ComBio2013 s Perth, Western Australia s 29 September - 3 October, 2013 Page 25 SYMPOSIA MONDAY SYM-01-01 SYM-O1-02 RAB GTPASES IN TRAFFICKING AND SIGNALING Sponsored by ENVIRONMENTS IN MACROPHAGES The University of Western Australia Stow J.L., Wall A., Luo L., Yeo J. and Condon N. HOW LIPID VARIANTS IMPACT CELL SIGNALING FOR Institute for Molecular Bioscience, University of Queensland, Brisbane ANIMAL DEVELOPMENT AND BEHAVIOR 4072, Australia. Zhu H.1, Kniazeva M.1, Shen H.1, 2, Sewell A.1, Wang Y.1, 2 and Han M.1 1 2 Macrophages are sentinels and front-line innate immune cells, HHMI and University of Colorado at Boulder. Department of charged with detecting and destroying pathogens. Accordingly, the Chemistry and IBS, Fudan University, Shanghai. macrophage cell surface is highly dynamic in order to accommodate cell migration, environmental sampling, protein secretion and receptor Fatty acids (FAs) are highly variable in structures, contributing greatly to activation. Gram negative bacterial lipopolyscaccharide (LPS) activates the vast diversity in lipid structures. The fact that omega-3 FA supplements macrophages through Toll-like receptor 4 (TLR4) – complexes within are a multi-billion dollar business indicates a strong belief about the impact special domains on the cell surface. We have used a suite of GFP-Rab of certain types of FAs on our health. However, despite sporadic reports GTPases to define membrane domains at and near the macrophage linking FA variants with human diseases and development, their functional cell surface that are shaped by exo- and endocytosis and which specificities are generally poorly studied, and we know little about the function to support TLR4 signaling. Specific domains defined by Rabs mechanism by which these variants contribute to the lipid composition in 5, 8, 35 and 31 are compared and contrasted by live cell imaging. specific tissues for cellular events. Our lab has been addressing the above Specific effectors for each of these Rabs have been defined and their questions by combining complex genetics with biochemistry. Specifically, roles in pathogen-induced phagocytosis and TLR4 signaling have been we have uncovered spectacular impacts of the rarely studied monomethyl explored by siRNA or genetic inactivation. Our results show a highly branched-chain FAs (mmBCFAs) on cell signaling, development and dynamic series of Rab-defined membrane domains that acutely control behavior in C. elegans. mmBCFAs are synthesized in humans and richly innate inflammatory responses. present in our daily diet, but their physiological functions were unknown. We discovered that mmBCFAs are essential for postembryonic growth and development, as well as proper neuronal behaviors. We showed that this profound role is mediated by an mmBCFA-containing glucosylceramide and the TORC1 signaling pathway in the intestine. Strikingly, activation of TORC1, by either mutating the NPRL2/3 complexes or introducing hyperactive mutant transgenes of TORC1 components, can bypass the need of this lipid for this function. In another study, we revealed that ACS family enzymes critically regulate the incorporation of mmBCFAs into specific phospholipids (PL) in the somatic gonad so that proper phospholipid composition is achieved in the zygote. Imbalance of mmBCFA-containing PLs compromises IP3 signaling, leading to dramatic disruption of membrane dynamics and embryogenesis. We will report some of these findings with the emphasis on the physiological significance. SYM-01-03 SYM-01-04 REGULATION OF PHOSPHOINOSITIDE SIGNALLING BY PHOSPHORYLATION OF αSNAP IS REQUIRED INOSITOL POLYPHOSPHATE 5-PHOSPHATASES FOR SECRETORY ORGANELLE BIOGENESIS IN TOXOPLASMA GONDII Mitchell C.A., Dyson J., Davies M., Gurung R., McGrath M., Eramo M., 1, 2 1, 2 1, 2 3 3 Conduit S. and Ooms L. Stewart R.J. , Bradin C.H. , Wu H.J. , Dekiwadia C. , Ralph S.A. , Baum J.1, 2 and Tonkin C.J.1, 2 Department of Biochemistry and Molecular Biology, Monash University, 1 2 Wellington Road, Clayton, VIC 3800, AUSTRALIA. The Walter and Eliza Hall Institute of Medical Research. The Department of Medical Biology, University of Melbourne. 3Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute. Phosphoinositides are membrane bound signalling molecules that interac t with a plethora of ef fec tor proteins to regulate vesic ular traf fi c k ing, The phylum Apicomplexa encompasses a number of important human metabolism, actin dynamics, cell proliferation and survival. The parasitic pathogens. Of specific note arePlasmodium spp., causative agent generation and turnover of specific phosphoinositide species is achieved of Malaria and responsible for up to 1 million deaths per year, and Toxoplasma by the activity of both phosphoinositide kinases and phosphatases, gondii, the most ubiquitous apicomplexan infecting approximately 30% of the worlds population. Apicomplexan parasites are obligate intracellular which phosphorylate and dephosphorylate respectively phosphates pathogens that absolutely require invasion into host cell for survival and from the inositol head group of phosphoinositide signalling molecules. as such have evolved specialized invasion machinery. During intracellular Phosphoinositide 3-kinase (PI3K) generates the signalling molecule replication, these parasites utilize the secretory pathway for the de novo PI(3,4,5)P3 which can be dephosphorylated by the inositol polyphosphate synthesis of the apical invasion organelles, as well as of inner membrane 5-phosphatases to generate PI(3,4)P2 which is in turn dephosphorylated complex (IMC) and plasma membrane (PM) biogenesis. As a part of at the plasma membrane and on early endosomes by 4-phosphatases the secretory pathway, vesicle trafficking and membrane fusion events to generate PI(3)P. Both PI(3,4,5)P3 and PI(3,4)P2 are required for the are mediated by a suite of proteins that are highly conserved throughout activation of the serine threonine kinase Akt, which in turn activates a eukaryotes. Membrane specific tethering and priming brings vesicle and plethora of down-stream signalling cascades that promote cell survival, target membranes in close proximity allowing trans interactions between proliferation, migration, angiogenesis and metabolism. There are ten corresponding vesicle (v)-SNAREs and target (t)-SNAREs. Further protein mammalian 5-phosphatases and recently several of these enzymes interactions and membrane modifications lead to fusion of membranes have been implicated in embryonic development. Genetic mutations in and release of vesicle contents resulting in cis-pairing of v-SNAREs and the 5-phosphatase, INPP5E, are causative of the ciliopathy syndromes t-SNAREs in the same membrane. αSoluble NSF Attachment Protein of Joubert and MORM, which are associated with mental retardation, (αSNAP) is required for recycling of cis-SNARE complexes, through abnormal neuronal development, polydactyly and other abnormalities. recruitment of ATPase N-ethylmaleimide sensitive factor (NSF), to recharge Deletion of murine Inpp5e causes mid-gestation lethality with ciliopathy the cellular pool of SNAREs for further trafficking events. Utilising T.gondii, phenotypes including neural tube defects, exencephaly, polydactyly modulation of key phosphorylation sites on αSNAP allows us to interfere and polycystic kidneys providing an ideal model to examine its role in with endogenous αSNAP processes with a regulated dominant negative ciliopathies. The molecular mechanisms by which 5-phosphatases by system. We have shown that ablation of normal αSNAP functions leads to degrading membrane bound phosphoinositide signalling molecules in severely disrupted secretory systems lethal to the parasite. Cytokenesis, turn regulate embryonic and post-natal development of specific tissues de novo apical organelle, PM and IMC biogenesis are all disordered while secretory-independent mechanisms, such as karyokenesis are not will be explored. directly affected. Understanding of molecular trafficking mechanisms in Apicomplexa, especially those related to the unique invasion organelles, can aid our understanding of these parasites and inform future drug and vaccine designs. Page 26 ComBio2013 s Perth, Western Australia s 29 September - 3 October, 2013 SYMPOSIA MONDAY SYM-01-05 SYM-O2-01 ACTO-MYOSIN-II CONSTRICTING RINGS THE CP12 PROTEIN FAMILY: A REDOX-MEDIATED CONTRIBUTES TO ACTIVITY-DEPENDENT BULK METABOLIC SWITCH? ENDOCYTOSIS Raines C.A.1, Lopez Calcagno P.1 and Howard T.2 Nguyen T.H., Gormal R.S., Wen P.J. and Meunier F.A. 1School of Biological Sciences, University of Essex, Colchester CO3 The University of Queensland, Queensland Brain Institute. 4JE UK. 2Biosciences, College of Life and Environmental Sciences, Geoffrey Pope Building, University of Exeter, EX4 4QD, UK. In chromaffin and neurosecretory cells, the cortical actin network is emerging as an active player in neuroexocytosis. However, its role in CP12 is a small, redox-sensitive protein, representatives of which are compensatory endocytosis is unclear. In this study, we used time-lapse found in most photosynthetic organisms, including cyanobacteria, confocal microscopy of chromaffin cells transfected with Lifeact, a diatoms, red and green algae, and higher plants. The only function fluorescently labelled actin binding peptide, to investigate the role of the that has been clearly demonstrated for CP12 in any organism is in the actin cortical network in activity-dependant bulk endocytosis. We first regulation of the C3 photosynthetic cycle by