschutz Medical Campus, Aurora, CO 3Molecular Biology, University of Colorado - Anschutz Medical Campus, Aurora, CO

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized by the BCR-ABL fusion gene. Bcr-Abl tyrosine kinase inhibitors including Imatinib have been used to treat CML in patients. These inhibitors fail to eliminate all types of Bcr-Abl+ leukemia especially in advance and blast crisis phases. In this study, we identified that inhibiting mitochondrial MONDAY metabolism with oligomycin A, an inhibitor of mitochondria ATP-synthase, sensitizes CML cells to Imatinib. The combination of 1 µM Imatinib and 5-10 nM oligomycin A resulted in elimination of CML cells without inhibition of mitochondrial respiration. Although Annexin V/Propidium Iodine analysis showed that most of the cells died by apoptosis, the exact mechanism by which the combination therapy successful eliminated these cancer cells is unknown since the doses of OA used are ~100-fold lower than the IC50. The level of HIF-1α and downstream targets of Bcr- Abl including p-Akt, p-ERK, and p-STAT5 were not affected by the treatment. The combination therapy increased intracellular superoxide levels and induced expression of the antioxidant response downstream of NFE2L2 expression. Normal and cancerous cells protect themselves from the damage caused by high levels of superoxide by activating their antioxidant responses. These responses include the induction of the transcription factor nuclear factor (erythroid- derived 2)-like 2 (NFE2L2), which in turns regulates the expression of its downstream target genes including superoxide dismutase-2 (SOD2), gamma-glutamyltransferase 1 (GGT1), glutamate-cysteine ligase catalytic subunit (GCLC), and heme oxygenase (decycling) 1 (HMOX1). The combination therapy inhibits mitochondrial metabolism without inhibiting mitochondrial respiration and leads to increases in superoxide levels and compensatory increases in antioxidant responses.

2180 Genome-Wide RNAi Screen for Tetraploid-Specific Lethality in Cancer Cells. T. S. Kuroda1, R. K. Dagher1, D. Pellman1; 1Pediatric Oncology, Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Boston, MA

Cancer cells are often genetically unstable, resulting in aneuploid genomes. The consequences of aneuploidy are poorly understood, but it has been suggested to play roles in tumor aggressiveness, chemotherapy resistance, and metastasis.

One important route to an aneuploid genome may be through doubling of the genome, leading to unstable tetraploid cells. Many cell division errors ultimately manifest as a failure of cytokinesis. Especially in cells defective in p53 function, tetraploid cells proliferate, but generate both high rates of whole chromosome aneuploidy and defects in the maintenance of chromosome structural integrity. Our laboratory has demonstrated that in p53-null primary cells, tetraploidy promotes tumor development and the resulting tumors display markedly abnormal genomes (Fujiwara T. et al. Nature 2005, 437: 1043). It is now known that several human cancer-inducing mutations-- e.g. RB loss or loss of the adenomatous polyposis coli tumor suppressor-- also predispose cytokinesis failure and tetraploidy. These findings highlight a need to understand in detail the physiological changes that accompany tetraploidy, which might provide with a new strategy for cancer therapeutics.

To gain an understanding of physiological alterations associated with tetraploidy in cancer cells, we have established tetraploid cell lines derived from a near diploid human colorectal cancer cell line HCT116. Compared to diploid cells, anaphase FISH showed that tetraploid cells have a higher rate of chromosome mis-segregation—with levels similar to chromosomally unstable cancer cells.

Using these paired tetraploid and diploid cell lines, we have performed a genome-wide RNAi screen to identify genes whose knockdown selectively affect the viability of chromosomally unstable tetraploid cells. After secondary validation of the screen, we have identified 61 tetraploid-selective lethality genes. Consistent with the known p53-dependent barrier to proliferation of tetraploid and aneuploid cells, and validating the overall approach, one tetraploid-selective lethality gene is MDM2, an E3 ligase for p53. MONDAY

2181 Growth Inhibitory Effects of Death Receptor 5 Agonist, Drozitumab alone and in Combination with the Novel Spirocyclic Lignan Ramonanin A. K. J. Chavez1, B. Stone1, S. Lipkowitz1; 1Laboratory of Cellular and Molecular Biology, National Institutes of Health, National Cancer Insititute, Bethesda, MD

Clinically, breast cancers can be divided into distinct subtypes that express estrogen receptors (ER), progesterone receptors (PR), those that have amplification of HER2/Neu, and those that lack expression of ER or PR and lack amplification of Her2/Neu (so called triple negative breast cancer or TNBC). TNBC has a poor outcome compared to the other subtypes of breast cancers. Previously, we have shown that a recombinant GST fusion protein with TNF-related Apoptosis Inducing Ligand (GST-TRAIL) selectively kills triple-negative breast cancer cells with mesenchymal features by activation of TRAIL receptor 2 (TRAIL-R2). In this study we have characterized the growth inhibitory effects of a clinically relevant agonistic antibody to TRAIL- R2, referred to as Drozitumab. To determine the selectivity of Drozitumab, we chose a panel of 15 breast cancer cell lines including 3 ER/PR positive cell lines, 4 HER2/Neu amplified cell lines, and 8 triple-negative cell lines. Treatment of these cells with Drozitumab selectively killed TNBC cell lines with mesenchymal features. ER positive, HER2/Neu amplified and TNBC cell lines with epithelial features were resistant to Drozitumab induced cell death. Drozitumab induced caspase activation (measured by activation of the initiator caspase 8, activation of the downstream caspases 3/7, and PARP cleavage) and the toxicity of Drozitumab was blocked by the pan-caspase inhibitor z-VAD-FMK. This was identical to the pattern seen with GST-TRAIL. Cross-linking with an anti-FC antibody enhanced the efficacy of Drozitumab as shown by the more rapid appearance of cleaved caspase 8, greater caspase 3/7 activity, and more rapid PARP cleavage. Furthermore, we show that a novel family of Spirocyclic lignans from Guaiacum sp. (Ramonanins) enhances Drozitumab -induced apoptosis in MBA-MD231and HCC38 triple-negative breast cancer cell lines. In conclusion, our data indicates that Drozitumab selectively inhibits growth of mesenchymal phenotype triple-negative breast cancer cell lines in a caspase dependent fashion and that cell death is enhanced in the presence of Ramonanin A. This data provides evidence that TRAIL-R2 targeted therapies may have therapeutic potential in the treatment of TNBC with mesenchymal features.

2182 The effective inhibition of growth rate and induction of apoptosis in adherent and suspension cancer cell lines by the administration of various Panax (Ginseng) species. N. Yousaf1, A. Luiso1, T. Zeleke1, A. Bettica1; 1Biology, Manhattanville College, Purchase, NY

Ginseng (Panax) is a perennial plant with fleshy roots that contains Rg1, Rg2, Rf and other ginsenoside groups. A popular traditional medicine for centuries, ginseng is believed to enhance memory, stimulate the immune system, reduce cholesterol levels, and fight depression and insomnia. Various Panax species, such as Korean, American, and Chinese Red or White Ginseng, have been shown to enhance memory and stimulate the immune system. Ginseng extracts have also been considered as adaptogens, natural substances that aid in homeostatic balance. The red ginseng, in its various forms, differs from white ginseng in its processing. The Korean ginseng, grown in cooler climates, has higher ginsenosides/saponin count than Chinese ginseng. Panax red ginseng, either Chinese or Korean, is considered to be more potent than the American ginseng, Panax quinquefolium. To determine the anticancer effects of the various ginseng species, the inhibition of growth rate and the induction of apoptosis is determined for both an adherent pancreatic adenocarcinoma cell line (PANC-1) and a suspension B-cell leukemic cell line (SUP-B15). B cell chronic lymphocytic leukemia (B-CLL) has long been considered a disease in which B-CLL cells accumulate due to a presumed defect in their MONDAY apoptotic mechanism. Previous morphologic observations suggest that B-CLL cells are resting cells with rarely detected cell cycle activity in the blood. For single and combination treatments, cells will be seeded at a density of 5x104 cells/well in 96-well plates. For suspension cultures, round bottom 96 well plates are used. When seeding round bottom well plates, the cells are allowed to sediment, forming a relatively loose monolayer. All plated cells are allowed one to two population doubling times (PDT) before treating. Single drug 24 hour treatments for dose response curves in ten fold dilutions determine effective IC50 values for each of the ginseng species. Results from these preliminary dose response studies determine whether consecutive (24 hours of one species followed by 24 hours of second species at effective doses) or additive drug trials (1/2 effective dose of each species together for 24 hours), is considered. The treatment course is followed by 1-2 PDT and assayed by one of two different methods. All treatment plates are repeated in triplicate. To determine inhibition of growth rate and cell viability, the MTT cell proliferation assay is performed with adequate washing. The optical density is read between 490-550nm and adjusted for background. The Caspase-3 colorimetric protease assay quantifies caspases that recognize the amino acid sequence DEVD, and since caspase 3 is an effector caspase, the level of apoptosis under the test conditions can be determined. All data are statistically analyzed using a one-way ANOVA with Tukey’s post hoc tests. Treatments with American Ginseng showed protection in the nanogram ranges. Korean Ginseng was more effective individually and, in combination treatments, the Chinese Red/Chinese White Ginseng extracts were most effective overall. Considering the growth rate inhibition and induction of apoptosis on adherent and suspension cancer cultures, ginseng species are potential anticancer agents.

2183 Isoform specific function of the formin FMNL2. C. Péladeau1, A. Heibein1, G. L. Lahaie1, S. Copeland1, J. Copeland1; 1Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada

Cancer cell metastasis is induced by actin-dependent cell migration and is consequently affected by the regulated function of cytoskeletal remodelling proteins. Formins, a highly conserved family of actin remodelling proteins, have been shown to provoke motility during metastasis and invasion. Indeed, formin-like 2 (FMNL2) has been shown to increase motility and facilitate invasion when overexpressed in colorectal cancer cells (CRC). Furthermore, knocking down the expression of FMNL2 inhibits amoeboid-style invasion by melanoma cells in vitro. FMNL2 mRNA is subject to alternative splicing and results from our lab, and others, suggest that the resulting encoded proteins are likely to differ in their regulation, subcellular localization and activity. The main objectives of this study are to identify the FMNL2 splice forms expressed in various cancer cell lines and determine if FMNL2 activity is required by these cells for invasion. In this study we have used RT-PCR to identify four FMNL2 splice forms (ITM, YHY, PMR and TQS) expressed in non-invasive (SW480) and invasive (SW620) colorectal cancer cells, as well as in highly invasive A375 melanoma cells. Preliminary data from cell-based assays suggest that there are functional differences between our identified FMNL2 isoforms in terms of both auto-regulation and their effects on actin dynamics. In addition, our qPCR data suggests that an “invasive” isoform of FMNL2 may be preferentially expressed in highly invasive cells. The data obtained in this study will lead to further understanding of the role of FMNL2 in invasion and metastasis as well as identify specific targets for the development of future anti- metastatic therapies.

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2184 Profilin-1 downregulation promotes transendothelial migration of breast cancer cells through increased VEGF secretion. M. Joy1, P. Roy2; 1Bioengineering, University of Pittsburgh, 2Bioengineering and Pathology, University of Pittsburgh, Pittsburgh, PA

Transendothelial migration of cancer cells is necessary for both intravasation and extravasation steps of tumor metastasis. Clinical correlation and xenograft studies from our laboratory have revealed that downregulation in the expression of profilin-1 (Pfn1: an actin-binding protein) is associated with increased metastatic potential of breast cancer cells. The aim of the present study was to evaluate the effect of loss of Pfn1 expression on transendothelial migration of breast cancer cells. We found that stably silencing Pfn1 expression through shRNA increases the ability of MDA-MB-231 breast cancer cells to transmigrate through a confluent monolayer of human vascular endothelial cells (VEC), and this can be rescued by re-expression of an RNAi- resistant version of Pfn1. Immunostaining for beta-catenin (a component of adherens junctions) showed that Pfn1 knockdown facilitates tumor cell-induced disruption of cell-cell junctions in VEC. Biochemical analyses of conditioned media derived from MDA-231 cells revealed that secretion of several factors that promote tumor metastasis including VEGF (a potent disruptor of endothelial cell-cell junctions) is significantly increased upon loss of Pfn1 expression. Silencing VEGF expression abrogates the stimulatory effect of Pfn1 KD on transendothelial migration thus suggesting that Pfn1 downregulation promotes transendothelial migration of breast cancer cells through increased VEGF secretion. These findings set the stage for future in vivo studies examining Pfn1’s role in regulating tumor cell dissemination into the vasculature.

2185 Microarray analysis of differentially expressed genes in MDA-MB-231 breast cancer cells treated with the laminin-derived peptide C16. E. S. Santos1, V. M. Freitas1, M. S. Menezes1, G. M. Machado-Santelli1, R. G. Jaeger1; 1Departament of Cell and Developmental Biology, Institute of Biomedical Sciences of The University of Sao Paulo, Sao Paulo, Brazil

Human breast cancer constitutes a worldwide health care problem. During breast cancer development and progression, tumor cells are engaged in a complex interplay with the surrounding microenvironment. In this scenario, extracellular matrix molecules play important roles influencing malignant behavior. An increasing number of evidences have shown that peptides derived from laminin cleavage are involved in tumor progression. Among them, C16 (KAFDITYVRLKF), derived from laminin-111 gamma-1 chain, is a cell-adhesive peptide that increases cell migration and invasion, enhances metastasis, and promotes angiogenesis. This prompted us to analyze whether C16 would regulate gene expression in cells derived from human breast cancer (MDA-MB-231). Cells were treated with C16 or scrambled peptide control (FKLRVYTIDFAK) for 24 hours. After treatment, gene expression was analyzed by microarray. Total RNA was extracted, biotin-labeled cDNA was generated and hybridized to Human Gene 1.0 ST Array (Affymetrix). GeneChips were stained and scanned. Expression Console software converted gene fluorescence intensities into numerical values. We found that C16 regulates expression of 80 genes in MDA-MB-231, including genes directly involved in processes associated with cancer. Among those genes, FGFR3, GPNMB and SPOCK1 expression was increased by C16 treatment, as confirmed by quantitative Real-Time PCR. FGFR3 encodes a tyrosine kinase receptor, which regulates various biological processes, including cell migration and angiogenesis. GPNMB encodes a transmembrane glycoprotein involved in angiogenesis and promotion of migration, invasion and bone metastasis of breast cancer cells. SPOCK1, on the other hand, encodes a proteoglycan associated with cell adhesion and increased migration. MONDAY

To correlate gene expression with function, we analyzed whether C16 would regulate invasion, proliferation and apoptosis in MDA-MB-231 cells. Functional results also indicated that C16 significantly increases MDA-MB-231 cell invasion, as shown by assays in Boyden chambers coated with Matrigel. However, no effect on cell proliferation and apoptosis was observed. We concluded that laminin-derived peptide C16 regulates gene expression and enhances invasion of metastatic MDA-MB-231 breast cancer cells.

2186 Laminin-derived peptide C16 induces invasion and invadopodia activity in human oral squamous cell carcinoma and fibrosarcoma cells. A. S. Siqueira1, R. Rafael1, M. P. Pinto1, V. M. Freitas1, R. G. Jaeger1; 1Cell and Developmental Biology, Institute of Biomedical Sciences - University of Sao Paulo, Sao Paulo, Brazil

Our Laboratory has been studying the effects of laminin-derived peptides in tumor biology. Here, we addressed whether peptide C16 (KAFDITYVRLKF, gamma 1 chain) would stimulate invasion activity in cell lines derived from oral squamous cell carcinoma (OSCC) and fibrosarcoma (HT1080), two malignant tumors with different origins and poor prognosis. Using Boyden chambers coated by Matrigel, we observed that C16 increased invasion rate in both cell lines compared to scrambled control peptide (C16SX). Tumor cells that actively invade surrounding tissues depend on invadopodia to degrade extracellular matrix barriers. Invadopodia are actin-rich membrane protrusions associated with proteolytic activity. Therefore, we analyzed the role of C16 on invadopodia activity of OSCC and HT1080 cells, through a fluorescent substrate degradation assay. Measurement of digestion spots (black areas in fluorescent background) showed that C16 significantly increased invadopodia activity of both neoplastic cell lines compared to C16SX. Invadopodia dynamics in C16-treated cells was futher explored using time-lapse 4D fluorescence microscopy. Time-lapse videos of living cells transfected with GFP-cortactin and cultured on fluorescent gelatin were acquired at 5 min interval (total 1-2 hours). Invadopodia extensions through the fluorescent substrate were evaluated with acquisition of at least ten Z sections per time point, using a piezoelectric device coupled to the microscope objective. Measurements of degraded areas showed that C16 increased invadopodia activity over time compared to C16SX. We also searched for signaling pathways related to C16 effects in OSCC and HT1080 cells. This peptide increased p-ERK expression in both cell lines compared to control.Furthermore, inhibition of ERK signaling pathway with MEK inhibitor U0126 decreased C16-related invasion and invadopodia activity. These results demonstrated that C16 effects in these cell lines may be related to ERK 1/2 pathway. We conclude that C16 increases invasion and invadopodia activity in cells derived from oral squamous cell carcinoma and fibrosarcoma, probably through ERK signaling pathway. Support: FAPESP (08/57103-8; 09/17336-6) and CNPq (304986/2009-7).

2187 ADAMTS-1 regulating migration, invasion, and invadopodia formation in breast cancer cells. V. M. Freitas1, J. B. Amaral1, E. S. Santos1, R. G. Jaeger1, F. R. Mangone2, M. A. Nagai2, G. M. Machado-Santelli1; 1Department of Cell and Developmental Biology, Institute of Biomedical Sciences of The University of Sao Paulo, Sao Paulo, Brazil, 2Department of Radiology, School of Medicine of The University of Sao Paulo, Sao Paulo, Brazil

ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs) is a member of the ADAMTS family of metalloproteases. This enzyme is related to pathological processes such as inflammation and cancer. In spite of ADAMTS-1 biological relevance, the mechanisms underlying its involvement in tumor biology remain elusive. Here we sought to investigate the MONDAY role played by ADAMTS-1 regulating migration and invasion of malignant mammary cell lines (MDA-MB-231 and MCF7). We observed variable levels of ADAMTS1 mRNA expression in a series of primary breast tumors. MDA-MB-231 cells with reduced expression of ADAMTS-1 exhibited increase in migration and invasion. Cancer cells depend on invadopodia to degrade and invade surrounding extracellular matrix. Invadopodia are membrane protrusions with enzymes required for extracellular matrix components (ECM) degradation. Silencing of ADAMTS-1 by siRNA increased invadopodia activity in MDA-MB-231 cells, as shown by fluorescent degradation substrate assays. Cortactin and MT1-MMP are important invadopodia proteins. Immunoblot of cells transfected with ADAMTS-1 siRNA showed augmentation of cortactin and MT1-MMP expression compared to controls. Regulatory mechanisms underlying ADAMTS-1 effects may be related to VEGF, a growth factor involved in migration and invasion. MDA-MB-231 cells with silenced ADAMTS-1 showed increase of VEGF concentration in the conditioned medium compared to control. This conditioned medium also induced HUVEC tubulogenesis. Furthermore, MBA-MB-231 showed high expression of VEGF receptor (VEGFR2) compared to MCF7 cells. We may assume that ADAMTS-1 effects in tumor invasiveness may be related to VEGF and VEGFR expression. We suggest that decrease of ADAMTS-1 stimulates migration, invasion and invadopodia formation in breast cancer cells. These effects are probably regulated by VEGF and VEGFR.

2188 Analysis of endothelium mimicry by the CSC-like cells derived from iPS cells. S-I. Matsuda1, A. Mizutani1, T. Kasai1, A. Satoh1, T. Kudoh1, L. Chen1,2, M. Seno1; 1Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan, 2Tianjin Central Hospital of Bynecology Obstetrics, China

Cancer stem cells (CSCs) are capable of self-renewal and differentiation, which has been considered to be responsible for tumor initiation and to contribute to cancer resistance. In addition, CSC-like cells in glioblastoma have also been demonstarted to mimic or differentiate into vascular endothelium. Eradication of these CSCs is considered critical part of successful anti-cancer treatment, but detailed analysis of CSCs is still slow because of their rarity and difficulty of maintenance in culture. Recently, we have established stable CSC-like cells from mouse induced pluripotent stem (miPS) cells (Mizutani et al., 2011 ASCB meeting), which were cultured in the conditioned medium of several kinds of mouse cancer cell lines P19, Lewis lung carcinoma (LLC), B16 and MC.E12. In particular, miPS cells converted to CSC-like cells in the conditioned medium of Lewis lung carcinoma (miPS-LLCcm) cells exhibited extensive angiogenesis in vivo. In this study, we evaluated these miPS-LLCcm cells for the potency to mimic vascular endothelium. First we inquire about their stemness by assessing the expression of stemness marker genes, Oct3/4, Sox2, Klf4 and Nanog. Compared with miPS, these markers except Oct3/4 tend to be up-regulated in the obtained CSC-like cells implying some difference from normal stem cells. Simultaneously, the expression of vascular endothelial markers, VE-cadherin and VEGFR2, were also found upregulated in miPS-LLCcm and miPS-B16cm cells. However, only miPS- LLCcm cells exhibited tube formation on MatrigelTM, which is consistent with the results of tumorigenicity in vivo. Since this in vitro tube formation was observed in the absence of VEGF, VEGFR2 might not directly be involved in the angiogenesis. When miPS-LLCcm cells were cultured in the presence of puromycin, the tube formation was not observed. This impotency was recovered when puromycin was removed from the culture media. Taking these into consideration miPS-LLCcm cells should have potential of differentiation, which is responsible for vasculogenic mimicry in vivo, maintaining the heterogeneous population in the culture. This speculation was supported by the flow cytometoric analysis for GFP expression in miPS-LLCcm cells showing approximately 30 % was GFP positive. Thus, miPS-LLCcm cells in this study MONDAY have successfully been shown to have CSC-like characters, such as cancer stemness, malignant behavior and capacity to mimic vascular endothelium. Our CSC-like cells might be a good model to explain the mechanism of tumor angiogensis in detail not limiting in glioblastoma.

2189 The HT29 and DLD1 colorectal cancer cell lines contain cancer stem cells. C. B. Penny1, B. L. Milner1, V. E. Gibbon1, P. Ruff1; 1Oncology, University of Witwatersrand Med Sch, Johannesburg, South Africa

Cancer stem cells (CSCs) similar to embryonic stem cells are defined by their abilities to self- renew and additionally retain the ability to form tumours ectopically. They have been identified in several human malignancies, recently being associated with functional properties of drug resistance, invasion and migration. This resistance to conventional chemotherapies may lead to tumour relapse and the arisal of new tumours. Using confocal microscopy, we have identified putative colon cancer stem cells (CSC) in the HT29 and DLD-1 colon cancer derived cell lines. These lines representative of stage 3 colorectal adenocarcinoma, co-express the cell surface markers, CD133 (prominin 1) and epithelial cell adhesion marker (EpCam). Upon FACS analysis of these cell lines, using CD133 (PE-Miltenyi Biotech) and EpCam antibodies (FITC-BD Biosciences), two distinct cell populations were gated; within the HT29 cell line, 64.63% of gated events were positive for EpCam and 35.30% expressed both EpCam and CD133; and in the DLD1 cell line, 46.77% of gated events were positive for EpCam expression and 52.83% for both markers. Subsequently, magnetic cell separation was used to isolate pure populations of these cells from each cell line, with a CD133 antibody labeled with superparamagnetic particles (Miltenyi Biotech). The purified CD133+ cells were cultured in StemPro human embryonic stem cell medium (serum free) (GIBCO), to minimize the possibility of differentiation. The cells were grown both as adherent cultures and as three dimensional spheroids, these being more representative of in vivo tumours. To further investigate the “stemness” of these cells, the expression of the pluripotency markers Nanog, Sox 2 and Oct3/4 were assessed by confocal microscopy. All of these transcription factors essential to stem cell identity are present in the CD133+ cells. In summary, the CSCs isolated here, represent a novel population of cells for evaluating cancer therapies directed against pluripotent cells.

2190 Floating spherical cell colonies from human adenocarcinoma of the lung are enriched in stem-like phenotypic markers. A. Tesei1, C. Arienti1, G. Paganelli1, A. Pasini2, G. Brigliadori1, D. Calistri1, E. Giordano2, W. Zoli1; 1Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (I.R.S.T.), Meldola (FC), Italy, 2Laboratory of Cellular and Molecular Engineering, University of Bologna - Campus of Cesena, Cesena (FC), Italy

It is presently widely accepted that tumor growth rely on a niche of so-called cancer stem cells (CSCc), or tumor-initiating cells (TICs), endowed with self-renewal properties. We recently showed that a subset of normal lung epithelial-specific stem cells might reside within the bronchoalveolar duct junction (Tesei A., et al. Cell Proliferation 2009; 42: 298-398). The aim of the present work was to isolate and characterize CSCc/TICs from a human adenocarcinoma cell line (RAL) that we originally established at the Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (I.R.S.T.) at Meldola (Gasperi-Campani A., et al., Cancer Genetics and Cytogenetics 1998; 107:11–20) and from surgical human tissue samples. Single-cell suspensions derived from the RAL cell line were cultured in ultralow attachment plates in a serum-free medium. Neoplastic lung tissue samples, derived from patients affected by adenocarcinoma at different stages, were mechanically and enzymatically dissociated before MONDAY expansion in culture. Expression of stemness related genes in cell clusters was evaluated with RT-PCR and Real Time RT-PCR. Single-cell suspensions gave rise to floating spherical colonies within about ten days. Lung-spheres obtained by our originally established lung adenocarcinoma cell line RAL, resulted enriched in CD133 and OCT-3/4 transcripts. RT-PCR analysis of tumor spheres obtained from surgical tissue samples showed a higher expression of BCRP-1, CD133, BMI-1, OCT-3/4, Lef-1, CD44 and Slug, when compared to original tissue. A gradual loss of CD133 expression was evident as the tumors become more undifferentiated. Evaluation of the level of CpG methylation in OCT-3/4 and CD133 gene promoters was also performed by means of bisulfite sequencing in cells growing in floating spherical RAL cell colonies and in the original adherent cell culture. They both showed a constant hypermethylated status suggesting that this level of transcriptional control is not involved in modulating the expression of these stem-like phenotypic markers.

2191 Generation of Cancer Stem Cell Model from Mouse iPS Cells. A. Mizutani1, S-I. Matsuda1, T. Kasai1, T. Kudoh1, L. Chen2, M. Seno1; 1Okayama University, Okayama, Japan, 2Tianjin Central Hospital of Bynecology Obstetrics, China

Objective; Heterotopic transplantation of embryonic stem cells and induced pluripotent stem (iPS) cells have been shown to form teratoma, a benign tumor exhibiting heterogeneous but normal phenotype of differentiated cells, but not teratocarcinoma. Since the stem cells niche has been thought to play a significant role for the maintenance and the differentiation of stem cells, we hypothesized that cancer niche could drive stem cells into malignant cells, particularly into cancer stem cells (CSCs). The presence of CSCs in tumor has been considered as a reason for a poor prognosis. Until now, no model of cancer stem cell line is established as an effective tool to study CSC. To investigate CSC in detail, we tried to generate cancer cells from mouse iPS (miPS) cells, in which green fluorescent protein (GFP) has been inserted into the 5’ untranslated region of the Nanog gene. Methods and Results;A ccording to our hypothesis, miPS cells were cultured in the conditioned medium of various cancer derived cells (P19, LLC, B16, and MC.E12) for 4 weeks. The survived population of the cells was evaluated for tumorigenicity in nude mice. The miPS cells cultured in the conditioned medium from Lewis lung carcinoma cells (miPS-LLCcm) developed tumor with rapid growth and exhibited remarkable angiogenesis. Histology of the tumor derived from miPS- LLCcm showed the malignancy, while that of teratoma derived from miPS cells showed no malignancy. Morphologically stem-like cells expressing GFP was observed in the primary culture of the malignant tumor derived from miPS-LLCcm cells. These cells were found to grow as spheroid with GFP expression in suspension culture. The spheroid cells were also found to be highly tumorigenic in nude mice. The expression of the stem cell marker genes, such as Nanog, Cripto, Oct3/4, Sox2, Klf4, were detectable in miPS-LLCcm cells as much as in miPS cells. Conclusion; In this study, we cultured miPS cells in the conditioned mediun of cancer derived cells, which was regarded as cancer niche converting the normal stem cell into CSC cells. The resultant cells formed spheroids in suspension culture and developed malignant tumor in vivo exhibiting extensive angiogenesis. In addition, these cells showed a capacity of self-renewal and expression of the markers associated with stem cell properties of undifferentiated state. Thus we concluded these cells as a model of CSC. This model should critically be helpful for the CSC study and for the development of effective cancer therapies.

MONDAY

Cell Biology of Microbes and Parasites

2192 Molecular mechanism underlying MinE-induced membrane deformation. Y-L. Shih1,2; 1Institute of Biological Chemistry, Academa Sinica, Taipei, Taiwan, 2Institute of Biochemical Sciences, National Taiwan University, Taiwan

The Min system of E. coli mediates placement of the cell division septum at midcell. We recently reported that MinE can directly associate with the cell membrane through its N-terminal domain. An interesting observation is the ability of MinE to induce membrane tubule formation in vitro, a property similar to several eukaryotic proteins involved in membrane trafficking, such as dynamin and Bar-domain proteins. To investigate the mechanism underlying the MinE-induced membrane deformation, we used time-lapse fluorescence microscopy to characterize the membrane deformation in vitro. We recorded massive tubulation from giant liposomes and initiation of membrane tubule formation and lipid clustering on the supported lipid bilayers. Furthermore, we found that residues 2-9 are capable of forming an amphipathic helix of 1-2 helical turns when interact with the membrane.We used the tryptophan blue shift assays to determine the helical face that interacts with the membranes. CD spectral analyses suggested that conformational changes in MinE1-31 occurred in the presence of liposomes. We conclude that the alpha helix of MinE2-9 appears to work in concert with the charge residues R10/K11/K12 to mediate the membrane association of MinE. It is known that insertion of an amphipathic helix into the membranes can induce local curvature changes that are able to destabilize the cell membranes and initiate membrane tubule formation. In conclusion, MinE not only shares common structural motifs to proteins involved in membrane trafficking, but is also active in inducing membrane deformation. We therefore uncovered the mechanism of how MinE may sculpt the membrane curvature.

2193 Biochemical and functional characterization of GlpQ homolog of Xenorhabdus nematophila. P. Singh1,2, R. Bhatnagar1, N. Banerjee2; 1School of Biotechnology, Jawaharlal Nehru University, Delhi, India, 2Insect Resistance Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India

Xenorhabdus nematophila is a gram negative bacteria living in symbiotic association with a soil nematode of the genus Steinernema and as a pathogen stage in insect hosts. The bacteria produces different exoenzymes which help in degradation of macromolecules to provide nutrients necessary for development of both the bacteria and the nematode. In the present study, we have characterized an extracellular enzyme glycerophosphodiester phosphodiesterase (GDPD) encoded by glpQ gene of X. nematophila. The DNA sequence encoding the enzyme was cloned, expressed and purified. We used the recombinant protein for biochemical characterization. The enzyme was able to hydrolyse glycerophosphodiesters like glycerophosphorylcholine, glycerophosphoethanolamine and to a lesser extent serine and inositol esters of phospholipids. The enzyme is thermostable at 50°C for upto 4 hours, retaining 85% of initial activity. It has an optimum pH of 9.5. The enzyme activity was stimulated by Ca2+ions. Activity profile of GDPD in different growth phases of X. nematophila showed maximum activity in mid log phase. The enzyme was largely localised in periplasm and extracellular medium while negligible activity was detected in cytosol. It is also displayed on the cell surface of the bacteria, as treatment with pronase abolished all the enzyme activity from the outer membrane preparation. Based on the crystal structure of GlpQ of E. coli a homology model of Xenorhabdus GlpQ was created. The residues Glu-59, Asp-61 and His-74 located in MONDAY the active site of the enzyme were changed to alanine using site directed mutagenesis. Enzyme activity was abolished in all the three protein variants validating the crucial role of these residues in the active site of the enzyme. Since the bacteria is exposed to different unfavourable environments in the hosts so we determined glpQ profile in nutritional stress conditions. Transcriptional analysis of glpQ specific mRNA has shown upregulation of the gene in response to phosphate starvation. Similarly enzyme activity was increased by 1.6 folds when the cells were starved for phosphate upto 7 hours. To further analyse it with the growth phase of bacteria it was shown that upregulation was mainly associated with the early log phase of the bacteria. Stationary phase cells did not respond to phosphate starvation. Bioinformatic analysis of the upstream region of glpQ showed presence of two promoters. This was confirmed by primer extension analysis which clearly indicated promoters P1 and P2 about ~80 and ~565 nucleotides upstream of the start codon of the gene. To explore the function of both the promoters in phosphate regulation, lacZ fusion constructs were prepared and promoter activity was determined by β-galactosidase assay which indicated that promoter located 80 bp upstream of the start codon has a role in phosphate regulation of glpQ gene.

2194 The role of a bacterial SMC in chromosome segregation. M. Schwartz1, L. Shapiro1; 1Developmental Biology, Stanford University, Stanford, CA

The bacterial genome must be completely replicated and segregated prior to the completion of cell division in order to ensure that each daughter cell receives one complement of the genome. The Structural Maintenance of Chromosomes (SMC) protein is conserved from bacteria to humans and is thought to play important roles in chromosome organization and segregation. We have identified a point mutation (SMC-E1076Q) in the ATPase domain of Caulobacter SMC that disrupts chromosome segregation and cell division. This mutation caused a dominant- negative phenotype in which DNA replication was able to proceed, but segregation was inhibited, resulting in an accumulation of parS centromeres at one pole. Surprisingly, DNA was still deployed throughout the cell and the cellular positions of other chromosomal loci were in the wild-type order relative to the parS centromere. Loss of ATP hydrolysis, either by this point mutation or by the utilization of non-hydrolyzable ATP caused abnormally stable binding of SMC to DNA in vitro. We propose that SMC spuriously links the duplicated chromosomes immediately after passage of the replication fork when the two chromosomes are in very close proximity. In wild type cells, ATP hydrolysis opens the SMC dimer, freeing one chromosome to segregate to the opposite pole. SMC is more likely to trap DNA from a single chromosome once the replicated chromosomes are physically separated, thereby condensing the chromosome. In this model, the loss of ATP hydrolysis causes the SMC-E1076Q dimer to remain bound to both chromosomes, inhibiting segregation. The linking of the two chromosomes can be reinforced by additional mutant dimers binding elsewhere on the neighboring chromosomes.

2195 Binding of recombinant soluble Influenza A hemagglutinins to cell surface sialic acids of cultured cells and tissue sections. A-K. Sauer1, M. Bohm1, C. Schwegmann-Weßels1, G. Herrler1; 1Institute of Virology, University of Veterinary Medicine Hannover, Hannover, Germany

The major viral envelope glycoprotein of influenza A viruses is the hemagglutinin (HA) which is responsible for host cell recognition and virus entry. It is synthesized as an uncleaved precursor in the host cell secretory pathway where it is posttranslationally modified and forms trimers. The receptor determinants for influenza A viruses are sialic acids of glycolipds and/or glycoproteins. Avian influenza strains preferentially bind to sialic acids linked to galactose in α2,3-conformation MONDAY whereas human strains prefer α2,6-linked sialic acids. To distinguish between both forms, the plant lectins MAA and SNA, respectively, are used in many studies. Due to the huge diversity of oligosaccharide structures and the different hemagglutinin subtypes, those two plant lectins are not sufficient to characterize the binding properties of influenza hemagglutinins. To get a closer insight into the cellular interaction partners of influenza A viruses we generated soluble hemagglutinins that can be used as lectins for detection of those sialoglycoconjugates that are recognized by influenza viruses. Connection of the ectodomain to the Fc-component of a human IgG and to a modified GCN4 leucin zipper motif as trimerization domain resulted in chimeric proteins that are secreted into cell culture supernatant after transfection and were purified by FPLC. The binding of the soluble HAs can be demonstrated by immunofluorescence- based assays.. These recombinant soluble HAs of the avian subtypes H7 and H9 were found to bind to the surface of different cell lines, trachea sections of chicken and turkey and sections of the porcine lung. On all permanent mammalian cell lines tested, the H9 shows stronger binding compared to the H7 protein. On trachea sections of chicken and turkey a 2fold higher amount of H9 is needed to establish an equal binding pattern compared to that of H7 despite the presence of both sialic acid linkage types as indicated by the lectin stain. Porcine lung sections show more SNA staining on the surface whereas MAAII stains cells in basal layers of the epithelium. Only H9 binds to porcine lung sections but not as widely distributed over the surface as the SNA stain. These experiments show that a lectin staining alone cannot sufficiently depict the distribution of the cellular interaction partners of influenza A viruses. Soluble hemagglutinins promise to be a valuable tool to visualize potential influenza binding sites on cells and tissues.

2196 Adaptation of avian influenza viruses of the subtype H9N2 to avian and human respiratory epithelial cells. M. Erdt1, M. Bohm1, H. Petersen2, C. Winter1,2, S. Rautenschlein2, C. Schwegmann-Weßels1, G. Herrler1; 1Institute of Virology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany, 2Clinic for Poultry, University of Veterinary Medicine Hannover, Foundation

Influenza viruses initiate infection by binding of the haemagglutinin (HA) to sialic acid residues presented by cell surface components. Viruses isolated from different host species may differ in their preference for the type of sialic acid, e.g. N-acetylneuraminic acid (Neu5Ac), or for a linkage type connecting the sialic acid molecule to the neighboring sugar. Influenza viruses from mammalian hosts prefer α-2,6-linked sialic acids whereas most avian influenza viruses prefentially recognize Neu5Ac attached via an α-2,3-linkage to galactose. Avian viruses of the H9 subtype are an exception, because several strains of H9 influenza viruses have been shown to favor α-2,6-linked sialic acids. This finding was surprising because in some avian species α- 2,6-linked sialic acids are expressed in very low amounts, at least in the respiratory epithelium, a primary target for influenza virus infection We analyzed the adaptation of an egg-grown avian influenza virus of the subtype H9N2 (strain A/chicken/SaudiArabia/CP7/1998) to the respiratory epithelium of chicken and turkeys. Tracheal organ cultures (TOC) served as a culture system which preserves the setting of epithelial cells as present in the avian trachea. The parental virus was passaged four times in either chicken or turkey TOCs. The progress of infection was monitored by determining the viral titer induced by this virus at various time points post infection. In the course of the passages, the virus underwent an adaptation process indicated by an increase in the viral titer. Infection of chicken TOCs by the chicken TOC- adapted virus resulted in a 3.5-fold increase in the viral titer compared to the egg-grown virus. MONDAY

By contrast, infection of turkey TOCs with the turkey TOC-adapted virus was characterized by a nearly 10-fold increase in viral titer. Furthermore, Calu-3 cells, a permanent human cell line with features of the bronchiolar epithelium when grown under air-liquid interface conditions (ALI), was infected with these avian viruses. The influence of several passages in these cells was monitored and analysis of cell culture supernatants showed that avian H9N2 viruses in Calu-3 cells reached higher titers than in the TOC system. In the future these experiments should be extended to primary human respiratory epithelial cells.

2197 Peritoneal Macrophages Employ Predominantly αxβ2 But Not αMβ2 For Candida albicans Recognition. S. Jawhara1, D. Soloviev1; 1Molecular Cardiology, Cleveland Clinic Lerner Research Institute, Cleveland, OH

Candida albicans is an opportunistic yeast pathogen that represents the fourth most frequent cause of nosocomial bloodstream infections in the United States. Recently, we demonstrated that the integrin αMβ2 is the principal leukocyte receptor for the filamentous form of C. albicans. In the present study, we explored the role of another β2 integrin, αxβ2 (CD11c/CD18, p150,95), in the model of antifungal defenses. Although αxβ2 is highly homologous to the αMβ2, its role remains unclear in the immune response to fungal infections. Employing αx-knock out (KO) mice in the murine model of systemic candidiasis, we first demonstrated that mice deficient in αxβ2 had significantly increased susceptibility to the invasive fungal infection. We observed that C. albicans induced mortality in αx-KO mice more rapidly (2- to 3-fold faster) than when the same inoculum was used in wild-type mice. Notably, the fungal burden was significantly higher in both the brain and kidneys of αx-KO than wild-type mice. Subsequently, the histopathology examination of both kidneys and brain from C. albicans-infected αx-KO mice showed a higher presence of filamentous growth and tissue damage than in wild-type mice. Elimination of αxβ2 significantly impedes peritoneal macrophages recruitment to the place of infection and their capacity for anti-fungal activity, while having virtually no effect on the neutrophil`s function. In contrast, αM-elimination mostly affected the antifungal activity of neutrophils but not that of peritoneal macrophages. Additionally, αxβ2 elimination increased the expression of interleukin- 6, but not the tumor necrosis factor alpha (TNFα). Taken together, these data indicate that αxβ2 is essential for C. albicans elimination by macrophages and plays a role in innate antifungal immunity.

