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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.

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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 mediating the formation show, using a high molecular weight dextran uptake assay, that bulk of a complex between glyceraldehyde-3-phosphate dehydrogenase endocytosis is the predominant form of compensatory endocytosis in (GAPDH) and phosphoribulokinase (PRK) in response to changes chromaffin cells in response to secretagogue stimulation. Importantly, in light intensity. Under low light the GAPDH-PRK-CP12 complex we then show that in chromaffin cells expressing Lifeact, actin rings forms and the activity of both enzymes is reduced; under high light form concomitantly with and precisely surround these dextran-positive conditions the complex dissociates, mediated by thioredoxin, resulting bulk endosomes. Further analysis revealed that these rings were in increased GAPDH and PRK activity. Although the role of CP12 in the contractile (maximum diameter ~ 1 μm) and formed through dynamic non-enzymatic, redox-mediated formation of a multiprotein complex is changes of the cortical actin network, characterized by a reorganization clear, a number of studies now provide evidence that the CP12 proteins of actin fibers following a phase of depolymerization. FRAP analysis may have a wider role. CP12 is expressed in non-photosynthetic A. of Dextran-positive compartments at different stages of the actin ring thaliana tissues, and anti-sense suppression of tobacco CP12 disrupts constriction revealed differential fluorescence recovery, suggesting a a variety of developmental processes. Furthermore, in addition to the role for these rings in narrowing the neck of bulk endocytic structures higher plant genomes which encode up to three forms of CP12, analysis prior to their pinching off from the plasma membrane. The contractile of cyanobacterial genomes has revealed that not only are there multiple nature of these actin rings points to the involvement of myosin-II in their forms of the CP12 gene, but that in these organisms CP12 is also found formation. We confirmed that myosin-II is indeed required for both bulk fused to cystathionine-β-synthase domain containing proteins. In this uptake of dextran and actin ring formation as both these processes review we present the latest information on the CP12 protein family and are sensitive to blebbistatin treatment or myosin-II shRNA knockdown. explore the possibility that CP12 proteins form part of a redox-mediated Collectively, our results point to a selective role of acto-myosin II in metabolic switch, allowing organisms to respond to rapid changes in promoting activity-dependent bulk endocytosis in neurosecretory the external environment. chromaffin cells, which may represent a universal process that present in central neurons and at the neuromuscular junction.

SYM-02-02 SYM-02-03 WRKY TRANSCRIPTION FACTORS: POINTS OF RUBISCO FINE-TUNING TO INCREASE GLOBAL CROP CONVERGENCE BETWEEN CHLOROPLAST AND YIELD MITOCHONDRIAL STRESS RESPONSES Kapralov M.V. and Whitney S.M. Van Aken O., Zhang B., Law S.R., Narsai R. and Whelan J. Australian National University. ARC CoE Plant Energy Biology, University of Western Australia, 35 Stirling Highway, 6009 Crawley, WA. Ribulose-1,5-bisphospate carboxylase/oxygenase (Rubisco) serves as a gateway for inorganic carbon to enter metabolic pathways in most Throughout the life of a plant, biogenesis and fine-tuning of energy ecosystems on Earth. As the performance of Rubisco can greatly affect organelles is essential both under normal growth and stress conditions. crop yield, substantial efforts have been made to study its structure Communication from organelle to nucleus is thus essential to adapt and function. However despite substantial progress over the past few gene regulation and protein synthesis specifically to the current needs decades, better enzymes for crops are yet to be delivered. Recent of the plant. This organelle-to-nuclear communication is termed findings showed that Rubisco properties are continually being adjusted retrograde signaling and has been studied extensively over the last by natural selection to better fit gaseous and thermal environments decades. Using large-scale gene expression data sets relating to the enzyme is found at in different species. Human mediated artificial perturbations of chloroplast and mitochondrial function, we have shown selection has created a vast variety of crop cultivars in relatively short a highly significant overlap between gene expression changes triggered time. Many crops nowadays are grown in climates different from the by chloroplast and mitochondrial perturbations. These overlapping ones experienced by their ancestors, and hence Rubisco possessed by gene expression changes appear to be common with general abiotic, some of modern crop cultivars might not be optimal for the conditions it biotic, and nutrient stresses. However, retrograde signaling pathways experiences. Thus there is a potential for artificial improvement of crop are also capable of distinguishing the source of the perturbation and Rubiscos to better fit new climates as well as for new faster and/or more subsets of genes are only responding to malfunction of each specific specific crop Rubiscos resulting in increase of the global crop yield. We organelle. Our analysis suggested that WRKY transcription factors analysed sequences of genes encoding the large subunits of Rubisco, play a coordinating role on the interface of both organellar signaling where the active sites are located, for amino acid replacements that pathways. Therefore we screened the 72 annotated WRKY transcription may be responsible for changes in Rubisco properties. However, these factors of Arabidopsis thaliana for binding to the promoters of genes replacements could be lineage specific and produce different results in responding to organellar dysfunction. Transgenic overexpression and different plant species. So, further we assessed possibility of assembly knockout lines for multiple binding WRKY factors were generated and of foreign Rubisco large subunits from major cereal, legume, tuberous tested for altered expression of the marker genes during normal and and vegetable crops with tobacco small subunits in tobacco plants. The stress conditions. This analysis demonstrated that AtWRKY40 and effects of particular amino acid replacements on Rubisco kinetics and AtWRKY63 are particularly involved in regulating the expression of ability of foreign Rubisco large subunits assembly with tobacco small genes responding commonly to both mitochondrial and chloroplast subunits are discussed. dysfunction, but not of genes responding to either mitochondrial or chloroplast perturbation. In conclusion, this study establishes the role of WRKY transcription factors in the coordination of mitochondrial and chloroplast function during environmental stress conditions.

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SYM-02-04 SYM-O2-05

CHLOROPLAST GENE EXPRESSION IN C4 CLEOME DIURNAL LIGHT AND FLUORESCENCE PROFILES IN ARTIFICIAL RICE CANOPIES: BUILDING A DYNAMIC Tanz S.K., Kajala K. and Small I.D. MODEL OF PHOTOSYNTHESIS FOR ORYZA SATIVA ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia. Meacham K.G.1, 2, Furbank R.1, Von Caemmerer S.2 and Sirault X.1 1High Resolution Plant Phenomics Centre, CSIRO Plant Industry, 2 The C4 cycle enhances photosynthesis by concentrating CO2 from Canberra, ACT 2601, Australia. Plant Science Division, Research mesophyll (M) cells into RuBisCO-containing bundle sheath (BS) cells. School of Biology, Australian National University, Canberra, ACT, Cell-specific accumulation of proteins, especially in the chloroplast, is Australia. critical for an efficient C4 cycle. Although the mechanisms governing cell-specific expression of nuclear genes have been extensively Traditionally, photosynthesis has been characterized using gas investigated, little is known about the mechanisms that give rise to exchange measurements, but this procedure is slow and laborious M- and BS-specific expression of chloroplast genes, despite the and impractical for mapping photosynthetic parameters at the canopy primordial role of chloroplast-encoded RuBisCO in C4 photosynthesis. or whole plant level. In this study, photosynthesis is characterised in To complete our understanding of the processes that generate cell- relation to plant architecture and heterogeneity of dynamic canopy specific accumulation of proteins used in the C4 pathway, we have light environment. Investigation of evolving canopy irradiance, and analysed transcript abundances across the whole chloroplast genome the photosynthetic and architectural response is examined. First, in both M and BS cells in Cleome, the genus containing the closest using single genotype (Nipponbare) with leaves at different canopy known C4 relatives of Arabidopsis. Chloroplast transcripts show up to levels, through vegetative and reproductive growth, and repeated 35-fold differences in leaves of the closely related species C. gynandra using 4 genotypes with contrasting morphology with similar Harvest (C4) and C. hassleriana (C3). However, these differences are mostly not Index. Weekly 3D imaging with a novel digitization platform is used to due to M- or BS-specific accumulation of chloroplast transcripts. The characterize key plant architectural traits and phenology. Gas exchange similarity of the M and BS chloroplast transcriptomes in C. gynandra was measured with LiCor 6400, and diurnal measurements of leaf contrasts with the striking differences at the protein level and allows chlorophyll fluorescence were made with a multi-head pulse modulated us to rule out transcriptional regulation and RNA degradation as chlorophyll fluorometer by applying saturating flashes every 30 minutes major determinants of cell specific expression, as they would lead to in a glasshouse environment. Irradiance at varying canopy levels was observable differences in steady-state RNA levels. Most likely, cell- tracked throughout growth. Dynamically, as intercepted irradiance specific chloroplast gene expression in C. gynandra is regulated at the reduces per leaf over time due to self shading, photosynthesis and post-transcriptional or translational level. leaf chlorophyll consequently reduces. However, the diurnal relaxation kinetics of the photoinhibition response to variation in intercepted irradiance at varying canopy level show clear variation across genotypes, and at different canopy levels in a single genotype. We are presenting results from the 3D imaging platform, characterization of photosynthesis from fluorescence using curve fitting techniques to model ETR as a function of Irradiance, and incorporation of these aspects into a functional dynamic model of photosynthesis.