2198 The role of TrpM7 in Salmonella pathogenesis. A. M. Young1, J. E. McCombs1, S. Van Engelenburg1, W. W. Blakeslee1, A. E. Palmer1; 1Chemistry and Biochemistry, University of Colorado Boulder, Boulder, CO

Salmonella subspecies are enteropathogenic gram-negative bacteria responsible for food-borne illness in humans ranging from gastroenteritis to typhoid fever. The infection of a range of host cells by Salmonella is driven by the injection of bacterial effector proteins through a needle-like type III secretion system (T3SS). Effector proteins hijack host cell machinery and intracellular signaling pathways. These interactions promote invasion of the cell through cytoskeleton rearrangement and the engulfment of the bacteria into a Salmonella containing vacuole (SCV). Salmonella invasion is known to induce an elevation in host cell Ca2+, however the molecular mechanism of this event has remained undefined. Fluctuations in Ca2+ activate a diverse array of downstream signaling processes including gene transcription, actin polymerization, and endocytosis. Given these roles, it is likely that Salmonella manipulates and utilizes host cell Ca2+ MONDAY signaling during invasion. In this work, we investigate the origin and nature of the Ca2+ elevation by examining Salmonella invasion of non-phagocytic cells at the single cell level. Localized Ca2+ elevation was observed to result from influx through a transient receptor potential cation channel (TRPM7) at sites of invasion. This study identifies a novel mechanism by which TRPM7 is activated by a Salmonella effector, SopB, through its ability to modulate phosphoinositides, indicating that TRPM7 can be gated by rare signaling phospholipids.

2199 Defining the roles of inclusion membrane microdomain proteins in chlamydial pathogenesis. J. Mital1, E. Lutter1, T. Hackstadt1; 1Rocky Mountain Labs/NIH, Hamilton, MT

Chlamydia trachomatis is the leading cause of sexually transmitted disease in the Western world and of infectious blindness in the world. Chlamydiae are obligate intracellular bacteria that reside within a membrane bound vacuole termed the inclusion, which does not fuse with the endosomal pathway but selectively interacts with vesicular pathways to acquire lipids and nutrients from the host cell. The inclusion is extensively modified by insertion of chlamydial proteins (Incs), which are ideally positioned to mediate host-pathogen interactions. There is little functional information regarding Inc proteins. Once internalized, the nascent inclusion repositions itself at the centrosome in a process that is dependent upon chlamydial protein synthesis, microtubules and dynein. Inclusion repositioning is independent of the cargo linker p50 dynamitin, indicating that a bacterial effector may directly link the inclusion to dynein. We previously described a subset of Incs (B, 101, 222, 850) that colocalize with host Src-family kinases in microdomains in the inclusion membrane. These microdomains are closely apposed to host centrosomes, indicating a probable role in microtubule interaction. Knockout and inhibitor studies show that Src family kinases are involved in the microtubule dependent processes of inclusion repositioning, sphingomyelin trafficking and are essential for normal chlamydial development. Here we report studies of the functional roles of the microdomain Incs in chlamydial pathogenesis. When ectopically expressed in HeLa cells, Inc850 consistently colocalizes with host centrosomes. Inc850 contains a dynein light chain binding domain and has been shown by yeast two-hybrid assays to interact with dynein light chain subunits. This suggests that inclusion microdomains comprised of chlamydial and host proteins are directly involved in microtubule dependent trafficking of the nascent chlamydial inclusion. The full role of these microdomains in pathogenic processes remains to be elucidated.

2200 The PDZ1 domain of MAGI-1 rescues CAREx8 from MAGI-1 mediated degradation and augments adenoviral infection. A. O. Kolawole1, R. Yan1, P. Sharma1, K. J. Excoffon1; 1Biological Sciences, Wright State University, Dayton, OH

A major factor in virus entry into cells is localization and abundance of the primary receptor. The Coxsackievirus and adenovirus receptor (CAR) is the primary receptor for group B coxsackievirus and many serotypes of adenovirus. In most epithelia, a seven exon isoform of CAR (CAREx7) is exclusively localized at the basolateral surface where it behaves as a homophilic adhesion protein and is inaccessible for viral infection. However, in well- differentiated human airway epithelia, we recently discovered an alternatively spliced, low abundance eight exon isoform of CAR (CAREx8) that is localized apically, where it may initiate apical viral infection. The two isoforms differ only in the last 26 (CAREx7) or 13 (CAREx8) amino acids of the cytoplasmic domain, which suggests that some intracellular interactions may differ. One such differential interaction involves MAGI-1, an essential PDZ domain containing protein MONDAY with 6 distinct PDZ domains (PDZ0-5), known to be involved in cell polarization and cancer. The molecular basis of the CAR-MAGI-1 interaction was investigated by adenovirus infection, immunocytochemistry, MAGI-1 siRNA knockdown, yeast two hybrid assay, co- immunoprecipitation, in vitro translation, and fluorescence resonance energy transfer. In non- polarized cells, MAGI-1 caused a loss of CAREx8 at the protein level and reduced adenovirus infection. In contrast, MAGI-1 siRNA knockdown increased adenovirus infection, suggesting that endogenous MAGI-1 regulates CAREx8 levels. The key PDZ-based interactions between CAR and MAGI-1 were then identified and confirmed. Both CAR isoforms strongly interacted with PDZ3. CAREx8 also interacted with PDZ1. Co-expression of CAREx8 with isolated MAGI-1 PDZ3 significantly reduced CAREx8 cell surface expression and adenovirus infection but did not reduce total CAREx8 protein levels. These data suggest that MAGI-1 PDZ3 is responsible for retaining CAREx8 within the cell but degradation of CAREx8 likely requires MAGI-1 interacting proteins/domains.Co-expression of the isolated PDZ1 domain of MAGI-1 with CAREx8 did not affect adenovirus infection. However, PDZ1 was able to rescue CAREx8 from MAGI-1-mediated degradation, thereby supporting adenovirus infection via CAREx8 in the presence of MAGI-1. These data suggest that molecules directed at facilitating or inhibiting the interaction between PDZ1 and CAREx8 may be able to regulate the levels of CAREx8 and hence increase or decrease epithelial susceptibility to adenoviral infection.

2201 The VP1 Subunit of JC Polyomavirus Drives Trafficking to the ER for Productive Viral Infection. A. Derdowski1, C. Nelson1, M. Maginnis1, B. O'Hara1, W. Atwood1; 1Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI

There are at least nine different human polyomaviruses. Two of these, JC polyomavirus (JCV) and BK polyomavirus (BKV), are long-established as human pathogens in immunocompromised hosts causing progressive multifocal leukoencephalopathy (PML) and nephropathy, respectively. The remaining human polyomaviruses have all been discovered within the last decade, establishing this class of viruses as emerging opportunistic pathogens. Polyomaviruses are coated with 72 pentamers of the viral attachment protein VP1. VP1 is responsible for attachment to the host cell surface and implicated in trafficking to the ER and nucleus for productive infection. The VP1 subunit of JCV was bacterially expressed and purified by FPLC. VP1 pentamers were then directly labeled and viral entry and trafficking were analyzed by confocal microscopy in real-time. We demonstrate for the first time that the JCV VP1 pentameric subunit drives viral trafficking through Rab 5 and 7-positive vesicles to the ER for productive infection, and interference of this trafficking with pharmacological inhibitors reduces infection. This work not only highlights the importance the viral VP1 pentamer in polyomavirus trafficking, but also establishes a viral trafficking model for emerging polyomaviruses that lack cell culture systems.

2202 Inhibition of Rab5 activation by Exotoxin S of Pseudomonas aeruginosa. S. Mustafi1, N. Rivero1, M. Barbieri1; 1Florida International University, Miami, FL

Pseudomonas aeruginosa is a human pathogen that frequently causes severe hospital-acquired infections. Exotoxins secreted from Type III secretion system of Pseudomonas aeruginosa are critical molecules to disrupt mammalian epithelial cell membrane trafficking and signaling. One of them also known as Exotoxin S, is a bi functional protein which shows ADP ribosyltransferase activity and targets several intracellular proteins, including Ras and Rab small GTPases. Here, we demonstrate that Rab5, but not Rab4 and Rab7, play a critical role during early steps of the MONDAY invasion of Pseudomonas aeruginosa. We show that invasion of live Pseudomonas aeruginosa, but not heat inactivated Pseudomonas aeruginosa, down-regulates of Rab5 activity in macrophages. In addition, invasion of live Pseudomonas aeruginosa lacking Exotoxin S was unable to inactivate Rab5. Furthermore, we also found that overexpression of Rin1 partially reverses the inactivation of Rab5 during invasion of live Pseudomonas aeruginosa. These observations are consistent with a model, in which the presence of Exotoxin S may interfere with the Rab5 cycle activity in macrophage

2203 The ATPase activity of EccC is required for ESX-1 secretion in Mycobacterium tuberculosis. D. Dovala1, O. Rosenberg1, J. Cox1; 1University of California, San Francisco, San Francisco, CA

The human pathogen Mycobacterium tuberculosis (Mtb) utilizes a specialized type VII secretion system, ESX-1, to circumvent host defenses during macrophage infection. ESX-1 mutants are severely attenuated, highlighting the importance of this secretion system. While many substrates and components of ESX-1 have been identified, the mechanistic details of how these substrates are pumped into the host cell remain elusive. The Mtb proteins EccCa and EccCb contain several putative FtsK-like ATPase domains and form a complex which is required for ESX-1 secretion. FtsK domains in other proteins are known to exert force on substrates, and thus may serve as the pump in the ESX-1 translocon. We show that the EccC complex forms an ATPase with three functional ATPase domains. We show that all ATPase domains are required for successful secretion of ESX-1 substrates from Mtb, and genetic approaches have identified the roles of each domain in complex formation and substrate binding.

2204 Host Innate Receptor Regulation is Mediated by Influenza Virus Proteins. D. J. Vigerust1,2, V. L. Shepherd1,2; 1VanderbIlt University SOM, Nashville, TN, 2Department of Veteran Affairs, Nashville, TN

Influenza virus A (IVA) infection is a significant cause of morbidity and mortality each year. Interactions between the influenza virus and cells of the innate immune system – specifically macrophages – may exacerbate acute symptomology and immune evasion and disease outcome, but the interaction between influenza and the macrophage remains poorly understood. Host-pathogen dynamics may prevent the proper function of the macrophage in the clearance of harmful pathogens. Our current hypothesis is that host innate defense molecules such as the cell surface mannose receptor (MR) may be utilized as a receptor of entry for viruses such as influenza, and that molecules such as MR and MHC-I are targeted for down-regulation by influenza A viruses leading to dysregulation of macrophage function. In previous work from our laboratory the HIV-1 Nef protein was shown to interact with the MR tail to down-regulate the surface receptor expression. The interaction between the MR and Nef was localized to two specific regions of Nef. Sequence comparison of the known interacting domains of the HIV-1 Nef protein and the sequence of influenza A NS1 and PB1 revealed similarity in several motifs that indicate a potential for MR regulation by influenza. Our initial data suggest that infection of MR-expressing cells results in a significant decrease in surface expression of MR by 48 hours post-infection. Further, in vitro transient transfection of macrophages with influenza proteins leads to removal of MR from the surface at levels approaching 30% for NS1 and PB1. Removal of MHC-I from the cell surface was also seen as early as 5 hours post-transfection. These data support the hypothesis that influenza proteins regulate two important host innate defense molecules in a fashion that is similar to that seen with HIV-1 Nef.

MONDAY

New Technologies and Frontiers

2205 High-throughput cellular assays using a well-less plate format. G. Quinones1, K. Nicholes1, M. Lye2, N. Kim2, J-P. Stephan1; 1Genentech, South San Francisco, CA, 2Curiox Biosystems, Singapore

Cellular assays represent a great opportunity for researchers to test various molecules in a more biologically relevant context than biochemical assays. Many of these cell-based assays could be adapted for high-throughput and high content screening assays, providing investigators the opportunity to interrogate large number of samples and conditions. Despite significant technical advances made during the last few years, high-throughput/high content cellular assays still suffer from key limitations in working with a large number and variety of cell lines. A key limitation is the use of suspension cell lines, especially when multi-step staining procedures are required. Here, we demonstrate that the use of a well-less plate system which significantly improves the flexibility of our high content screening platform. The well-less format utilizes surface tension to maintain the cell population on the glass surface of plate in 2.5mm diameter drops, while a gentle buffer exchange allows the cells to remain on the plate surface throughout a variety of experimental procedures. We performed cell viability experiments in the presence of the antimitotic agent monomethyl auristatin E (MMAE) on the suspension cell lines U-937 and K-562 in immunofluorescence and chemiluminescence viability assays, showing that in both assays we achieve an EC50 of 4.4uM and 9.1uM respectively. We also performed immunofluorescence-based, protein-protein interaction assays using a large library of single transmembrane, multi-transmembrane and secreted proteins expressed in COS7 cells. The expressed library was incubated with tagged bait proteins to interrogate novel protein-protein interactions at the cell surface. This resulted in the successful expression and binding of several known ligands to their respective receptors, i.e. PD-1 binding to PD-L1, NGF to NGFR, PVRL2 to PVRIG, and HVEM binding to BTLA. We also examined changes in cell morphology using both established and primary human cell lines infected with fluorescent organelle-specific baculovirus constructs. Live imaging of the infected cells revealed alterations in the trafficking patterns of EEA1-labeled early endosomes in the presence of the dynamin inhibitor Dynasore. We also observed changes in the actin and tubulin cytoskeletal structure in the presence of Cytochalasin D and Blebbistatin. The added flexibility in terms of cell lines and readouts enables the screening platform to truly perform high-throughput and high content assays to examine a wide variety of cellular processes.

2206 Solutions for the preparation of samples for integrated correlative microscopy. M. A. Karreman1,2, A. V. Agronskaia1, E. G. Van Donselaar2, K. E. Vocking2, H. C. Gerritsen1; 1Molecular Biophysics, Utrecht University, Utrecht, Netherlands, 2Biomolecular Imaging, Utrecht University, Utrecht, Netherlands

The Integrated Laser and Electron Microscope is a novel tool for correlative microscopy, combining a fluorescence microscope (FM) and a Transmission Electron Microscope (TEM) within one set-up. Here, a specially designed laser scanning fluorescence microscope is mounted into one of the side ports of a conventional Tecnai 12 TEM. First, the region of interest is identified based on fluorescence labeling of the sample. This region can then effortlessly be relocated in the TEM and subsequently investigated at high resolution. Due to the integrated nature of this set-up, the sample (eg. sections of cells on a grid) needs to be suitable both for FM and TEM. Unfortunately, the heavy metal stain employed to contrast the biological material for TEM has a quenching affect on the fluorophore required for FM imaging. Furthermore, most MONDAY fluorophores are optimized for an aqueous environment and lose a large part of their brightness in a dry environment, which is needed for the TEM.

Here, we demonstrate an optimized sample preparation protocol for iLEM. By creating a distance between the metal stain and the fluorophore, the quenching effect can be prevented. This is achieved by performing a block staining, so the biological material in the section is already contrasted. The fluorescent label on the section is then physically separated from the stain in the section. We show that the intensity and the lifetime of the fluorophore are not affected by the heavy metal stain bound to the biological material in the section. Furthermore, the choice of fluorophores for iLEM imaging is critical. Although in wet conditions Alexa 488 fluoresces brighter than the TRITC dye, in a dry environment the opposite is the case. We have found that the lifetime and intensity of Alexa 488 is severely reduced in a dry environment whereas the TRITC dye is unaffected.

Therefore, we can conclude that the sample preparation can be optimized by physically separating the heavy metal stain from the fluorophore. Also, the choice of fluorophores is of high importance due to their different properties in a wet or dry environment.

2207 Imaging promoter activity with Intracellular MultiAptamer Genetic tag (IMAGEtag). J. Ray1, I. Shin1, M. Zhao2, M. Ilgu1, V. Gupta3, J. Beasley3, L. Peng2, G. Kraus3, M. Nilsen- Hamilton1; 1Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, 2Optical Sciences, The University of Arizona, Tucson, AZ, 3Chemistry, Iowa State University, Ames, IA

A detailed understanding of the processes involved in gene transcription is necessary to answer many fundamental biological questions. Imaging techniques have been developed in the last decade with the capability of exploring the elements of gene expression and monitoring the fates of selected mRNAs. In this study we describe an aptamer-based system to monitor promoter activity in living cells and in real time, which we have named as IMAGEtag (Intracellular MultiAptamer Genetic tag). We have cloned and expressed a string of multi- aptamers downstream of the GAL1 promoter in Saccharomyces cerevisiae and have incubated these cells with fluorescent-labeled aptamer target molecules followed by induction of the promoter. This technology uses FRET (Förster resonance energy transfer) to tell us that when the promoter is active and provides a dynamic transcriptional profile of the cells. In this work we have used three different types of multiaptamers (viz. PDC aptamer, tobramycin aptamer and neomycin aptamer) with their respective ligands, which have been individually labeled with cy3 and cy5 dyes. With this noninvasive technology we are able to measure gene expression in real time of individual living cells. This allows an evaluation of the intercellular variation in gene expression. We have also used this method to measure the rate of transcriptional elongation. The IMAGEtag system has the potential of being used to track the real-time levels of gene expression at the cellular level in a broad range of cell types.

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2208 In Situ Microscopic Visualization and Relative Quantification of Inorganic Polyphosphate Stores by 4',6-Diamidino-2-Phenylindole (DAPI)-stainning. F. M. Gomes1,2, I. Ramos1,2, W. Girard-Dias2, E. Machado1,3, K. Miranda2,3; 1Laboratorio de Entomologia Medica, Instituto de Biofísica Carlos Chagas Filhos, UFRJ, Rio de Janeiro, Brazil, 2Laboratorio de Ultraestrutura Celular Hertha Meyer, Instituto de Biofisica Carlos Chagas Filho, UFRJ, Rio de Janeiro, Brazil, 3Laboratorio de Biotechnologia, Diretoria de Programas, INMETRO, Rio de Janeiro, Brazil

Inorganic polyphosphate (polyP) is a widely distributed biological polymer composed of phosphate residues linked by phosphoanhydride bonds. They play key biological roles such as phosphate and energy reservoir, metal homeostasis, regulation of transcription factors and transcription fidelity, as well as regulation of several enzyme activities [1]. Nevertheless, polyP has remained poorly studied mainly due to the limited methods for polyP quantification and in situ visualization. 4',6-diamidino-2-phenylindole (DAPI) is a commonly used nuclei stain that binds to double stranded DNA, exhibiting a fluorescence emission maximum around 450 nm. Interestingly, it has been shown that polyP is able to shift DAPI emission maximum to a higher wavelength around 525-550 nm displaying a greenish-yellowish color distinct from the blue nuclei-signals [2]. This DAPI-polyP interaction has been shown to be specific and to generate proper quantum yield for microscopic observation. Nevertheless, although a few studies have focused on the spectroscopic properties of analytical grade sodium polyphosphate-DAPI fluorescence, there are no consistent reports concerning methods of polyP visualization using DAPI. In addition, the lack of a general protocol for polyP staining hinders its wider utilization as a polyP-visualization tool. In the present study, we report a systematic evaluation of different protocols of DAPI-staining for the detection of polyP stores in different biological models. Suspensions of subcellular fractions of insect eggs and parasites containing polyP granules showed a clean DAPI-polyP fluorescence after brief incubation with DAPI, without previous treatments. Staining of Poly P stores in intact cells usually required aldehyde fixation and/or detergent permeabilization, as exemplified in DAPI-polyP fluorescence obtained from Eimeria parasites. DAPI-polyP signals were also detected from cryosections of mild-fixed, OCT- embedded midgut of insects. In addition, a DAPI-polyP fluorimetric method was applied to quantify polyP mobilization during early egg development of the insect Rhodnius prolixus. The method was validated by the parallel measurement using a more widely accepted polyP quantification protocol that uses a recombinant yeast exopolyphosphatase. Taken together, our results strongly support the broader use of DAPI as a valuable tool for both polyP visualization and quantification in different biological models. Nevertheless, one should take into account that sample preparation may vary according to the model used and that care should be taken when localizing and quantifying polyP from different biological models.

[1] Rao, N.N., M.R. Gómez-García, and A. Kornberg, Inorganic polyphosphate: essential for growth and survival. Annual review of biochemistry, 78, 605-647 (2009) . [2] Aschar-Sobbi, R., et al., High Sensitivity, Quantitative Measurements of Polyphosphate Using a New DAPI-Based Approach. Journal of Fluorescence, 18 (5), 859-866 (2008).

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2209 Teachable tool for standardization of human induced pluripotent stem cell colony selection from live cell microscopy image sequences. S-J. J. Lee1, H. Lai1, W. T. Hendriks2, Z. A. Kenyon1, S. V. Alworth1, C. Nakada3, L. Daheron4, Y. Kiyota3, L. L. Rubin2, C. A. Cowan2; 1DRVision Technologies LLC, Bellevue, WA, 2Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Cambridge, MA, 3Instruments Company, Nikon, Yokohama-city, Kanagawa,Japan 4Harvard University, Harvard Stem Cell Institute, Cambridge, MA

In a 2006 breakthrough study, Yamanaka and Takahashi proved that somatic cells could be reprogrammed to an embryonic-like state using four transcription factors. The induced pluripotent stem cell (iPSC) field has since progressed at a remarkable pace impacting basic research, drug discovery, bioproduction, drug screening, personalized medicine and cell therapy. One of the key challenges facing the industry is that iPSC, or iPSC-like colony selection remains technically challenging, and thus state-of-the-art protocols in a broad range of iPSC applications are often of low yield and un-predictable quality. Tools and techniques for the standardization of colony selection in real time systems, such as microscopy incubators are particularly valuable, to reduce the variability of outcomes and lower costs. In particular, tools that can predict future outcomes are useful because it allows productive colonies to be selected.

We developed a teachable image recognition tool that can be taught to perform the quantitative analysis and classification of phase contrast and fluorescence images automatically. To enable the standardization of iPSC colony selection we created an image analytic by teaching. The analytic performs fully automated colony detection, colony feature measurement and iPSC colony classification in human iPSCs. Importantly, the classification step in the analytic is updateable by teaching. This allows the tool to be iteratively improved over time as the assay protocol is improved and colony true outcomes are determined by monitoring the complete re-programming process.

The objective of this initial study was to validate our tool for the prediction of human iPSC colony outcomes. Registered images covering the whole well were acquired on the Nikon BioStation CT and stitched in our tool to create a large (~20,000 x 20,000 pixels) composite image sequence of the whole well include phase constrast and a viral-GFP construct that is silenced upon full reprogramming. We validated the tool by predicting week four outcomes using image frames up to week three. Ground truth is established by a loss of the GFP reporter. Our hypothesis is that the prediction using images up to week three can accurately predict each colonies' week four outcome as quantified by a loss in GFP fluorescence. We test the hypothesis by assessing the sensitivity and specificity of the week three prediction accuracy. We conclude that the hypothesis is supported with high prediction accuracy. Future studies will look towards real time colony selection and future outcome (e.g. differentiation) prediction.

2210 The Use of Discrete Polyethylene Glycol Linkers to Reduce Non-Specific Staining in Immunofluorescence. R. S. Brody1, M. Vermillion1, A. A. Rampersaud1, P. D. Davis2, D. Moothard3; 1QuantaBiodesign, Columbus, OH, 2QuantaBiodesign, Powell, OH, 3American Qualex, San Clemente, CA

The objective of this study was to develop fluorescein conjugated antibodies for tissue and cell imaging that are brighter and show less non-specific staining than standard fluorescein isothiocyanate (FITC) conjugates. This objective was achieved by conjugating fluorescein to an antibody through a discrete polyethylene glycol (dPEG®) linker. This study will be extended to MONDAY the use of dPEG® linkers to conjugate proteins with other fluorescent dyes with different excitation and emission wavelengths.

METHODS.Fluorescent antibody conjugates were prepared by attaching fluorescein (FL) to one end of a dPEG® linker that contains 12 ethylene glycol units and a terminal N- hydroxysuccinimide ester (FL-dPEG®12-NHS). FL-dPEG®12-NHS was conjugated to goat anti-rabbit IgG (GAR) and goat anti-mouse IgG (GAM) using standard NHS ester reaction conditions followed by purification over size exclusion columns. The same antibodies were also labeled with FITC for comparison. Cell based images were made using paraformaldehyde-fixed MCF-7 cells that were treated with 0.3% Triton X-100 and then incubated with a primary mouse monoclonal antibody against a golgi protein (GM130) followed by incubation with either FL- dPEG®12-GAM or FITC-GAM. Cell nuclei were labeled with DRAQ5 and imaging was done with a Zeiss 510 scanning laser confocal microscope. Fixed tissue sections were investigated by incubating them with a rabbit monoclonal antibody against a cancer bio-marker protein followed by incubation with either FL-dPEG®12-GAR or FITC-GAR. Fluorescence was detected using a Nikon TE2000-E inverted epi-fluorescent microscope. For both the cell and tissue analyses, the intensity of the specific labeling and the fluorescence background for each conjugate was evaluated.

RESULTS. At high incorporation levels, fluorescein that is attached to an antibody through a dPEG12® linker shows much less self-quenching than fluorescein attached to an antibody via a FITC label. The fluorescence of FL-dPEG®12 labeled antibodies in solution increases almost linearly with fluorescein incorporation until approximately 20 fluorescein molecules are attached to each antibody. FL-dPEG®12-GAM was found to bind specifically to the target mouse monoclonal in the MCF-7 cells, giving images with moderate intensities and low backgrounds. FITC labeled GAM gave images with similar intensities but much higher backgrounds. Cancerous cells could be clearly imaged in tissue sections using FL-dPEG®12-GAR. In contrast, FITC-GAR gave very high backgrounds with similar sections that made detection of cancerous cells problematic.

2211 Computational Quantitative Image Analysis of Tissue Elongation in the Drosophila Embryo. M. Westacott1, A. Zommer1, T. Blankenship1, D. Loerke1; 1University of Denver (DU), Denver, CO

Elongation of the Drosophila embryonic epithelium is driven by cell intercalation, which causes the tissue to narrow in one dimension and lengthen in the other. The remodeling of the epithelial sheet during this process is a powerful system to illuminate the role of cell-cell adhesion and membrane traffic in generating the necessary forces for re-organization of the cellular architecture, and 4-D in vivo imaging of the Drosophila embryo allows us to measure cell interface remodeling dynamics on the second timescale. We have developed an automated computational assay to quantitatively measure the dynamics of epithelial sheet remodeling. This assay is based on the segmentation of cells/interfaces to yield a 'skeletonized' representation of the sheet, the tracking of features across planes and in time, and the in silico reconstitution of the 3D spatial structure and network topology of the epithelial sheet and its temporal remodeling. This assay allows quantitative measurement of interface dynamics and cellular reorganization, as well as an unbiased measurement of protein localization asymmetries within the epithelial sheet in multi-color imaging experiments. We have measured the temporal dynamics of interface contraction/elongation, and observe the propagation of remodeling MONDAY dynamics both laterally within the sheet and in the vertical direction (from apical to basolateral plane).

2212 A simple, versatile method for GFP-based single molecule localization microscopy. J. Ries1, C. Kaplan1, E. Platonova1, H. Eghlidi1, H. Ewers1; 1Institute of Biochemistry, ETH Zurich, Zurich, Switzerland

Single molecule localization-based superresolution microscopy methods such as PALM or STORM, have been breakthrough techniques of the last years. Until now however, they require special fluorescent proteins to be cloned or high-affinity antibodies to be generated for specific labeling. On the other hand, many laboratories will have most of their constructs in GFP form and entire genomes are available as functional GFP-fusion proteins. Here, we report a method that makes all these constructs available for superresolution microscopy by targeting GFP with tiny, high-affinity antibodies coupled to blinking dyes. It thus combines the molecular specificity of genetic tagging with the high photon yield of organic dyes and minimal linkage error. Direct STORM on microtubules labeled with our novel antibodies showed that indeed the linkage error was minimal, whereas the large size of standard antibodies resulted in an additional error of >10 nm in immunolabeling. The brightness of our labels enabled us to perform rapid time-lapse dSTORM and sptPALM on living neurons expressing the outer membrane protein GPI-GFP. Three-dimensional dSTORM on microtubules using the bi-plane approach allowed us to distinguish overlapping microtubules with an axial separation of ~100 nm. Using a budding yeast GFP-tag genomic library we could readily image several GFP-tagged proteins targeted to specific intracellular locations. In summary, targeting of GFP-labeled constructs with tiny antibodies provides fast and simple access to superresolution microscopy of virtually any known protein in cells. Since for several organisms the entire genome is available as GFP-tagged constructs, all these proteins are immediately accessible without the requirement for cloning or the generation of antibodies. Finally, due to a simple one-step labeling protocol, our technique opens the door to high- throughput localization analysis of entire genomes at the nanoscopic level in cells.

2213 Next Generation Sequencing Analysis of the Evolutionary Impact of Ploidy. A. Selmecki1, P. A. Richmond2, R. Dowell2,3, D. Pellman1; 1Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, 2Molecular, Cellular, Developmental Biology, University of Colorado, Boulder, CO, 3Biofrontiers Institute, University of Colorado

The role of polyploidy—increased copy number of chromosomes relative to an organism’s accepted euploidy—on cells under environmental stress is poorly understood. Ploidy is common in cancer so it is important to understand how ploidy impacts evolution. We performed an evolution experiment on strains of Saccharomyces cerevisiae with different ploidy (haploid, diploid, and tetraploid) under selection for growth on a low-glucose, high raffinose media. After 240 generations, the parental strain and seven of the surviving strains (2 diploid and 5 tetraploids) were sequenced via next generation sequencing technology. We analyzed the sequenced strains to identify mutations causal for survival and compared the mutations between the strains of different ploidy. Preliminary results reveal more mutations in the tetraploid strains relative to their diploid counterparts. In addition, we have benchmarked MONDAY variation-calling software with both “in silico” generated synthetic datasets, as well as our evolved strains for calling SNPs and indels at higher ploidy (N > 2).

2214 An automated method for tracking punctate objects in live cell imaging data. A. Ma1, J. Metz2; 1Albert Einstein Coll Med, Bronx, NY, 2Albert Einstein College of Medicine, Bronx, NY

Continuing advances in fluorescence optical microscopy are yielding an unprecedented surge in live cell imaging data, leaving manual analysis intractable. We have developed a general and model-free algorithm for automatic tracking of single-particle objects in crowded and noisy environments in live cell time-lapse imaging data. In this algorithm, we divided objects in the imaging data into two categories: those with unique image patterns and those without. We first use multi-scale bidirectional template matching procedure to identify objects with unique image patterns. From object linkings made for these objects, we can extract valuable information of the dynamics of the system. Such information will be used as inputs to the dynamic model based procedure that we use to track the objects without uniquely recognizable image patterns. The versatility and robustness of the method were demonstrated by both synthetic and experimental time lapsed imaging data.

2215/L124 Enabling Biology with the Structural Biology Knowledgebase. M. Gabanyi1, J. Westbrook1, D. Micallef1, Y-P. Tao1, R. Shah1, W. McLaughlin2, T. Schwede3, P. Adams4, W. Minor5, H. Berman1; 1Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, 2The Commonwealth Medical College, Scranton, PA, 3Swiss Institute of Bioinformatics, University of Basel, Switzerland, 4Physical Biosciences, Lawrence Berkeley National Labs, 5University of Virginia Medical School, Charlottesville, VA

The PSI Structural Biology Knowledgebase (SBKB, http://sbkb.org) is a scientific search portal that returns comprehensive biological, structural, and methodological information about proteins. Information related to 3D protein structures and the sequences targeted by worldwide structural genomics efforts are combined with links to open biological resources to give an integrated view of a protein. Using the information found on the SBKB, researchers can make informed decisions about their projects and what to do next. It is created by the Protein Structure Initiative in collaboration with the Nature Publishing Group, and features the latest research and technology advances each month to enable researchers in a broad range of biomedical fields.

2216 Identification of Ca2+/CaM as a target protein of a new hypoxia-inducible factor 1-alpha inhibiting small molecule using phage display biopanning. B. Kim1, K. Lee2, H. Jung1, H. Kwon1; 1Chemical Genomics National Research Laboratory, TRCP, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea, 2Dongguk University, Seoul, Korea

Hypoxia inducible factor-1 alpha (HIF-1α) is an essential transcription factor subunit that regulates expression of proteins involving in angiogenesis and metastasis such as VEGF. Accordingly, HIF-1α represents an emerging therapeutic target for development of novel anticancer drugs. We recently developed a new synthetic small molecule inhibiting HIF-1α (YCG-6), which results in decrease of VEGF mRNA expression. Here, the anti-proliferative and the anti-angiogenic activity of the compound on human umbilical vascular endothelial cells MONDAY

(HUVECs) were validated through MTT assay and in vitro tube formation and chemoinvasion assays. Furthermore, identification of the target protein of the compound was conducted by using phage display biopanning. As the result, calmodulin (CaM) was isolated as a plausible target protein and Ca2+ is required for binding of the compound to CaM. Moreover, direct interaction of the CaM and compound was confirmed by Surface Plasmon Resonance analysis. Virtual molecular docking study suggested that the compound acts as a CaM antagonist by binding to hydrophobic pocket of the protein. Moreover, the cell proliferation was arrested by the compound as like other CaM antagonizers did. The effect of YCG-6 on Ras/ERK signaling and prostate specific antigen expression in LNCaP androgen-sensitive cells also clearly reveal that YCG-6 acts as HIF-1α inhibitor by targeting CaM.

2217 Exploring Atomic Force Microscopy for Single Cell Manipulations. R. Afrin1, U. S. Zohora2, H. Uehara2, S-I. Machida1, T. Watanabe-Nakayama2, M. Saito1, A. Ikai1; 1Innovation Laboratory, Tokyo Institute of Technology, Yokohama, Japan, 2Life Science, Tokyo Institute of Technology, Yokohama, Japan

The atomic force microscope (AFM) is a versatile tool for imaging, force measurement and manipulation of proteins, DNA and living cells basically at the single molecular level. In the cellular level manipulation, extraction and identification of mRNA’s from defined loci of a cell, insertion of plasmid DNA and pulling of membrane proteins, for example, have been reported from our group [1]. We used AFM as a novel tool to create holes at defined loci on the cell membrane for visualization of intracellular structure through the hole and for targeted gene delivery into living cells as the ultimate purpose of the application of nano-medicine. Targeted gene delivery was successfully performed by inserting an AFM probe that was coated with the Monster Green Fluorescent Protein phMGFP Vector for transfection of the cell. Following targeted transfection, the gene expression of GFP was observed and confirmed by the fluorescence microscope with high accuracy. Using this AFM probe technology as a fishing nano-device we successfully harvested intra-cellular mRNA’s and study mRNA expression in single living cells without damage to the cells and analyzed by PCR method. For this, an AFM tip was inserted into a living cell to extract mRNAs, which were then analyzed for the number of adhered mRNA through multiplication by RT-PCR and quantitative PCR [2]. We performed quantitative measurement of mRNA at different loci within individual living cells before and after stimulation with fetal bovine serum. Before activation, mRNAs were found mostly near the nucleus, whereas after activation, they were also found in the frontal regions of locomotive cells. The results agreed with previous experiments of FISH [3]. Asymmetric localizations of cellular proteins and mRNAs are important for cell functions such as division, differentiation and development. The localization of speci?c mRNA generates cell polarity by controlling the translation sites of speci?c proteins and thereby restricting their locations to appropriate cellular regions. This method can also be applied to study such important aspects of mRNA distribution within living cells. References: 1. Afrin R. et al., (2009) J. Mol. Recognit. 22:363-372. 2. Uehara H. et al., (2009) Methods Mol Biol. 544:599-608. 3. Kislauskis EH et al., (1997) J. Cell Biol. 136:1263-1270 .

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2218 High Throughput Passive Rheology Assay for Cancer Cell Mechanics. L. D. Osborne1, J. Cribb1, V. Swaminathan2, R. Spero1, E. O'Brien1, R. Taylor3, R. Superfine1; 1Dept. of Physics & Astronomy, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 2Laboratory of Cell and Tissue Morphodynamics, National Heart, Lung, and Blood Institute, 3Dept. of Computer Science, The University of North Carolina at Chapel Hill

While the dynamic nature of cellular structure enables a cell to sense and respond to environmental forces, this complexity introduces a significant degree of variability in the mechanical properties of individual cells within a population. Understanding the composition of cell populations is critical as cells become cancerous and their mechanics change due to alterations in cytoskeletal structure. To address this need, we are developing a high throughput system that utilizes passive microbead rheology to characterize cancer cell mechanics. Here, we report on the progress towards a parallel array of 12 independently functioning imaging systems capable of gathering mechanical measurements for a 96-well specimen plate in under 10 minutes. Using our single imaging system prototype, we show that the thermal diffusion of 1um beads connected to integrin surface receptors via fibronectin can distinguish ovarian cancers with varying metastatic potentials. With a sampling of over 500 beads, we report moduli differences between cancer cells and within cancer cell populations. Additionally, we show differences in cell moduli caused by substrate stiffness. Our results support previously documented work describing the inverse relationship between mechanical stiffness and invasion behavior. This demonstrates the value of our high throughput passive rheology assay as a screening tool for studying specific signaling pathways involved in mechanotransduction.

2219 WITHDRAWN

2220 Toxic metal detection in foodstuff. Synthetic biology approach used to create biosensors. A. Bartos1,2, B. Wang1, M. Buck1, J. Schumacher1; 1Imperial College London, London, United Kingdom, 2Technical University of Lodz, Poland

The objective of the presented research was to explore possibilities of using redesigned E.coli genetic regulatory systems to quantify foodborne metals for analytical purposes. Trace amounts of exogenous metals are known food contaminants. Metallic ions such as mercury, nickel, led, copper, cadmium, mercury etc. are amongst most toxic agents found in commercially available food products. Adsorbed and accumulated over years they lead to chronic health disorders. Ordinarily used instrumental chemistry methods (graphite furnace atomic absorption spectrometry, inductively coupled plasma atomic emission spectroscopy, inductively coupled plasma mass spectrometry) can go down to ng/dm3 in their limits of detection, but have a set of drawbacks. We designed a microbiological transcription-responsive biosensor to identify trace amounts of toxic metals. Such recombinant senses a number of metals, resulting in transcription of genes regulated by inducible protein factors: ZraR in the case of Zn2+, Pb2+; CusR for Cu2+, NikR for Ni2+ etc. We have combined the promoter sequences of GlnK, ZraP, NorV, PspA, PrpB, CusC to the coding sequence of GFP to obtain metal responsive fluorescent outputs. Conducted research provides scope and prospects of recombinant E.coli as biosensors, with emphasis of detection limits confined by cells responsibility to molecular signalling and viability to grow in toxic environment. NCM3722, MC1061 and Top10 strains were induced at OD600=0.05 and, separately, at OD600=0.4 with 10 metal solutions known to activate the corresponding signalling pathways. Results were obtained with the use of plate reader in the form of fluorescence and OD600 measurements over time past induction. They indicate MONDAY successful use of bacterial cultures to sense metals with detection limits below the milimolar range.

2221 Integrated Laser and Electron Microscopy: in search of the causes of FSHD Muscle Dystrophy. M. Karreman1,2, E. van Donselaar2, S. Agronskaia1, W. Voorhout3, T. Verrips2, H. Gerritsen1; 1Science Faculty, Molecular Biophysics, Utrecht, Netherlands, 2Dept. of Biology, Biomolecular Imaging, Utrecht, Netherlands, 3Life Sciences Division, FEI Company, Eindhoven, Netherlands

Facio Scapulo Humeral Dystrophy (FSHD) is the third most common type of muscle dystrophy, and affects 1:20,000 people per year worldwide. Currently, much is known about the genetic background of this disease, and how FSHD affects the patients. However, the cellular processes involved in FSHD are hitherto not identified.

We set out to study the cellular causes and effects of FSHD with a novel tool for correlative microscopy. The Integrated Laser and Electron Microscope (iLEM) combines a fluorescence microscope (FM) and a transmission electron microscope (TEM) within one set-up. This allows for the localization of regions of interest over a large field of view with the FM, and subsequent ultrastructural analysis of these areas with the TEM. Since both microscopes are integrated within one set-up, the correlation between the FM and the TEM is extremely fast and very accurate.

FSHD is known to be a heterogeneous disease; its expression can vary from muscle to muscle and from cell to cell. Ultrastructural studies of affected cells will help us to gain more insight in the processes involved in this disease. The iLEM is employed to navigate to the FSHD affected regions, so that these areas can subsequently be studied at high resolution. We aimed to localize FSHD affected cells with the iLEM in muscle biopsies from patients. At a certain stage of muscular dystrophy, creatine kinase forms crystalline structures in the mitochondria. This protein was used as a marker to navigate to the FSHD affected cells in the biopsy; next the crystalline structures and their cellular context was studied at high resolution.

In conclusion; due to the heterogeneity of the expression of FSHD, the iLEM is a unique tool to rapidly navigate to the cells of interest and study the effects of this disease with high resolution.

2222 A novel single chain Rac biosensor for the study of tumor cell migration. Y. Moshfegh1; 1Albert Einstein College of Medicine, Bronx, NY

The p21 Rho family of small GTPases are heavily involved in cell motility and migration. Specifically, Rac is critical for cell protrusion at the leading edge, and has been shown to be overexpressed in several types of tumors. However, the exact role of Rac in relation to the other Rho GTPases still remains unclear, because previous approaches for the study of these molecules are limited due to their lack of sub-cellular spatiotemporal resolution. By developing genetically encoded, single-chain fluorescent biosensors, we are able to bypass this limitation and visualize protein activation as well as localization in real time, making this innovation a necessary and powerful tool for the study of small G-proteins and cell migration. To study the function of the Rac during cell protrusion, and also its relationship to the other Rho GTPases, we developed a novel, genetically-encoded fluorescent Rac1 biosensor, based on an intra-molecular design. This new biosensor is a massive improvement over the previous- generation Rac biosensor because there is an equimolar distribution of FRET donor and MONDAY acceptor and this produces a more accurate readout. Our design maintains the correct C- terminal lipid modification of full-length Rac1, enabling proper interaction with upstream regulators, including GEFs, GAPs, and GDIs. We constructed this new single-chain biosensor by modifying the placement of the binding domain in relation to the first fluorescent protein, to achieve the proper orientation for optimal interaction between the GTPase and its binding domain. In addition, we incorporated an internal autoinhibitory mechanism into our design, to modulate the binding domain affinity. This new biosensor will allow the real-time visualization of the spatiotemporal dynamics of Rac in live carcinoma cells. In particular, we will be able to explore the molecular basis for the mechanism of Rac, Rho, and Cdc42 coordination at the leading edge.

2223 An inducible, reversible system for the rapid and complete destruction of proteins in mammalian cells. D. Fachinetti1, A. J. Holland2, D. W. Cleveland3; 1Department of Cellular and Molecular Medicine, Ludwig Institute for Cancer Research, La Jolla, CA, 2Department of Cellular and Molecular Medicine, University of California, Ludwig Institute for Cancer Research, La Jolla, CA, 3Department of Cellular and Molecular Medicine, University of California, Ludwig Institute for Cancer Research

Inducible degradation in living cells is a powerful approach for identifying the function of a specific protein or protein complex. Here we demonstrate that an auxin-inducible degron (AID) system is capable of controlling the stability of AID-tagged proteins that are in either nuclear or cytoplasmic compartments and even when incorporated into protein complexes. Induced degradation occurs rapidly after addition of auxin with protein half-life reduced to as little as 9 minutes and proceeding to completion with first order kinetics. Auxin-inducible, degron-mediated instability is demonstrated to be rapidly reversible. Induced degradation is shown to initiate and continue in all cell cycle phases, including mitosis, making it especially useful for identifying function(s) of proteins of interest during specific points in the mammalian cell cycle.

2224 An Application Specific Microfluidic Format for Dynamic Live Cell Studies. M. Allen1, P. Hung1, T. Gaige1, P. Lee1; 1CellASIC Corp., Hayward, CA

Studying living mammalian cells in vitro is critical to understanding basic biology, signaling pathways, drug effects, and disease models. While the tools and techniques for cellular analysis have advanced rapidly over the past decades, the cell culture platform has remained largely undeveloped since the Petri dish. In light of growing evidence that the cellular culture environment is critical for sustaining physiologically relevant phenotype and behavior, we have developed a microfluidic cell culture platform to control various cellular microenvironment parameters including reagent perfusion, temperature, and gas composition, all through a universal control system used in tandem with different application specific microfluidic plates. Each plate fits a standard microplate format containing four independent micro-fabricated PDMS culture chambers which can be addressed by up to 6 different reagents. The microfluidic plate houses all necessary solutions in simple layout of wells that are readily accessed during experimental setup using a pipette. A 170-micron thick glass coverslip bottom enables high magnification imaging on an inverted microscope. Dynamic environment changes within plates are controlled through software that integrates the universal control system with real-time image analysis to form a closed feedback loop. The utility of the platform was demonstrated with a number of applications. First, automated immunostaining was demonstrated using HT-1080 fibrosarcoma cells. Cells were cultured in the microfluidic plate to the desired density, then MONDAY automatically washed, fixed, permeablized, blocked, and fluorescently stained with antibodies using a series of solutions and programmed exposure times, significantly simplifying the traditional protocol. Second, promyelocytic HL-60 cells differentiated into granulocytes were tracked to show a chemotactic response to a dynamic chemical gradient for live cell migration studies. Third, real-time transfection optimization was performed using HeLa cervical carcinoma cells revealing expression of GFP-tagged tubulin via live cell fluorescence microscopy. Fourth, 3D cell culture was demonstrated with MCF10A mammary epithelial cells cultured in Matrigel with continuous perfusion to track acinus formation. This application specific design philosophy promises to bring dynamic cell microenvironment control within reach for a large range of user- generated cell biology applications to complement current advanced cellular analysis techniques.

2225 Use of Short Tandem Repeats for Rapid Authentication of Human Cell Lines. T. Reid1, K. Wilson2, C. Kraemer3, M. Baird4; 1DNA Diagnostics Center, Cincinnati, OH, 2University of Cincinnati, OH 3DNA Diagnostics Center, 4DNA Diagnostics Ctr, Fairfield, OH

Human cell lines are used worldwide in biological and biomedical research, and accurate data interpretation depends on unambiguous identity of a cell line relative to its original source. Contamination and misidentification of human cell cultures has been identified as a long- standing issue within the scientific community, and steps are now being taken to address the problem. Short-tandem-repeat (STR) DNA profiling provides an accurate, reliable, and standardized method for authentication of human identity. Currently, the ATCC is considering proposed standards for cell line authentication utilizing human STR typing, and many journals have adopted the NIH-recommended policy and mandate cell line authentication as a requirement for publication.

Here we demonstrate the validation and use of two different STR systems to authenticate human cell lines as well as detect contamination by mouse feeder cells and other human cell lines. Threshold levels for detection of complete profiles as well as contamination are compared between the systems, and typical typing issues specific to cell lines are discussed.

2226 Monitoring Changes in NF-kB Pathway Regulation Using Highly Sensitive Multipex Bioluminescent Reporter Assays. D. Hughes1, M. dobbs1, J. Narahari1, J. Choi1, A. Deshpande1, B. Webb1; 1Thermofisher Scientific, Rockford, IL

The study of complex cellular signaling pathways requires powerful and specific tools to monitor changes in gene activation or repression. In order to accurately monitor these processes, reporter gene assays are commonly used. We have developed a series of next generation multiplexed luciferase reporters for studying gene regulation. These reporters were developed to improve the sensitivity and convenience of conventional luciferase reporter systems. First, we have used two naturally secreted luciferase genes, Gaussia luciferase from the Marine copepod Gaussia princeps and Cypridina luciferase from the Marine ostracod Cypridina noctiluca to develop a novel dual secreted reporter system. This Gaussia/Cypridina dual system enables monitoring transcriptional regulation of two promoters within tissue culture media without the need for cell lysis using sequential addition of luciferase substrates. Importantly, both Gaussia luciferase and Cypridina luciferase are considerably brighter than traditional Firefly luciferase reporters. Second, we have utilized a mutant form of the Italian Firefly Luciferase from Luciola MONDAY cruciata that has a red-shifted emission spectrum to develop a dual luciferase assay with Gaussia luciferase in which the light output of the two luciferases are spectrally resolvable. This Gaussia/Red Firefly dual spectral assay allows simultaneous monitoring of two promoters in a single read assay through addition of both substrates and then spectral interrogation of the resulting light output. In the present study, we utilized both techniques, multiplexing by spectral separation using Gaussia/Red Firefly, and multiplex assays relying on sequential addition of substrates two secretory luciferases, Gaussia/Cypridina to monitor changes in NFkB promoter activity in response to small molecule agonists. Our results demonstrate the utility of dual secreted luciferase assays for sensitive real time monitoring of NFkB reporter activity in the media and simultaneous detection of spectrally resolvable luciferases using filter based detection.

2227 Optimization of Protein Purification Using Small-Scale Separation Columns. R. Tobias1, L. Hoang1, C. Suh1, D. Gjerde1; 1PHYNEXUS, INC., San Jose, CA

Biophysical and functional characterization of therapeutic candidates requires that proteins are well purified and enriched post-expression. Currently the process for adequate preparation requires that sufficient quantities of material be scaled up and processed in a time consuming manner using expensive chromatography equipment. As improvements in functional and analytical assays increase throughput and reduce the quantity of protein required for analysis, the availability of efficient small-volume protein purification methods would be of high value to researchers in earlier stages of drug discovery and development. Recent advances in the area of miniaturized, high-throughput tools for purification, enrichment and desalting of proteins eliminate bottlenecks associated with traditional protein purification processes. By performing high-performance functional protein separations on small samples in parallel, it is now possible to obtain more relevant data in a completely automated format. Data from protein separations in micro-scale chromatography columns is presented here along with optimized conditions enabling functional and analytical characterization of therapeutic proteins purified by this unique format.

2228 Alternative Digital Microfluidic Devices for Cell Assays. N. Thorne1, B. Demchak1, L. Pontiggia1, S. Freire1; 1University of the Sciences, Philadelphia, PA

Digital microfluidics (DMF) is a technique for transport of droplets by an electric potential applied to an array of electrodes, and has appeared as an alternative to the conventional fluid transport in microchannels. DMF enables automation and control of droplets unparalleled by any other technique of fluid transport, without the need for pumps, tubes, or valves (1). In particular, we are interested in developing DMF platforms to study ciliary motion.

Usually, the fabrication of DMF devices requires access to well-equipped facilities and the training of students in a variety of techniques (e.g, metal deposition, photolithography, etc.). This is often expensive and time consuming, limiting the access to this technique, particularly for researchers at undergraduate institutions.

We developed an alternative method for fabrication of DMF devices (2). One key aspect is that miniaturized interelectrode gaps, thought as essential for optimal performance, are not a requirement for droplet actuation; altogether, the strategy enables fast fabrication of robust and reliable devices at low costs.

MONDAY

However, this technique requires relatively high voltages for DMF operation (typically 500 VRMS), which contrasts with the low voltages (~120 VRMS) used in conventional, clean-room based devices. This has been a point of criticism, with the claim that high voltages might be detrimental to biological specimens.

Tetrahymena thermophila (SB 255) cells, well suited for research purposes due to the large number of cilia, were used for tests. Droplets (10 µL) containing cells were actuated on DMF devices. We will present results showing that no changes were observed in the vitality and proliferation of these cells after transported in the alternative DMF devices. This corroborates previous results showing that the voltage across the droplet in a DMF device is an insignificant fraction of the total applied voltage, indicating very little, if any, effects on cells (3).

We are currently evaluating non-specific adsorption of analytes to surfaces, which effects device performance. We have chosen a simple and well-established assay, cell deciliation with dibucaine. We will show that the minimum concentration of dibucaine for a complete and almost instantaneous loss of cilia is close to the used in conventional, off-chip deciliation (1.3 mM)(4). This indicates little non-specifc adsorption of dibucaine to surfaces in this assay.

Since adsorption to surfaces might be analyte dependent, we are also looking for collaborations to develop other assays of interest, to further characterize the devices. However, these preliminary results pave the way to on-chip deciliation. Future studies will focus on characterizing motion of extracted cilia, directly on a microfluidic device. This work has been done by undergraduate students (Biological Sciences and Physics).

References:

1 Fair, R. B. "Digital microfluidics: is a true lab-on-a-chip possible?" Microfluidics and Nanofluidics 2007, 3, 245-281. 2 Thorne, N.; Lamberto, M.; Mazza, A.; Freire, S. L. S. "An Alternative Strategy for Fabrication of Robust and Flexible Digital Microfluidic Devices" Pacific Northwest Journal of Undergraduate Research and Creative Activities 2011, 2 3 Barbulovic-Nad, I.; Yang, H.; Park, P. S.; Wheeler, A. R. "Digital microfluidics for cell- based assays" Lab Chip 2008, 8, 519-526. 4 Thompson, G. A., Jr.; Baugh, L. C.; Walker, L. F. "Nonlethal deciliation of Tetrahymena by a local anesthetic and its utility as a tool for studying cilia regeneration" J Cell Biol 1974, 61, 253-257.

2229 Deciphering the Intermediate Filament Structure(s) using cryo Electron Microscopy and Novel Metal Labels. R. Kirmse1, C. Bouchet-Marquis1, M. Pagratis1, A. Hoenger1; 1MCDB, University of Colorado at Boulder, Boulder, CO

Intermediate filaments (IFs) are critical building blocks of the cell´s cytoskeleton. Together with actin filaments and microtubules they are responsible for various cell functions such as mechanical stress resistance, signal transduction, and material transport. In contrast to actin and tubulin a detailed 3D structure of IF proteins and grown filaments remains elusive. Crystallization of IF proteins for example is extremely difficult caused by their elongated shape. Regardless, cryo electron microscopy and tomography provides the opportunity to observe proteins in 3D. By rapid freezing we are able to keep the IFs hydrated and as close to their native state as possible. Subsequently cryo tomography allows reconstructing the 3D structure MONDAY of the observed sample in great detail. In addition we are developing new labeling methods utilizing cloneable high-density markers to further label the IF substructure. These markers will enable us to draw conclusion about the basic underlying arrangements of the IF proteins in the mature filaments.

TUESDAY

TUESDAY, DECEMBER 6- Late Abstracts 3

Actin Cytoskeleton

2230 Depletion of Arp2/3 complex reveals critical role of lamellipodia in haptotaxis, but not chemotaxis. C. Wu1, J. E. Bear2,3; 1Cell and Developmental Biology, UNC-Chapel Hill, Chapel Hill, NC, 2UNC-Chapel Hill, Chapel Hill. CA 3HHMI

Lamellipodia are sheet-like, leading edge protrusions in firmly adherent cells that contain Arp2/3-generated dendritic actin networks. Although lamellipodia are widely believed to be critical for cell motility, this notion has not been rigorously tested. Using fibroblasts derived from Ink4a/Arf-deficient mice, we generated a stable line depleted of Arp2/3 complex that lacks lamellipodia. This line shows defective random cell motility and relies on a filopodia-based protrusion system. Utilizing a microfluidic gradient generation system, we tested the role of Arp2/3 complex and lamellipodia in directional cell migration. Surprisingly, these cells respond normally to shallow gradients of PDGF indicating that lamellipodia are not required for chemotaxis. Conversely, these cells cannot respond to a gradient of extracellular matrix (haptotaxis). Consistent with this finding, cells depleted of Arp2/3 fail to globally align focal adhesions suggesting that one principle function of lamellipodia is to organize cell-matrix adhesions in a spatial coherent manner.

2231 A Peptide Mimetic of Heat Shock Protein Beta 6 Alters 3t3 Migration through a Cofilin- Dependent Mechanism. C. Smoke1, D. N. Derkach1, D. Eng1, J. Uhlenkamp1, K. B. Perkins1, M. R. Sheller1; 1Capstone Therapeutics, Tempe, AZ

Prior research has shown that AZX100, a 24-amino acid peptide mimetic of Heat Shock Protein β6 (HSPβ6), reduces filamentous actin and focal adhesions in myofibroblasts, cells instrumental in the process of dermal scar healing. It has also been shown that HSPβ6 binds to 14-3-3, leading to dephosphorylation of cofilin. Dephosphorylated cofilin severs filamentous actin and increases the pool of globular actin. Investigating the manner in which AZX100 affects cofilin dephosphorylation, alters actin dynamics and influences migratory behavior in myofibroblasts could elucidate the role of AZX100 in scar healing.

The present study used 3T3 fibroblasts treated with transforming growth factor β-1 (TGFβ-1) alone or in the presence of varying concentrations of AZX100 for one hour. Fibroblasts were treated with TGFβ-1 to induce differentiation to myofibroblast-like cells. Cell lysates were resolved by 2-dimensional gel electrophoresis to determine the cofilin-to-phospho-cofilin expression levels. AZX100 treatment of the 3T3 cells significantly increased the percent of dephosphorylated cofilin compared to cells treated with TGFß-1 alone (73% vs. 40% respectively; p =0.006). Additionally, live cell imaging was performed to study the effect of various doses of AZX100 on the actin cytoskeleton. Treatment with 1 and 10 μM AZX100 produced the greatest change in the actin cytoskeleton as measured by overall reduction in cell area. Doses of AZX100 greater than 10 μM and lower than 1 μM did not significantly alter the actin cytoskeleton compared to control cells. Finally, myofibroblast migration was analyzed through the Electric Cell-substrate Impedence Sensing (ECIS) system, which measured changes in resistance over time. AZX100 treatment resulted in a dose-dependent reduction of migration over one hour compared to TGFβ-1 alone, with a maximal reduction of 53%. In TUESDAY conclusion, AZX100 increases cofilin dephosphorylation inmigratory cells. The associated migration pattern was also altered, which may be due to morphological changes resulting from the liberation of dephosphorylated cofilin from 14-3-3.

2232 Biophysical Linkage between Local Mechanical Properties and Global Cellular Responses in Living Fibroblasts: Actin Cytoskeleton Perspectives. H-C. Harn1, Y-K. Wang2, H-H. Lin3, C-M. Cheng4, M-J. Tang1,3; 1Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan, 2Dept. of Medicine, Skeleton-Joint Research Center, National Cheng Kung University, 3Department of Physiology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, 4Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu, Taiwan

Each organ in our body has different mechanical properties—ranging from 200 Pa (brain) to 310 MPa (Achilles tendon)—serving as the structural-based scaffolding and the inherent force source of mechanical stimulation for single cells. The cellular response under mechanical stimulation is highly tuned to influence a cell’s structure provided by the cytoskeleton. The cytoskeleton governs a diversity of cellular behaviors such as cell division, motility, and morphology. Solon et al. have observed that fibroblasts adjusted their spreading areas and actin cytoskeleton organization in order to match the change of an external environment. We therefore hypothesize that actin cytoskeleton could be a key determinant to the changes in cell’s mechanical properties in response to different mechanical stimuli. In this study, we attempted to explore the mechanical properties of an individual actin filament in living fibroblasts (NRK49F), and to determine its contributions to the elasticity of the cell as a whole with external physical stimuli. We used a biological atomic force microscope to (i) scan a living fibroblast in a liquid environment, (ii) indent around the periphery in a living fibroblast, and (iii) indent on top of the nucleus in a living fibroblast to determine Young’s moduli of specific locations, respectively. We also constructed cell’s 3D images via confocal fluorescent microscopy, in order to probe the morphological responses of actin cytoskeleton with mechanical stimuli. The results showed that a fibroblast changed its local and global elasticities under substratum stiffness. The organization of actin filaments—the number of actin filaments at a specific location that we probed in a living fibroblast—were strongly affected by the change with substrate stiffness. Furthermore, even for a single fibroblast seeded on a stiffer substrate showed a distinct difference (20 and 200 kPa) in the elasticity of actin filaments; this could correspond to the variations in the thickness of actin filaments and the distribution of actin filaments around the cell periphery. This study connects the local mechanical properties of actin filaments to the overall elasticity of a living cell in response to mechanical stimulation. It highlights the important contribution of actin filaments could play in regulating the behavior and function of a cell.

2233 Stereocilia F-actin polymerization is coupled to the mechanotransduction machinery in the auditory hair bundles. V. Michel1,2, E. Caberlotto1, D. Weil1, C. Petit1,3; 1Pasteur Institut-INSERM, Paris, France, 2Université Pierre et Marie Curie, Paris, France, 3Collège de France, Paris, France

In the hair bundles of the auditory hair cells, the mechanotransducer channels are gated by tip- links, extracellular filaments that interconnect the stereocilia and stretch from the tips of stereocilia in the short and middle rows to the sides of neighboring, taller stereocilia. Tip-links consist of cadherin-23 and protocadherin-15, products of the Usher syndrome type 1 genes USH1D and USH1F, respectively. In this study, we found that upon the post-natal disappearance of the tip-links, caused by the deletion of one of its components, cadherin-23, or TUESDAY of one of its anchoring proteins, sans, stereocilia from the short and medium but not of the high stereocilia row undergo a dramatic reduction in length. This indicates that the mechanotransduction machinery has a positive effect on F-actin polymerization. The loss of the tip-links and the resulting loss of functional MET channels is what stops the renewal of the short and middle rows of stereocilia in these mutant mice. We can conclude that the tip-link plays an unsuspected, direct or indirect role on the undergoing actin polymerization processes in the different stereocilia rows, which ensures the development and the dynamic maintenance of the hair bundle’s staircase pattern.

2234 Mutual regulations of actin cytoskelton remodeling and peroxisome proliferator-activated receptor-γ on early adipocyte differentiation. H. Nobusue1, N. Onishi2, Y. Oki1, T. Shimizu2, T. Chiyoda2, H. Saya2, K. Kano1; 1Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, Fujisawa, Japan, 2Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Shinjuku-ku, Japan

Adipocyte differentiation at an early stage is directly regulated by gene expression of a master regulator such as peroxisome proliferator-activated receptor-γ (PPARγ). During adipogenesis actin cytoskelton remodeling defines important events of the differentiation process; this is characterized by the conversion of filamentous actin from stress fibers to cortical actin structures. Here, we examined the regulatory relationship between actin cytoskelton remodeling and PPARγ in early adipocyte differentiation. Depending on adipogenic induction, actin stress fibers were immediately disrupted prior to the expression of PPARγ. And treatment with cytoskeletal fixation agent phalloidin maintained actin fiber structures even after adipogenic induction, and caused a down-regulation of PPARγ. In addition, ectopic expression of activated RhoA, which is known to promote the formation of actin stress fibers, inhibited the disruption of actin stress fibers and PPARγ expression after adipogenic induction, and these effects were recovered by treatment with the RhoA kinase inhibitor Y-27632. Moreover, treatment of the actin polymerization inhibitor cytochalasin D in active RhoA-expressing cells caused the disruption of actin stress fibers, and as a result, PPARγ expression was rescued. On the other hand, introduction of the PPARγ-specific shRNA resulted in maintained the high-level expression of fibronectin (FN) matrix and inhibited the reorganization into adipocyte-specific cortical actin structures during adipocyte differentiation. And transfection of FN-specific siRNA in PPARγ- knockdown cells after adipogenic induction caused the formation of cortical actin structures. In conclusion, our findings indicate that actin stress fiber disruption directly induces PPARγ expression, and that PPARγ regulates the reorganization into adipocyte-specific cortical actin structures through the down-regulation of FN matrix, and provide a novel insight into the mutual regulatory mechanisms between actin cytoskelton remodeling and adipogenic transcription factors in early adipocyte differentiation.

2235 The Carboxy Termini of Human Myopodin Isoforms Influences Non-Muscle Myosin II- Dependent Myopodin Subcellular Localization. F. Kai1, R. Duncan1,2; 1Department of Microbiology, Dalhousie University, Halifax, NS, Canada, 2Department of Biochemistry & Molecular Biology and Department of Pediatrics, Dalhousie University, Halifax, NS, Canada

More than 80% of patients with invasive prostate cancer have deletions within their myopodin gene, suggesting that myopodin is a reliable predictive marker of prostate cancer metastasis. However, the functions of myopodin in cancer metastasis remain unknown. Biochemical studies TUESDAY have revealed that myopodin is an actin-binding protein. Since actin rearrangement is an essential process involved in cell migration, it has been postulated that myopodin might suppress cancer metastasis by modulating the actin cytoskeleton. Presently, five human myopodin (hMYO) splicing variants, each with a unique amino and/or carboxy-termini, have been identified. However, it is unclear whether the unique sequence of each isoform differentially regulates myopodin function. To examine if these splicing variants function distinctly, we ectopically expressed myopodin isoforms in invasive prostate cancer cells (PC3) and benign prostatic hyperplasia cells (BPH-1). Immunofluorescent staining revealed that the different myopodin isoforms induce distinct actin structures within the cell body. Each isoform interacts distinctly with these actin structures, but none of the isoforms colocalize with actin near the cell periphery. Truncation analysis indicated the unique carboxy-terminal sequences regulate the distinct localization of myopodin isoforms with the actin cytoskeleton. Furthermore, the restrictive subcellular localization of some myopodin isoforms was abrogated by inhibitors that affect downstream effectors in the activated RhoA pathway (i.e. the ROCK inhibitor Y- 27632 and the non-muscle myosin II inhibitor blebbistatin). Taken together, these results indicate the carboxy-termini of the human myopodin isoforms regulate their ability to differentially alter and co-localize with actin cytoskeletal structures in a non-muscle myosin II- dependent manner.

FuiBoon Kai is supported by a trainee award from The Beatrice Hunter Cancer Research Institute with funds provided by The Terry Fox Foundation Strategic Health Research Training Program in Cancer Research at the Canadian Institutes of Health Research.

2236 Surface-bound VASP controls Arp2/3 complex-dependent actin gel growth. P. Noguera1, A. Lamora1, J. Plastino1; 1Physico-chimie Curie (PCC) - UMR 168, Institut Curie, Paris, France

The Drosophila Enabled/Vasodilator Stimulated Phosphoprotein (Ena/VASP) protein family is involved in numerous actin based processes such as filopodia formation and lamellipodia protrusion, but the precise function and mode of action of Ena/VASP proteins remain controversial. To address this question, we use an in vitro assay composed of beads coated with actin polymerization activators, incubated in a protein mix that supports the growth of an actin gel on the bead surface. Over time, the symmetrical gel breaks open to form a comet tail that propels the bead forward, mimicking in simplified conditions the dynamics of the actin cytoskeleton at the plasma membrane of moving cells. Here we use beads that activate the actin-related protein (Arp) 2/3 complex, an actin polymerization nucleator, and additionally recruit wild-type and mutant forms of murine VASP to the bead surface. We show that VASP increases the rate of actin comet growth. We further show that this activity requires the F-actin binding capacity of VASP, its tetramerization domain and its profilin recruiting site, but that the G-actin binding site is dispensable. When VASP is not targeted to the bead surface, it has no effect on bead speed, despite the fact that it is present throughout the actin comet. In another series of experiments, we provide evidence that VASP focuses polymerization near the bead surface in the absence of capping protein via its F-actin binding capacity. Overall these results shed light on the molecular mechanism of Ena/VASP proteins, and on how these proteins enter into the actin dynamics equation in vivo.

TUESDAY

2237 Rho-GTPase mediated regulation and membrane targeting of human leukocyte formins FMNL1 and FMNL2. S. Kuehn1, J. Block2, K. Rottner2, M. Geyer1; 1Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany, 2Institute of Genetics, University of Bonn, Bonn, Germany

Dynamic actin filament structures required for cell motility and migration are regulated by nucleation and elongation factors, such as members of the formin family. The large, multidomain proteins FMNL1 and FMNL2 belong to the subgroup of Diaphanous-related formins (DRFs). The FH2 domain-mediated actin assembly activity of DRFs is inhibited by an intramolecular interaction between the N-terminal regulatory region and the C-terminal DAD domain. This inhibition is released via binding of Rho-GTPases to the GBD and FH3 domain. Additional cofactors might be required for full activation, localization or membrane targeting. We here show that human FMNL2N interacts specifically with GppNHp-bound Cdc42 in the micromolar affinity range. Cdc42 binds to FMNL2N via switch I, switch II as well as the insert helix that is unique for Rho-GTPases. In FMNL2, binding to Cdc42 involves the GBD and all armadillo repeats of the FH3 domain. The structure thus provides for the first time an effector- specific binding of the Rho-specific insert helix. We could determine crucial residues, which can, if mutated, switch the specificity of FMNL2 for Cdc42 to Rac1. We identified a basic insert that disrupts the canonical FH3 repeat fold. This insert binds acidic phospholipids and negatively charged liposomes in vitro, suggesting plasma membrane association through electrostatic interactions. Additionally, FMNL2 is N-terminally myristoylated, a fatty acid modification that drives membrane binding. Together with the C-terminal prenylation of the GTPase, a large membrane binding surface of the dimeric formin complex assembles that sustains membrane association during migration. In vivo experiments reveal that both, the basic insert and the myristoyl moiety, are responsible for plasma membrane targeting of the actin polymerization factor.

2238 Watching the motions of the cell wall synthesis machinery and underlying cytoskeleton in B. subtilis with high precision particle tracking. E. C. Garner1, X. Zhuang2, W. Wang3, R. Bernard4, D. Rudner4, T. MItchison5; 1Harvard University, Cambridge, MA, 2Chemistry and Chemical biology, 3Physics, Harvard University, Cambridge, MA, 4Microbiology, Harvard University, Cambridge, MA, 5Systems Biology, Harvard University, Cambridge, MA

Rod-shaped bacteria elongate by the action of cell-wall synthesis complexes linked to underlying dynamic MreB filaments, but how these proteins function to allow continued elongation as a rod remains unknown. To understand how the movement of these filaments relates to cell wall synthesis, we characterized the dynamics of MreB and the cell wall elongation machinery using high-resolution particle tracking in Bacillus subtilis. We found that both MreB and the elongation machinery move in linear paths across the cell, moving at similar rates (~20nm / second) and angles to the cell body, suggesting they function as single complexes. These proteins move circumferentially around the cell, principally perpendicular to its length. We find that the motions of these complexes are independent, as they can pause and reverse, and also as nearby complexes move independently in both directions across one surface of the cell. Inhibition of cell wall synthesis with antibiotics or depletions in the cell wall synthesis machinery blocked MreB movement, suggesting that the cell wall synthetic machinery is the motor in this system. We propose that bacteria elongate by the uncoordinated, TUESDAY circumferential movements of synthetic complexes that span the plasma membrane and insert radial hoops of new peptidoglycan during their transit.

2239 WITHDRAWN

2240 Computational models of embryonic wound healing. M. A. Wyczalkowski1, V. D. Varner1, L. A. Taber1; 1Biomedical Engineering, Washington University in St. Louis, St. Louis, MO

The objective of this work is to characterize experimentally the healing of circular and slash wounds in epithelia of early stage chick embryos and to develop a finite element model of wound healing dynamics.

Early stage chick embryos were harvested and cultured ex ovo. Circular and slash wounds (~100um in size) were made in the blastoderm and allowed to heal. The healing process was captured with time lapse microscopy, and wound area and shape were characterized as functions of time. The contractile response of the tissue to wounding was implemented in a finite element model, and the results were critically compared to experimental results for both wound types.

The embryonic wounds displayed a two-phase behavior: rapid wound healing lasting about a minute, followed by a more gradual healing to full closure within tens of minutes. This behavior, as well as more complicated wound geometry trends (e.g., aspect ratio), were reproduced by the model in which 1) wounding induces a rapid isotropic contraction of the tissue surrounding the wound followed by 2) a slower formation and contraction of a relatively thin fiber ring bordering the wound edge.

The computational model is able to capture the non-trivial healing dynamics of wounds in embryonic epithelia, lending support to a hypothesis where wound healing is governed by two distinct cellular mechanisms.

2241 Nuclear protein MARVELD1 regulates activity of Rho GTPases and inhibits cell adhesion and cell motility. S. Wang1, J. Hu1, F. Han1, M. Shi1, J. He1, S. Liu1, L. Yue1, Y. Li1; 1Harbin Institute of Technology, Harbin, China

A number of cytoplasmic factors regulate cell adhesion on an extracellular matrix (ECM), which plays a crucial role in cell motility, invasion, cell signal transduction and gene expression. Activity of Rho GTPase regulates F-actin dynamics. The focal adhesions and associated F-actin structures reorganization led to the alteration of focal adhesion anchored actin stress fibers. However, its regulation via nuclear proteins remains obscure. In this study, we identified a nuclear protein, MARVELD1 that could suppress cell spreading and movement. Over- expression of MARVELD1 in A549 cells decreased the expression level of integrin β1 and vinculin, and further led to dephosphorylation of FAK at Tyr 397. Also, knockdown of MARVELD1 in HeLa cells and H520 cells could upregulate integin β1 and vinculin, and promote FAK Tyr397 phosphoylation. Moreover, we found that activity of Cdc42/Rac1/RhoA was regulated. And MARVELD1 partially colocalized with SC35 and interacted with nuclear cap binding protein subunit 2, CBP20. In addition, we explored that pre-mRNA processing of several focal adhesion proteins, including vinculin and actinin, was affected by MARVELD1. TUESDAY

Our studies demonstrate that the nuclear protein MARVELD1 plays a role in pre-mRNA processing of some important FAC genes and regulates activity of Rho GTPases, and thereby inhibits cell adhesion and cell motility. These studies provide a novel regulatory mechanism of cell-ECM adhesion by nuclear protein in tumor cells.

Molecular Motors

2242 The Tail region inhibits the motor activity of human myosin VIIA at molecular and cellular level. T. Sakai1, R. Ikebe1, M. Ikebe1; 1Microbiology and Physiological Systems, University of Massachusetts Medical school, Worcester, MA

Myosin VIIA is a responsible gene for human Usher Syndrome type 1B, which causes hearing and visual loss, and plays a role in human auditory function. We previously reported that Drosophila myosin VIIA is a monomer and the tail domain inhibits the actin activated ATPase activity under low Ca2+ condition. However, it is unknown whether the tail-inhibition mechanism is operating in cells. Another critical issue is whether or not mammalian myosin VIIA is regulated by similar mechanism since it has been known that regulation mechanism of myosin is often quite different between vertebrate myosin and its non-vertebrate counterpart. To address these questions, we studied the regulation mechanism of human myosin VIIA (HuM7A) at both molecular and cellular level. We studied the inhibitory function of the tail by examining the actin-activated ATPase activity of HuM7A. The C-terminal deletion (-40 amino acids) of the full-length HuM7A significantly increased the ATPase activity. This result suggested that the C-terminal end of tail region inhibits the motor activity. The result is similar to the study of Drosophila myosin VIIA, suggesting that tail-inhibition mechanism is also operating in HuM7A. Our earlier study showed that the dimer formation of GFP-HuM7A HMM or GFP-HuM7A full- length induces the filopodia tip localization of human myosin VIIA in cells, suggesting that HuM7A moves along actin bundles in filopodia to the tips. Using this system, we examined the inhibitory activity of HuM7tail in cells. We co-expressed the tail with the forced-dimer of HuM7A HMM, and studied the effect of the tail on HuM7AHMM translocation to the filopodial tips. Expression of the entire tail significantly inhibited the translocation of HuM7A HMM to the filopodial tips. We also found that translocation of the forced-dimer of full-length HuM7A is significantly attenated and this was facilitated by deletion of the C-terminal 40 amino acids. These results are consistent with the tail-inhibition of the ATPase activity in vitro. Present results suggested that the tail-induced regulatory mechanism is common between vertebrate and non- vertebrate myosin VIIA and is operating in vivo.

2243 Myosin-Va is implicated in cell adhesion and spreading. A. Ramao1, G. P. Nader1,2, E. M. Espreafico1; 1Cellular and Molecular Biology, FMRP-USP, Ribeirão Preto, Brazil, 2Pathology and Cell Biology, Columbia University, New York, NY

The main objective of this work was to assess the role of myosin-Va in cell adhesion and spreading. Here we show that MYO5A mutant melanoma cells exhibit deficient adhesion and spreading abilities, and lack the usual organization of the actin cytoskeleton. Myosin-Va expression in these cells restored actin bundles, induced lamelipodial/filopodial growth and led to redistribution of focal adhesions towards the cell cortex. It is well known that depolymerization of microtubules in quiescent fibroblasts leads to an intense formation of focal adhesions TUESDAY followed by dissociation of these structures after microtubule repolymerization. To investigate a possible role for myosin-Va in the formation step or dissociation of focal adhesions, human fibroblasts from a healthy individual (control) or from a patient with deleterious mutation in the MYO5A gene were left 48 hours in quiescence and treated with nocodazole for 4 hours. Confocal microscopy analysis of treated cells stained for the focal adhesion marker, talin, showed no differences between mutant and wild-type cells in the ability of focal adhesion formation nor in the disassemble of focal adhesions observed concomitantly to microtubule regrowth, 60 minutes after washing out the nocodazole. The same assay was performed with myosin-Va knocked-down WM793 human melanoma cells. Knock-down was mediated by a lentiviral vector carrying a short-hairpin RNA targeted to MYO5A mRNA (shMYO5A) or, as control, an irrelevant shRNA against bacterial LacZ. We observed that, after treatment with nocodazole, lamelipodia from WM793-shLacZ cells had numerous peripheral focal adhesions, as expected, whereas most of myosin-Va knocked-down cells rather showed ruffled edges and lacked focal adhesions. These data suggest that myosin-Va is required for formation of focal adhesions in melanocytic cells, but not in fibroblasts. Our laboratory has previously showed that the MYO5A-null fibroblasts used here overexpress myosin-Vb and Vc. Therefore, future studies should be conducted to answer whether in fibroblasts the formation of focal adhesions is compensated by one of the other two myosin-V paralogs, and also to uncover the mechanisms by which myosin-V functions in focal adhesion formation in melanocytic cells.

Financial support: FAPESP, CNPq, CAPES, FAEPA

2244 Characterization of the kinesin KIF9 in mammalian cell cycle progression. J. E. Hoke1, M. E. Rivera2, A. M. Billow1, L. Alsina1, N. J. Quintyne1; 1Harriet L. Wilkes Honors College, Florida Atlantic University, Jupiter, FL, 2Florida Atlantic University-Honors Coll, Jupiter, FL

The kinesin family of microtubule motors is divided into subfamilies based upon structure and function. KIF9 is the founding member of the Kinesin-9 family, a largely uncharacterized group of kinesins. It was originally identified by sequence homology to other kinesins and shown to interact with the Ras-like GTPase Gem (Piddini et al., 2001). Subsequent studies have indicated that KIF9 is vital for flagellar movement in Trypanasoma brucei, likely via interaction with dynein (Demonchy et al., 2009) as well as podosome regulation (Cornfine et al., 2011). Additionally, Kinesin-9 family members have also been proposed to function in proper microtubule organizing center (MTOC) positioning and timely mitotic entry. We have examined KIF9 function in mammalian cells by using siRNA-mediated knockdown and overexpression. Our analysis has focused on normal progression through S phase and subsequent transition to, and progression through, mitosis. We have seen several effects on cell cycle progression. First, a transfected, synchronized population of cells shows a notable delay in mitotic entry. Second, there is a delay in normal S phase progression. Third, we see that there is a change in mitotic index and decrease in the number of anaphase and telophase cells. Finally, we see an increase in the rate of multinuclearity, a hallmark of failure of cytokinesis. Taken together, we propose that KIF9 is required for normal entry and completion of mitosis, possibly via regulation of the contractile ring.

TUESDAY

2245 Kinesin-5 promotes disassembly of long kinetochore microtubules. E. S. Tubman1, A. Claas1, D. J. Odde1; 1Biomedical Engineering, University of Minnesota, Minneapolis, MN

Kinesin-5 is a plus end directed, homotetrameric motor protein that is well known for its role in mitosis. It is widely accepted that the motor protein crosslinks antiparallel microtubules, where it walks towards the plus end of each, sliding them apart and creating a bipolar spindle. Additionally, kinesin-5 in budding yeast plays a role in maintaining a spatial gradient of kinetochores (protein complexes that link chromosomes to dynamically assembling microtubule plus ends) along the metaphase spindle. Kinesin-5 establishes this gradient by promoting disassembly of long kinetochore microtubules. We are interested in determining if kinesin-5 also controls the spatial gradient of kinetochore microtubule assembly in other organisms. To test this hypothesis we used the infectious yeast Candida Albicans imaged by confocal fluorescence microscopy to measure the fluorescence distribution of the tubulin in control cells and mutant cells, where one of the two copies of the KIP1 gene (kinesin-5 in Candida) was knocked out. In control cells, a bilobed fluorescence distribution indicated that the ends of the kinetochore microtubules are drawn to an attractor zone located between the spindle pole and the spindle equator, consistent with the distribution in budding yeast. In mutant cells, we found a homogeneous fluorescence distribution, indicating a loss of the attractor zone. Our initial results here suggest that kinesin-5 acts as a kinetochore microtubule length-dependent disassembly promoter in C. Alibcans to mediate the proper alignment of chromosomes across the spindle equator during metaphase.

2246 Casein Kinase 2 Reverses Tail-Independent Inactivation of Kinesin-1. J. Xu1, B. Reddy2, P. Anand2, Z. Shu2, S. Cermelli2, M. Mattson2, S. Tripathy2, M. Hoss2, N. James2, S. King3, L. Huang2, L. Bardwell2, S. Gross2; 1University of California, Merced, Merced, CA, 2University of California, Irvine, Irvine, CA, 3University of Central Florida, Orlando, FL

Kinesin-1 is a plus-end microtubule-based motor, and defects in kinesin-based transport are linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a direct head-tail interaction, but is believed to be active otherwise. Here we report a tail-independent inactivation of kinesin, reversible by the disease-relevant signaling protein, casein kinase 2 (CK2). The majority of initially active kinesin (native or tail-less) loses its ability to bind/interact with microtubules in vitro, and CK2 reverses this inactivation (~ 4-fold) without altering kinesin’s single motor properties. This activation pathway does not require motor phosphorylation, and is independent of head-tail autoinhibition. In cultured mammalian cells, reducing CK2 expression, but not kinase activity, decreases the force required to stall lipid droplet transport, consistent with a reduction in the number of active motors. These results provide the first direct evidence of a protein kinase up-regulating kinesin-based transport, and suggest a novel pathway for regulating the activity of cargo-bound kinesin.