SYM-03-01 SYM-03-02 EVOLUTION OF THE WAKE-UP CALL FROM SMOKE HOW SEED DEVELOPMENTAL PATTERNS VARY ACROSS SPECIES Smith S.M. University of Western Australia, WA 6009. Hay F.R. International Rice Research Institute. The 2004 discovery of the primary chemical component of burning vegetation that stimulates seed germination, has opened up a new area This paper will consider how patterns of seed development vary for of fundamental plant biology. The original compound, karrikinolide, is species from diverse habitats. While orthodox seeds by definition, one of a family of related compounds named karrikins. By employing acquire desiccation tolerance during seed development, the relative Arabidopsis thaliana as a model genetic system to study karrikin mode timing of this varies and in some species, seeds are dispersed before all of action, three different genes required for the karrikin response the seeds are desiccation tolerant. In contrast, recalcitrant seeds never have been discovered. One such gene is also required for the action acquire the same levels of desiccation tolerance as orthodox seeds, of strigolactone hormones, while the other two are close relatives of although desiccation sensitivity may somewhat less at natural dispersal. genes required strigolactone action. Studies of these genes suggest For many orthodox seeds, seed quality (in particular, seed longevity), that early land plants probably employed an ancestral version of this will continue to increase, even after seeds have acquired desiccation genetic system as part of their developmental program. With the tolerance. Hence, it is recommended that seeds of shattering plants emergence of seed plants, elements of this genetic system apparently are harvested when they are about to disperse naturally. If seeds are became replicated and specialised to mediate aspects of the control of harvested prematurely, placing them under conditions that simulate root and shoot architecture by strigolactone hormones. Today, some natural environmental conditions may mean that developmental events parasitic plants exploit this modern strigolactone hormonal system will continue. Using published examples, the implications for storing to trigger germination of their seed, while fire-followers probably use seeds for ex situ conservation or for use in restoration or for species elements of the more ancient genetic system to enable karrikins to reintroduction will be discussed. trigger germination of their seed.