2247 Protein-Protein Interactions within Dynactin’s Shoulder/Sidearm. S. A. Ketcham1, T. A. Schroer1; 1Biology, Johns Hopkins University, Baltimore, MD

Dynactin is a multi-subunit complex that acts as a cargo adaptor and processivity enhancer for the microtubule-based motors dynein and kinesin-2. Dynactin interacts with these motors through the p150Glued subunit, which along with dynamitin and p24 comprise the shoulder/sidearm. Disruption of dynactin with the chaotropic salt KI yields two subcomplexes, TUESDAY the shoulder, which contains p24 and dynamitin, and the shoulder/sidearm, which contains p24, dynamitin and p150Glued. Although the shoulder/sidearm is released from dynactin as a stable subcomplex, the protein-protein interactions that determine its structure remain undefined. Our current work suggests that p24 serves as a bridge between dynamitin and p150Glued. Recombinant p24 is insoluble, but solubility can be restored by stepwise dialysis out of denaturant in the presence of either dynamitin or a fragment of p150Glued. Without either binding partner, p24 remains insoluble when subjected to this procedure. We have taken advantage of the necessity of a binding partner for p24 solubility to explore p24 structure. Renaturation of a series of p24 fragments with dynamitin or p150Glued, coupled with gel filtration and velocity sedimentation to analyze the complexes that form, has allowed us to map the domains of p24 necessary for its interactions with its binding partners. These results are beginning to yield a better understanding of the structure of the shoulder/sidearm and will be exploited to gain insight into how the shoulder/sidearm anchors to the Arp1 minifilament.

Cell Division

2248 Cellular Size Checkpoints During G1/S- and G2/M-Transition Mediate Contact Inhibition Of Proliferation. S. J. Streichan1, C. R. Hoerner1, D. Holzer1, T. Schneidt1, L. Hufnagel1; 1Cell Biology & Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

Tissue and organ development is characterized by tightly controlled patterns of coordinated cell growth, cell division, and cell rearrangements. Non-cancerous epithelial cells in culture show a similar behavior in terms of regulated proliferation: initially, they proliferate at high rate and compensate for their reduced size after cell division by cell growth. Then, after reaching confluence cells undergo size-reductive divisions and the rate of cell division drops markedly, a phenomenon called contact inhibition of proliferation (CI). However, it remains elusive how cells coordinate growth and division and how during CI the cell cycle of individual cells is regulated according to the cell density in the cell collective. Here we use fluorescent time-lapse live cell imaging, quantitative image analysis, and biophysical modeling to study the link between cell growth and cell proliferation in Madin Darby canine kidney (MDCK) cells. Based on the predictions of our physical description we perform biophysical tests including active and passive manipulation of cell size. First, we find that cells have a critical size when entering S phase. Second, reduced cell size after reentry into the cell cycle results in prolonged G2 phase. Third, direct manipulation of cell size by stretching of a contact-inhibited cell collective is sufficient to reverse CI as cells reenter the cell cycle and progress through mitosis. We suggest that mechanical constraints in tissues may delay cell cycle reentry and progression by activating size checkpoints acting during the G1/S- and G2/M-transition.

2249 Concentration-dependent Correlation of Cell Cycle and Apoptosis Impacts of Anti-Cancer Compounds. K. Tran1, K. Gillis1, A. Khan1, J. Clor1, K. Tyagarajan1; 1EMD Millipore, Millipore Corporation, Hayward, CA

Recent research has demonstrated the delicate relationship between the opposing processes of cell cycle progression and apoptosis. Several anti-cancer compounds have been demonstrated to cause apoptosis and subsequently have been shown to cause arrests in specific phases of TUESDAY the cell cycle. The study of cell cycle impacts in addition to apoptosis can thus bring meaningful understanding of the diverse effects of cellular modulators and compound treatment and provide deeper understanding of the inter-relationship between cell cycle and apoptosis. In this study, we evaluated a series of anti-cancer compounds with known apoptotic activity to determine the sequence of impact on cell cycle versus apoptosis induction for multiple cell types. Jurkat and HeLa cells were treated with multiple concentrations of compounds and the impact on cell cycle and apoptosis (based on Annexin V binding) on different dosages of treatment were evaluated. Our studies demonstrate that multiple compounds could modulate different cell cycle phases depending on the concentration and cell type used. Further the sequence of impacts on cell cycle and apoptosis could be very different. Our results here demonstrate that treatment with Etoposide, a DNA topoisomerase II inhibitor at low concentrations results in the arrest of cells in the G2M stage under conditions where little apoptosis is observed. With increased concentration of etoposide, increase in the % of cells in the S phase of cell cycle is observed along with increased apoptosis. Thus for etoposide, cell cycle impacts were observed at significantly lower concentration than impacts on apoptosis. Staurosporine demonstrated both an arrest in G2/M phase with concurrent impacts on apoptosis at low concentrations, higher concentrations showed increase in the percentage of cells in the G0/G1 phase. Gambogic acid, a potential anti-cancer agent resulted in significant apoptosis and death but no impacts on cell cycle were observed. Nocodazole demonstrated impacts on cell cycle and apoptosis concurrently. Parallel analysis of apoptosis data along with cell cycle effects will be critical for understanding the linkage between these processes and provide for a more complete understanding of impacts of modulators of cell cycle and cell death and the design of effective cellular study models.

2250 Phospho-regulation of the anillin-related scaffolding protein Mid1 in fission yeast cytokinesis. A. K. DeWitt1, E. Kranz1, J. Phelan1, B. Nader1, K. L. Gould2, D. M. Clifford Hart1; 1Cell and Molecular Biology, Grand Valley State University, Allendale, MI, 2Department of Cell and Developmental Biology, Howard Hughes Medical Institute & Vanderbilt University School of Medicine, Nashville, TN

During cytokinesis, physical separation of one cell into two identical cells occurs through constriction of a protein-rich ring structure, called the contractile ring. The contractile ring is composed of F-actin, type II myosin and more than 100 cytokinetic ring proteins. In fission yeast, the anillin-related protein Mid1 plays a critical role in organizing the early steps of contractile ring formation and functions as a scaffold to bridge the cell cortex with the contractile ring. Cells lacking mid1 form off-centered, highly disorganized ring structures and exhibit severe cytokinesis defects. Coincident with its cortical accumulation, Mid1 becomes hyper- phosphorylated. Our previous research demonstrates that cyclin-dependent kinase, Cdc2, and the polo-like kinase, Plo1, directly phosphorylate Mid1. In addition to consensus phosphorylation motifs for Plo1 and Cdc2, Mid1 contains several RXXS motifs, which fit the phosphorylation consensus sequence for Sid2 kinase. Sid2 is the most downstream kinase in the Septation Initiation Network signaling cascade, which signals from the spindle pole body to trigger constriction of the contractile ring. To identify specific sites of phosphorylation, a combination of site-directed mutagenesis (serine to non-phosphorylatable alanine) with mass spectroscopy and two-dimensional phosphopeptide mapping were used. Both methods identified multiple Sid2 phosphorylation sites within Mid1. To analyze the significance of Sid2 phosphorylation in vivo, phospho-site mutants were generated at the endogenous mid1 locus and examined for cell division defects. Interestingly, phospho-site mutants displayed cell division defects, including sensitivity to low dose latrunculin A and disorganized actin localization. While phospho-site TUESDAY mutants maintained the mitotic spindle checkpoint, the majority of cells exhibited severe polarity phenotypes and Mid1 protein levels increased when compared to checkpoint activated cells expressing wild-type Mid1. Given that Mid1 departure from the contractile ring coincides with Sid2 relocalization to the division site, Sid2 may temporally regulate the interaction of Mid1 with the membrane or other contractile ring components; our initial experiments favor Sid2 regulating Mid1 dissociation from the cell cortex.

2251 Regulation of Myosin-II Dynamics and its Implication in the Assembly of the Cytokinesis Machine in S. cerevisiae. C. Wloka1,2, E. Bi1; 1Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, 2Institut für Chemie und Biochemie, Freie Universität Berlin, Berlin, Germany

Cytokinesis in animal and fungal cells utilizes a contractile actomyosin ring (AMR) to power the ingression of the cleavage furrow. The AMR consists of myosin-II and actin filaments. However, how the AMR is assembled and disassembled during the cell cycle is not clearly understood in any organism. We have analyzed the dynamics of 13 cytokinesis proteins during the cell cycle, including the sole myosin-II, Myo1, in the budding yeast Saccharomyces cerevisiae by fluorescence recovery after photo-bleaching. We found that Myo1 is dynamic until the onset of anaphase when it becomes progressively immobile. Further analysis indicates that the immobility of Myo1 during cytokinesis does not require its motor domain and actin filaments but requires the putative assembly domain near its C-terminus, which regulates Myo1 localization and cytokinesis. Thus, the putative higher-order assembly is important for Myo1 localization, dynamics, and its role in cytokinesis. These data also indicate that the AMR is progressively disassembled during its constriction without high turnover. We also found that proteins involved in AMR assembly and septum formation, such as Iqg1 (IQGAP), Hof1 (a F-BAR protein), and Inn1 are immobile whereas those involved in actin ring assembly (formin and tropomyosin) are highly dynamic during cytokinesis. Strikingly, the immobility of all examined proteins depends on the presence of Myo1. This and the observation that Myo1 is required for actin ring assembly suggest that Myo1 plays a scaffolding role in the assembly of the cytokinesis machine.

2252 Orbit/CLASP plays a role as linker connecting cortical microtubules and actomyosin ring in Drosophila male meiosis. D. Kitazawa1, D. Hayashi1, C. Miyauchi1, Y. Inoue1; 1Kyoto Institute of Technology, Insect Biomedical Research Center, Kyoto, Japan

Microtubules (MTs) near the cell cortex are essential for continuous constriction of the contractile ring in cytokinesis. However, the way in which MTs interact with the contractile apparatus has not been completely unveiled. In previous studies, we have shown that Orbit is required for stabilization of the MT populations essential for cytokinesis. Therefore, it is possible to speculate that Orbit plays an important role in cleavage furrow (CF) initiation in Drosophila. A body of evidence indicating that Orbit possesses a binding activity to both MTs and F-Actin in Drosophila and Mammalian cells implies another role in initiation of cytokinesis. In this study, we carefully examined localization of GFP-tagged Orbit in Drosophila male meiosis. We showed that Orbit changes its localization from around the spindle matrix towards prospective CF regions at late anaphase. It continuously stayed on the ring in the middle of the cell. Although formation of the Orbit ring depends on both MTs and F-Actin, Orbit maintains its localization on the CF even in the absence of Myosin II. We showed that Orbit directly binds to F-Actin and the heavy chain of Myosin II on the CF. Surprisingly, ectopic localization of Orbit on the cortex TUESDAY induces contractile ring formation in situ and it was followed by CF ingression. Accumulation of Orbit on the CF is dependent on MTs and F-Actin. In order to look for transport proteins conveying the Orbit toward the CF, we then performed knockdown experiments to examine whether all known MT-based motors and proteins displaying a genetic interaction with Orbit are required for cytokinesis. Among several genes that we have shown are critical for cytokinesis, we especially focused on CLIP190 and its relationship with Orbit. We found that CLIP190 is required for transport of Orbit to the prospective CF site independent of the MT binding domain of Orbit. Our current data suggests that Orbit plays a central role in contractile ring formation as a linker connecting MTs with Actomyosin ring.

2253 Fission yeast cyclin-dependent kinase, Cdc2, regulates the Mid1 scaffolding protein during cytokinesis. J. B. Hackett1, J. Phelan1, K. L. Gould2, D. M. Clifford Hart1; 1Cell and Molecular Biology, Grand Valley State University, Allendale, MI, 2Howard Hughes Medical Institute, Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN

Cytokinesis is the final stage of cell division and utilizes a highly conserved actin-myosin contractile ring that forms medially around the cell to pinch the cell membranes together resulting in physical separation of one cell into two. The conserved anillin homolog, Mid1, is a foundational part of the fission yeast contractile ring, functioning as the initial organizer and anchoring the structure to the cell membrane. Mid1 mutants show severe defects in contractile ring assembly and medial placement of the division site. Coincident with contractile ring formation, Mid1 is hyper- phosphorylated and our previous research identified at least two mitotic kinases that directly phosphorylate Mid1. Mid1 phosphorylation by the cyclin-dependent kinase Cdc2 at Threonine 517 facilitates association between the polo-like kinase, Plo1, and the C-terminus of Mid1. To identify additional Cdc2 phosphorylation sites within Mid1, we implemented a combination of mutagenesis and two-dimensional phosphopeptide mapping. Mutation of Serine 28 to a non-phosphorylatable alanine residue reduced phosphorylation of Mid1 N-terminus and this site fits a consensus motif for Plo1 binding. Fission yeast cells expressing Mid1 phospho-site mutations show sensitivity to low dose latrunculin A treatment and bypass the mitotic spindle checkpoint. Surprisingly, phosphosite mutants with an activated mitotic spindle checkpoint continue through mitosis but accumulate as septated cells. This research highlights a regulatory role for Plo1 and Mid1 in early and late stages of cytokinesis.

2254 Centrosomal Protein 55 Stability Is Negatively Regulated BY P53 Through POLO-Like Kinase 1. Y-C. Chang1,2, P. Ouyang3,4; 1Graduate Institute of Clinical Medicine Sciences, Chang Gung University, Guei-San, Taiwan, 2Nuclear Medicine and Molecular Imaging Center, Chang Gung Memorial Hospital, Guei-San, Taiwan, 3Anatomy, Chang Gung University, Guei-Shan, Taiwan, 4Molecular Medicine Research Center, Chang Gung University, Guei-San, Taiwan

Centrosomal protein 55 (Cep55), which is localized to the centrosome in interphase cells and recruited to the midbody during cytokinesis, is a regulator required for the completion of cell abscission. Upregulation of Cep55 and inactivation of p53 occur in the majority of human cancers, raising the possibility of a link between these two genes. In this study, we evaluated the role of p53 in Cep55 regulation. We demonstrated that Cep55 expression levels are well correlated with cancer cell growth rate, and that p53 is able to negatively regulate Cep55 protein and promoter activity. Downregulation of expression of Cep55 was accompanied by repression of polo-like kinase 1 (Plk1) levels due to p53 induction. Overexpression of Plk1 and knockdown TUESDAY of p53 expression both enhanced the post-translational protein stability of Cep55. BI 2356, a selective Plk1 inhibitor, however, prevented Cep55 accumulation in p53 knockdown cells, while persistently keeping Plk1 levels elevated. Our results, therefore, indicate the existence of a p53- Plk1-Cep55 axis in which p53 negatively regulates expression of Cep55 through Plk1 which, in turn, is a positive regulator of Cep55 protein stability.

2255 Cleavage furrow organization requires PIP2 mediated recruitment of anillin. J. Liu1, D. Ceccareli2, G. Fairn3, F. Sicheri1,2, A. Wilde1; 1Molecular Genetics, University of Toronto, Toronto, ON, Canada, 2SLRI, Toronto, Canada, 3Hospital for Sick Children, Toronto, ON, Canada

Anillin is a key component of the cytokinetic machinery that is essential for the successful completion of cytokinesis. Anillin is a multi-domain protein that has the potential to link the actomyosin and septin cytoskeletons. It also has a PH domain involved in targeting anillin to the cleavage furrow. We sought to examine the mechanism by which the PH domain targets anillin to the cleavage furrow. We find that the PH domain of anillin interacts with phosphatidylinositol phosphate lipids (PIPs), including PI(4,5)P2, which is enriched in the furrow. Reduction of cellular PI(4,5)P2 or mutations in the PH domain of anillin that specifically disrupt the interaction with PI(4,5)P2, interfere with the localization of anillin to the furrow. Reduced expression of anillin disrupts symmetric furrow ingression in the middle of the cell. However, symmetrical furrow ingression and division plane stability can be rescued in anillin depleted cells by targeting ectopically expressed anillin to the furrow using an alternate PI(4,5)P2 binding module. These data demonstrate an anillin-PI(4,5)P2 interaction targets anillin to the cleavage furrow and is required for stable, symmetrical ingression of the furrow during cytokinesis.

2256 Sauron is the Drosophila orthologue of the oncogene GOLPH3. S. Sechi1, V. Mattei2, G. D. Raffa3, G. Belloni3, G. Colotti1, M. G. Giansanti1; 1Istituto di Biologia e Patologia molecolari c/o Università Sapienza, CNR, Roma, Italy, 2Consorzio Universitario Sabina Universitas Rieti, Rieti, Italy, 3Dipartimento di Biologia e Biotecnologie, Università Sapienza, Roma, Italy

Drosophila male meiosis provides an excellent cell system for the molecular dissection of cytokinesis. Drosophila spermatocytes are considerably larger than most somatic cells and the spindle assembly checkpoint is not stringent in spermatocytes, allowing the characterization of genes whose products are required for multiple stages of cell division in addition to cytokinesis. In the course of a large screen for mutants defective in spermatocyte cytokinesis, we have isolated sauron (sau), a male sterile mutant with defects in both central spindle formation and contractile ring assembly. A P element lethal insertion (l(2)s5379) in the CG7085 gene failed to complement the sau1 mutation. DNA sequencing of the sau1 allele revealed a single point mutation in CG7085, resulting in a Glu-Lys amino acid substitution at the C-terminus of the predicted protein. These results together indicate that sau encodes a polypeptide of 294 amino acids which is 70% identical to human GOLPH3 (alias Gpp34, Gmx33, MIDAS). GOLPH3 is a Golgi protein which has recently been recognized as a potent oncogene amplified in many human cancers and implicated in cellular transformation via changes in the activity of mTOR. We have generated transgenic flies that express GFP-Sau under the control of tubulin promoter. The GFP-Sau protein rescues the defects of sau1, confirming that the cytokinesis phenotype is the consequence of alterations in the Drosophila GOLPH3 ortholog. We have also raised polyclonal antibodies against Sauron and started immunofluorescence analysis in fixed spermatocytes. Immunostaining of primary spermatocytes revealed that the endogenous protein TUESDAY is enriched in the Golgi of prophase spermatocytes and accumulates at the cleavage site during early telophase suggesting an early role in cytokinesis.

2257 Microtubule-free cytokinesis in vertebrate somatic cells: the roles of Plk1 and Aurora B kinase. E. H. Hinchcliffe1; 1Hormel Institute, University of Minnesota, Austin, MN

Cytokinetic furrow formation requires the initiation of cortical contractility, and its restriction to the cell’s equator. Following anaphase onset spindle microtubules (MTs) and the mitotic kinases Aurora B (Aur B) and Polo-like kinase 1 (Plk1) are thought to drive furrow formation, but how these activities are coordinated remains poorly understood. Here we use live-cell imaging and a temperature-based spindle MT depolymerization/re-growth assay to examine cytokinesis in vertebrate somatic cells. Spindle MTs disassembled in either early anaphase (<2 min), mid anaphase (2 – 4 min) or later anaphase (4 – 6 min) reform upon warming, build a midzone, and complete cytokinesis. When Plk1 or Aur B kinase activity is inhibited in these assays, astral MTs reform, but midzone MTs do not, and no cleavage furrow forms. When spindle MT re-assembly is blocked by nocodazole (Noc) beginning in early anaphase, cortical contractility is initiated, but a defined furrow does not form. This contractility requires Plk1 activity, but not Aur B. When MT re-assembly is blocked in late anaphase cells, a single equatorial furrow assembles upon re- warming. However, if Plk1 activity is also blocked, these cells form a “half-furrow”, where only one surface of the equatorial cortex ingresses; ingression is slowed and these cells fail cleavage. Together, these studies reveal important insights into the early stages of cytokinesis, and how the activities of Aur B, Plk1 and spindle MTs are coordinated in this process.

2258 Dissecting the biomechanical mechanism underlying the actomyosin ring contraction for cytokinesis in budding yeast. I. M. Pinto1, B. Rubinstein1, A. Kucharavy2, J. R. Unruh1, R. Li1; 1Stowers Institute for Medical Research, Kansas City, MO, 2Ecole Polytechnique, Palaiseau, France

Animal and yeast cells use a contractile ring that is attached to the plasma membrane to create a cleavage furrow that partitions one into two in the latest step of the cell cycle. The contractile ring is a network of actin and myosin-II motor (Myo1 in budding yeast) filaments embedded in a complex and compact protein core structure at the cell division site. The molecular and biophysical mechanisms underlying Myo1 function at the cell division site with actin ring contraction are not clear. In this work, using a mathematical model and experimental analysis of actin and Myo1 kinetic profiles during contraction we show that depolymerization of actin filaments, as opposed to the classical motor-driven actin sliding model, provides the major driving force for actomyosin ring constriction in budding yeast cell division.

2259 WITHDRAWN

TUESDAY

2260 Delivery of the cytokinetic signal to the plasma membrane – analysis of the RhoGEF Ect2. K-C. Su1, T. Takaki1, M. Petronczki1; 1Cell Division and Aneuploidy, Cancer Research UK - London Research Institute, Clare Hall, Potters Bar, England

Cytokinesis is the final stage of cell division and leads to the birth of two individual daughter cells. During the process of cytokinesis, the ingression of cleavage furrow divides the cytoplasm of the mother cell.

In animal cells, cytokinesis is controlled by activation of the small GTPase RhoA, which initiates the formation of the contractile ring at the equatorial cell cortex during anaphase. It is well established that the mitotic spindle determines the position and activity of the contractile machinery at the plasma membrane. However, the precise mechanism by which the microtubule-associated signalling complexes control the cytokinetic machinery at the membrane is poorly understood.

The conserved RhoGEF protein epithelial cell transforming sequence 2 (Ect2) is essential for cleavage furrow formation and RhoA activation at the equatorial cortex. Ect2, through its N- terminal tandem BRCT domains, interacts with the centralspindlin subunit MgcRacGAP leading to Ect2 recruitment to the spindle midzone. In its C-terminal region, Ect2 contains a guanine nucleotide exchange factor domain and a pleckstrin homology domain, the detailed function of either remain to be understood. We are using genetic, cell biological, and biochemical assays to address how Ect2 initiates cytokinesis at the plasma membrane in a spatially and temporally controlled manner.

Our analysis will provide new insights into the molecular mechanism that delivers the cytokinetic signal to the equatorial cortex during anaphase in animal cells.

2261 Control of Mitotic Exit Network (MEN) signaling in S. cerevisiae meiosis. M. Attner1, A. Amon1,2; 1David H. Koch Institute for Integrative Cancer Research, Dept of Biology, Massachusetts Inst Technology, Cambridge, MA, 2Howard Hughes Medical Institute

When cells exit from mitosis, cyclin dependent kinases (CDKs) are inactivated. In budding yeast, Cdc14, a protein phosphatase that is released from its inhibitor in the nucleolus during anaphase, triggers CDK inactivation. Cdc14 release is controlled by the mitotic exit network (MEN), an essential GTPase signaling cascade. How CDK activity is regulated in meiosis is less clear. During meiosis, a diploid cell undergoes two rounds of chromosome segregation following one round of DNA replication, resulting in the formation of four haploid gametes. At the meiosis I-meiosis II transition, CDK activity is thought to be low enough to allow the meiosis I spindle to dissemble but high enough to prevent DNA re-replication. To understand how the cell achieves this balance at the meiosis I-meiosis II transition, we have characterized the mitotic exit network (MEN) in meiosis. We have found that the MEN is not required and not active at the meiosis I- meiosis II transition. However, the MEN becomes active during anaphase II and is required for timely exit from meiosis II. We further show that MEN signaling in meiosis is regulated differently from MEN signaling in mitosis. Whereas localization to the spindle pole body (SPB) is critically important for MEN signaling in mitosis, meiotic MEN signaling relies on the regulated binding of downstream MEN components and does not depend on SPB components. Taken together, our data suggest that exit from meiosis I and II are regulated by different mechanisms than exit from mitosis, which may help establish permissive conditions for the meiotic divisions. TUESDAY

2262 A midzone-based ruler adjusts chromosome compaction to anaphase spindle length. G. E. Neurohr1,2, A. Naegeli2, I. Titos1, D. Theler2, B. Greber2, Y. Barral2, M. Mendoza1; 1Center of Genomic Regulation (CRG), Barcelona, Spain, 2Institute of Biochemistry, ETH Zurich, Zurich, Switzerland

For successful partitioning of the genetic material during cell division, mitotic chromosomes must condense enough to be segregated by the fully elongated spindle, while spindles must elongate enough to segregate the bulkiest chromosome. Although both maximal spindle length and chromosome size can vary even within a given species, we know little about how cells cope with these changes to ensure faithful segregation of the genetic material under such conditions. We have managed to fuse the two longest chromosomes of the budding yeast S. cerevisiae to generate a yeast strain containing an oversized chromosome. The long arm of this chromosome exceeds the longest wild type arm by 50%. Remarkably, these cells were able to cope with the presence of the long chromosome without delaying anaphase or increasing spindle length, and without slowing down their growth rate. Instead, cells adapted through increasing the condensation of the compound chromosome specifically. Consistently, cells carrying the fused chromosomes became more sensitive to loss of condensin activity. In addition we observed that length-dependent stimulation of condensation took place during anaphase and depended on the spindle midzone, aurora/Ipl1 activity, and phosphorylation of histone H3 on Ser10. We propose that the spindle midzone functions as a ruler that adapts the condensation of long chromosome arms to spindle length to promote their faithful segregation during anaphase, regardless of variations in chromosome or spindle length.

2263 Identifying Novel Kinases Involved in Mitotic Progression Using Kinome-wide Screens. H. Chen1, H. Ma1, Y. Tsang1, C. Cheung1, S-S. Ng1, J. Zhang1, R. Poon1; 1Division of Life Science, Hong Kong University of Science and Technology, Hong Kong, China

The main purpose of mitosis is to segregate sister chromatids into two nascent cells, such that each daughter cell inherits one complete set of chromosomes. Protein phosphorylation is one of the main post-translational regulation mechanisms of mitosis. A number of protein kinases such as cyclin-dependent kinases, Aurora kinases, Polo-like kinases (PLKs), NIMA-related kinases, and spindle assembly checkpoints kinases BUB1, BUBR1 and MPS1, are known to be involved in the control of various mitotic processes. Dysfunction of phosphorylation events regulated by these protein kinases would cause abnormal chromosome segregation as well as genome instability. However, the whole repertoire of mitotic kinases probably remains to be deciphered. In this study, we sought to identify novel mitotic kinases with RNAi approaches. Mouse fibroblasts NIH3T3 modified with histone H2B-GFP expression was transfected with a mouse kinome siRNA library to deplete individual kinases. Novel kinase candidates essential for mitosis progression were identified according to an increased mitotic index. Subsequently, the candidate mitotic kinases were compared with a previous screen in Drosophila. Several matched candidates from the mouse and Drosophila screens were chosen for further analysis in human cancer cells. Approaches including flow cytometry, live cell imaging, and immunostaining were applied to confirm the functional impact of these candidates in mitosis. These results indicate that in addition to the set of well-studied kinases, several novel protein kinases may also play critical role in mitosis.

TUESDAY

2264 A stochastic model of kinetochore–microtubule attachment accurately describes chromosome segregation. G. Gay1, T. Courtheoux1, C. Reyes, 1, S. Tournier1, Y. P. Gachet1; 1LBCMCP, CNRS, Toulouse, France

Erroneous attachments of spindle microtubules to kinetochores are frequent in early mitosis. Most of these are corrected before the onset of anaphase by a mechanism involving the protein kinase Aurora B which destabilizes kinetochore microtubules in the absence of tension between sister chromatids. Here, we describe a minimal mathematical model of mitotic chromosome segregation based on stochastic attachment and detachment of kinetochore microtubules. The model accurately reproduces the process and timing of correct chromosome bi-orientation and segregation seen in fission yeast. Prevention of attachment defects requires both appropriate kinetochore orientation and an Aurora B-like activity. The model also reproduces abnormal chromosome segregation behavior (due, for example, to inhibition of Aurora B). It predicts that, in metaphase, merotelic attachment is prevented by a kinetochore orientation effect and corrected by an Aurora B-like activity whereas in anaphase it is corrected through unbalanced forces applied to the kinetochore. These unbalanced forces are sufficient to prevent aneuploidy and the ‘cut’ phenotype.

2265 High Throughput imaging of cell based assays and cellular models using microplate cytometry. P. Wylie1, D. Caracino2, D. Onley1, W. Gaisford1; 1TTP Labtech Ltd, Royston, United Kingdom, 2TTP Labtech, Cambridge, MA

Microscope-based, high-content instruments are used for many cell based assays in high content screening (HCS). Using Acumen eX3® with its wide field objective lens and “on-the-fly” laser scanning capabilities, it is possible to rapidly scan entire wells in a wide range of cell based assays relevant to the drug discovery industry including cell cycle analysis, cell surface markers and cell migration.

In this poster, data is shown demonstrating the use of Acumen to assess the effect of inhibitory compounds on the mitotic index and cell cycle. In addition, with its high depth of field, it has also shown to be capable of scanning larger organisms such as C.elegans and even Zebrafish to provide information on multi-cellular drug interactions at an early screening stage. For efficient and rapid resolution most assays require the use of higher resolutions which entail lengthy read times, using single colours and analyzing only a small percentage of the total number of cells in a well to keep plate read times at a minimum. Recent studies also demonstrate that Acumen is capable of rapidly analysing complex cellular or animal models, such as angiogenic tube formation, C. elegans or drosophila larvae, thus enabling the development of whole organism or tissue based assays models for HCS.

Built in software offers the flexibility of exporting whole well open source TIFF images for batch processing by third party image analysis software packages. This new screening paradigm represents a major breakthrough in how microplate cytometry can be applied to complex cellular models since rapid cytometric analysis can now be combined with image-processing methodology.

TUESDAY

2266 Condensin structures chromosomal DNA through topological links. S. Cuylen1, J. Metz1, C. H. Haering1; 1Cell Biology & Biophysics Unit, EMBL, Heidelberg, Germany

The multisubunit condensin complex is essential for the structural organization of eukaryotic chromosomes during their segregation by the mitotic spindle, but the mechanistic basis for its function is not understood. To address how condensin binds to and structures chromosomes, we have isolated from Saccharomyces cerevisiae cells circular minichromosomes linked to condensin. We find that either linearization of minichromosome DNA or proteolytic opening of the ring-like structure formed through the connection of the two ATPase heads of condensin’s structural maintenance of chromosomes (SMC) heterodimer by its kleisin subunit eliminates their association. This suggests that condensin rings encircle chromosomal DNA. We further show that release of condensin from chromosomes by ring opening in dividing cells compromises the partitioning of chromosome regions distal to centromeres. Condensin hence forms topological links within chromatid arms that provide the arms with the structural rigidity necessary for their segregation.

2267 Antioxidants rescue carcinogen induced mitotic defects in both chromosomally stable and unstable cells. I. S. Griffin1, T. J. Yates1, N. J. Quintyne1; 1Honors College, Florida Atlantic University, Jupiter, FL

Tumor cells are characterized by an increase in genomic instability, brought about by both chromosomal rearrangement and chromosomal instability. Both of these broad changes can be induced by exposure to carcinogens. During mitosis, cells can exhibit lagging chromosomes, multipolar spindles or anaphase bridges, all of which contribute to genomic rearrangement. We have studied the link betweenexposure to carcinogen and prevalence of mitotic defect in both chromosomally stable and unstable cell lines. We have exposed UPCI:SCC103 oral cancer cells, MES-SA uterine cancer cells and RPE normal retinal cells to vinyl chloride,tamoxifen and aflatoxin at varying concentrations. When treated with carcinogens, we see a preferential increase in the frequency of lagging chromosomes, although rates of other mitotic defects are also higher. In addition, we have examined the effects of antioxidants on decreasing the frequency of mitotic defect: analysis on chromosomally unstable cells with or without carcinogen treatment as well as chromosomally stable cells after carcinogen treatment indicates that Beta- carotene, vitamin C and vitamin E are all effective at reducingrates of mitotic defect. We are also investigating whether pre-treatment with the antioxidants will weaken the effects of carcinogen exposure in these cell lines.

2268 Bod1 inhibits Protein Phosphatase 2A-B56 during mitosis. I. M. Porter1, K. Schleicher1, J. R. Swedlow1; 1Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dundee, UK

Bod1 is a small, 22 kDa, protein that is required for resolution of syntelic attachments and proper chromosome alignment during mitosis and proper phosphorylation of MCAK by Aurora B in mitosis (Porter et al., 2007). Here we demonstrate that Bod1 has sequence similarity to Ensa and Arpp-19, two proteins recently identified as inhibitors of PP2A-B55, which are required for proper entry into mitosis (Mochida et al., 2010; Gharbi-Ayachi et al., 2010). We have now shown that Bod1 binds to the PP2A-B56 holo-enzyme during mitosis and is required for mitotic TUESDAY progression. Binding of Ensa and Arpp-19 to PP2A-B55 requires a conserved Asp residue and phosphorylation by Greatwall kinase at a conserved Ser adjacent to the Asp. The Asp residue is conserved in Bod1 (D98), whereas the Ser is not, but a consensus CDK1 site (T95) is present immediately upstream. We show that Bod1 is phosphorylated in vitro by CDK1 kinase. Expression of Bod1 D98A or T95A mutants fail to rescue Bod1 depletion whereas the T95E mutations rescues as well as expression of wt Bod1. siRNA depletion of Bod1 from HeLa cells increases localisation of PP2A-B56 to kinetochores and loss of Sgo1 and Plk1 from kinetochores. Overall, these data show that when phosphorylated by CDK1, Bod1 binds PP2A- B56 and suggest that this binding inhibits the activity of PP2A. In the presence of hyper-active kinetochore-associated PP2A, Plk1 activity is compromised and phosphorylation of downstream targets is decreased. Together Bod1, Ensa and Arpp-19 form a new class of PP2A inhibitors.

2269 Daxx and USP7: novel regulators of genomic stability, mitosis and taxanes sensitivity. S. Giovinazzi1, V. Morozov1, W. C. Reinhold2, A. M. Ishov1,3; 1Anatomy and Cell Biology, University of Florida, Gainesville, FL, 2Genomics & Bioinformatics Group, National Cancer Institute, NIH, Bethesda, MD, 3Shands Cancer Center, University of Florida, Gainesville, FL

Daxx is a multifunctional protein that plays a pivotal role in both physiological and pathological cellular processes. We previously demonstrated that cells with low levels of Daxx have reduced sensitivity to taxanes, powerful chemotherapeutic agents, by persisting in a pro-metaphase block that allows cells to escape taxane-induced cell death. In this study we dissected the mechanisms of Daxx-dependent taxanes resistance that also suggests function of this protein in mitotic progression. We show that Daxx interacts and cooperates with Ubiquitin Specific processing Protease-7 (USP7) to regulate mitosis. We demonstrate that depletion of USP7 promotes stabilization of cyclin B, aneuploidy and mitotic anomalies, as it was previously observed for Daxx. We further demonstrate that USP7 depletion results in reduced stability of the mitotic E3 ubiquitin ligase Checkpoint with Forkhead and RING finger (CHFR). Consequently cells depleted by USP7 accumulate CHFR substrate, Aurora A kinase that has a crucial role in mitotic progression. We conclude that Daxx and USP7 are necessary to regulate proper execution of mitosis and their effects are at least partially mediated by CHFR and Aurora A kinase. Results from colony formation assay and in silico analysis show that USP7 expression negatively correlates with response to taxanes in cancer cell lines indicating that this protein can be used as predictive factor for taxanes response in cancer patients.

2270 Effects of actin and myosin inhibitors on PtK2 spindle length changes induced by laser microbeam irradiations across the spindle. R. Sheykhani1, M. W. Berns2, A. Forer1; 1York University, Toronto, ON, Canada, 2University of California San Diego, San Diego, CA

Cutting spindle microtubules across the entire half spindle using a 532 nm pulsed picosecond laser microbeam causes the irradiated spindle pole to move inward (N. Baker PhD thesis, University of California, San Diego 2010). This usually is followed by the inward movement of the un-irradiated pole. We tested whether the inward movement of the poles might be due to the function of actin and myosin: we pharmacologically poisoned the actin-myosin system using anti-actin drugs Cytochalasin D (CD), Latrunculin B (LatB), or Jasplakinolide (JASP), or anti- myosin drugs BDM and Y-27632, and then irradiated across the entire half spindle. All these drugs altered the inwards movements of both poles. In some cells neither pole moved, in some cells only one pole moved, in some cells movements of the poles were delayed, and in only a few cells did the poles move as far as in non-treated cells. This suggests that, as in Xenopus TUESDAY cells (Woolner et al., 2008), there is an interaction between actin, myosin and microtubules to control spindle length, though in our case it might be that actin/myosin forces are antagonistic to microtubule forces.

2271 HDAC3 regulates NudC acetylation in mitosis. C. Chuang1, J. Pan2, D. H. Hawke3, S-H. Lin3, L-Y. Yu-Lee1,2; 1Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, 2Medicine, Baylor College of Medicine, Houston, TX, 3Department of Molecular Pathology, MD Anderson Cancer Center, Houston, TX

Mitosis is a highly regulated process in which errors can lead to genomic instability, a hallmark of cancer. During this phase of the cell cycle, transcription is silent and RNA translation is globally inhibited. Thus, mitosis is largely driven by post-translational modification of proteins, including phosphorylation, methylation, ubiquitination, and sumoylation. Recent proteomics studies suggest that protein acetylation is as prevalent as protein phosphorylation. However, very little is known about the role of acetylation in mitotic progression.

Using anti-acetyl-lysine immunoprecipitation of mitotic HeLa cell lysates followed by mass spectrometry, we identified 51 unique non-histone proteins, including proteins involved in RNA binding and processing, and cell cycle regulation (1). One cell cycle protein identified is NudC, a highly-conserved dynein/dynactin associated factor that plays a role in mitosis and cytokinesis. I confirmed that NudC is acetylated in mitosis by immunoprecipitation followed by immunoblot. Moreover, NudC acetylation in mitosis increased upon treatment with the HDAC inhibitor apicidin that shows specificity towards HDAC3, suggesting that NudC acetylation is regulated by HDAC3.

Interestingly, comparing unperturbed HeLa cells to mitotically-enriched HeLa cells, NudC is more acetylated outside of mitosis. Utilizing a double thymidine block and release protocol, I enriched for HeLa cells in S, G2, early mitosis (prometaphase-like), late mitosis (anaphase), and G1. I found that NudC associates with HDAC3 throughout mitosis, which may explain its lower level of acetylation during mitosis. Immunofluorecense staining confirmed that NudC and HDAC3 co-localize during early mitosis.

Mass spectrometry analysis of NudC immunoprecipitated from unperturbed HeLa cell lysate identified one acetylation site on NudC at K39. I generated NudC K39 acetyl-deficient (K39R) and acetyl-mimetic (K39Q) mutants and will decipher the role of NudC K39 acetylation in mitotic progression. Additionally, how NudC acetylation/deacetylation regulates its association with various mitotic regulators to promote mitotic progression are under analysis.

These studies provide a framework from which to address how dynamic acetylation/deacetylation regulates the function and/or localization of proteins that drive mitotic progression and cell division. Understanding acetylation of cell cycle protein networks in cell cycle regulation is likely to reveal new paradigms for anti-cancer therapy.

1. Chuang et al. J. Proteome Res. 2010, 9, 4554-4564.

TUESDAY

2272 Attenuation of mitotic RanGTP gradient in normal somatic cells. K. Hasegawa1, S. Ryu1, P. Kalab1; 1LCMB, National Cancer Institute, NIH, Bethesda, MD

Studies in variety of organisms showed that mitotic segregation of chromosomes requires regulation of spindle assembly factors (SAFs) by small GTPase Ran. Essential feature of such regulation is thought to be the chromosome-centered concentration gradient of free RanGTP which arises in mitotic cells due to chromosome binding of RCC1, the guanine nucleotide exchange factor for Ran and cytoplasmic localization of RanGAP1. Close to chromosomes, RanGTP releases spindle assembly factors (SAFs) from their inhibitory complexes with importins, thus providing a spatial clue to mitotic spindle assembly. Using live cell microscopy with Forster resonance energy transfer (FRET) biosensors, we observed that in contrast to HeLa, normal human primary cells divide in the absence of a steep mitotic RanGTP gradient or a gradient of activated SAFs. Consistent with proposed Ran functions in mitotic spindle assembly, the absence of a steep mitotic RanGTP gradient in primary cells correlated with extended prometaphase, although no mitotic defects were observed in such cells. As expected, we found that RCC1 abundance and its binding to chromatin were among the key factors controlling the steepness of mitotic RanGTP gradient. These results show that a steep RanGTP gradient is not required for mitosis in all normal cells and suggest that attenuated mitotic Ran function may be characteristic to homeostatic cell proliferation of normal tissues in vivo.

2273 Molecular mechanisms governing extrinsic forces in mitotic spindle organization. M. Kwon1,2, M. Bagonis2, G. Danuser2, D. Pellman1,2; 1HHMI/Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, 2Cell Biology, Harvard Medical School, Boston, MA

Precise control of centrosome number is crucial for bipolar spindle assembly and accurate segregation of chromosomes to daughter cells. Cancer cells often contain supernumerary centrosomes that create the potential for catastrophic multipolar divisions. Despite the presence of extra centrosomes, many cancer cells successfully divide because of mechanisms that suppress multipolar mitoses by clustering their extra centrosomes. Using functional genomics and follow-up studies, we have previously identified key pathways contributing to centrosome organization in cancer cells. In addition to spindle intrinsic forces, we found that cell adhesion patterns can determine the fate of mitosis whether they divide into two (bipolar) or more (multipolar). Yet, the mechanism by which actin and adhesion-dependent forces to control the fidelity of mitosis remain unclear at the molecular level. One model proposes that sites of strong cell matrix adhesion are imprinted as an actin rich structure called retraction fibers during mitosis, and the retraction fibers serve as cortical cues to concentrate force generators and regulators to pull centrosomes. By imaging cancer cells plated on fibronectin micropatterns, here we demonstrate that Myo10, an unconventional myosin identified from our genome-wide screen, is an essential component linking retraction fiber-mediated forces to astral microtubules (MTs) during spindle organization. Myo10 specifically localizes along and to the tips of the retraction fibers. When cells with extra centrosomes were plated on Y-shaped fibronectin micropatterns, control cells divide tripolar toward Y-adhesion axis; however, cells depleted of Myo10 undergo bipolar division independent of adhesion and retraction fiber positions, suggesting that Myo10 is a force coupler from retraction fibers to centrosomes. We have hypothesized that intrinsic properties of MTs adjacent to retraction fibers are altered to favor stable interaction between astral MTs and cell cortex. To uncover heterogeneity of MT dynamics in relation to retraction fibers, we have performed live cell imaging of GFP-EB3 with high spatio- temporal resolution in cells plated on fibronectin micropatterns where retraction fiber positions are precisely manipulated. We have developed a system that quantifies and visualize MT TUESDAY dynamics in different subregions of cells by an automated computer program capable of tracking GFP-EB3 comets. This approach uncovered heterogeneity of MT dynamics around retraction fiber regions. Interestingly, our analyses have revealed that Myo10 is in part responsible for promoting the long-lived and long distance travelling MTs preferentially at adhesion sites. Taken together, these results suggest that Myo10 is a key adhesion-dependent regulator that confers heterogeneity of mitotic cortex by coupling cell geometry/adhesion to mitotic fidelity and genome stability.

2274 Terminating Mitotic Checkpoint Surveillance at the Anaphase Onset. M. D. Vazquez Novelle1, M. Petronczki1; 1Cancer Research UK (Clare Hall Labs), Potters Bar, England

The mitotic checkpoint monitors the attachment of kinetochores to microtubules and delays anaphase onset until all sister kinetochores have become attached to opposite poles. Correct bipolar attachment leads to kinetochore deformation and tension that silences the checkpoint. What prevents mitotic checkpoint reactivation when sister centromeres are split and tension is lost at anaphase onset? Aurora-B kinase, the catalytic subunit of the chromosomal passenger protein complex (CPC), acts as a sensor at inner centromeres for the status of attachment. Phosphorylation of Aurora-B targets at erroneously attached kinetochores elicits the correction of these attachments and the activation of the mitotic checkpoint. At anaphase the CPC leaves the centromeres and relocates to the spindle midzone. This Cdk1-controlled iconic translocation might prevent the checkpoint from re-engaging after anaphase onset. To test this hypothesis we experimentally retained Aurora-B and the CPC at the centromere throughout anaphase in human cells. Preventing CPC translocation caused the untimely recruitment of mitotic checkpoint proteins (Bub1, BubR1 and Mps1) to kinetochores at anaphase in an Aurora-B kinase activity dependent manner. Our results suggest that the relocalization of the CPC, an evolutionarily conserved event in eukaryotes, is a key mechanism that prevents mitotic checkpoint activation at anaphase. However, the retention of Aurora-B at centromeres after anaphase onset does not suffice to trigger a full mitotic checkpoint engagement in response to the loss of tension due to sister chromatid splitting. Mad1 and Mad2 recruitment to kinetochores is suppressed after anaphase onset independently of CPC relocalization. What regulates these events at the metaphase-to-anaphase transition in human cells? Experiments in yeast, flies and frogs suggest that Cdk1 activity is required for mitotic checkpoint activity and the error correction pathway. Thus, we are investigating whether inactivation of Cdk1 at the metaphase-to- anaphase transition could play an important role in terminating mitotic checkpoint surveillance. Our preliminary data are consistent with the notion that Cdk1 is a master regulator of the mitotic checkpoint in human cells. In conclusion, together our results suggest that Cdk1 inactivation prevents mitotic checkpoint re-engagement at anaphase onset, through the translocation of CPC and other events that control Mad1/2 recruitment to kinetochores.

2275 A mechanism linking microtubule destabilisation to maturation of chromosome- microtubule attachments. R. L. Shrestha1, N. Tamura1, I. Zulkipli1, V. M. Draviam1; 1Department of Genetics, University of Cambridge, Cambridge, United Kingdom

Selective destabilization of erroneous chromosome-microtubule attachments is critical to prevent the mis-segregation of chromosomes. Initially, chromosomes attach to lateral walls of microtubules and then convert this attachment to the ends of microtubules through a poorly understood maturation process. We demonstrate here a role for microtubule destabilization in TUESDAY the maturation of attachments made to microtubule walls, and describe the consequence of persistent immature attachments. TAO1 kinase mediates microtubule destabilisation during mitosis and cells depleted of TAO1 display persistent laterally attached kinetochores that fail to congress properly. By briefly exposing cells to mild microtubule stabilizing drugs, we demonstrate that suppressing microtubule dynamics is sufficient to deter the maturation of lateral attachments. Laterally attached kinetochores, under microtubule stabilizing conditions, fail to retain Mad2 or MPS1 and compromise error-correction in cells released from monastrol treatment. Together our findings provide first insight into the fate of unresolved lateral attachments, and highlight the importance of maturation in ensuring the accurate segregation of chromosomes.

Signal Transduction and Signaling Networks II

2276/L50 Regulation of Notch1 signaling by Jagged-1 intracellular domain. J-S. Ahn1, E-J. Ann1, J-H. Yoon1, H-S. Park1; 1School of Biological Sciences and Technology, Chonnam national university, Bukku, Gwangju, Korea

Notch signaling involves the proteolytic cleavage of the transmembrane Notch receptor after binding to its transmembrane ligands. Jagged-1 also undergoes proteolytic cleavage by gamma-secretase and releases an intracellular fragment. In this study, we have demonstrated that the Jagged-1 intracellular domain (JICD) inhibits Notch1 signaling via a reduction in the protein stability of the Notch1 intracellular domain (Notch1-IC). The formation of the Notch1-IC- RBP-Jk-Mastermind complex is prevented in the presence of JICD, via a physical interaction. Furthermore, JICD accelerates the protein degradation of Notch1-IC via Fbw7-dependent proteasomal pathway. These results indicate that JICD functions as a negative regulator in Notch1 signaling via the promotion of Notch1-IC degradation.

2277 Role of R-Ras GTPases in the Wnt-Planar Cell Polarity pathway. N. Hartig1, M. Muñoz-Alegre1, L. Young1, P. Rodriguez-Viciana1; 1UCL Cancer Institute, London, United Kingdom

Ras genes are mutated in about 30% of human tumours and likely play a role by indirect activation (e.g. receptor tyrosine kinases) in an even larger number of cancers. The closely related R-Ras GTPases share many of the properties of Ras genes including the ability to behave as oncogenes. Despite sharing biochemical and biological properties, the R-Ras subgroup also have distinct functions. In order to identify novel R-Ras effector proteins potentially accounting for these differences, we employed a Tandem Affinity Approach (TAP) using activated R-Ras as bait. This approach lead to the identification of Wnt-Planar Cell Polarity (PCP) protein Vangl1 as a novel R-Ras interacting protein.

The Wnt pathway plays a key role in development and disease. In addition to the better studied ß-catenin dependent pathway, Wnt ligands can also activate the separate ‘non-canonical’ or PCP pathway, which coordinates complex cell migration during development. Perturbations in the PCP pathway contribute to the pathogenesis of a variety of diseases including cardiac and neuronal tube defects, cilia based disorders and to the invasiveness of cancer cells.

We have shown that Vangl1 functions as a true R-Ras effector and mapped the interaction to the Vangl1 C-terminus. We have identified several Vangl1 interacting proteins that confirm it’s TUESDAY conserved role in polarity and conducted biological assays that show that Vangl1 is critically required in migration. We have also shown that certain Frizzled receptors cooperate with the Ror2 tyrosine kinase to selectively stimulate Vangl1 function and possibly activation of R-Ras GTPases.

In conclusion, the identification of Vangl proteins as novel effectors of R-Ras GTPases provides an exciting new link between Ras signalling and the Wnt-PCP pathway.

2278 Loss of serine protease HtrA1 potentiates transforming growth factor-β signaling and bone formation. J. R. Graham1, A. Chamberland1, X. Li1, D. Dai1, Q. Lin2, M. S. Ryan3, W. Zeng1, T. Blanchet1, M. A. Rivéra-Bermúdez1, C. R. Flannery1, Z. Yang1; 1Inflammation & Remodeling Research Unit, Pfizer Inc, Cambridge, MA, 2Global Biotherapeutics Technologies, Pfizer Inc, Cambridge, MA, 3Immunology & Autoimmunity Research Unit, Pfizer Inc, Cambridge, MA

HtrA1 is a member of the High Temperature Requirement (HtrA) family of serine proteases. Genetic and gene expression studies have implicated HtrA1 in the progression of several pathologies, most notably in conditions with aberrant deposition of extracellular matrix. The inhibitory activity of HtrA1 on the transforming growth factor (TGF)-β family pathway has been reported, partially explaining the function of HtrA1 in physiological or disease processes. This study characterized the molecular mechanism by which HtrA1 regulates TGF-β signaling and examined the effect of HtrA1 gene deletion on musculoskeletal tissues in mice.

Overexpression of HtrA1 inhibited the TGF-β-mediated transcription of a Smad-responsive promoter. Incubation of HtrA1 with several candidate substrates in the TGF-β pathway revealed that this protease was capable of efficiently cleaving type II and III TGF-β receptors (TβRII and TβRIII), but not TβRI or TGF-β itself. Furthermore, cleavage increased with the addition of CPII, an HtrA1 agonist. Overexpression of HtrA1 decreased the cell surface levels of both TβRII and III, but not TβRI. Conversely, silencing HtrA1 expression resulted in significantly more TGF-β cell surface binding, increased Smad2 phosphorylation, and enhanced TGF-β-regulated gene expression. To better characterize the role of HtrA1, we generated mice with targeted gene deletion. MicroCT analysis showed that deletion of the HtrA1 gene resulted in a marked increase in bone volume in the distal femur and lumbar vertebrae (48% and 20%, respectively). Embryonic fibroblasts from HtrA1 knockout mice exhibited increased TGF-β-induced expression of three genes known to promote bone formation, Runx2, CTGF and PAI-1.

Our results reveal that the cleavage of two TGF-β receptors (TβRII and III) is a novel mechanism for HtrA1 inhibition of TGF-β signaling. Deletion of HtrA1 in mice leads to a dramatic increase in bone formation, most likely due to enhanced signaling by TGF-β or TGF-β family proteins.

TUESDAY

2279 Cellular and Biochemical Activities of the Non-Canonical Wnt Receptors ROR1 and ROR2. T. W. Bainbridge1, V. De Almeida2, C. Chalouni3, R. Kelly1,4, J. Goldsmith2, A. Majeed1,5, B. Pan6, G. Quinones1, J. Lill1, W. Sandoval1, A. Izrael-Tomasevic1, D. Arnott1, M. Costa2, P. Polakis2, B. Rubinfeld2, J. A. Ernst1,7; 1Protein Chemistry, Genentech, South San Francisco, CA, 2Cancer Pathways, Genentech, South San Francisco, CA, 3Center for Advanced Light Microscopy, Genentech, South San Francisco, CA, 4University of California, Los Angeles, CA, 5Materials Science & Engineering, Stanford University,Palo Alto, CA, 6Structural Biology, Genentech, South San Francisco, CA, 7Early Discovery Biochemistry, Genentech, South San Francisco, CA

The receptor tyrosine kinases, ROR1 and ROR2, activate non-canonical Wnt signaling and planar cell polarity pathways in response to Wnt5a. Various mutations within the intracellular domain (ICD) of ROR2 have been linked to Robinow syndrome and brachydactyly type B, while increased expression of ROR1 has been associated with acute lymphoblastic leukemia, chronic lymphocytic leukemia and mantle cell lymphoma. Previous studies have shown phosphorylation of ROR2 in response to Wnt5a stimulation, although autophosphorylation has not been demonstrated. The human ROR kinase domains lack conservation of the P-loop consensus sequence and are expected to be deficient in phosphotransferase activity, but studies with isolated ROR have provided conflicting results in kinase activity assays. CAM-1, the C. elegans ortholog of ROR, retains the P-loop consensus sequence, but the kinase activity has not been evaluated in vitro. In this work, we investigate the cellular role of the ROR ICD and compare the in vitro kinase activities of the isolated ICDs of ROR1, ROR2, CAM-1, and the most closely related human kinase, MuSK.

2280 Serotonin Receptor Signaling in JC Virus Infection. M. Maginnis1, W. Atwood1; 1Molecular Biology, Cell Biology & Biochemistry, Brown University, Providence, RI

The JC polyomavirus (JCV) is a prevalent human pathogen and the causative agent of the fatal, demyelinating disease Progressive Multifocal Leukoencephalopathy (PML). JCV infection is initiated by interactions with cell-surface alpha 2,6-linked sialic acid on lactoseries tetrasaccharide c (LSTc) and serotonin 5-hydroxytryptamine2A receptor (5-HT2AR). Engagement of viral receptors can activate intracellular signaling pathways that regulate key steps in the virus life cycle. The objective of this study is to define whether cellular signaling events emanate from the G-protein coupled-receptor (GPCR) 5-HT2AR in JCV infection. 5-HT2AR activates the Ca2+/calmodulin pathway leading to activation of the ERK/MAPK signaling pathway and phosphorylation of ERK. To determine whether ERK is phosphorylated following engagement of 5-HT2AR, cell lysates were harvested at early time points following JCV infection and analyzed by immunoblot analysis for ERK phosphorylation. ERK was phosphorylated as early as 5 minutes post-infection consistent with receptor engagement. Furthermore, treatment of cells with inhibitors of MEK and ERK abolished JCV infection in a dose-dependent manner suggesting that activation of the ERK/MAPK signaling pathway is critical for JCV infection. To define whether Ca2+/calmodulin signaling is activated during JCV infection, cells were treated with inhibitors of calcium release and calmodulin kinase, both of which decreased JCV infection. 5-HT2AR contains two calmodulin binding sites expressed in the second transmembrane loop and carboxyl terminus. Mutations in key hydrophobic residues in the calmodulin binding sites were generated in a 5-HT2AR construct and expressed in poorly permissive HEK cells. Expression of a wild-type 5-HT2AR rescues infection in HEK cells, but a mutation in the TUESDAY calmodulin-binding site in the carboxyl terminus resulted in a decrease in JCV infection. These findings provide new information regarding the role 5-HT2AR in JCV infection, and suggest that signals initiated upon engagement of the 5-HT2AR are essential for a productive viral infection.

2281 A genetic approach to identify new regulators for Smoothened. H-T. Shih1, W-Y. Chen1, J-T. Wu1; 1Institute of Molecular Medicine, National Taiwan University, Taipei City, Taiwan

G protein-coupled receptors (GPCRs) are seven-transmembrane receptors that initiate signaling processes of many physiological responses upon ligand stimulation. In Drosophila, Smoothened (Smo) belongs to GPCR family and transduces Hedgehog signaling that is critical in embryonic and larval development. Whereas the strength of GPCR-mediated signaling could be modulated at the level of GPCR by desensitization and internalization of the GPCRs, it is not clear whether the level of Smo is also subjective to any regulation that eventually changes the strength of Hedgehog signaling. To test this possibility, we performed an in vivo RNAi screen in a smo loss- of-function mutant background to identify regulators for Smo expression that also modulate the strength of Hedgehog signaling. First, we used a MS1096-Gal4 driven USA-smo5A to generate a fused L2 and L3 wing vein, the characteristic smo deficient phenotype in adult wing. Next, we crossed MS1096>smo5A to RNAi or dominant negative stocks for small G proteins, Rac1, Cdc42, RhoA, and Rab, and GTPase-activating proteins (GAPs). Our preliminary data showed that ectopic expression of small G proteins caused a marked wing shrinkage or lethality. But dominant negative form of small G proteins enhanced Smo deficient phenotype. Interestingly, most GAPs had no obvious effect on the smo deficient phenotype, suggesting a functional redundancy among GAPs. Our long term goal is to understand the distinct regulation impinged on Smo as oppose to those on conventional GPCRs.

2282 Dihydrotestosterone activates Src to increase EGF-induced Erk activation in fetal lung fibroblasts. M. K. Lee1, S. M. Smith1, S. Murray2, L. D. Pham2, H. C. Nielsen2; 1Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, CA, 2Newborn Medicine, Tufts Medical Center, Boston, MA

Neonatal respiratory distress syndrome has long been recognized as being more severe in premature male infants. This observation has been ascribed to differences in lung maturation, which is regulated by interactions between mesenchymal and epithelial cell populations. Fibroblast-type II cell communications are, in turn, modulated by epidermal growth factor (EGF)- mediated Erk1/2 activation. Because dihydrotestosterone (DHT) has been implicated in delayed lung maturation, we hypothesized that androgens could affect tyrosine kinase signaling in fetal lung fibroblasts. Androgens act by both translation-dependent and independent mechanisms, and we further hypothesized that DHT regulates fetal lung fibroblast signaling by inducing cytosolic Src to phosphorylate EGF receptors, thereby enhancing their subsequent activation by EGF. To assess these postulates, fetal lung fibroblasts were isolated from either pregnant mice treated with subcutaneous timed-release dihydrotestosterone (DHT) pellets or from untreated timed-gestation mice at 16 days gestation. Cells were maintained in culture containing 100 nM DHT for up to 4 d prior to overnight serum starvation and stimulation with 100 ng/ml EGF for up to 30 m. Cells were lysed in RIPA buffer, resolved by SDS-PAGE, and transferred to nitrocellulose for Western analysis. Membranes were probed with antibodies against phosphoErk1/2, phosphoEGF receptor, and phosphoSrc. To attenuate Src activity, selected cells were transfected with siRNA against Src, treated with the pharmacological Src inhibitor TUESDAY

PP2, or infected with adenoviruses expressing a dominant negative Src. Both long-term (in utero plus in culture) and short-term (in culture only) DHT exposure increased peak Erk activation by EGF, and this increase was evident with as little as 60 min of DHT exposure. The phosphorylation of Src and EGF receptor was increased by DHT. EGF-induced Shc phosphorylation and Grb2 association with Shc were similarly increased by prior DHT exposure, confirming pathway activation. Finally, Src silencing, PP2 treatment, and dominant negative Src adenoviruses all prevented DHT from increasing EGF-mediated Erk1/2 phosphorylation. These results suggest that DHT activates Src to increase the activity of EGF receptors and, hence, the EGF signaling pathway leading to Erk activation in primary fetal lung fibroblasts.

2283 Regulation of calcium-dependent calcineurin activity by inositol sphingolipids. S. A. Jesch1, S. A. Henry1; 1Molecular Biology and Genetics, Cornell University, Ithaca, NY

Inositol-containing sphingolipids and phosphoinositides at the plasma membrane play important roles in regulating stress response pathways, which are critical for lipid homeostasis and cell viability. We previously showed that inhibiting inositol sphingolipid metabolism triggers protein kinase C signaling and subsequent activation of the MAP kinase, Slt2p/Mpk1p, by regulating the Stt4p-dependent pool of phosphatidylinositol-4-phosphate (PI4P) at the plasma membrane. To identify additional lipid-mediated signaling networks that are activated by a similar mechanism, we carried out gene expression profiling following interruption of inositol sphingolipid synthesis by starving cells for inositol. We found that calcineurin-dependent genes are highly up-regulated following inositol starvation. Previous work has demonstrated that calcineurin, a calcium- dependent protein phosphatase, regulates the activities of the PH-domain proteins Slm1p and Slm2p, which are necessary for normal sphingolipid metabolism. Consistent with these data, calcineurin, stt4, and slm mutants exhibit inositol auxotrophy. Moreover, cells lacking Csg2p, a calcium binding regulator of mannosyl-inositolphosphorylceramide synthesis, constitutively activates calcium-dependent calcineurin signaling. This activation requires stt4, slt2/mpk1, and the plasma membrane-localized calcium channel Cch1p, which is a known target of Slt2p/Mpk1p. Together, these results suggest a feedback mechanism for regulating sphingolipid mediated signaling by regulating plasma membrane pools of PI4P and intracellular calcium levels.

2284 Anti-Inflammatory Effect of Benzylideneacetophenone Derivative on Thyroid-Associated Ophthalmopathy through Suppression CXCL10/IP-10 Production. S. Lee1, Y-H. Choi1; 1Ewha Woman's University, Seoul, Korea

Thyroid-associated ophthalmopathy (TAO) is an autoimmune inflammatory disorder of the orbital fatty tissue/connective tissue and the extra ocular muscles that is closely associated with Graves’ hyperthyroidism. Recent experimental evidence has shown that CXCL10/IP-10 is elevated in patients with TAO and plays an important role in the initial phases of TAO. Yakuchinone B from Alpinia oxyphylla,that belongs to ginger family has been used in oriental herbal medicine. It has been reported as one of anti-inflammatory phytochemicals which regulate COX-2 expression. To search for new candidate of anti-inflammatory compound at the inflammatory stage of TAO, benzylideneacetophenone compound JC3, (2E)-3-(4-hydroxy-3- methoxyphenyl)phenylpro-2-en-l-one), was synthesized from Yakuchinone B and their potential to suppress CXCL10/IP-10 production was evaluated. IFN-γ strongly increased the level of CXCL10/IP-10 in orbital fibroblasts with TAO and normal. JC3 compound revealed the potent inhibitory effect on IFN-γ-induced CXCL10/IP-10 protein expression. JC3 compound effectively decreased mRNA expression level of CXCL10/IP-10 and exhibited no toxicity in orbital TUESDAY fibroblasts at the 10μM concentration. Moreover, the result obtained from gel shift assay showed that JC3 suppressed IFN-γ-induced DNA binding of STAT-1. These results suggest that JC3 modulates high levels of CXCL10/IP-10 protein in response to IFN-γ, indicating that JC3 may be an attractive therapeutic agent to reduce the inflammatory stage of TAO.

2285 IL-15-induced IL-10 increases the cytolytic activity of human natural killer cells. T-D. Kim1, J. Park1, I. Choi1; 1Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea

Interleukin 10 (IL-10) is a multifunctional cytokine that regulates diverse functions of immune cells. Natural killer (NK) cells express the IL-10 and IL-10 receptor, but little is known about the function of IL-10 on NK cell activation. In this study, we show the expression and role of IL-10 in human NK cells. Among the cytokines tested, IL-15 was the most potent inducer of IL-10, with a maximal peak expression at 5 h after treatment. Furthermore, IL-10 receptor was shown to be expressed in NK cells. IL-10 alone had a significant effect on NK cytotoxicity which additively increased NK cell cytotoxicity in the presence of IL-15. Neutralizing IL-10 with anti-IL-10 antibody suppressed the inductive effect of IL-10 on NK cell cytotoxicity; however, IL-10 had no effect on IFN-γ or TNF-α production or NK cell activatory receptor expression. STAT signals are implicated as a key mediator of IL-10/IL-15 cytotoxicity response. Thus, the effect of IL-10 on NK cells is particularly interesting with regard to the STAT3 signal that was enhanced by IL-10 or IL-15.

2286 Anthrax Toxin Receptor 2 Carboxyl-terminal 12-residues are Important for Toxin Entry into Cells. R. Jimenez1, G. J. Chaudry1, S. Manam1; 1South Texas Center for Emerging Infectious Disease, Department of Biology, Univeristy of Texas at San Antonio, San Antonio, TX

Anthrax toxin is secreted by the Gram-positive, spore-forming, rod-shaped bacterium Bacillus anthracis. The toxin is one of two virulence factors of the organism, the other being its antiphagocytic poly-D-glutamate capsule. Three secreted proteins make up anthrax toxin: protective antigen (PA), edema factor (EF), and lethal factor (LF). EF is an adenylyl cyclase that harms cells by producing excessive amounts of cAMP. LF is a Zn-dependent metalloprotease that cleaves MAP kinase kinases. PA delivers EF and LF to cytosol, but itself is without any toxic catalytic activity. Thus, active anthrax toxins are PA+EF and PA+LF. The toxins enter cells by receptor-mediated endocytosis. PA utilizes two receptors for entry, tumor endothelial marker 8 (TEM8; ANTXR1) and capillary morphogenesis gene 2 protein (CMG2, ANTXR2). The two membrane-bound forms of CMG2 that function as PA receptors comprise 488 and 489 residues, and they differ only in the last 12 residues. Our analysis shows that absence of these residues results in a receptor that affords PA binding and its cleavage by furin, but does not support intoxication by the toxin. The block to intoxication appears at the level of the SDS- resistant heptamer, a structure that forms upon exposure to acidic pH in endosomes. Functional analysis by alanine-scanning mutagenesis revealed that essentially all 12 residues are important for receptor function. Surprisingly, deletion of the entire cytoplasmic portion of CMG2 left the truncated receptor fully functional for anthrax toxin. Thus, the 12-residue C-terminal segment must remain intact for the full-length receptor to function, but the segment is dispensable when the rest of the cytoplasmic domain is also absent. The results also suggest that different mechanisms underlie toxin entry via the full-length CMG2 and the receptor lacking the cytoplasmic domain.

TUESDAY

2287 Rho A signaling contributes to statin-induced osteogenesis in bone marrow mesenchymal stem cells. I-C. TAI1,2, M-L. Ho2,3, J-K. Chang2,4; 1Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, 2Orthopaedic Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan, 3Department of Physiology, Kaohsiung Medical University, Taiwan, 4Department of Orthopedic Surgery, Kaohsiung Medical UniversityHospital, Taiwan

Statins, 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, reduce cholesterol synthesis and prevent cardiovascular disease. Previous in vitro and in vivo studies showed that statin stimulated bone formation. They also have been found to inhibit prenylation of Rho proteins in recent decade. Previous reports showed that statins inhibited protein prenylation and decreased the active form of Rho A in osteoclasts. Others reports indicated that statins inhibited protein prenylation but increased the active form of Rho A in human erythroleukemia cells. Therefore, the role of statins regulate Rho A activity remains unclear. Rho GTPases act as molecular switches to regulate mesenchymal stem cell differentiation. Previous study showed that transfected constitutively active-form of RhoA into human mensenchymal stem cells (hMSCs) which leaded differentiation of hMSCs into osteoblasts. On the other hand, dominant negative RhoA leaded differentiation of hMSCs into adipocytes. According to the description above, we want to investigate whether Rho A signaling contributes to statin-induced osteogenesis in BMSCs. Pluripotent mesenchymal cells, D1, which were cloned from Balb/c mouse bone marrow cells and purchased from ATCC. For all experiments, cells were seeded at a density of 80% confluence and treated with or without simvastatin. The mRNA expression of Rho A was detected and quantified by real time PCR. And the mineralization effect on rat bone mesenchymal stem cells (rBMSCs) was tested by Alizarin Red S Staining. The Alizrin red S staining assay showed that the simvastatin were potentially enhanced the cell mineralization on BMSCs. The data of mRNA expression of Rho A showed that simvastatin 1uM significantly increased Rho A gene expression on first day. However, there were no significantly different between control and treatment group on third and fifth days. The protein level of Rho A increased with 0.5uM and 1uM but not 0.1uM simvastatin treatment on first day. From these results, we suggest Rho A signaling may contribute to simvastatin-enhanced osteogenesis in rat bone mesenchymal stem cells (rBMSCs).

2288 Magnetic Manipulation of signaling “hotspots” inside living cells shows context- dependent amplification of the Rac pathway. F. Etoc1, M. Coppey1, D. Lisse2, Y. Bellaiche3, J. Piehler2, M. Dahan1; 1LKB-IBENS, Paris, France, 2biophysics, Osnabruck University, Germany, 3Curie Institute, France

In vivo imaging has shown how the establishment and maintenance of a polarized cellular state relies on complex mechanisms by which signaling cascades become activated and regulated at sub-cellular levels. It has recently led to the concept of spatially-restricted signaling modules. Yet, it remains unclear which ingredients and processes specify a module. In this context, we used a new tool to locally perturb and probe signaling pathways associated to the small Rho- GTPases.

First, magnetic nanoparticles (500nm), functionalized with constitutively active Rho-GTPases (Rac1 and cdc42) were microinjected inside living cells. We observed the induction of downstream signaling at the particle surface by visualization of effectors binding (Pak and N- WASP) and the local polymerization of actin structures, thereby demonstrating that these particles behave in the cytosol as signaling "hotspots". Using FRAP, we further measured in TUESDAY vivo dissociation rates between the immobilized GTPase and its direct effector. Next, the nanoparticles were manipulated with a magnetic tip to position their signaling activity at different subcellular locations. With nanoparticles functionalized in situ with Tiam (a known Rac1 activator), we were able to specifically activates Rac1 signaling by bringing the nanoparticles in contact with the plasma membrane. The Rac1 activation occurred regardless of the subcellular localization at the membrane. However, the signal was non-linearly amplified only when the signaling activity was carried to protrusive areas of the cell. This result reinforces the concept of subcellular signaling modules and shows that a module could be specified by its ability to amplify rather than just linearly transduce incoming signals.

2289 Implications of RhoA regulation in Hela cells exposed to stress radiation. J. H. Osaki1, G. E. Silva1, F. L. Forti2; 1Biochemistry, Institute of Chemistry, University of Sao Paulo, 2Biochemistry, Institute of Chemistry, University of Sao Paulo, Sao Paulo, Brazil

The protein RhoA GTPase belongs to the superfamily of small GTPases related to Ras. RhoA are enzymes acting as molecular switches to control a wide variety of signal transduction pathways in eukaryotic cells such as remodeling of actin cytoskeleton, cell cycle, and cell death. RhoA overexpression is found in aggressive breast cancer and cervix carcinomas, where the enzyme is usually constitutively activated. Ionizing radiation (gamma and UV) causes single and double strand breaks in DNA, and also pyrimidine dimers lesions, damages usually repaired by homolog recombination, non-homologous end-joining and nucleotide excision repair mechanisms. In this study, using HeLa cells and mutants expressing dominant-negative RhoA (HeLa-RhoA-N19) and constitutively-active RhoA (HeLa-RhoA-V14), the RhoA protein was analyzed as a possible player in survival, senescence, migration, DNA damage response and repair after irradiation of cells with low doses of gamma and UV light. Results show that RhoA activity was downregulated by the highest energy ionizing. The HeLa-RhoA-V14 presents an increase in cell proliferation, survival and migration rate compared to HeLa-RhoA-N19. Furthermore, in RhoA-V14 clones either the DNA damage is decreased or the repair mechanisms are increased resulting in attenuated cellular senescence after the different ionizing treatments. In conclusion, these data confirm the essential role of RhoA protein in proliferation and migration and more intriguingly point to possible involvements of this enzyme in mechanisms of DNA repair and cell senescence after genotoxic stress induced by ionizing radiation. Supported by Fapesp: Project # 2008/58264-5.

2290 Rho small GTPases activates PI3K via a cooperative positive feedback loop. H. Lee1, H. Yang1, M-G. Shin1, S. Lee1, W. Park1, J-R. Kim1, K-H. Cho1, T. Meyer2, W. Heo1; 1Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2Stanford University

Phosphoinositide3-kinases (PI3Ks) and Ras and Rho family small GTPases are key regulators of processes such as cell polarization, motility and chemotaxis. These signaling components may control each other¡¯s activities by direct and indirect feedback processes that are only partially understood. Here, we used a live cell screen to evaluate the role of 100 human small GTPases in the regulation of PI3K and found that 21 small GTPase homologs activate PI3K. Using a protein-protein interaction assay that we recently developed, we found that K-Ras, H- Ras and 5 other homologs function upstream of PI3K and directly bind to the PI3K catalytic subunit, p110, while three Rac isoforms and 11 other small GTPase homologs activate PI3K by indirect feedback mechanisms. Furthermore, live cell experiments and in silico analysis revealed that significant level of PI3K activation can be generated by cooperation of multiple TUESDAY activators in endogenous level. We demonstrate two modes of PI3K activation that is initiated by upstream activators and is further enhanced by the cooperation of PI3K activators.

2291 Arl4A complexes with ELMO to promote actin cytoskeleton remodeling. T-C. Chiang1, M. Patel2,3, V. Tran2, F-J. Lee1, J-F. Cote2,3; 1Institute of Molecular Medicine, Taipei, Taiwan, 2Institut de Recherches Cliniques de Montreal, 3Departement de Medicine, Université de Montréal

The prototypical DOCK protein, DOCK180, is an evolutionarily conserved Rac regulator and is indispensable during processes such as cell migration and myoblast fusion. The biological activity of DOCK180 is tightly linked to its binding partner ELMO. We previously reported that auto-inhibited ELMO proteins regulate signaling from this pathway. One mechanism to activate the ELMO/DOCK180 complex appears to be the recruitment of this complex to the membrane via the Ras-binding domain (RBD) of ELMO. In the present study, we aimed to identify novel ELMO-interacting proteins to further define the molecular events capable of controling ELMO recruitment to the membrane. To do so, we performed two independent interaction screens: one specifically interrogated an active GTPase library while the other probed a brain cDNA library. Both methods converged on Arl4A, an Arf-related GTPase, as a specific ELMO interactor. Biochemically, Arl4A is constitutively GTP-loaded and our binding assays confirm that both wild- type and constitutively active forms of the GTPase associate with ELMO. Mechanistically, we report that Arl4A binds the ELMO RBD and acts as a membrane localization signal for ELMO. In addition, we report that membrane targeting of ELMO via Arl4A promotes cytoskeletal reorganization including membrane ruffling and stress fiber disassembly via an ELMO/DOCK180/Rac signaling pathway. We conclude that ELMO is capable of interacting with GTPases from Rho and Arf families, leading to the conclusion that ELMO contains a versatile RBD. Furthermore, via binding of an Arf family GTPase, the ELMO/DOCK180 is uniquely positioned at the membrane to activate Rac signaling and remodel the actin cytoskeleton.

2292 New biosensor for Cdc42-GDI complexation reveals dynamic regulation of GDI-Cdc42 binding by Src-kinase. D. Spiering1, L. Hodgson1, M. Sabouri2, C. der Mardirossian2, G. Danuser3, K. Hahn4; 1Albert Einstein College of Medicine, Bronx, NY, 2The Scripps Research Institute, La Jolla, CA, 3Harvard Medical School, Boston, MA, 4University of North Carolina at Chapel Hill, Chapel Hill, NC

Signaling by the p21 Rho family GTPases is critically important in a broad range of cell behaviors. They have been most extensively studied in motility and polarization, where their activation (GTP binding) is tightly coordinated in space and time to generate cell morphological changes. Guanosine Dissociation Inhibitors (GDI) sequester inactive (GDP bound) GTPases in the cytosol, and mediate localization of the activated GTPase in the plasma membrane. The role of GDI in controlling the precise placement and timing of GTPase activation remains poorly understood. To investigate this, we constructed fluorescent biosensors reporting the interaction of endogenous RhoGDI with either Cdc42, Rac1 or RhoA. Using the Cdc42-GDI biosensor together with the meroCBD biosensor detecting the activation of endogenous Cdc42 in MEFs, we imaged simultaneously the Cdc42–endogenous GDI complexation dynamics and the endogenous Cdc42 activation dynamics in single living cells. Using the morphodynamics mapping and computational multiplexing technology, we show directly for the first time that the Cdc42 uptake rate by the GDI is regulated through Src-kinase-mediated phosphorylation.

TUESDAY

Proteostasis, Cell Stress, and Aging

2293 Is the ubiquitin ligase itch phosphorylation modulating its binding and ligase activity towards all substrates? R. Forget1, A. Angers1; 1Biological Sciences, University of Montreal, Montréal, Québec, Canada

Itch is an ubiquitin ligase closely related to Nedd4. These enzymes bind to their substrate proteins with their central region and transfer ubiquitin through their C-terminal catalytic HECT domain. By promoting the proteasomal degradation of substrate proteins, ubiquitin ligases are involved in a variety of process like endocytosis, apoptosis, inflammation, cell signaling, etc. Recently, the regulation of the activity of ubiquitin ligases themselves has received a lot of attention. Itch is the target of the cJun N-terminal kinase (JNK) which phosphorylates three residues in a region overlapping with a proline-rich region of Itch that mediates recognition by SH3-domain proteins. Phosphorylation of Itch by JNK has been shown to regulate its activity towards itself and substrates cJun and JunB. An inhibitory intramolecular interaction abrogated by JNK-phosphorylation has been proposed to mediate this effect. Here, we evaluate if this model can be generalized to other Itch substrates, most specifically endophilin that has been shown to interact with Itch through its SH3 domain, distinct from most Itch known substrates. Using site-directed mutagenesis, we created mutants of Itch that could not be phosphorylated by JNK, and a phosphomimic mutant. These mutants were tested for their ability to interact with endophilin, and to mediate endophilin ubiquitylation and proteasomal degradation. We found that Itch phosphorylation by JNK has no effect on endophilin recognition by Itch, nor on the ligase ability to promote endophilin ubiquitylation. Although Itch phosphorylation is not required for proteasomal degradation of endophilin, a small facilitating effect of Itch phosphorylation could be observed. In conclusion, if JNK- mediated phosphorylation of Itch can slightly potentiate its ubiquitin- ligase activity, it has no incidence on the interaction between Itch and SH3 proteins like endophilin. Therefore, the regulation of Itch activity seems to differ according to the selected substrate.

2294 Potentiation of ER stress-mediated apoptotic pathway by protein tyrosine kinase p56lck in proteasome inhibitor MG132-induced apoptosis human Jurkat T cells. C. Han1, H. Park1, D. Jun1, H. Woo1, J. Lee1, Y. Kim1; 1School of Life Science and Biotechnology, Kyungpook National University, Daegu, Korea

Exposure of human Jurkat T cells to MG132 caused apoptosis along with upregulation of Grp78/BiP and CHOP/GADD153, activation of JNK and p38MAPK, activation of Bak, mitochondrial membrane potential (¥Ä¥÷m) loss, cytochrome c release, activation of caspase- 12, -9, -3, -7, and -8, cleavage of Bid and PARP, and DNA fragmentation. However, these MG132-induced apoptotic events, with the exceptions of upregulation of Grp78/BiP and CHOP/GADD153 and activation of JNK and p38MAPK, were abrogated by Bcl-xL overexpression. Pretreatment with the pan-caspase inhibitor z-VAD-fmk prevented MG132- induced apoptotic caspase cascade, but allowed upregulation of Grp78/BiP and CHOP/GADD153 levels, activation of JNK and p38MAPK, ¥Ä¥÷m loss, and cleavage of procaspase-9 (47 kDa) to active form (35 kDa). Further analysis using selective caspase inhibitors revealed that caspase-12 activation was required for activation of caspase-9 and -3 to the sufficient level for subsequent activation of caspase-7 and -8. MG132-induced cytotoxicity, apoptotic sub-G1 peak, Bak activation, and ¥Ä¥÷m loss were reduced in part by p38MAPK inhibitor, but not by JNK inhibitor. MG132-induced apoptotic changes, including upregulation of Grp78/BiP and CHOP/GADD153 levels, activation of caspase-12, p38MAPK and Bak, and TUESDAY mitochondria-dependent activation of caspase cascade were more significant in p56lck-stable transfectant JCaM1.6/lck than in p56lck-dificient JCaM1.6/vector. The cytotoxicity of MG132 toward p56lck-positive Jurkat T cell clone was not affected by the Src-like kinase inhibitor PP2. These results demonstrated that MG132-induced apoptosis was caused by ER stress and subsequent activation of mitochondria-dependent caspase cascade, and that the presence of p56lck enhances MG132-induced apoptosis by augmenting ER stress-mediated apoptotic events in Jurkat T cells.

Key words: proteasome inhibitor, apoptosis, ER stress, mitochondria-dependent caspase cascade, p56lck

2295 Translation elongation factor eEF3 and translation termination factor eRF3 are novel constituents of heat-induced stress granules in Saccharomyces cerevisiae. T. Grousl1, P. Ivanov2, I. Malcova1, I. Frydlova1, P. Pompach1, R. Slaba1, P. Vasicova1, L. Novakova1, J. Hasek1; 1Inst. of Microbiology of ASCR, v.v.i., Prague 4, Czech Republic, 2Faculty of Biology, Moscow State University, Moscow, Russia

In response to severe environmental stresses eukaryotic cells shut down translation and form transient mRNA assemblies called stress granules (SGs). Stress granules are often referred to as dominant accumulations of stalled translation preinitiation complexes since their typical components are the translation initiation factor eIF3 and the small ribosomal subunit 40S. We have shown previously that in the budding yeast Saccharomyces cerevisiae the SGs containing the eIF3 complex and the 40S ribosomal subunit are formed upon the robust heat shock at 46°C only. The formation of these SGs is independent of phosphorylation of eIF2alpha by Gcn2 kinase and also of the scaffolding proteins of P bodies Edc3 and Lsm4. Here we present the evidence that the robust heat shock-induced SGs of S. cerevisiae contain also the translation elongation factor eEF3 (Yef3). In addition, distinct accumulations of Yef3GFP were observed in the edc3lsm4C mutant even upon a mild heat shock at 42°C and they pre-determined accumulations of eIF3a upon the robust heat shock at 46°C. Furthermore, some of the robust heat shock-induced stress granules co-localized with accumulations of Sup35 suggesting that also translation termination factor eRF3 is a constituent of SGs. While the N-terminal domain of Sup35 is essential for [PSI +] prion formation its absence did not affect assembly of SGs. Our data indicate that SGs containing translation preinitiation complexes and termination factors are successively assembled on accumulations of the elongation factor eEF3 (Yef3) that was released from translation during heat shock. This work was financed by grants CSF 204/09/1924, LC545 and IRCAV0Z50200510.

2296 Pathogenic missense mutations located in distinct domains affect the folding and function of dystrophin. D. M. Strandjord1, D. M. Henderson1, B. Li1, J. M. Ervasti1; 1University of Minnesota, Minneapolis, MN

Dystrophin is a 427-kDa protein encoded by the DMD gene on the X chromosome. This structural protein localizes to the plasma membrane of muscle cells, where it establishes a mechanical link between the cytoskeleton and the extracellular matrix. The N-terminus and a segment of the middle rod domain in dystrophin interact with cytoplasmic actin filaments while modules near the C-terminus interact with the transmembrane protein -dystroglycan. Dystrophin and -dystroglycan, along with several other proteins, function as a complex known TUESDAY as the dystrophin-glycoprotein complex, or DGC, which is thought to stabilize the sarcolemma against mechanical stress. Males with a mutation in their only copy of the gene encoding dystrophin are diagnosed as having either Duchene Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD). Many patients have mutations that cause a dystrophin-deficiency in cells and develop extreme muscle deterioration in their first decade of life. More recently, sequencing has revealed that some patients have mutations in the dystrophin gene that result in a single amino acid change in the protein. Less is known about the molecular pathomechanism of these missense mutations. We recently demonstrated that missense mutations in the N-terminal actin-binding domain (ABD1) of dystrophin had minimal effect on actin binding activity, but caused thermal instability and aggregation in vitro (D.M. Henderson et al., 2010 PNAS). Additional pathogenic missense mutations have been identified in the -dystroglycan binding domain (DgBD) of dystrophin. To determine the effect of these pathogenic changes in a location distinct from ABD1, we engineered the disease-causing missense mutations D3187G, C3207R, F3228L, A3311P, C3313F, D3335Y into dystrophin. Both wild-type dystrophin and E3032K, a non- synonomous Single Nucleotide Polymorphism (SNP) found in the population, served as controls. When expressed in insect cell culture, quantification of the protein in the soluble versus insoluble fractions of the cell lysate revealed that some missense mutations significantly decreased the solubility of dystrophin. Circular Dichroism melt curves of purified proteins revealed that missense mutations decreased the cooperative unfolding transition which is indicative of a misfolded, heterogeneous population of protein. Supported by the Torske Klubben Graduate Fellowship and a grant from the National Institutes of Health (AR042423).

2297 Two distinguishable aging states found in an isogenic population at a single chronological age. D. M. Eckley1, S. Rahimi1, N. V. Orlov1, S. Mantilla1, C. E. Coletta1, J. D. Delaney1, Y. Zhang2, K. Becker2, W. Iser3, M. Wilson3, I. G. Goldberg1; 1IICBU, Laboratory of Genetics, National Institute on Aging (NIA), National Institutes of Health, Baltimore, MD, 2Research Resources Branch, NIA-Intramural Research Program, 3IMGU, Laboratory of Neuroscience, NIA

We present the initial molecular characterization of a transition between two early aging states. An age score reflecting physiological age was developed using a machine classifier. Images of C. elegans populations at fixed chronological ages throughout their lifespan were used for training. The distribution of age scores identified three stable post-developmental states and transitions. We examined the transition that occurs at day 5 post L4 molt, because a significant percentage of that population exists in both State I and State II. We measured the temperature dependence of the timing of the transition (Q10 ~ 1.17). This value is too low to be explained by a step-wise process with an enzymatic or chemical rate-limiting step, implicating a more complex timing mechanism. Individual animals at day 5 were sorted into State I and State II groups by the machine classifier and analyzed by microarray expression profiling. Despite being isogenic, grown for the same amount of time, and indistinguishable by eye, these two morphological states were confirmed to be molecularly distinct by hierarchical clustering and principal component analysis. The distinction demonstrates that pharynx morphology must reflect the aging state of the whole organism. Our expression profiling also yielded a gene set that showed significant overlap with those from three previous age-related studies and identified several genes not previously implicated in aging. A highly represented group of genes unique to this study is involved in the ubiquitination pathway, including Skip1-related (SKR) as well as F- box-containing adaptors which target specific proteins for degradation.

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2298 Glucose Stress Reduces Fertility and Impairs Mating in Caenorhabditis elegans. A. K. Engstrom1, M. R. Liggett1, M. A. Mondoux1; 1Department of Biology, College of the Holy Cross, Worcester, MA

Diabetes is a chronic disease marked by insulin resistance and the inability of cells to correctly respond to high levels of blood glucose. Diabetes currently affects 25.8 million people in the United States, and has been shown to lead to increased potential for birth defects, miscarriages, and infertility. Caenorhadiditis elegans has a conserved insulin-signaling pathway, making it an ideal model organism for studying the effects of excess glucose exposure. In previous studies, glucose stress in C. elegans was shown to lead to both a developmental delay and a reduction in hermaphrodite fertility; however the cause(s) of these defects is unknown. By exposing worms to high glucose during either development or adulthood, and then studying their reproductive profile and brood size, we found that the developmental delay is separable from the decrease in fertility. Feeding high glucose only during development led to a two-day reproductive delay but had no effect on fertility. Likewise, glucose stress during adulthood reduced fertility as much as lifetime exposure to high glucose. We assayed post-fertilization phenotypes under high glucose conditions and saw no change in external or internal hatching, suggesting that glucose stress causes defects in the adult germline. We also tested whether glucose stress affects male fertility and mating and find that glucose stress interferes with mating, leading to decreased brood sizes and decreased numbers of male offspring. Preliminary evidence suggests that male fertility is also decreased by glucose stress. We are currently investigating the roles of the insulin-signaling pathway and apoptosis in the response to glucose stress in hermaphrodites and determining which components of the male fertility and mating pathways respond to glucose stress.

2299 Differences in neuropeptide Y and Tirosine Hydroxylase levels related to ageing and gender in Locus Coeruleus. K. L. Farizatto1, M. F. Ferrari1, D. R. Chadi2; 1Faculdade de Medicina, Fisiopatologia Experimental, Universidade de Sao Paulo, Sao Paulo, Brazil, 2Universidade de Sao Paulo, Sao Paulo, Brazil

Age is a important factor for emerging of some diseases, as neurodegeneration and hypertension. Locus Coeruleus (LC) is a brain area involved with autonomic functions and behavior wich play a role in differences of blood pressure between males and females during lifespan. The goal of this study was to analyze the NPY and TH levels in the LC of spontaneously hypertensive (SHR) rats during aging in males and females. Methods: All the procedures and protocols were performed in accordance with the Institutional and International Guidelines for Animal Experimentation. We have employed SHR rats of: 3 months, 1 and 2 years (N=6) old. Animals were decapitated, the brains were frozen to achieve coronal sections from pons for the analysis of LC, through in situ hybridization and imunohistochemistry techniques. Afterwards, sections were analyzed by means of a semiquantitative microdensitometric procedure. Results are showed as mean ± standard error in arbitrary units. Two-way ANOVA was used for statistical analysis. Results: NPY mRNA expression in the LC is increased at 2 years females (0,056 ± 0,005) as compared to 1 year (0.036 ± 0.004) and decreased at 1 year (0.036 ± 0.004) as compared to 3 months old female rats (0.090 ± 0.010). In males group is decreased at 2 years (0.031 ± 0.002) as compared to 3 months old male rats (0.090 ± 0.016), (p<0.05). The peptide is increased at 1 year (100.0 ± 4.0) as compared to 2 years (79.0 ± 3.5) and 3 months old male rats (76.5 ± 4.5), (p< 0.001). In females peptide did not significant difference. The TH mRNA expression there is not significant difference. The TUESDAY peptide is increased at 2 years females (80.5 ± 2.5) as compared to 1 year (68.0 ± 2.5) and 3 months old female rats (60.0 ± 1.5). In males group is decreased at 2 years (54.5 ± 3.0) as compared to 3 months old male rats (65.5 ± 2.0), (p<0.01). There is a significant difference between the 2 year-old female and the 2 year-old male (p< 0.001). Discussion: The present study suggests that age and sexual dimorphism promote important changes can be influencing TH and NPY mRNA and peptide synthesis as well intracellular mechanisms regulation during aging. Females showed increased neurotransmitter levels compared with males, which might affect cardiovascular regulation as well others physiologic response during aging. Financial Support: CAPES, FAPESP and CNPq.

2300 Aged muscle stem cell function is not rescued by a young environment. J. D. Bernet1, T. McClure1, B. Olwin1; 1University of Colorado-Boulder, Boulder, CO

Skeletal muscle mass and strength begin to decline in both middle-aged mice and humans. Aged muscle regenerates poorly, which may be due to the impairment of aged muscle stem cells, called satellite cells, which are required for muscle regeneration. Satellite cells typically reside in a quiescent, non-proliferative state, between the basement membrane and the plasma membrane of the myofiber. Upon muscle injury, satellite cells activate, express the myogenic regulatory factor MyoD, enter the cell cycle, undergo cell division, and eventually differentiate and fuse to repair the damaged myofibers in a process called myogenesis. Satellite cells must self-renew to replace the quiescent satellite cell population for future rounds of muscle repair. Consistent with the reduced regenerative capabilities of aged msucle, aged satellite cells from two-year-old mice exhibit aberrant progression through myogenesis in comparison with young satellite cells from three-month-old mice. Our work and that of others have shown that cultured, aged satellite cells exhibit a decrease in clonal expansion and proliferation, but exhibit an increase in differentiation rate compared with young satellite cells. However, the addition of Fibroblast Growth Factor-2 (FGF-2) to cultured satellite cells increases clonal expansion and proliferation in both young and aged satellite cells. To further investigate this phenomenon, we first cultured both aged and young satellite cells and their associated myofibers in the absence of FGF-2 and observed a reduction in satellite cell proliferation, number of quiescent cells and expression of muscle lineage markers in aged cells compared to young cells. Upon addition of FGF-2 to aged satellite cell- myofiber cultures, we observed an increase in Pax7 expression, which marks cells capable of self-renewal, in satellite cells as well as an increase in markers of myogenic progression, with no alteration in proliferation. Together, these results suggest that there may be a cell autonomous defect in aged satellite cells. To rigorously test for a cell autonomous defect in aged cells, we transplanted myofibers with their associated satellite cells from young or aged transgenic GFP expressing mice into young, wild-type hosts. Thirty days post-transplantation, we observed fewer engrafted GFP+ satellite cells in the muscle of mice transplanted with aged myofibers compared with muscle transplanted with young myofibers. Overall, our studies demonstrate a cell autonomous defect in aged satellite cells that cannot be rescued by a young muscle environment. This cell autonomous age-associated defect in aged satellite cells may result in a reduction in self-renewal, thereby limiting the size of the functional aged satellite cell population available to maintain skeletal muscle homeostasis.

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2301 Depletion of ARMS enhances reactive oxygen species-induced autophagy. Y-H. Liao1,2, P-H. Huang3,4; 1Department of Dermatology, National Taiwan University College of Medicine, Taipei, Taiwan, 2Department of Dermatology, National Taiwan University Hospital, Taipei, Taiwan, 3Institute of Pathology, National Taiwan University College of Medicine, Taipei, Taiwan, 4Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan

Increased resistance to oxidative stress in melanoma cells plays an important role for melanoma development and contributes to the ineffectiveness of treatment aimed at oxidative stress-induced cell death. Autophagy is a self-digestion process in response to stimuli including oxidative stress. We have previously found that depletion of ankyrin repeat-rich membrane spanning (ARMS) protein in melanoma cells caused significantly increased autophagosomes. To determine the role of ARMS in oxidative stress-induced autophagy, we established siRNA- mediated ARMS-knockdown and control B16F0 stable melanoma cell lines. We found that depletion of ARMS significantly enhanced autophagosome formation, as evidenced by autophagic vacuolization examined by electron microscopy, punctate translocation of GFP-LC3 (microtubule-associated protein light chain 3) by immunofluorescence, and increased LC3-II by Western blotting. Significantly increased intracellular reactive oxygen species production in ARMS-RNAi cells at baseline and in time course of hydrogen peroxide treatment was revealed by DCHF-DA flow cytometry. Moreover, the percentage of cells with GFP-LC3 puncta was suppressed by treatment with antioxidant N-acetyl-L-cysteine or tiron in ARMS-RNAi cells. In vivo knockdown of ARMS in Drosophila imaginal eye discs also showed robust autophagy. In conclusion, these results suggested that ARMS silencing increased ROS production, which led to enhanced autophagy.

2302 Physiological role of the membrane contact site:Nucleus-Vacuole (NV) junctions. C-C. Lin1, D. S. Goldfarb1; 1Biology, University of Rochester, Rochester, NY

Nucleus-Vacuole (NV) junctions are membrane contact sites (MCSs) where perinuclear ER (nuclear envelope) and vacuole membrane come into close apposition. NV junctions are formed by the direct interaction between outer nuclear membrane protein Nvj1 and the vacuole membrane protein Vac8. A unique autophagic process in yeast Saccharomyces cerevisiae called ‘Pieacemeal microautophagy of the nucleus (PMN)’ emanates from NV junctions. Osh1 and Tsc13, which are involved in lipid homeostasis, are recruited to NV junctions and are essential for normal PMN biogenesis. Here, we report another protein that is sequestered to NV junctions-Nvj2. Unlike Osh1 and Tsc13, Nvj2 is neither required for NV junctions formation nor PMN process. Nvj2 localizes on ER, and is enriched on NV junctions upon Nvj1 expression is up regulated. Nvj1-Vac8 complex and the Y321 of Nvj1 are essential but not sufficient for the enrichment of Nvj2 on NV junctions. The absence of NVJ2 confers fungicide resistance, and overexpression of Nvj1, which sequesters Nvj2 to NV junctions, phenocopy the deletion of NVJ2. These results suggest an alternative role of NV junctions for the fungicide resistance other than PMN process.

2303 Premature aging of yeast Saccharomyces cerevisiae RAS2val19mutant. A. Pichová1, K. Sigler1; 1Institute of Microbiology, Academy of Sciences of the Czech Republic, Praha 4, Czech Republic

Aging and apoptosis, multifactorial processes involving a complex network of regulations, are conveniently studied on single cell models. Yeast, especially S. cerevisiae, is suitable for this TUESDAY purpose because it exhibits mother cell specific aging, where the cell divides asymmetrically, giving rise to a next-generation large mother cell and a smaller daughter cell that resets the clock to zero while mother cell accumulates all aging-connected changes. This asymmetry is disturbed in the oncogene mutant RAS2val19. This strain, known as a short-lived mutant, shows also a higher sensitivity to starvation and heat shocks. Micromanipulations revealed a high percentage of virgins unable to form micro- and macro-colonies. Young cells, mostly virgins, separated by centrifugal elutriation show markers of apoptosis, such as having a high load of ROS (reactive oxygen species) detected by DHR (dihydrorhodamine); most cells appeared with DNA breaks stained by the TUNEL test and their nuclei also appeared to have aberrant morphology. Quantitative comparison of the amount of extrachromosomal DNA rings (ECR) in separated young and old cells of the wild type, RAS2val19 mutant and a strain with delayed aging together with other data point to the premature aging being connected with symmetric aging, where the daughter cell is already inheriting some features of aging.

2304 The effects of OYE2 and ZWF1 overexpression in furfural stressed Saccharomyces cerevisiae. N. Carlson1, R. McRae1, S. Raehtz1, S. W. Gorsich1; 1Biology, Central Michigan University, Mount Pleasant, MI

A renewable fuel source is needed as economical, environmental, and security concerns continue to rise regarding the use of fossil fuels. Bio-ethanol is one potential solution. In the U.S. bio-ethanol is mainly produced by fermenting agricultural products such as corn starch. However, the use of corn starch is problematic due to concerns with quantity of of corn supply and the competing use of corn as a food source. Alternatively, biomass waste products such as lignocellulose (e.g. corn stover, wood chips) could be used. Currently, lignocellulose has not been optimized for fermentation. One major barrier to using lignocellulose is that during its hydrolysis many growth inhibitors are produced. Furfural is a major inhibitor generated. Furfural has been shown to inhibit growth and metabolic enzymes, reduce ethanol yield, and increase reactive oxygen species (ROS) that go onto damage internal membranes, actin filaments, and chromatin. Furfural can be removed using various chemical and physical methods, but at the expense of increased cost and environmental waste production. Our method to reduce the effect of furfural is to genetically engineer yeast that are robust enough to tolerate high concentrations of furfural while optimizing ethanol yield. For instance, overexpressing either ZWF1 (Zwf1p produces NADPH from the pentose phosphate pathway) or OYE2 (Oye2p is an NADPH-dependent reductase linked to oxidative stress tolerance) individually improves tolerance to furfural and reduces ROS accumulation and damage to cellular structures. In the present study we show how overexpressing both OYE2 (pRS425-MET25-OYE2) and ZWF1 (pRS426-ZWF1) in the same yeast strain effects overall furfural tolerance as assayed by growth (absorbance A600), reactive oxygen species (ROS) accumulation (dihydroethidium stain), membrane damage (mitochondrial targeted-GFP and FM 4-64), and chromatin damage (DAPI stain). Our strategy of genetic engineering fermenting microorganisms is valuable in improving the technology needed to optimize bio-ethanol fermentation from lignocellulose.

2305/L80 Vitamin C recycling and glutamate effects in astrocytes. P. Cisternas1, K. Oyarce1, P. Llanos1, C. Silva-Alvarez1, F. Nualart1; 1Department of Cell Biology, University of Concepcion, Concepcion, Chile

Specialized cells transport vitamin C in its reduced form using sodium-dependent cotransporters (SVCT1 and SVCT2). Additionally, different cells transport the oxidized form of vitamin C, TUESDAY dehydroascorbic acid (DHA), through glucose transporters (GLUTs). Vitamin C recycling associated with neuron-astrocyte interactions needs specific AA uptake in neurons and DHA uptake in astrocytes. SVCT2 is expressed principally in neurons mediating temperature- and sodium- dependent AA uptake. GLUT1 is expressed in astrocytes mediating glucose-DHA uptake. It has been suggested that glutamate increases vitamin C recycling. However, the specific mechanism has not yet been defined. In this work we analyzed the expression of GLUT1 and GLUT3 primary cultures of astrocytes and neurons obtained from rat brain, and assessed their differential capacities to incorporate AA or DHA. Using HPLC and radioactive pulse tracing we defined the effect of glutamate in the regeneration of AA in neurons and astrocytes. Our results confirmed that, in vitro, astrocytes only express GLUT1 and neurons preferentially express GLUT3. Functional data indicate that astrocytes transport mainly dehydroascorbic acid, using the glucose transporter GLUT1. Additionally, we defined that glutamate stimulates intracellular reduction of DHA in astrocytes of 15 and 30 DIV. In parallel, we observed that neurons have a lower reductive capacity, which increases only in the presence of high concentrations of glutamate. In this study we determined that glutamate is directly involved in the recycling of vitamin C in the CNS. This recycling model may work as an efficient system for the salvage of vitamin C by avoiding the hydrolisis of DHA produced by antioxidative protection.

Grants Support by FONDECYT 1100396.

2306 ER Calcium Depletion and ER Stress by Palmitate in Mouse Podocyte. S. Xu1, J-H. Kim1, R. Das1, X. Quan1, S-K. Cha1,2, K-S. Park1,2; 1Physiology, Yonsei University Wonju College of Medicine, Wonju, Korea, 2Institute of Lifestyle Medicine

Podocytes play a major role in glomerular filtration barrier, but also participate in pathogenic process of diabetic nephropathy. Diabetic patients have elevated plasma levels of saturated free fatty acid (FFA) that induces ER stress and apoptosis in different cell types. In this study, we investigated the cytotoxic mechanism of FFA in immortalized mouse podocyte. Incubation with palmitate, a saturated FFA, elicited apoptotic cell death in a dose dependent manner (50~500M). Palmitate elevated cytosolic or mitochondrial reactive oxygen species (ROS) production measured by fluorescence imaging system, which was blocked by N-acetyl-cystein (NAC). However, a mono-unsaturated FFA oleate itself or combination with palmitate did not increase the ROS level in podocyte. ER stress proteins such as GRP78/Bip, spliced Xbp1 and CHOP were strongly upregulated by palmitate, but not by oleate or oleate with palmitate. Palmitate decreased the luminal Ca2+ level in ER measured with ER Ca2+ sensing protein D1ER. In the absence of extracellular Ca2+, cytosolic Ca2+ increase induced by blocking ER Ca2+ uptake with cyclopiazonic acid (CPA) was markedly reduced in palmitate-treated cells, which could be recovered by NAC pretreatment. These data suggest that palmitate as the predominant circulating saturated FFA leads to ER stress and apoptosis in podocyte, which might be mediated by ROS-triggered ER Ca2+ depletion.

2307 Involvement of yeast homolog MOH1 of human YPEL5 gene in DNA damage-induced apoptosis in the budding yeast Saccharomyces cerevisiae. J. Lee1, D. Jun1, Y. Kim1; 1School of Life Science and Biotechnology, Kyungpook National University, Daegu, Korea

The homolog of Drosophila yippee protein is known to be highly conserved among eukaryotes from yeast to human. For five members of the human YPEL family, YPEL5 is evolutionarily TUESDAY distinct from other four members, YPEL1 through YPEL4, especially. However, their functional roles are still poorly understood. To understand the role of yippee protein, we have employed the budding yeast (Saccharomyces cerevisiae) mutant possessing disrupted yeast homolog (MOH1; YBL049W) of yippee protein gene, and compared its physiological characteristics with those of the wild-type strain. The mutant for the MOH1 gene exhibited an enhanced resistance to UVC (254 nm) irradiation and genotoxic agents as compared to the wild-type. This phenomenon was abrogated when the disrupted MOH1 gene in the mutant was restored by transformation of the functional gene. The level of MOH1 mRNA in the wild-type was upregulated in response to UVC irradiation and genotoxic agents. And exposed phosphatidylserine was detected by reaction with annexin V-FITC and PI staining after UVC treated. Our findings provide evidence for functional conservation of YPEL5 protein between lower and higher eukaryotes, which can confer apoptotic cell death to DNA damage in S. cerevisieae. Key word: MOH1, YBL049W, Saccharomyces cerevisiae, complementation test, YPEL5, UVC irradiation, genotoxic agent

Development and Morphogenesis II

2308 Two periods of testicular regression, not directly linked to apoptosis, are peculiar events of the annual reproductive cycle of Myotis nigricans (Chiroptera: Vespertilionidae). M. R. Beguelini1, C. C. Puga2, F. F. Martins2, S. R. Taboga1, E. Morielle-Versute2; 1Biology, UNESP - University of Estadual Paulista, São José do Rio Preto, Brazil, 2Zoology and Botany, UNESP - University of Estadual Paulista, São José do Rio Preto, Brazil

The reproductive data available for Myotis nigricans, an endemic Neotropical species of vespertilionid bat, are few and controversial; and indicate that its reproductive cycle is geographically variable, presenting an active pattern throughout the year in some regions and a hibernating pattern in others. Thus, this study aimed to evaluate the seasonal modifications in testicular morphology in a tropical environment. Twenty-eight mature specimens, collected in São Paulo State, Brazil (49W22'45" 20S49'11"), between September 2008 and August 2009, were submitted to morphometric and immunohistochemical (cell proliferation-PCNA; apoptosis- TUNEL; androgen receptor-AR) analysis. Morphological observations revealed that the testes present two periods of regression (TR) during the year: in late spring (November) and in early winter (June); and peaks of spermatogenic activity in early spring (September-October) and middle autumn (April). The TR in spring begins in October, with a sharply decrease in testicular epithelium and in the amount of PCNA+ cells; however, the amount of TUNEL+ and AR+ cells were basal in this period, indicating that the regression did not occur by apoptosis. In November the testes were totally regressed but, they already presented patterns of reactivation, as the increase in expression of AR and PCNA. In December was noted the greatest expression of PCNA and a great expression of AR, indicating an accelerated development, which can lead to errors that are eliminated by apoptosis, thus explaining the great amount of TUNEL+ cells. The recrudescence process extended from December to the production of the first spermatozoa only in March, with this period presenting a gradually decrease in the AR, PCNA and TUNEL expressions, culminating in the second period of TR in June. The TR in June was less accentuated than the first (November), presenting a more rapidly reactivation of the spermatogenesis (July). This reactivation was induced by the greatest AR expression and culminated in the greatest occurrence of apoptotic cells. All data demonstrated the presence of two periods of TR in the reproductive cycle of M. nigricans, a pattern never previously observed TUESDAY in bats; and that the apoptosis was not linked to the TR, but with the elimination of errors caused by the rapidly reactivation of the spermatogenesis.

2309 Reactive oxygen species cause reduced spore development and spore fitness in the budding yeast, Saccharomyces cerevisiae. M. M. Postema1, T. Stokes1, M. Scott1, S. W. Gorsich1; 1Biology, Central Michigan University, Mount Pleasant, MI

The generation of spores in yeast is essential for sexual reproduction and survival of the organism. When diploid yeast cells undergo meiotic division it is important that four optimally fit spores are produced. During this specialized division the four resulting spores not only have a haploid copy of nuclear chromosomes, but also a complete complement of organelles, such as mitochondria. Mitochondria undergo temporally regulated fusion and fission events to assure that mitochondria are represented equally in each of the resulting spores. When this mitochondrial network is not maintained, due to mutations in mitochondrial fission genes (eg. dnm1/dnm1), it has been shown that fewer spores survive and the ones that do survive are more likely to have reduced respiratory fitness. We hypothesized that environmental factors could also influence spore development in wild-type diploid yeast. In the present study, we demonstrated that hydrogen peroxide phenocopied spore development phenotypes associated with mitochondrial fission mutants. Wild-type yeast exposed to hydrogen peroxide have mitochondrial morphology and distribution defects during spore development, reduced spore viability, and decreased respiratory competency just as seen in dnm1/dnm1 fission mutants. Next we demonstrated that reactive oxygen species (ROS) sensitive mutants have increased ROS accumulation during spore development. In addition spore development defects associated with these mutants and wild-type cells exposed to hydrogen peroxide were reduced when treated with various antioxidant strategies. These findings suggest that the ability of yeast to produce optimal numbers of fit spores is heavily influenced by their ability to protect themselves from exogenous or endogenous ROS.

2310 A Role of Xenopus Zygote Arrest 2 (Xzar2) in Translational Regulation of Key Cell Cycle mRNAs. J. M. Cook1, T. M. Yamamoto1, A. Charlesworth1; 1Department of Integrative Biology, University of Colorado Denver, Denver, CO

Zygote arrest proteins, Zar1 and Zar2 (aka Zar1-like), have been implicated in the oocyte to embryo transition, zygotic genome activation, pre-implantation development and epidermalization. However, the mechanism of action of Zar proteins is unknown: transcriptional regulation, chromatin remodeling and RNA metabolism have all been suggested. Early Xenopus development is coordinated through the translational regulation of maternal mRNAs and is mediated by a variety of cis-elements in the 3’ untranslated region (3’UTR) of the mRNA. The recently described translational control sequence (TCS) is one such cis-element found in mRNAs, such as Wee1 and Pcm1. Here, we show that Xenopus Zar2 (Xzar2) binds the TCS of maternal mRNAs and can regulate translation in a progesterone responsive manner. In an electrophoretic mobility shift assay (EMSA), the C-terminal portion of XZar2 (CXzar2) bound a fluorescent labeled wild-type Wee1 3’UTR probe, resulting in a shifted protein-RNA complex. This band was super-shifted with a Zar2 antibody, verifying the presence of Zar2 protein in the band. Complex formation was inhibited by the addition of unlabeled Wee1 3’UTR or Pcm1 3’UTR probe, but was not inhibited if the TCSs were mutated, indicating the TCS is required for RNA binding. In a dual luciferase reporter (DLR) assay, the N-terminal domain of TUESDAY

Xzar2 fused to an MS2 tethering protein (NXzar2-MS2) was injected into oocytes and allowed to incubate with or without the addition of progesterone. NXzar2-MS2 repressed translation of the reporter mRNA in immature oocytes in a dose responsive manner. Furthermore, that repression was relieved during progesterone-induced meiotic maturation. Endogenous Zar2 levels were evaluated during maturation via Western blot, and it was found that levels drop dramatically during oocyte maturation. These data suggest that Xzar2 protein binds developmentally important mRNAs in a sequence specific manner and exerts translational control over those mRNAs differentially during meiotic maturation. We propose that one role of Xzar2 in early development may be to modulate the synthesis of maternal cell cycle regulatory proteins at the translational level in the maturing oocyte.

2311 Zygote Arrest Proteins Bind Maternal mRNAs. K. D. Silva1, T. Khat1, T. M. Yamamoto1, A. Charlesworth1; 1Department of Integrative Biology, University of Colorado Denver, Denver, CO

Zygote arrest proteins, Zar1 and Zar2 (aka Zar1-like), are important in early development. Zar1 has been implicated in the activation of the zygotic genome and progression past the two-cell stage. However, the mechanism of Zar protein action has yet to be characterized. The product of the maternal Wee 1 mRNA has also been identified as a regulator of early development. Xenopus laevis Wee 1 mRNA contains a newly characterized cis-element in its 3’untranslated region (UTR) called the Translation Control Sequence (TCS), which regulates mRNA translation during early development. The protein that binds to the TCS has yet to be identified. The objective of this study is to show that Zar proteins bind to Wee 1 mRNA. Zar1 and Zar2 proteins possess a highly conserved C-terminal region containing five pairs of invariable cysteine residues, four of which are thought to make up a putative atypical FYVE/plant homeodomain (PHD) zinc finger motif. Our data shows that Zar proteins use the conserved C-terminal region to bind the TCS of Wee1 maternal mRNA. To characterize binding to mRNA, Xenopus laevis Zar protein binding mutants, with point mutations at specified cysteine residues, were designed and used to perform electrophoretic motility shift assays (EMSA) with a Cy5 labeled Wee 1 3’ UTR probe. We show that cysteine to alanine mutation at any of the invariable cysteines disrupted Zar binding to Wee 1 mRNA. To test if zinc was a requirement for binding interactions, Zar protein was purified and an EMSA was performed in a zinc free buffer. This EMSA showed that without zinc, Zar proteins did not bind to Wee 1 mRNA. This data suggests that Zygote arrest proteins contain a new type of zinc finger motif that constitutes an mRNA binding domain, providing support to the hypothesis that Zar proteins play an important role in translational regulation of maternal mRNAs.

2312 Three-dimensional cell cultures of human urothelium by using a collagen gel scaffold. C. Wasén1, M. Levin2,3, D. Giglio1,3; 1Pharmacology, Neuroscience and Physiology, Göteborg, Sweden, 2Wallenberg Laboratory, Sahlgrenska Center for Cardiovascular and Metabolic Research, Göteborg, Sweden, 3The Sahlgrenska University hospital, Jubileumskliniken, Göteborg, Sweden

In vivo, urothelial cells create a 3-6 cell thick epithelial layer in the urinary bladder. In vitro, urothelial cells are most often grown two-dimensionally (2D) forming a one-cell layer. Three dimensional cell culture (3D) is an alternative to classic 2D culture. In 3D, epithelial cells form in vivo-like structures. In the present study, we investigated if urothelial cells may be grown three- dimensionally in a collagen gel scaffold. We used the UROtsa cell line, which is an immortalized TUESDAY normal human bladder urothelial cell line (Petzoldt et. al., 1995). UROtsa cells grown three- dimensionally in a collagen I gel formed spherical multicellular formations after 7 days. The cells expressed occludin and laminin indicating presence of tight junctions and basal membrane, respectively. The present findings reveal a new and promising way to study urothelial function in vitro by growing urothelial cells three-dimensionally in a collagen gel scaffold.

2313 The 3Ds - development, differentiation and death, of yeast colony. L-C. Liou1, Q. Ren1, Z. Zhang1; 1Zoology and Physiology, University of Wyoming, Laramie, WY

The budding yeast Saccharomyces cerevisiae has long been used as a model system to study all aspects of biology. Majority of the studies, however, are conducted in liquid culture. Yeast cells, when growing on solid medium, form organized colony structure. Cells inside the colony develop and differentiate into a mature structure under normal conditions. Colony cells may die when the colony encounters severe stress (internal, such as mutation or external, like drought). The objective of this study is to understand the mechanism of the development, differentiation and death of yeast colony. Using transmission electron microscopy (TEM), we showed that for a 4 day-colony, the dividing cells are mostly located at the periphery and the bottom of the colony, where cells are in direct contact with the nutrient. Dividing cells are also observed at the top surface of the colony, where cells are far away from the nutrient. The non-dividing cells are mostly located at the middle portion of the colony. TEM analyses of mutants that are defective of colony growth and development, including chc1 and sro7/sro77 double deletion, revealed that cell death occurs mostly in the middle portion of the colony. Our results suggest the cells in the middle portion of the colony play an important, supporting role for the yeast colony development and differentiation. They likely cease to divide, or even die, to support the proliferation of other cells inside the colony.

2314 An immunoglobulin domain-containing protein, IGP, is involved in trafficking of FGFR1. S. Kim1, J. Liu1, G. Yagnik1, A. Stoner1, C. Stevens1, E. Cherkez1, J. kim1, S. Boyadjiev1; 1UCDMC MIND Inst, Sacramento, CA

Fibroblast growth factor receptors (FGFRs) are critical for cell growth, differentiation, and animal development. Mutations in this receptor family are associated with craniofacial defects and cancer. In this study, we characterize an immunoglobulin domain-containing protein (IGP) that influences stability and function of FGFR1. When IGP and FGFR1 are coexpressed, their levels are increased compared to individual expression. In addition, IGP enhances conversion of FGFR1 from ER forms to Golgi forms. Furthermore, IGP enhances phosphorylation of PLC- gamma, but does not affect other FGFR1 signaling components such as AKT and ERK1/2. Interestingly, IGP predominantly exists as ER forms. These results indicate that IGP facilitates ER export of FGFR1 and activates the FGFR1-mediated PLC-gamma signaling pathway. Because aberrant activation of the FGFR signaling pathways is implicated in craniosynostosis (CS, premature fusion of cranial sutures), we have screened IGP for rare mutations from a cohort of nonsyndromic CS patients (431 individuals) who do not carry mutations in the previously known CS-causing genes. We have identified three rare missense mutations from this screening. Strikingly, one of these mutant IGPs further enhances phosphorylation of PLC- gamma. In summary, IGP plays a critical role in ER export/maturation/signaling of FGFR1. Furthermore, our results further suggest that IGP mutations may be a risk factor for CS.

TUESDAY

2315 Sonic Hedge Hog Pathway Expression in Normal and Megabladder Mice During Development. K. R. DeSouza1, M. Saha1, A. R. Carpenter1, K. M. McHugh1; 1Center for Molecular & Human Genetics, The Research Institute at Nationwide Children's Hospital, Columbus, OH

In this study, we present the first comprehensive assessment of the expression pattern of the Sonic Hedge Hog (shh) signaling pathway in the bladders of normal mice at consecutive stages during development. Additionally, we compared the expression pattern of the shh signaling pathway in megabladder (mgb-/-) mice to wild type mice to determine if this critical signaling pathway is altered in the mutant bladders of these animals. We characterized the expression pattern of the shh pathway’s ligand (Shh), receptor (Ptch), and transcription factor (Gli) and the key smooth muscle regulatory gene, Myocardin (Myocd), in the bladders of male and female wild type and mgb-/- mutant mice at embryonic days 13.5, 14.5, 15.5 and 16.5 (E13.5-E16.5), by in situ hybridization analysis. This study provides evidence that the shh pathway exhibits a canonical pattern of expression in wild type bladders, with each molecule displaying a specific spatial localization within the bladder. Shh expresses exclusively in the urothelium of the developing bladder. Ptch expresses in the proximal mesenchyme immediately adjacent to the urothelium, while Gli expresses in the distal mesenchyme, where smooth muscle cells eventually develop. Myocd, a key smooth muscle regulatory gene, expresses in the distal mesenchyme, where the presumptive smooth muscle develops in the bladder. Our study shows the shh pathway is functional in mgb-/- mutant bladders but shows a potential developmental delay with poorly restricted signal boundaries. In contrast, our results indicate that Myocd is minimally expressed in mgb-/- bladders when compared to controls. This study represents the first comprehensive analysis of the Shh signaling pathway during normal bladder development in mice. In addition, we show the shh pathway is functional in mgb- /- bladders but the key smooth muscle regulator, Myocd, is drastically reduced or absent. This finding suggests that the lack of detrusor smooth muscle development in mgb-/- bladders is not due absent short axis patterning but appears to result from a dramatic reduction in the expression of Myocd.

2316 Imbalance on cell proliferation and basal cell frequency of senile gerbil prostate: exposure effects of ethinylestradiol during the puberty. A. P. Perez1, M. F. Biancardi1, B. F. Gonçalves1, L. R. Falleiros Junior2, F. C. Santos3, S. R. Taboga2; 1Biology, Unicamp, Campinas, Brazil, 2Biology, IBILCE/UNESP, São Joé do Rio Preto, Brazil, 3Morphology, Federal University of Goias, Goiânia, Brazil

Endocrine disruptor chemicals (EDCs) are exogenous compounds with the potential to disturb hormonal regulation. Male and female rodent exposed to EDCs during critical time points of development, such as prenatal and neonatal as well as puberty, affect the reproductive tract of both sexes. Some studies show that the prostate is a highly sensitive gland to the EDCs action that might increase the risk of emergence of prostatic pathologies with aging. Thus, this study examined the ethinylestradiol effects during puberal development on histological differentiation, cell proliferation and p63-positive cells frequency of senile gerbil male ventral prostate (VP) and female prostate. For this, male and female gerbil (Meriones unguiculatus) with 42 days old received by gavage 15µg/kg/day of 17α–ethinylestradiol (EE- component oral contraceptive) diluted in mineral oil during one week (Pub group) and control group (males and females) were remained untreated. When males and females of control and Pub groups completed 12 months of age, they were sacrificed and male VP and female prostate were retired, processed and TUESDAY histological sections for each experimental group were subjected to immunohistochemistry for proliferating (PCNA) and basal (p63) cells. Puberty exposure to ethinylestradiol promoted different alterations in the senile gerbil male VP and female prostate. The Pub group male VP presented a significant increase in percentage cell proliferation (25.65±3.19) compared to control group (10.57±1.37) mainly in regions with invasive lesions. While this increase was not observed in the Pub group female prostate. Regarding the prostatic basal cells frequency, was observed a significant increment in female Pub group (23.24±1.18) compared to control group (12.82±0.91), indicating difference in proportion of main cell populations in acinar epithelium. The Pub group male VP did not presented significant difference in this frequency. In conclusion the synthetic estrogen exposure during puberty causes aberrant proliferation in prostatic cells of senile male, while in female prostate was observed basal cells stratification, mainly in regions with epithelial hyperplasia. Although prostatic epithelium proliferation is regulated by androgen, the estrogen is also able to cause this event. Thus the synthetic estrogen exposure during prostatic morphogenesis can favor the emergence of invasive lesions mainly in senile male prostate.

2317 F3 ENU genetic screen – a quest for endothelial cells directional movement cues. S. Krispin1, J. Yu2, T. Mulligan2, A. Davis2, D. Castranova2, B. M. Weinstein2; 1Program in Genomics of Differentiation, National Institute of Child Health and Human Development, NIH Bethesda, MD, 2 National Institute of Child Health and Human Development, NIH Bethesda, MD

Blood vessels are essential for tissue homeostasis within the complex bodies of vertebrates. Studies in developing vertebrates have uncovered many genes crucial for embryonic endothelial specification and for blood vessel differentiation and growth, but we still know little about what guides the patterning of developing blood vessels and determines the anatomical architecture of the vascular system. The metameric arrangement and relative anatomical simplicity of the zebrafish trunk primary angiogenic network makes it ideally suited to study the cues and mechanisms leading to the formation of vascular network. Of particular interest are the intersegmental vessels (ISVs), which emerge from the dorsal aorta (DA) and migrate dorsally along somites boundaries through angiogenesis. The cues that control this dorsal migration are still not understood. We have been using genetic screens to identify novel genes involved in endothelial cell (EC) directional movement. We used ENU (N-ethyl-N-nitrosourea) to induce random, single-base-pair mutations in the zebrafish genome, followed by a large-scale F3 genetic screen to uncover the homozygous vascular phenotypes of induced mutations. We facilitated the screening process by visualizing alterations in the vasculature primary network formation using a stable germline transgenic line, fli1-EGFP, which expresses enhanced green fluorescent protein in endothelial cells in vivo under the control of the fli1 promoter. We identified a new mutant, y210, in which trunk ISVs fail to migrate dorsally properly. We are in the process of mapping and positionally cloning the affected gene from the mutant, and then study its functional role in EC directional movements. Addressing this question will provide insight into one of the basic enigmas in developmental/vascular biology: what cues guide vessel positioning relative to other tissues and organs.

TUESDAY

2318 Novel antibodies to track neurogenesis in planarians. K. G. Ross1, M. Taylor1, R. K. Munday1, A. Hubert1, R. M. Zayas1; 1Biology, San Diego State University, San Diego, CA

Neural stem cells proliferate and integrate into the adult central nervous system (CNS) of diverse organisms, however there are gaps in our knowledge of the precise molecular mechanisms controlling these events. Planarian flatworms are amongst the simplest organisms to possess a CNS. Their nervous system is surprisingly complex at the molecular level, containing numerous diverse cell types, many of which demonstrate functional similarity with those found in vertebrates. Planarians maintain a population of adult pluripotent stem cells (neoblasts) that proliferate and differentiate in response to injury or during normal tissue homeostasis, enabling them to regenerate all of their tissues, including a complete nervous system. These features make planarians excellent models for in vivo studies of stem cell based CNS regeneration. However, many neurons in planarians have not been fully characterized and little is known about the spatial distribution of differentiating cells and temporal sequence of cell replacement during their regeneration. To improve our understanding of cell turnover and regeneration in the planarian CNS, we have performed a monoclonal antibody screen for neuronal markers in the planarian Schmidtea mediterranea. We have produced nine monoclonal antibodies thus far, each of which is specific to different planarian tissues, including discrete neuronal populations. We have characterized the staining patterns of these antibodies in intact and regenerating animals and are using them in combination with bromodeoxyuridine pulse-chase experiments to track neural differentiation in regenerating planarians. Our work has yielded new markers to help visualize CNS architecture and quantify differentiation of neuronal subpopulations. These analyses will help us understand how these animals maintain and reconstruct their nervous system and provide useful tools for phenotypic screening of gene knockdown experiments in planarians. This work was supported by CIRM Grant RN2-00940-1 to RMZ.

2319 Imaging Epithelial Morphogenesis in the Developing Hair Placode. L. Ahtiainen1, S. Lefebvre1, P. Lindfors1, I. Thesleff1, M. L. Mikkola1; 1Institute of Biotechnology, University of Helsinki, Finland

Development of different ectodermal appendages (hair follicle, mammary gland, tooth) shares similarities at the morphological and molecular level. All epidermal appendages begin as a thickening of the epithelium, a placode. The key signaling pathways regulating placode formation are rather well known, but the cellular mechanisms involved are still poorly understood. Two hypotheses have been proposed: local cellular proliferation or cell migration. Only very recently light microscopy imaging techniques have advanced to enable tracking of the fate of single cells in live tissue. This study aims at establishing and developing 3D and 4D live tissue imaging methodology for studying cellular processes regulating epithelial morphogenesis, in particular the role of the Ectodysplasin (Eda)/Eda receptor(Edar)/NF-κB signaling pathway in skin appendage development and disease. We have established laser scanning confocal imaging methodology that allows live visualization of E13.5-E14.5 mouse embryonic skin whole mount explants for up to 24h during the first wave of hair placode formation. As a tool we use K17-GFP embryonic mouse skin, where GFP expression driven by the K17 promoter marks emerging placode cells. We provide evidence indicating that epithelial cell migration is associated with hair placode formation and that inhibition of migration and actin remodeling suppresses placode formation. Imaging studies in the Fluorescent Cell Cycle Indicator (Fucci) TUESDAY mouse suggest that proliferation has a less prominent role in placode formation. Accordingly, inhibition of proliferation had no gross effect on timing or morphology of forming placodes.

2320 Multiparametric image analysis of lung branching morphogenesis. C. Schnatwinkel1, L. Niswander1; 1Pediatrics, Universtiy of Colorado, Denver/HHMI, Aurora, CO

Morphogenesis remains a fascinating problem in biology and proper morphogenesis is required for the shape and function of all organs and tissues. However, the morphogenetic mechanisms by which the genome moulds developing tissue masses into specific shapes remain poorly understood. To provide the first insights into the cellular dynamics of lung branching, we developed a life imaging set up to study cellular dynamics during branching morphogenesis. This approach helped in many ways to understand the interpretation of various phenotypes we have observed in mutant lungs. In this study, we performed a systematic time-lapse analysis of two of the recently postulated branching subroutines domain branching and planar bifurcation of epithelial cells in both lung explant- and organotypic three-dimensional (3D) culture. Our analysis revealed morphological and molecular distinct stages during branching morphogenesis that occurred in normal cultured lungs. We have classified a single bifurcation event into four morphological and molecularly distinct stages. Throughout the stages, we noticed a proximal to distal increase in proliferation, as well as a consecutive reduction in bud size before undergoing another round of bifurcation. This phenomenon may be a potential reason why lungs in culture only have a limited branching capacity. The stages are the following: (1) The bud stage: the size of the initial epithelial bud is increased due to proliferation, which appears to occur throughout the epithelium. (2) The flattening stage: Initially the distal tip cells apply mechanical tension along the basal membrane, whereas the surrounding mesenchyme acts partially as a stabilizing scaffold. This step may be a checkpoint stage and is required to precisely position and determine the cleft region for the subsequent step. (3) The bifurcation stage: epithelial cells on the two sides of the bud move slightly outward and then towards the distal end of the lung explants. This movement is partially provided by proliferation along the epithelial axis, which leads to the extension of the monolayered epithelium. Cells in the cleft region seem not to respond to migrational cues and remain in place. (4) The outgrowing stage: The cells at the two sides of the bifurcating bud continue their directional growth. Previously lateral cells become tip cells and cleft cells stay behind and localize to what becomes the stalk. To address the role of distinct cellular behaviors, we applied pharmacological inhibitors. Lung explants treated with an inhibitor of cell proliferation and apoptosis, prevented bud growth but maintained the capability to branch. On the other hand, treatment of explants with inhibitors against MLCK to break force generating processes in the cell allowed the bud to continue growing but failed to bifurcate if the inhibitor was applied before the flattening stage. However, new formation of a bud from the stalk region (domain branch) was still present. In fact, blocking the ATPase activity of myosin by blebbistatin increased the number and location of domain branches. Among other findings, this suggests that proliferation is a key step in driving domain branch formation, whereas local changes in force generating mechanisms mold and divide the distal bud into two.

Our finding defined a new set of parameters that can be applied to future studies with various types of mutant lungs as well as indicated that force generating mechanisms play an important role in the progression through the stages of branching morphogenesis.

TUESDAY

2321 In vitro assessement of bioactivities of rmGDF-9 and rhBMP-15. L. Niu1, V. Hernandez1, J. Li1, V. Kalabokis1, G. Wu1, J. Humphrey1, R. Hao1; 1R&D Systems, Minneapolis, MN

Growth differentiation factor -9 (GDF-9) and bone morphogenetic protein -15 (BMP-15) are oocyte-derived growth factors that are well known to play important roles in regulating folliculogenesis and female fertility. However, recent publications implicating their biological functions in human cancers raised the questions of whether GDF-9 and BMP-15 exert diverse biological functions in non-ovarian tissues. We have purified recombinant mouse GDF-9 and human BMP-15 from Chinese Hamster Ovary cells and evaluated their bioactivities using an array of bioassays. As expected, both mouse GDF-9 and human BMP-15 bind to the recombinant human/mouse BMP-RII/Fc extracellular domain with Kd values of 1.4 nM and 2 nM respectively, in a functional ELISA. In addition, the two proteins activate Smad 2/3 in a mouse embryonic teratocarcinoma P19 cells. Furthermore, we discovered that the recombinant mouse GDF-9, similar to other members of the transforming growth factor -βfamily, is able to induce apoptosis of mink lung epithelia MV1Lu and prostate carcinoma DU145 cells. In contrast, the recombinant human BMP-15, like other BMP family members, is osteogenic, promoting differentiation of MC3T3-E1 cells to osteoblasts. We also found that human BMP-15 is very potent in supporting the survival and proliferation of NIH3T3 cells in nutrient deprived conditions. These in vitro differential functions of GDF-9 and BMP-15 provide the stepping stones for new research initiatives and also open a whole new avenue to explore their functions in cancer biology, in addition to the area of reproductive physiology. These studies in turn may eventually lead to an expanded role in their therapeutic potential.

2322 Reg1 and Reg3β expression in the pancreas of adult diabetic Goto-Kakizaki (GK) rats. J-C. Irminger1, M-H. Giroix2, S. Calderari3, J. Ehses4, J. Coulaud2, K. Rickenbach1, P. Serradas3, F. Homo-Delarche2; 1University of Geneva, Geneva, Switzerland, 2University of Paris-Diderot, Paris, 3University Pierre et Marie Curie, Paris, 4University of British Columbia, Vancouver, BC, Canada

Reg protein expression is associated with islet development, β-cell damage, diabetes and pancreatitis. We previously reported that islets of 4-month-old (4-mo) GK rats, a spontaneous model of type 2 diabetes, overexpress Reg1, 3α, 3β and 3γ vs age-matched Wistar control islets. Reg1 and 3β are involved in cell growth/survival control and inflammation, respectively. Diabetic GK islets also exhibit progressive inflammation reaction, consisting of high CCL2 (MCP-1), CCL3 (MIP-1α), CXCL-1 (murine IL-8 analog) and IL-6 expression/release and mostly peri-islet macrophage infiltration. Importantly, Reg1 gene promoters contain IL-6-responsive elements. Here we analyzed in greater detail the pancreatic expression of Reg1 and Reg3β in diabetic GK rats. Isolated pancreatic islets and acinar tissue from male Wistar and GK rats were used for quantitative RT-PCR analysis. Islet IL-6, CCL2, CCL3 and CXCL1 release was measured by LuminexTM technology after a 48h islet culture on collagen. Pancreatic Reg immunohistochemistry (IHC) was performed on paraffin sections with a rabbit anti-human polyclonal antibody (Ab) and a mouse anti-rat monoclonal anti-Reg1 Ab. Macrophage infiltration was detected on cryostat sections using CD68 and MHC class II antibodies. Islet macrophage+ area was quantified and expressed as % of corresponding islet area. Statistical analyses used the Student’s t-test for unpaired data. The exocrine/endocrine ratio of Reg mRNA expression in 4-mo normoglycemic Wistar and diabetic GK rats was: 1) Reg1: 41.3±2.4 and 5.0±1.5, respectively (n=3 different TUESDAY isolations/group, p<0.005); 2) Reg3β: 74.2±17.0 and 7.0±4.9, respectively (n=3, p<0.02). Furthermore, Reg1 and 3β were overexpressed in GK vs Wistar islets (x11.2±1.4, p<0.005 and 77.9±16.3, p<0.01, respectively, n=3 in both cases). Next, the polyclonal Reg Ab stained most islet insulin+ cells in 4-mo Wistar pancreas but much less so in GK pancreas. By contrast, the monoclonal anti-Reg1 Ab stained just a few peri-islet exocrine cells in Wistar pancreas but many more in GK rats, particularly around large islets. Since IL-6 stimulates Reg1 expression, we compared cytokine/chemokine release by small and large 2.5-mo Wistar and GK islets. Large GK islets released significantly more IL-6 and CCL3 than large Wistar islets (x4.7±0.9, p<0.02 and 2.7±0.2, p<0.005, respectively, n=3 in both cases). Concomitantly, CD68+ and MHC II+ peri-islet macrophage infiltration correlated with islet size in 2.5-mo GK rats (r=0.57 and r=0.95, respectively). Reg1 and 3β genes are strongly expressed in the exocrine pancreas of control rats. The two genes are markedly overexpressed in islets of diabetic GK vs control Wistar rats, probably reflecting an adaptive stress response. While the polyclonal Ab mainly stains islet insulin+ cells, Reg1 protein is the hallmark of acinar tissue around large GK islets. Its peri-insular localization might result from increased islet release of IL-6 (a factor stimulating Reg1 expression) and is similar to that of macrophage infiltration.

2323 Morphometric changes in the testis of Gallus domesticus during aging. E. Soria-Castro1, G. González-Móran2; 1Patología, Instituto Nacional de Cardiologia Ignacio Chavez, Mexico City, Mexico, 2Biología Animal Comparada, UNAM, Mexico City, Mexico

The male reproductive system in mammals has been extensively studied, but investigations on birds testes have focused mainly in testes of sexually mature fowl and during the embryonic stages. Therefore, the aim of this paper was to contribute to knowledge of the biology of aging through the histomorphometric methods in testes from mature (12-month-old, sexual maturity) and aged (48 month-old, cessation of reproductive function) chickens; of these animals left testes was dissected and testes measurements were recorded (length, width and thickness). The volume of the organ were determined according to Ahmed et al., 1969. The central portion of the testes was fixed in 2.5% glutaraldehyde, followed by osmium tetroxide 2% washed with cacodylate buffer and dehydrated in acetone and embedded in Epon 812. Semi-thin were obtained from 800 nm for histological observations and stereological evaluations. The total volumes of the interstitial tissue and of the tubular compartment, and the diameter of seminiferous tubules were estimated. Results indicate that in mature chick testes, the total volume occupied by the tubular compartment is greated than that occupied by the interstitial tissue, and all stages of spermatogenesis in seminiferous tubules were found, but in the aged chick testes the total volume occupied by the tubular compartment is almost equal to that occupied by interstitial tissue, and exhibited damage and deterioration of seminiferous tubules and interstitial tissue, containing cellular fragments and fibrous material. Seminiferous tubules presented deformations with monolayer spermatic epithelium with some Sertoli and germ cells. This findings indicate that there are marked age-dependent changes in chicken testes histology, and may explain the cessation of reproductive function in these aged animals.

2324 Investigating the process of single lumen formation in the zebrafish gut. A. Alvers1, M. Bagnat1; 1Duke University Medical Center, Durham, NC

Tubes are a key element in the design of many organs including the lungs, vasculature and the gut. While these tubes are assembled through very diverse developmental processes they always possess a single lumen. To uncover cellular processes controlling single lumen TUESDAY formation we are using the zebrafish gut as model system. The zebrafish gut tube forms from a solid rod of endodermal cells that differentiate and start to polarize and assemble tight junctions which give rise to multiple small lumens. These small lumens subsequently enlarge though the accumulation of fluid and eventually coalesce to form a single lumen. However, other cellular processes such as the remodeling of cell contacts must also be involved to facilitate coalescence into a single lumen. We are interested in these additional cellular processes that regulate the transition into a single lumen. Analysis of lumen formation in wild-type (WT) embryos revealed that the coalescence of multiple small lumens into one involves the formation of a double lumen intermediate. Interestingly, we found that smoothened (smo) mutants often fail to properly form a single lumen and instead display a double lumen phenotype. In smo mutants, cells are frequently found in between the two lumens and display mislocalization of adhesion proteins which suggests a defect in the remodeling of adhesive contacts. We analyzed the transcriptome of smo mutants and found that several genes associated with intracellular trafficking were down-regulated. In light of this finding, we generated transgenic zebrafish lines to determine the function of trafficking and recycling in the process of single lumen formation. Our analyses suggest that that intracellular membrane recycling is required for single lumen formation. Together, our data suggest that smo regulates a transitional step in single lumen formation that facilitates intercellular contact remodeling through recycling.

Cancer Cell Biology II

2325 Rapamycin treatment increases mTORC2 activity and selectively activates the AKT1 isoform in epidermis by inhibition of the mTORC1-dependent feedback loop to IRS-1. K. Sully1, O. A. Akinduro1, M. P. Philpott1, C. A. Harwood1, R. F. O'Shaughnessy2, C. Byrne1; 1Cutaneous Research, Queen Mary University of London, London, United Kingdom, 2Immunobiology, UCL Institute of Child Health, London, United Kingdom

Epidermal squamous cell carcinoma (SCC) is dramatically increased in patients undergoing immunosuppression following organ transplantation. The mammalian target of rapamycin complex 1 (mTORC1) inhibitor, rapamycin, when used as a post-transplantation immunosuppressive, significantly reduces SCCs, though the mechanism is not understood. Epidermal SCC is associated with changes in AKT signaling. AKT2, associated with less differentiated keratinocytes, is upregulated in SCC and in response to ultraviolet (UV) radiation, suggesting a tumour-promoting role. In contrast, AKT1, associated with differentiated keratinocytes, is downregulated in SCC and after UV radiation, suggesting a tumour-suppressor role. In some tumour types rapamycin activates AKT signaling via inhibition of mTORC1- dependent negative feedback loops acting on the insulin receptor substrate-1 (IRS-1). It is unclear whether this feedback loop is active in epidermis. Upregulation of epidermal AKT signaling by rapamycin would have different effects on tumourigenesis depending on which AKT isoform is upregulated. The objective was to establish whether the mTORC1-dependent negative feedback loop to IRS-1 is active in epidermis and find which epidermal AKT isoform is affected. We show that rapamycin increases keratinocyte AKT phosphorylation at both S473 and T308, the two sites required for full activation, with a single treatment of rapamycin maintaining increased AKT phosphorylation for over 24 hours. Both acute and prolonged rapamycin treatment also increases epidermal mTORC2 activity, shown by upregulation of downstream targets. We demonstrate that the mTORC1-dependent negative feedback loop to IRS-1 is active in keratinocytes and responsible for rapamycin-induced mTORC2 activity and AKT phosphorylation. Rapamycin inhibits mTORC1-dependent phosphorylation of IRS-1 at TUESDAY

Ser636/639 and increases IRS-1 tyrosine phosphorylation, protein stability and downstream signaling to AKT. Epidermal AKT1 isoform, but not AKT2, is selectively upregulated via rapamycin’s effects on epidermal mTORC2 and inhibition of the mTORC1-dependent feedback loop to IRS-1. These findings suggest a mechanism for rapamycin’s anti-tumour activity in skin. We propose that rapamycin’s selective activation of epidermal AKT1 promotes AKT1’s tumour-suppressing role in epidermis and protects against epidermal tumourigenesis.

2326 Gab2 regulates the migratory behaviors and E-cadherin expression via activation of the PI3K pathway in ovarian cancer cells. Y. Wang1, Q. Sheng2, M. A. Spillman3, K. Behbakht3, H. Gu1; 1Department of Pathology, University of Colorado Denver Anschutz Med Campus, Aurora, CO, 2Department of Cancer Biology, Dana-Farber Cancer Institute, and Harvard Medical School, Boston, MA, 3Department of Obstetrics and Gynecology, University of Colorado Denver Anschutz Med Campus, Aurora, CO

Ovarian cancer, the most deadly gynecologic malignancy, is often diagnosed late and at the advanced stage when the cancer cells have already migrated and invaded into other tissues and organs. Better understanding of the mechanism of metastasis in ovarian cancer cells is essential to the design of effective therapy. In this study, we investigated the function of scaffolding adaptor protein Gab2 in ovarian cancer cells. Gab2 is found to be overexpressed in a subset of ovarian tumors and cancer cell lines. Gab2 expression mainly regulates the migratory behaviors of ovarian cancer cells. Overexpression of Gab2 promotes the migration and invasion, and down-regulates E-cadherin expression in ovarian cancer cells with low-Gab2 expression. Conversely, knockdown of Gab2 expression inhibits the migration and invasion, and promotes E-cadherin expression in ovarian cancer cells with high-Gab2 expression. By expressing Gab2 wild type and Gab2 mutants that are defective in activation the PI3K and Shp2-Erk pathways, we find that Gab2 inhibits E-cadherin expression and enhances the expression of Zeb1, a transcription factor involved in epithelial-to-mesenchymal transition (EMT), and cell migration and invasion through the activation of the PI3K pathway. Knockdown of Zeb1 expression blocks Gab2-induced suppression of E-cadherin expression and increase in cell invasion. LY294002 and GDC-0941, inhibitors of PI3K, or Rapamycin, an inhibitor of PI3K downstream target mTOR, can reverse the effects of Gab2 on migration and invasion. Overall, our studies reveal that Gab2 overexpression, via activation of the PI3K-Zeb1 pathway, promotes characteristics of EMT in ovarian cancer cells. Ovarian cancer cells with high Gab2 expression are potentially more metastatic in vivo and drugs that target the PI3K pathway may be used to treat a subset of ovarian cancer with high Gab2 expression.

2327 LRIG2 promotes PDGF induced experimental glioma. C. Holmlund1, T. Karlsson1, M. Faraz1, O. Casar Borota2,3, J. Miller4, B. Wang1, C. Sweeney4, R. Henriksson1, H. Hedman1; 1Radiation Sciences, Oncology, Umeå, Sweden, 2Medical Biosciences, Pathology, Umeå, Sweden, 3Laboratory Medicine, Pathology/Cytology, Umeå, Sweden, 4University of California, UC Davis Medical Center, Sacramento, CA

Leucine-rich and immunoglobulin-like domains protein 2 (LRIG2) is a transmembrane protein, whose expression is associated with poor survival of oligodendroglioma patients. This is in contrast with findings for the homologous protein LRIG1, a negative regulator of growth factor signaling, whose expression is associated with good survival in several different malignancies. Here, we address the role of LRIG2 in an experimental glioma model and its possible regulation TUESDAY of platelet-derived growth factor (PDGF) receptor (PDGFR). Gliomas were induced in Ntv-a mice by intracranial injection of PDGFB-expressing RCAS viruses. All injected Lrig2-wild-type mice developed oligodendroglioma-like brain tumors of grade II/III (82%) or glioblastoma-like tumors of grade IV (18%), whereas Lrig2-deficient mice only developed grade II/III tumors (77%) or no detectable tumors (23%). In transfected cells, LRIG2 and PDGFRα co-localized in intracellular vesicle-like structures. Intriguingly, LRIG2, in contrast to LRIG1, did not negatively regulate the protein levels of PDGFRα or PDGFRβ. Instead, LRIG2 up-regulated the protein levels of PDGFRα. In summary, we show that LRIG2 promoted the genesis and/or progression of PDGF-induced oligodendroglioma and positively regulated PDGFRα protein levels. This further supports the notion that LRIG proteins play important roles in cancer biology and suggests that LRIG1 and LRIG2 may have opposing functions in the regulation of PDGF signaling.

2328 The Study of Androgen Effects on LPA-induced VEGF-C Expression in Prostate Cancer Cell Lines. Y-C. Lin1, H. Lee2; 1Institute of Zoology, National Taiwan University, Taipei, Taiwan, 2Department of Life Science, National Taiwan University, Taipei, Taiwan

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that binds to a family of G protein- coupled receptors (GPCRs), termed LPA1~LPA5. It has been reported that vascular endothelial growth factor-C (VEGF-C) was related to the lymph node metastasis in human prostate cancer. Our previous study has indicated that LPA induces the expression of VEGF-C in human prostate cancer PC-3 cell line. Moreover, a well-known effector, androgen, modulating prostate cancer cell progression was also shown to negatively regulate VEGF-C expression in prostate cancer. Therefore, we attempt to investigate whether LPA-induced VEGF-C expression is affected by androgen in different prostate cancer cells. By real-time PCR analysis, we proved that LPA could trigger VEGF-C expression in dosage and time-dependent manners in different prostate cancer cell lines. In addition, our results further showed that androgen agonist decreased LPA-induced VEGF-C expression. On the contrary, androgen antagonist enhanced LPA-induced VEGF-C expression. In summary, our results demonstrated that androgen was involved in LPA-induced VEGF-C expression in human prostate cancer cells.

2329 Mitochondrial Genome Instability and ROS Enhance Intestinal Tumorigenesis in APCMin/+ mice. D. Woo1, P. Green2, J. Santos2, A. D’Souza3, Z. Walther3, D. Martin4, B. Christian3, N. Chandel5, G. Shadel3; 1Gyeongsang National University, Jinju, Korea, 2University of Medicine and Dentistry, New Jersey-New Jersey Medical School, Newark, NJ, 3Yale University School of Medicine, New Haven, CT, 4Emory University School of Medicine, Atlanta, GA, 5Northwestern Medical School, Chicago, IL

Alterations in mitochondrial oxidative phosphorylation have long been documented in tumors. Other types of mitochondrial dysfunction, including altered reactive oxygen species (ROS) production and apoptosis, can also contribute to tumorigenesis and cancer phenotypes. Furthermore, mitochondria contain mtDNA, mutation and altered amounts of which have been observed in cancer cells. However, how mtDNA instability per se contributes to cancer remains largely undetermined. Tfam is a mitochondrial transcription factor and mtDNA-packaging protein required for expression and maintenance of mtDNA. Tfam heterozygous knock-out (Tfam+/-) mice exhibit mild mtDNA depletion, but have no overt phenotypes. Here, we show that Tfam+/- mouse cells and tissues not only have less mtDNA, but also increased oxidative mtDNA TUESDAY damage. Crossing Tfam+/- mice to the APCMin/+ mouse cancer model revealed that mtDNA instability increases tumor number and growth in the small intestine. This was not due to enhancement of Wnt/-catenin signaling, but rather appears to involve a propensity for increased mitochondrial ROS production. Direct involvement of mitochondrial ROS in intestinal tumorigenesis was demonstrated by crossing APCMin/+ mice to those that have catalase targeted to mitochondria (MCAT), which resulted in a significant reduction in tumorigenesis in the colon. Altogether, these results show that mitochondrial genome instability and ROS can enhance tumorigenesis, and elucidate Tfam+/- mice as relevant model to address the role of mtDNA instability in disease states where mitochondrial dysfunction is implicated, such as cancer, neurodegeneration and aging.

2330 Upregulation of Plk1 in castration-resistant prostate cancer cells. A. Deeraksa1, J. Pan1, Y. Sha1, X. Liu1, A. C. Ferrari2, N. T. Eissa1, S-H. Lin3, L-Y. Yu-Lee1; 1Medicine, Baylor College of Medicine, Houston, TX, 2Medicine, New York University School of Medicine, New York, NY, 3Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, TX

Castration-resistant progression of prostate cancer (PCa) is a challenge in PCa treatment. A recent study found that PCa cells cultured in charcoal-stripped media (androgen-independent AI condition) have undergone a genetic reprogramming that selectively upregulated the expression of M-phase cell cycle genes. We found that the mitotic kinase Plk1 is upregulated during the reprogramming process in AI PCa cells. We employed a pair of PCa cells, LNCaP (androgen- dependent PCa cells derived from lymph node metastasis) and LNCaP-AI cells (AI PCa cells derived from LNCaP), to determine the role of Plk1 in castration-resistant progression of PCa.

We found that LNCaP-AI cells express 3 – 5 fold higher levels of Plk1 relative to LNCaP cells. Cell cycle proteins along the Plk1-regulated mitotic pathway, including Cdc25C (mitotic entry), Sgo2 (chromosome cohesion), PICH (chromosome architecture), BubR1 (spindle checkpoint), and NudC (cytokinesis) are all elevated in LNCaP-AI cells. Both LNCaP and LNCaP-AI cells were highly sensitive to a 5-day treatment with a small molecule inhibitor of Plk1, BI2536, with IC50 of 0.6 nM and 0.2 nM, respectively, compared to many other cell types with IC50 between 10 – 50 nM. Under charcoal-stripped culture conditions, LNCaP did not grow or respond to BI2536 treatment. In contrast, LNCaP-AI cells remained highly sensitive to BI2536 inhibition, suggesting that the upregulated Plk1 pathway is inhibited by BI2536 in LNCaP-AI cells.

We further examined whether growth inhibition by BI2536 leads to cell death. In full medium culture conditions, LNCaP cells responded to BI2536 with caspase 3 cleavage and membrane blebbing, characteristics of apoptosis. In contrast, LNCaP-AI cells did not show caspase 3 cleavage but exhibited PARP-1 cleavage, a common end point for cell death. Immunofluorescence microscopy showed unusual nuclear morphologies in BI2536-treated cells. LNCaP cells contained a single enlarged/lobular nucleus, a phenomenon previously observed with Plk1 inhibition in other cell types. In contrast, LNCaP-AI cells formed giant cells that contained up to 30 small and large independent nuclei within a single cell unit, likely due to nuclear fragmentation and cytokinesis failure. Live cell imaging indicate that these giant cells underwent membrane disintegration and cell death characteristic of necroptosis. These results suggest that LNCaP-AI cells utilize a caspase 3-independent cell death modality in response to BI2536 inhibition. These observations suggest that Plk1 has a novel role in cell growth/death regulation in LNCaP-AI cells and Plk1 may be targeted for therapy of castration-resistant PCa.

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2331 HPV E6 oncoproteins from high and low risk types associate with the proteasome independently of E6AP. L. M. Banks1, V. Tomaic1; 1ICGEB, Trieste, Italy

Human papillomavirus (HPV) E6 oncoproteins associate with components of the ubiquitin proteasome pathway, including the cellular ubiqutin ligases E6AP, EDD and HERC2. E6AP was shown to play a major role in E6’s degradatory activities, while more recent studies suggest that its presence is also critical for E6 protein stability. In addition, it has been suggested that E6’s association with EDD could have a profound effect upon the ability of E6 to target various substrates for proteolytic degradation and thereby directly influence the development of HPV- induced malignancy. To further investigate E6 function, we performed a mass spectrometric analysis on HPV-18 E6 and E6AP protein complexes. Besides identifying several known binding partners of these two proteins, we also identified several proteasome regulatory subunits as being associated with E6 and E6AP. We also show that both HPV-18 E6 and E6AP, independently of each other, interact with proteasome regulatory subunits in vitro. HPV-18 E6 preferentially interacts with S2 and S4 subunits, while E6AP interacts with S2, S4, S5a, S7 and S8 subunits, an interaction that is independent of E6AP’s catalytic activity. Furthermore, HPV-16 E6 and HPV-11 E6 also preferentially interact with the S2 and S4 subunits, although the HPV- 16 E6 interaction is significantly stronger than that with HPV-11 E6. In presence of endogenous E6AP HPV-18 E6 also binds strongly to the subunit S5a, while binding to S2 and S4 subunits is reduced. These results demonstrate that E6AP can recruit E6 to the S5a subunit. Ongoing studies will now aim to elucidate these aspects further and clarify how E6AP mediates E6s’ association with the proteasome.

2332 Cyclin B1 and B2 have oncogenic properties but drive tumorigenesis through distinct mechanisms. H-J. Nam1, J. van Deursen1,2; 1Pediaric and Adolescent Medicine, Mayo Clinic, Rochester, Rochester, MN, 2Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Rochester, MN

High cyclin B1 and B2 levels are observed in a large proportion of human tumors and correlate with poor clinical outcome. However, the molecular and cellular defects caused by increased expression of these mitotic cyclins and their roles in tumorigenesis are unknown. To address these questions, we generated transgenic mice that overexpress cyclin B1 or B2. These mice show high aneuploidy rates, with cyclin B1 overexpression inducing chromatin bridges and cyclin B2 overexpression lagging chromosomes. Furthermore, cyclin B1 overexpression leads to persistent inhibition of separase, while cyclin B2 overexpression does not. Conversely, cyclin B2 overexpression induces supernumerary centrosomes, whereas cyclin B1 does not. These results demonstrate that both cyclin B1 and B2 induce chromosome missegregation, although through distinct mechanisms. Furthermore, preliminary results indicate that both cyclin B1 and B2 transgenic mice are prone to spontaneous tumors, although the tumor spectra of the transgenes are remarkably different. In addition, it seems that cyclin B2 overexpression, but not cyclin B1 overexpression, predisposes mice to DMBA-induced cancers. Taken together, the data suggest that both cyclin B1 and cyclin B2 drive chromosomal instability and tumorigenesis, but through markedly different mechanisms and in distinct tissues.

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2333 Reactive oxygen species regulates CDK2 activity differently in normal and cancer cells to induce senescence and proliferation. C. Hwang1, S-M. Lee1, K-S. Kwon1; 1Aging Research Center, KRIBB, Daejeon, Korea

Reactive oxygen species (ROS) were previously shown to trigger cellular senescence through several pathways such as Ras, p38MAPK, p66shc, Rb, p53, and p21Cip1. However, the detailed mechanisms remain to be elucidated. Previously we demonstrated that p21Cip1 was degraded at an early phase after low dose H2O2 treatment in a variety of cell types. Here, we observed that low dose of H2O2 induced cellular senescence in HDFs and IMR90 cells but induced cellular proliferation in cancer cells, even though p21Cip1 was degraded in both cases. Interestingly, normal cells showed sustained inhibition of CDK2 activity upon H2O2 stimulation, which is a likely mechanism in cell cycle arrest and subsequent senescence. However, H2O2 stimulation resulted in temporary CDK2 activation in cancer cells, which might cause H2O2-induced proliferation. Still unclear are how ROS control CDK2 activity differently in the normal and cancer cells.

Cip1 Key words: H2O2, CDK2, p21 , senescence, proliferation, cell fate

2334 The nuclear translocation of hTERT is mediated by the classical importin alpha/beta- dependent import pathway. S. Jeong1, J. Lee1, I. Chung1; 1Department of Biology and Integrated Omics for Biomedical Science, WCU Program of Graduate School, Yonsei university, Seoul, Korea

The maintenance of eukaryotic telomeres requires telomerase, which is minimally comprised of a telomerase reverse transcriptase (TERT) and an associated RNA component (TERC). Telomerase activity is tightly regulated by expression of hTERT at both the transcriptional and posttranslational levels. In our previous study, we identified the region of bipartite nuclear localization signal (NLS) of hTERT. However, the molecular mechanism of nuclear translocation of hTERT remains unclear. To investigate clear nuclear translocation mechanism, we minimized the NLS region of hTERT, and then this NLS region was conjugated to Glutathionine S- transferase (GST). Clearly, GST protein was diffused at both cytoplasm and nucleus. In contrast to GST protein, GST-conjugated wild type NLS protein was predominantly localized in the nucleus and GST-conjugated mutant (7A+S227A) NLS protein was not. To prove the molecular mechanism of nuclear translocation, we co-immunoprecipitated GST-conjugated wild-type NLS or endogenous hTERT with importin α/β1. And we also measured telomerase activity after importin alpha, beta and Ran overexpression by TRAP assay. We next investigated cells selectively depleted of importin alpha and beta1 by transfection with siRNAs. We observed the effect of importin siRNAs on endogenous hTERT by immunofluoresence analysis, subcellular fractionation assay and TRAP assay. These findings demonstrate that the nuclear translocation of hTERT is mediated by the classical importin α/β- dependent import pathway.

2335 P300-mediated Acetylation of TRF2 is required for Telomere Maintenance. Y. Her1, I. Chung1; 1Department of Biology and Integrated Omics for Biomedical Science, WCU Program of Graduate School, Yonsei University, Seoul, Korea

Human telomeres are protected from DNA damage by a nucleoprotein complex that includes the repeat-binding factor TRF2. The Abundance of TRF2 protein at telomeres is tightly TUESDAY regulated by post-translational modifications, protein-protein interactions and protein stabilization. Here we report that TRF2 is physically interacts with p300 and its lysine 293 is acetylated by p300 in vitro and in vivo. Whereas overexpression of p300 increases the level of telomere-bound TRF2, depletion of endogenous p300 expression decreases the level of telomere-bound TRF2. We show that loss of TRF2 from telomeres results in ubiquitination and degradation of TRF2 by the proteasome. In addition, we found that p300-mediated TRF2 stabilization is dependent of p300 catalytic activity, using either the inactive form of p300 or the acetylation mutant TRF2-K293R. Depletion of p300 in cancer cells induced DNA-damage response foci at internal genome as well as telomeres as evidenced by telomere dysfunction- induced foci, indistinguishable from TRF2-K293R stably expressing cell lines. Some of the metaphase chromosomes showed no telomeric signal at chromatid ends, suggesting an aberrant telomere structure. Furthermore, we found that stable overexpression of K293R induces growth arrest in human cancer cells, suggesting that K283R may control cell proliferation. Taken together, these results suggest p300-mediated acetylation of TRF2 is required for the efficient telomere binding of TRF2 as well as telomere protection and represents a new pathway for telomere maintenance by modulating the level of TRF2 at telomeres.

2336 Human chromosomal passenger Borealin co-exists with active telomerase and regulates telomerase activity. P. Khadka1, J. Lee1, S. Beak1, I. Chung1; 1Department of Biology and Integrated Omics for biomedical science, WCU program of Graduate school, Yonsei University, Seoul, Korea

From different studies it has shown that the conventional DNA metabolism machineries directly or indirectly regulate telomerase activity and telomere, still it is not clearly known how DNA replication proteins regulate telomerase. Here, we report the role of borealin, member of chromosomal passenger, which binds with hTERT and regulates telomerase activity. Borealin is a member of the chromosomal passenger complex that plays important role as a regulator of DNA replication, chromosome alignment, and spindle assembly checkpoint. Here, we show that borealin co-exists with active telomerase in vivo via telomeric DNA in telomerase positive cells and telomerase-immortalized human somatic cells but not in human ALT cells. In the present study, we demonstrate that hTERT interacts with borealin both in vitro and in vivo. Here, we show that the association of borealin with hTERT is independent of RNA but requires DNA and also we show that N-terminal of hTERT binds to borealin. Furthermore, we show that borealin depletion by RNA interference reduces the telomerase activity inducing the cell growth arrest in MCF7 cells. Borealin has until now been described as a component of chromosomal passenger, mainly involved in DNA replication, here, we confirmed that about 30%-40% of borealin can bind to telomeric end. This is, to our knowledge, the first evidence that Borealin coexist with active telomerase and regulates telomerase activity, revealing a not yet recognized link of this CPC protein to telomere.

2337 PIAS1 and RNF4 regulate the homeostasis of SUMO-conjugated TRF2. J. Her1, I. Chung1; 1Department of Biology and Integrated Omics for biomedical science, WCU program of Graduate school, Yonsei University, Seoul, Korea

Eukaryotic chromosome end is composed of DNA and proteins, called telomere and forms T loop to protect chromosome ends. TRF2 is a major component of sheltrin complex and function to prevent loss of genetic materials and shelter chromosome end to avoid DNA damage machinery. Without TRF2, DNA damage signal occurs and cells go to apoptosis. To avoid it, TRF2 mostly binds to telomere and is very stable. So we think that there should be complex TUESDAY mechanism to degrade. Sheltrin complex proteins are regulated by several post translational modifications. Among these modifications, SUMOylation is one of essential modifications in ALT cells. It is known that TRF1 and TRF2 are sumoylated by MMS21 and their SUMOylation is required for APB formation in ALT cells but it is not clear what the function of TRF1 and TRF2 SUMOylation is in telomerase positive cancer cells. Here, we found that PIAS1 is another SUMO E3 ligase for TRF2 and SUMOylated TRF2 is regulated by proteosomal degradation. SUMO-specific E3 ubiquitin ligase, RNF4, is also involved in SUMOylated TRF2 degradtion and control the levels of SUMOylated TRF2.

2338 Disruption of CTCF at the miR-125b1 locus in gynecological cancers. E. Soto-Reyes1, R. Gonzalez1, F. Cisneros-Soberanis1, C. Castro2, D. Cantú1, F. Recillas- Targa3, L. A. Herrera4,5; 1Dirección de Investigación, Instituto Nacional de Cancerología, Mexico, Mexico, 2Genómica y Toxicología Ambiental, Instituto de Investigaciones Biomédicas UNAM, Mexico, 3Biología Molecular, Instituto de Fisiología Celular UNAM, 4Instituto Nacional de Cancerología, Mexico, 5Instituto de Investigaciones Biomédicas UNAM, Mexico

In cancer cells, transcriptional gene silencing has been associated with genetic and epigenetic defects. The disruption of DNA methylation patterns and covalent histone marks has been associated with cancer development. Until recently, microRNA (miRNA) gene silencing was not well understood. In particular, miR-125b1 has been suggested to be an miRNA with tumor suppressor activity, and it has been shown to be deregulated in various human cancers. Herein, we characterized the CCCTC-binding factor (CTCF) at the miR-125b1 locus in normal and breast cancer cells. The disruption of CTCF in breast cancer cells correlated with the incorporation of repressive histone marks such H3K9me3 and H3K27me3 as well as with aberrant DNA methylation patterns. To determine the effect of DNA methylation at the CpG island of miR-125b1 on the expression of this gene, we performed a qRT-PCR assay. We observed a significant reduction on the expression of miR-125b1 in cancer cells in comparison with controls, suggesting that DNA methylation at the CpG island might reduce miR-125b1 expression. These effects were observed in other gynecological cancers, including ovarian and cervical tumors. Our data strongly suggest that the loss of CTCF may contribute to the destabilization of chromatin permissive for transcription and the establishment of an aberrant repressive chromatin configuration, including DNA hypermethylation, that induce the epigenetic silencing of the miR-125b1 locus.

This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACyT: 83959) and Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica of the Universidad Nacional Autónoma de México (PAPIIT, IN213311).

2339 Cancer cell proliferation regulated by DOT1L histone methyltransferase. W. KIM1, R. KIM1, G. PARK1, J. KIM1; 1Kyung Hee University, Seoul, Korea

DOT1L, a histone methyltransferase for histone H3 K79, participates in the regulation of transcription, development, erythropoiesis, differentiation and proliferation of normal cells. With respect to cancer, DOT1L-mediated methylation contributes to development of MLL-rearranged leukemias. However, the role of DOT1L in cancer cell proliferation has not been fully elucidated. DOT1L siRNA-transfected A549 and NCI-H1299 lung cancer cells displayed a non-proliferating multinucleated phenotype. DOT1L-deficient cells also showed abnormal mitotic spindle formation and centrosome number, suggesting that DOT1L deficiency leads to chromosomal missegregation. This chromosomal instability led to cell cycle arrest at the G1 phase and TUESDAY induced senescence determined by enhanced activity of senescence-associated (- galactosidase activity. Meanwhile, overexpression of siRNA-resistant DOT1L, not a catalytically inactive mutant, substantially restores DOT1L siRNA-induced phenotypes. Overall, these data imply that downregulation of DOT1L-mediated histone H3 Lys79 methylation inhibits proliferation of cancer cells. Therefore, the inhibition of DOT1L activity might act as a barrier to aggressive growth of cancer cells.

2340 Class 3 semaphorins and their receptor complexes- neuropilin/plexins regulated by E2F1 and SOX4 in pancreatic ductal adenocarcinoma. H-Y. Huang1,2, Y-Y. Cheng1, P-H. Huang1,2; 1Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan, 2Institute of Pathology, National Taiwan University, College of Medicine, Taipei, Taiwan

Ligand/receptor co-expression can allow cells to respond to growth and survival demand through signal transduction in a certain circumstance, like Eph/Ephrin co- expression modulates axon guidance in the developing central nervous system Individual members of class 3 semaphorins (SEMA3), secreted cues for axonal growth cone guidance molecules, are frequently over-expressed with their receptor complexes plexins/neuropilins (PLXNs/NRPs) in tumors such as breast cancer, hepatocellular carcinoma, pancreatic cancer, etc. We thoroughly examined expression of SEMA3 and their receptors in pancreatic ductal adenocarcinoma (PDAC) and found that, unlike other epithelial malignancies that often over-express one or few specific members of SEMA3/PLXN family, combined expression of more than 5 members of SEMA3/Plexin family are noted in most human PDAC cases. Analysis of promoter regions of SEMA3, plexins and NRPs revealed that putative binding sites of E2F1 and SOX4 are almost always present in all SEMA3 and plexin promoters. With chromatin-immunoprecipitation, luciferase promoter activity assay, and electrophoresis mobility shift assay, E2F1 and SOX4 were demonstrated to directly bind the consensus sites at the promoter of each SEMA3 and plexin gene we examined to enhance the transcriptional activity. Statistical analysis further showed correlation between SOX4 expression and poor survival in human PDAC. Conversely, RNAi-knockdown of SOX4 resulted in concomitant decrease in expression level of SEMA3/plexin family members and accentuated tumor growth by affecting cell proliferation.

2341 microRNAs regulating matrix metalloproteinase 2 activities. S-Y. Choi1, J. Kim1, B. Shin1; 1Chonnam National University Medical School, Gwangju, Korea

MMP-2 plays an important role in tumor cell invasion and metastasis. Because microRNAs (miRNAs) have recently been reported to be implicated in these processes, we set out to determine a role for miRNAs regulating tumor cell invasion and metastasis. We screened 215 human miRNAs for upregulating MMP2 activities in human fibrosarcoma HT1080 cells. Transfection of HT1080 cells with miRNAs followed by zelatin zymography identified 14 miRNAs raising MMP2 activities. Of the 14 miRNAs, further analyses with Western blotting, luciferase assay and Matrigel invasion assay pointed to miR-105 and miR-128 as final candidates. We will perform in vivo pulmonary metastasis assay in mice under gain-of-function and loss-of-function contexts to validate the effect of miR-105 and miR-128 on tumor cell invasion and metastasis.

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2342 Profiling of linker histone variants in ovarian cancer. M. Medrzycki1, Y. Zhang1, J. McDonald1, Y. Fan1; 1Georgia Institute of Technology, Atlanta, GA

H1 linker histones play a key role in facilitating higher order chromatin folding. There are 11 H1 variants in mammals that are differentially regulated during development and cellular differentiation. Emerging evidence suggests that H1 and its specific H1 variants are important epigenetic factors in modulating chromatin function and gene expression. Ovarian cancer is a devastating disease, ranking the fifth leading cause of all women cancer death due to its poor prognosis and difficulty in early diagnosis. Although epigenetic alterations in ovarian cancers are being appreciated in general, the role of H1 in ovarian cancers has not been explored. Here, using quantitative RT-PCR assays, we systematically examined the expression levels of 7 H1 genes in 33 human epithelial ovarian tumors. We found that the expression of H1.3 is markedly increased, whereas the expression levels of H10, H1.1, H1.4 and H1x are significantly reduced in the malignant adenocarcinomas compared to benign adenomas. Strikingly, ovarian adenocarcinomas and adenomas exhibit characteristic expression patterns, and expression profiling of these 7 H1 genes in tumor samples can discriminate adenocarcinomas vs. adenomas. We identified 6 H1 genes as the minimum gene set required for correct discrimination of ovarian adenomas and adenocarcinomas with high accuracy. These findings indicate that the expression of H1 variants is exquisitely regulated and may serve as potential epigenetic biomarkers for ovarian cancer.

2343 Investigating the contribution of centrosome amplification in tumorigenesis. B. D. Vitre1, A. J. Holland1, Y. Wang1, D. W. Cleveland1,2; 1Ludwig Institute for Cancer Research, La Jolla, CA, 2Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA

As the major microtubule organizing centers, centrosomes play a central role in facilitating the formation of a bipolar mitotic spindle. Defects in centrosome biogenesis can induce an abnormal centrosome number and may lead to chromosome missegregation and subsequent aneuploidy. Although aneuploidy is an extremely common feature of tumor cells, its status as a cause or a consequence of cancer is highly controversial. In vertebrates and invertebrates, the conserved protein kinase Polo-like kinase 4 (Plk4) plays a key role in initiating centriole duplication and overexpression of Plk4 promotes the formation of extra centrosomes. Here we will describe the construction of two mouse models in which centrosome amplification can be induced through conditional overexpression of Plk4. We have made use of both a doxycycline-inducible promoter and the Cre-LoxP system to allow reversible and non-reversible expression of Plk4 in a tissue specific manner. These mice will be used to study the contribution of centrosome amplification in tumorigenesis.

2344 LEKTI re-expression up-regulates p53 and p21 in OSC-19, a metastatic squamous cell carcinoma. A. Iarrobino1,2, T. Shellenberger3, S. Klemann2,3; 1School of Medicine, University of Central Florida, Orlando, FL, 2Rollins College, Winter Park, FL, 3MD Anderson Cancer Center - Orlando, Orlando, FL

Lymphoepithelial Kazal Type Inhibitor (LEKTI) is a 15-domain serine proteinase inhibitor with constitutive expression in squamous mucosa and with reduced or absent expression in the majority of head and neck squamous cell carcinomas (HNSCC) and cell lines. The loss of TUESDAY expression may alter dynamics in the extracellular matrix of the tumor environment to favor local invasion and metastasis. Re-expression of LEKTI in HNSCC lines alters cell behavior in vitro and reduces local invasion in vivo. p53 is a well-characterized tumor suppressor gene with lost expression in many cancers, including HNSCC. In the context of examining LEKTI and matrix metalloproteinase expression in the HNSCC line OSC-19, we found the expression of p53 was restored upon stable transfection of OSC-19 with LEKTI to induce re-expression. Using OSC-19 parental, vector, and clones re-expressing LEKTI (LEKTI-17 and LEKTI-25), semi-quantitative and quantitative analysis demonstrate restored expression of p53 in OSC-19 cells after transfection with LEKTI and cultivation both in vitro and in vivo (murine flank and orthotopic tongue tumor models). With re-expression of LEKTI and the subsequent restoration of p53, the expression of p21 was in turn up regulated. Differential expression of LEKTI was noted for parental cells cultivated in vitro and in vivo. Whereas LEKTI expression did not take place in vitro, its expression took place in both tumor types. We found p53 and p21 expression in xenografts derived from parental cells and both OSC-19 clones transfected to express LEKTI. Whenever LEKTI was expressed, re-expression of p53 and p21 was observed. Lastly, in vitro treatment of parental and vector cell lines with conditioned medium containing secretory factors of LEKTI-25 cells resulted in the re-expression of p53 and p21. Our results demonstrate the integrity of signaling pathways through which extracellular factors act and highlight the importance of the tumor microenvironment in regulating downstream mediators involved in the biologic behavior of OSC-19. [The James and Esther King New Investigator Award of the Florida Biomedical Research Program (TS) and the Critchfield Fund of Rollins College (SK) funded this work.]

Normal and Diseased Organs and Therapeutics

2345 Therapeutic Multiple Exon-skipping Using Cell-penetrating Morpholinos for Dystrophic Dogs. T. Yokota1, T. Nagata2, A. Nakamura3, N. Urasawa4, T. Saito2, R. Kole5, P. Sazani5, T. Partridge6, E. Hoffman6, S. Takeda2; 1University of Alberta, Edmonton, AB, Canada, 2National Center of Neurology and Psychiatry, Japan, 3Shinshu University, Japan, 4Nagano Red Cross Hospital, Japan, 5AVI Biopharma, 6Children's National Medical Center, Washington, DC

Duchenne muscular dystrophy (DMD), the most common and fatal X-linked myopathy, and its milder form, Becker muscular dystrophy (BMD), are caused by mutations in the dystrophin (DMD) gene. Antisense-mediated exon skipping therapy is currently one of the most promising molecular therapies for DMD. The exon skipping leads to the production of internally deleted in- frame mRNA transcripts but the truncated quasi-dystrophin retains some functions like BMD. Previously we demonstrated the first successful exon-skipping treatment in body-wide skeletal muscles in Canine X-linked muscular dystrophy (CXMD) using a cocktail of phosphorodiamidate morpholino oligomers (PMOs, morpholinos) targeting exon 6 and exon 8 of dystrophin mRNA. However, unmodified (bare) morpholino injections led to inefficient delivery to the heart, and dystrophin induction was barely detectable in the cardiac muscle. Here, we sought to recover the dystrophin expression in cardiac muscles in dystrophic dogs using morpholinos conjugated with negatively charged arginine-rich cell-penetrating peptides (PPMOs). We demonstrated that the delivery moieties significantly improved dystrophin production in both skeletal and cardiac muscles. Intravenous injections with PPMOs restored dystrophin expression in cardiac muscles accompanied by ameliorated histology. No obvious toxicity was detected by blood tests and histology. Our results show the potential of PMO conjugates as therapeutic agents for DMD.

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2346 The Regenerative Response of Zebrafish Hearts to Long-Term Induced Exercise Stress. B. Schoffstall1, P. deVerteuil1, M. Jean1, N. Lopez1, J. Tapia1; 1Biology, Barry University, Miami Shores, FL

While adult human cardiomyocytes are capable of low level cell division, this hyperplastic response is not sufficient to repair damaged cardiac tissue. Danio rerio (zebrafish) cardiomyocytes have recently been shown to be capable of proliferating at high levels to completely repair hearts damaged by apex amputation or cryoinjury; thus, these fish have become a popular model to study heart regeneration. We hypothesized that exposure of zebrafish to long-term cardiac overload stress would elicit a similar response without invasive surgery, to provide us with a simple cardiac regeneration research model. In addition, this model may have useful applications to human stress-induced cardiac responses, which can lead to pathological cardiac hypertrophy and ultimately death. We stressed zebrafish cardiac systems using a 10-week forced swimming exercise regimen, then compared fully remodeled hearts to non-exercised controls for physiological function and histological evidence of cell proliferation. We sought to determine if the zebrafish heart response to cardiac overload stress results in heart enlargement, and whether hypertrophy or hyperplasia is involved. We also used a zebrafish full-genome expression microarray to screen for genes that may be key initiators of a regenerative response. Adult zebrafish were forced to swim twice daily against a strong water current. After a period of 10 weeks, individual fish were measured for overall size; hearts were filmed to determine heart rate, ventricular surface area, and percent shortening fraction; and hearts were extracted for histological tissue sectioning, cDNA preparation, and analysis of total protein. Our results indicate that fully remodeled exercised zebrafish heart ventricles are ~32% larger than non-exercised (p<0.01). We demonstrate that this heart enlargement does not result in any significant change in physiological function; thus, the process results in a normally- functioning compensated heart. Cardiomyocyte nuclei distribution within ventricular tissue was not significantly decreased in exercised fish hearts, indicating that enlargement was not due to individual cell hypertrophy, but instead due to regenerative hyperplasia. Microarray screening of 1,391 genes upregulated 2-fold or higher in exercised hearts identified 5 with human orthologs known to be involved in cell growth and division processes. These will be the target of future studies. Since the compensatory cardiac remodeling may have been complete before the end of the 10-week exercise regimen, future studies will examine hearts at various time points during the remodeling process, rather than at the end, to observe significant changes in physiological and molecular responses as cardiomyocytes actively undergo cell division. Full understanding of the zebrafish regenerative response to cardiac overload stress may lead to the identification of genes or proteins with therapeutic potential for the harmful effects of cardiac stress that lead to pathological hypertrophy in humans. Support: NIH MBRS RISE R25 GM059244

2347 Activin receptor IA/activin-like kinase-2 (R206H) mutation in Fibrodysplasia Ossificans Progressiva. R. Herrera-Esparza1, D. Pacheco-Tovar1, J-J. Bollain-y-Goytia1, F. Torres-del-Muro1, R. Ramírez-Sandoval1, M-G. Pacheco-Tovar1, E. Avalos-Díaz1; 1Univ Autonoma Zacatecas, Zacatecas, Mexico

Fibrodysplasia ossificans progressiva (FOP) is an exceptional uncommon genetic disease characterized by congenital malformations of the great toes and progressive heterotopic ossification (HO) in specific anatomical areas. Disease is caused by a recurrent mutation in activin receptor IA/activin-like kinase-2 (ACVR1/ALK2). Two characteristic criteria define FOP: TUESDAY

1) Malformation of great toes present at birth. 2) Progressive heterotropic ossification which starts at the first decade of life. Ossification is manifested by recurrent and painful swelling flare ups of soft tissues that involve fascias, ligaments, aponeuroses, tendons and skeletal muscles. A Mexican family with one member affected of FOP was studied. The patient with the illness was a 19 years old female, who started with this disease at the age of 8 years old; she developed spontaneous and painful swelling of the right scapular area accompanied of functional limitation for movement, among clinical laboratory abnormalities it was found alkaline phosphatase activity increased. By radiography there was abnormal calcification and "pseudo- exostosis dependent of ligament ossification at the site of attachment to the long bones. Mutation analysis was carried out using genomic DNA from peripheral blood lymphocytes; samples were assessed by PCR amplification using exon flanking primers (Shore et al, Nature Genetics 38; 525), PCR products were digested with respective restriction enzymes 1h at 37°C. Main results were obtained with the exon 4 flanking primers and Cac81 restriction enzyme, this gave a 253 bp product that carries the ACVR1 617→A mutation which causes aminoacid substitution of arginine by histidine in codon 206. ACVR1 gene encodes a type I bone morphogenic protein (BMP) transmembrane receptor, and mutation of this gene results in dysregulation of BMP signaling. This rare case is a novo mutation since her relatives were clinically healty and molecular analysis did not show ACVR1 617→A mutation.

2348 TGF-β and BAFF derived from CD4+CD25+Foxp3+ T cells mediate mouse IgA isotype switching. G-Y. Seo1, P-H. Kim1; 1Department of Molecular Bioscience, Kangwon National University, Chuncheon, Korea

TGF-β is generally accepted as the physiological IgA isotype switch factor. Nevertheless, it is virtually unknown what kinds of cells in mucus-associated lymphoid tissue (MALT) mainly provide this cytokine to B cells. Regulatory T cells (Tregs) has immune suppressive activity by secretion of inhibitory cytokine such as TGF-β and IL-10. Thus, it is plausible that Tregs may be involved in IgA class switching recombination (CSR) in MALT. We explored, in the present study, the possibility that CD4+CD25+ T cells facilitate IgA CSR in mouse B cells. CD4+CD25+Foxp3+ T cells were more stimulatory in IgA production by the cocultured splenic B cells than CD4+CD25-Foxp3- T cells, and this effect was markedly abrogated by anti-TGFβAb. This was paralleled by an increase of germ line transcriptα (GLTα), an indicative of IgA CSR. In contrast, CD4+CD25-Foxp3- T cells were more potent in induction of GLTγ1 and GLTε by the cocultured splenic B cells. Consistent to these results, phenotypic analyses revealed that TGF-β and IL-4 were predominantly expressed by CD4+CD25+Foxp3+ T cells and CD4+CD25-Foxp3- T cells, respectively. Further, we found that CD4+CD25+ T cells strongly express BAFF leading to AID expression in B cells. Taken together, our results suggest that CD4+CD25+ T cells may have an important effect on IgA isotype commitment through TGF-β and BAFF in MALT.

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2349 Anti-platelet activity of yuzu and its components is mediated by inhibition of TXA2 production. H. Yu1,2, S. Park3, I. Chung4, Y-S. Jung1,5; 1College of Pharmacy, Ajou University School, Suwon, Korea, 2Brain Korea 21 for Medical Sciences, Ajou University, Suwon, Korea, 3Molecular Biotechnology, Konkuk University, Seoul, Korea, 4Applied Life Science, Konkuk University, Seoul, Korea, 5Brain Korea 21 for Molecular Science and Technology, Ajou University, Suwon, Korea

The platelet activation and subsequent platelet aggregation play an essential role in the pathogenesis of atherothrombotic disease such as cardiovascular, cerebrovascular, and peripheral vascular diseases. Food can be one of the most important factors that influence risks for atherothrombotic diseases. Yuzu included in citrus is one candidate that may benefit the cardiovascular system. In this study, we investigated whether the methanolic extract of yuzu (yuzu ME) and its components (hesperidin and naringin) have anti-platelet activities. Yuzu ME and hesperidin inhibited collagen-, arachidonic acid (AA)-, ADP- and thrombin-induced rat platelet aggregation in vitro and ex vivo. Naringin also inhibited platelet aggregation induced by collagen, AA, or thrombin, but not aggregation induced by ADP. The oral administration of yuzu ME or hesperidin prolonged mouse tail vein bleeding time in a dose-dependent manner in vivo. In addition, yuzu ME including hesperidin and naringin inhibited collagen and thrombin-induced platelet aggregation by interfering with TXA2 production as established by the measurement of TXB2. These results suggest that yuzu ME and its component such as hesperidin and naringin have anti-platelet activity, and that intake of yuzu, which includes various flavonoids such as hesperidin, may be beneficial for individuals at high risk of cardiovascular diseases. This research was supported by the Technology Development Program (#308013-3) for ′Food′ Ministry for Food, Agriculture, Forestry and Fisheries, and the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ006986) Rural Development Administration, Republic of Korea.

2350 Dehydroepiandrosterone inhibits the activation and dysfunction of endothelial cells induced by high glucose concentration. E. Huerta García1, J. L. Ventura Gallegos2,3, M. E. Crescencio Victoriano1, A. Montiel Dávalos1,4, G. Tinoco Jaramillo5, R. López-Marure6; 1Biología Celular, Instituto Nacional de Cardiología “Ignacio Chávez”, México, 2Departamento de Bioquímica, Instituto Nacional de Ciencias Médicas y Nutrición "Salvador Zubirán", México, 3Departamento de Medicina Genómica y Toxicología Ambiental, Instituto de Investigaciones Biomédicas, UNAM, Mexico, 4Subdirección de Investigación Básica, Instituto Nacional de Cancerología, México, 5Jefatura de Ginecología, Hospital General de Zona 2A “Troncoso”, IMSS, México, 6Biología Celular, Instituto Nacional de Cardiología “Ignacio Chávez”, México City, Mexico

Dehydroepiandrosterone (DHEA), an adrenal steroid, has a protective role against diabetes; however, its mechanisms of action are unknown. Here, we focus on the effect of DHEA on the activation of endothelial cells induced by a high concentration of glucose. The adhesion on U937 cells, the expression of adhesion molecules, the production of ROS and NO, and the translocation of NF-[kappa]B were evaluated in human umbilical vein endothelial cells (HUVEC) treated with high concentrations of glucose, DHEA, and both. High concentrations of glucose (> 20 mM) induced an increase in adhesion, an increase in mainly E-selectin and PECAM-1 expression, ROS and NO production, translocation of NF-[kappa]B, and degradation of its inhibitor I[kappa]B-[alpha]. DHEA abolished adhesion and the increase of E-selectin, ICAM-1, VCAM-1, and PECAM-1 induced by glucose. In addition, DHEA completely blocked oxidative TUESDAY stress and decreased translocation of NF-[kappa]B and the degradation of I[kappa]B-[alpha] induced by glucose. These results suggest that DHEA protects against the activation of endothelial cells induced by high concentrations of glucose, indicating that DHEA could be useful in the treatment of hyperglycemia and diabetes.

2351 Activin A stimulates mouse antigen presenting cells to express APRIL and BAFF. G. Seo1, P-H. Kim1; 1Department of Molecular Bioscience, Kangwon National University, Chuncheon, Korea

The TNF family ligands APRIL and BAFF are primarily expressed by macrophages and dendritic cells and stimulate the differentiation and survival of B cells and their Ig production. In this study, we investigated the role of activin A in APRIL and BAFF expression by mouse antigen presenting cells and the signaling mechanism involved. Activin A markedly enhanced APRIL expression in mouse macrophages at both the transcriptional and protein levels. Overexpression of DN-Smad3 and SB431542 abrogated activin-induced APRIL transcription. Furthermore, activin A induced Smad3 phosphorylation. These results indicate that activin A enhances APRIL expression through both ALK4 and Smad3. In a subsequent analysis of activin A signaling, it was found that PD98059, an ERK inhibitor, eliminated activin A-induced APRIL expression. On the other hand, overexpression of CREB, a molecule downstream of ERK, augmented activin A-induced APRIL expression, and this effect could be abolished by PD98059. This finding that activin A induces ERK and CREB phosphorylation suggests that ERK and CREB act as intermediates in APRIL expression. Therefore, these results demonstrate that activin A can enhance APRIL expression through two different pathways, Smad3 and ERK/CREB. On the other hand, activin A also enhanced BAFF expression in mouse macrophages and dendritic cells at both the transcriptional and protein levels. Overexpression of DN-Smad3 and SB431542 abrogated activin-induced APRIL transcription. These results demonstrate that activin A can enhance BAFF expression through ALK4-Smad3 pathway. Taken together, these results suggest that activin A can modulate mouse APCs to express APRIL and BAFF.

2352 High-fat diet during sexual maturation influences the epithelium cell turnover kinetics in the adult rat ventral prostate. E. Z. Pytlowanciv1, D. L. Ribeiro2, R. M. Góes3; 1Biology Institute, State University of Campinas (Unicamp), Campinas, Brazil, 2Histology Sector, Institute of Biomedical Sciences, Federal University of Uberlandia (UFU), Uberlândia, Brazil, 3Department of Biology, University of Estadual Paulista (Unesp), São José do Rio Preto, Brazil

The obesity epidemic has been recognized as one of the major global health problems and is associated to several diseases including reproductive problems. Various consequences of obesity in the male genital tract have been related to changes in sex steroid hormones. Although this could be applied to prostate, the influence of obesity and high-fat ingestion to the prostate histophysiology is unknown. The aim of this study was to evaluate if a high-fat diet (HF) interferes on rat ventral prostate pubertal maturation and harm the histology of adult gland. Male Wistar rats (4w old) were fed for 3, 6 or 9 weeks (HF3, 6 and 9) with high-fat diet (20%fat, 4.9kcal/g) whereas control animals (C3, 6 and 9) received balanced chow (4%fat, 3.2Kcal/g). Ventral prostate was analyzed using stereological, immunocytochemical and western blotting methods. The relative proportion of tissue gland components were maintained in HF, except for an 1.1-fold increase in acinar epithelium volume at HF6. Morphological analyses indicated that acinar epithelium was significantly higher in HF groups, more pleated and exhibited mild diffuse TUESDAY epithelial hyperplasia. The high cellularity observed in acinar epithelium of all HF-treated groups is evidenced by a significant rise in cell densities. HF during sexual maturation promoted a 4- fold increment in cell proliferation as well as a 4-fold reduction in apoptosis rate and also elevated circulating testosterone serum levels in HF9 compared to C9 group. Immunocytochemistry indicated an increase in cells expressing AR under all HF conditions. However, the western blotting analyses surprisingly pointed to a tendency of 110kDa-AR band down expression and 45kDa band over expression in prostate crude protein extracts. This may be due to primary polyclonal antibody non-specific cross reaction and the small molecular weight bands found on prostate extracts possibly correspond to AR isoforms. Interestingly, there was an inverse correlation between 110kDa AR and smaller band expression in HF3 and HF9, which may suggest a fat dietary interference on differential expression of AR isoforms. The circulating androgen elevation, could explain the cell proliferation increment, apoptosis reduction and high prostate growth observed during HF treatment, since the androgens are the main factors responsible for maintaining the homeostasis of the prostate, through the activation of its receptor (AR). Finally, these experiments indicate that HF diet-treatment during puberty influences prostatic maturation causing hiperplastic alterations and higher gland volume at adulthood. In addition, the quantity of satured fat in diet interferes in the cell epithelium turnover kinetics in the adult prostate.

2353 Effect of peripheral serotonin on glucose uptake in murine brown adipocyte. H. Watanabe1, K. Saito1, R. Saito1, T. Nakano1, N. Okada1, K. Sumiyoshi1, S. Ohwada1, K. Watanabe1, H. Aso1; 1Tohoku University, Miyagiken Sendaishi, Japan

Serotonin is a neurotransmitter synthesized in the raphe nuclei of the brain stem and involved in the central control of food intake, sleep and mood. Serotonin is also a peripheral hormone produced by enterochromaffin cells in the intestine and involved in vasoconstriction, haemostasis and immune system. Serotonin is synthesized by two distinct tryptophan hydroxylase (TPH) rate-limiting enzyme in brain (TPH2) and in peripheral (TPH1). As serotonin is inability to cross the blood-brain barrier, there are two serotonin systems in brain and periphery with independent functions. We revealed that serotonin increased plasma glucose concentration through inhibiting glucose uptake from blood to tissue. However, the functions of serotonin in periphery have not yet been fully elucidated. The major thermogenic center is the brown adipose tissue (BAT), which is composed of multilocular lipid droplets and a large number of mitochondria. Thus, BAT is known as an important tissue in the regulation of body weight and energy metabolism. In this study, we have established a clonal murine brown preadipocyte (MBP) line and investigated the function of serotonin on glucose metabolism in MBP cells. After fasted mice were intraperitoneally injected with 1 mg serotonin, the glucose uptake in BAT was elevated. Next, we tried to reveal the effect of serotonin on glucose uptake in BAT using established MBP cells. MBP cells showed the characteristic of BAT followed induction of differentiation, such as multilocular lipid droplets and the expressions of BAT related genes including uncoupling protein 1 (UCP-1). Additionally, the stimulation of serotonin induced the increase of glucose uptake in MBP cells in a dose dependent manner. To determine what kind of serotonin receptors were related to the glucose uptake, we confirmed the expression of several serotonin receptors and measured the glucose uptake in MBP cells using three kinds of serotonin receptor antagonist: SB-204070 (5HT4), Ro- 04-6790 (5HT6) and methysergide (5HT1, 5HT2 and 5HT7). The pretreatment of serotonin antagonist SB-204070 inhibited the glucose uptake in MBP cells after serotonin treatment. These data indicate that serotonin regulates a glucose metabolism in BAT through serotonin 4 receptor.

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2354 Topical application of capsaicin induces adipokine secretion in visceral adipose tissues. G-R. Lee1, D-J. Yoon2, I-S. Han2; 1Medical Sciences, University of Ulsan, Ulsan, Korea, 2Biological Sciences, University of Ulsan, Ulsan, Korea

We attempted to determine whether topical application of capsaicin can protect fat accumulation in visceral adipose tissues. We first observed that the topical application of 0.075% capsaicin to male obese mice fed a high-fat diet significantly reduced weight gain and visceral fat compared to those of untreated obese control mice (p < 0.001). Fat cells were markedly reduced in mesenteric and epididymal adipose tissues of the mice treated with capsaicin cream. Capsaicin treatment lowered serum levels of fasting glucose, total cholesterol, and triglycerides. Immunoblot analysis and RT-PCR revealed increased adiponectin expression in capsaicin- treated mice. In addition, application of capsaicin to obese mice downregulated mRNA levels of TNF-Ą and IL-6 but upregulated other adipokines including PPARs,visfatin, and adipsin. These results indicate that topical application of capsaicin to obese mice limited fat accumulation in adipose tissues and may contribute to insulin sensitivity and anti-inflammation.

2355 Interleukin-6 / Stat3 axis mediates S100a9 expression in the colonic epithelial cells in a mouse model of ulcerative colitis. J-K. Lee1, M-J. Lee1, J. Choi1, C-H. Cho1, S-K. Ye1; 1Department of Pharmacology, Seoul National University College of Medicine, Seoul, Korea

In the intestinal epithelium lining, the mucosal surface is a key player in homeostasis. However, when the balance is disturbed, it might lead to intestinal inflammation, including ulcerative colitis (UC). Interleukin-6 (IL-6) and the signal transducer and activator of transcription 3 (STAT3) phosphorylation have been reported to be highly expressed in the colonic tissues in UC. Also, S100A9, as a fecal biomarker, is correlated with the severity of UC. However, the expression mechanism of S100A9 in colonic epithelial cells (CECs) remains elusive. Here, a mouse model of UC was developed to investigate the linkage of IL-6 and S100a9 in CECs. We injected sgp130Fc or siSTAT3/CH-NP into dextran sodium sulfate (DSS)–treated mice and observed a markedly decreased S100a9 expression in CECs compared to those treated with PBS or siNegative/CH-NP. Furthermore, the administration of JAK2 or STAT3 inhibitors post IL-6 stimulation into Caco-2 indicated that IL-6-mediated S100A9 expression occurs through STAT3 activation. Also, we found that STAT3 binds directly to the S100A9 promoter and enhances its activity. We provide insights on the role of S100a9, which may recruit granulocytes into inflamed colon tissues. Thus, we conclude that elevated S100a9 expression mediated by IL-6/STAT3 in CECs plays an important role in the manifestation of UC.

2356 Immune cell monitoring using a handheld cell counter. A. Cappione1, N. Thirumalapura2, E. Crossley2, D. Hoover1; 1EMD Millipore, Danvers, MA, 2University of Texas Medical Branch, Galveston, TX

Biological samples, such as primary isolates or cultured cells, are often comprised of a phenotypically heterogeneous population. The ability to discriminate subsets and determine their frequencies (and concentrations) is critical to many aspects of research. Cellular complexity is typically resolved by flow cytometry using panels of protein-specific fluorescent antibodies. Notably, many cell states are also uniquely distinguishable on the basis of size alone.

Cell counting devices, operating via the principle of Coulter-based particle detection, permit the determination cell diameter measurements at the submicron level, as well as providing accurate concentrations. We describe three examples where a handheld cell counter provided qualitative assessment of individual population frequencies in complex cell mixtures. These TUESDAY

include: (1) Determination of lymphocyte and monocyte concentrations and their relative frequency in PBMC isolates, (2) Assessment of T-cell activation as a function of cell size shift in PBMC cultures, and (3) Identification of changes in splenocyte population dynamics in a murine model of Human Monocytotropic Ehrlichiosis (HME).

Following cell harvest, single cell suspensions were prepared prior to acquisition. All samples were also stained with cell-specific fluorescent antibodies and analyzed by flow cytometry. In PBMC, the cell counter identified the lymphocyte and monocyte fractions as distinctly sized peaks (7.2 and 10.0µm, respectively). Across the 9 samples tested, Counter-derived frequency and concentration values for each subset were consistent with flow cytometry. In PBMC cultures co-stimulated with anti-CD3/CD28 antibodies, the counter confirmed the presence of a larger activated T-cell fraction (7.3 and 12.4 µm, respectively); the increase in cell size was coincident with expression of CD25. In the murine HME model, a change in the relative frequency of splenic erythroid (4.1µm) to lymphocyte (6.2µm) fractions was shown to be due to an increase in the Ter119+ erythroid fraction.

The availability of simplified methods for monitoring changes in culture dynamics is very useful for accelerating research in the areas of diagnostics and drug design. While flow cytometry provides a more sophisticated and quantitative cell analysis platform, our findings indicate that cell counting devices can fill this role providing in-line insight into cellular responses.

2357 Effect of IL-12 on Negative Selection in the TNC microenvironment. S-A. Miller1, S. C. Henley1, F. R. Davis1, R. H. Lewis1, G. C. Bernard1, S. Samuels1, M. T. Martinez1; 1Biology, Tuskegee University, Tuskegee, AL

Thymic nurse cells (TNCs) are cortical epithelial cells of the thymus that appear to be significant participants in the negative selection of thymocytes. TNCs express both class I and class II major histocompatibility (MHC) proteins on their cell surfaces. TNCs internalize thymocytes that are CD4+CD8+TCRlo (triple positive) into cytoplasmic vacuoles. These thymocytes are at the developmental stage where they undergo MHC restriction. In addition to thymocytes, TNC vacuoles also contain macrophages that interact intimately with the thymocyte subset. Greater than 95% of the TNC-interactive thymocyte population has been reported to undergo apoptosis within TNC vacuoles suggesting a strong role for TNCs in the negative selection of thymocytes. Triple positive thymocytes are reported to be non-responsive to a variety of cytokines including IL-6, IL-7, IL-10, IL-15 and IFN-γ. However, these thymocytes were found to be responsive to IL-12. Although IL-12 promotes pro-inflammatory responses in the periphery it was shown to significantly influence the deletion of the triple positive thymocyte subset in the thymus. We therefore hypothesized that within the TNC microenvironment IL-12 can safely facilitate negative selection. We used cOVA-TCR transgenic (Tg), D011.10 mice that recognize the cOVA 323-339 peptide to analyze negative selection within the TNC microenvironment. The Tg mice were injected with different combinations of anti-IL-12 antibody or rat IgG2a antibody and/or cOVA peptide over a four day period. TNCs and thymocyte populations were harvested and analyzed for apoptotis using Annexin V with flow cytometry and TUNEL with fluorescent microscopy. Unexpectedly, there were no observable differences in thymocyte apoptosis within the TNCs of mice injected with rat-IgG2a, anti-IL-12, or those that were not injected. These data suggest that IL-12 does not play a role in negative selection within the TNC microenvironment.

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2358 Overexpression of human alpha-1 antitrypsin (AAT) in PiZZ liver reduces the polymerization and facilitates secretion in vitro and in vivo. K. Xiao1,2, L. Wang1, Y. Lu3, R. Oshins1, R. L. Bridges1, E. J. McAndrew1, A. Huegel1, S. Song3, A. D. Fu4, C. Liu4, F. Rouhani1, M. L. Brantly1; 1Department of Medicine, University of Florida, Gainesville, FL, 2Genetics & Genomics Program, University of Florida, Gainesville, FL, 3Departments of Pharmaceutics, University of Florida, Gainsville, FL, 4Dept of Pathology, University of Florida, Gainsville, FL

The Alpha-1 Antitrypsin Deficiency (AATD) is one of the most prevalent inherited lung diseases next to the cystic fibrosis and also most common metabolic-genetic indication for pediatric liver transplantation. However, in heterozygote PiMZ patient, the liver damage is mild. To evaluate the effect of human wild (M) type AAT (MAAT) as a possible chaperone on the trafficking of secretion-incompetent PiZZ AAT protein (ZAAT), we established a PiZZ cell model from pediatric patient. Over-expression of human MAAT in this cell model prevents process of polymerization of ZAAT in the cytoplasma. The result was confirmed by AAT Western blot, immunostaining and electron microscope (EM). We further test this treatment on PiZ transgenic mouse through portal vein injection of 1 X 1011 vg of AAV8 that expressing MAAT. The characteristic accumulation of ZAAT as polymer in hepatocyte has decrease sharply by polymer specific Ab staining and ELISA. Furthermore, the secretion of ZAAT in the serum increases about 5-fold after 12 weeks of treatment. The secretion of ZAAT is dosage dependent on the MAAT secretion level. Secretion of one molecular of MAAT brings out approximately one molecular of ZAAT. The purified MAAT can also successfully block the formation of polymer of ZAAT in vitro which explains the mechanism of this treatment. The average serum SGOT level, which reflects liver function, decreased about 21% after the treatment. Our result may be further tested on clinical trial in patients with AAT liver diseases.

2359 Identification of Serum miRNAs as Potential Biomarkers for Acute Myocardial Infarction P-H. Chu1, H-C. Chen2, S-J. Chen2; 1Chang Gung Memorial Hospital, Taipei, Taiwan, 2Chang Gung Univeristy, Gueishan, Taoyuan

It is important to find specific biomarkers to understand the pathophysiology and identify patients with ST-segment elevation myocardial infarction (STEMI). Recent studies have revealed the role of microRNAs (miRNAs) in a variety of biological and pathological processes including acute myocardial infarction (AMI). We hypothesized that circulating serum miRNAs may be useful in the diagnosis of STEMI. The aim of this study was to establish the ability to detect the miRNAs, and then to elucidate the relationship between miRNAs and STEMI. We initially profiled the differential expressions of 270 serum miRNAs, and identified potential serum microRNAs from 8 STEMI patients and matched healthy controls. We then used the candidate miRNAs to evaluate 62 patients, including 31 STEMI and 31 normal patients. The t-test and receiver operating characteristic (ROC) curves were established to discriminate AMI patients from the normal patients. P values less than 0.05 were defined as statistically significant. We initially identified 12 up-regulated miRNAs and 13 down-regulated miRNAs showing at least a two-fold change. Serum miR-486-3p, miR-150*, miR-126, miR-26a and miR-191 were validated. ROC analysis using the expression ratio of miR-486-3p and miR-191 showed an area under the curve of 0.863. Our findings implicate that serum miRNAs may be used as potential diagnostic biomarkers for STEMI.

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2360 Therapeutic Potential of TGF-β2 -Treated Mesenchymal Stem Cells from Wharton′s Jelly in a Rat Myocardial Infarction Model. H-S. Wang1, P-C. Chu1; 1Anatomy, Natl Yang-Ming University, Taipei, Taiwan

Myocardial infarction (MI) is one of the leading causes of morbidity and mortality worldwide. The development of stem cell therapy for treating MI patients is a contemporary challenge in the field of cardiovascular medicine. In this study, left anterior descending artery ligation rats were used as a myocardial infarction model. The induction of left anterior descending artery ligation was confirmed by electrocardiogram, echocardiography, and the presence of c-troponin I in the serum. To investigate the improved myocardial function after transplantation of undifferentiated and TGF-β2-treated cells, electrocardiogram, echocardiogram, Masson′s Trichrome staining, and immunohistochemical staining of heart tissues at different time points were assessed in the infracted rat hearts. Our results indicated that transplantation of undifferentiated or TGF-β2- treated human umbilical cord mesenchymal stem cells from Wharton′s jelly both improve left ventricular systolic function, reduce the fibrotic area of heart, and increase the survival rate of MI rat model. These results suggested that deliver stem cells from human umbilical cord mesenchymal stem cells to sites of injury are able to ameliorate the dysfunctional heart failure.