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SYM-03-03 SYM-O3-04 EFFECTS OF PRODUCTION FARM ON GERMINATION GENETIC CHARACTERISATION OF SEED COAT AND EMERGENCE OF THREE PERENNIAL GRASS DEVELOPMENT IN PLANTAGO OVATA RESTORATION SPECIES Tucker M.R., Phan J., Shirley N.J. and Burton R.A. Espeland E.1, Horning M.2, Perkins L.3 and Johnson R.C.4 ARC Centre of Excellence in Plant Cell Walls, University of Adelaide, 1USDA-ARS Sidney MT USA. 2USDA-USFS Bend OR USA. 3South Waite Campus, Urrbrae, 5064, Australia. Dakota State University, Brookings SD USA. 4USDA-ARS Pullman WA USA. Seed development in flowering plants is initiated through the process of double fertilisation, which leads to the formation of an embryo and Plants in nonstressful environments tend to produce larger seeds that in endosperm. At the same time, residual maternal tissues surrounding turn may germinate quickly and produce initially larger plants. Also seed the filial structures differentiate to form a protective seed coat, which coats are comprised entirely of maternal tissues that affect dormancy resists stress and aids dispersion. Another common feature of seed and interact with the planting environment. Due to maternal effects coat development in species such as Arabidopsis and Plantago is in plants, it is likely that the production farm environment influences the development of specialised cells that contain a polysaccharide- subsequent performance. We tested interactions between production rich gel, termed mucilage, which is released upon contact with water. farm and planting environment for three improved varieties of perennial Hypotheses suggest that mucilage may function to aid seed hydration grass species commonly used for revegetation throughout the western and germination or to prevent or promote seed dispersal through United States: P-7 Bluebunch wheatgrass (Pseudoregneria spicata), adherence to soil or animals. We are studying seed coat development Lodorm Green needle grass (Nassella viridula), and Pryor Slender and mucilage production in Plantago ovata, a medicinally relevant wheatgrass (Elymus trachycaulus) from two to three production farms plant that is utilised commercially in human dietary fibre supplements. per species. We used four field environments spread across the western Using a combination of high-throughput RNA sequencing and gamma US and four laboratory environments set at different temperatures and mutagenesis, we have identified candidate genes and mutants involved daylengths. In the field, Elymus showed maternal effects; that is, one in mucilage cell development and polysaccharide biosynthesis. production farm demonstrated significantly different emergence among In addition, a detailed histological characterisation of mucilage all four planting environments. In the lab, Elymus exhibited maternal composition and development suggested that the amount, composition effects only for the other two production farms. Nassella did not show an and structure of mucilage varies between different Plantago species. interaction between production farm and planting environment in either We are using this information to characterise genes that influence seed the lab or the field, and Pseudoregneria showed an interaction from coat growth and development in Plantago and to provide insight into the only one production farm in the lab but not in the field. The production basis of natural variation in polysaccharide biosynthesis. farm effects on seed emergence in the field are species-specific, can be as large as 63%, and laboratory assays appear to be a poor estimate of the degree and direction of maternal effects. Understanding maternal effects in economically-important species may allow us to use them as a tool to manage risk in high-stakes plantings.

SYM-03-05 SYM-04-01 NON-MODEL DE NOVO TRANSCRIPTOMICS TO ABNORMAL EXPRESSION OF SEX BIASED GENES STUDY SEED PEPTIDE EVOLUTION IN PCDH19-FEMALE LIMITED EPILEPSY AND INTELLECTUAL DISABILITY (PCDH19-FLE) SUGGESTS Jayasena A.S.1, 2, Secco D.2, Whelan J.L.2 and Mylne J.S.1, 2 A ROLE FOR NEUROSTEROID HORMONES 1School of Chemistry and Biochemistry, The University of Western 2 Australia. ARC Centre of Excellence in Plant Energy Biology, The Tan C.1, Shard C.1, Hynes K.1, Douglas E.2, Buchanan G.1, Ranieri E.2, University of Western Australia (M310), 35 Stirling Highway, Crawley, Marini C.3, Berkovic S.F.4, Scheffer I.E.4 and Gecz J.1, 2 Perth, WA 6009, Australia. 1The University of Adelaide, North Adelaide, Australia. 2SA Pathology, North Adelaide, Australia. 3University of Florence, Florence, Italy. 4The Sunflower trypsin inhibitor 1 (SFTI-1) is a 14 amino acid, trypsin University of Melbourne, Melbourne, Australia. inhibitory cyclic peptide found in sunflower seeds (Luckettet al., 1999). SFTI-1 has an unusual genetic origin as it is buried within the open PCDH19-Female-Limited-Epilepsy (PCDH19-FLE) is an unusual reading frame of a much larger protein which also codes for a seed X-linked disorder that primarily affects females. PCDH19-FLE storage protein albumin. This gene was named PawS1 (Preproalbumin with SFTI-1) (Mylne et al., 2011). It is interesting to study this unique encompasses a broad clinical spectrum from early infantile epileptic and unusual dual-biosynthesis to appreciate whether dual-biosynthesis encephalopathy resembling Dravet syndrome to epilepsy with or is a more common genetic mechanism for producing new proteins without intellectual disability and behavioural problems, including than previously thought. PawS1 genes from a range of daisies (family autism. PCDH19-FLE is highly but not fully penetrant. We have Asteraceae) have defined a new class of seed peptides called “PawS tackled the questions of molecular pathogenesis of PCDH19-FLE by Derived Peptide (PDP) family”. The range of daisy family members examining the transcriptomes of primary skin fibroblasts of PCDH19- containing the PawS1 gene coupled with phylogenetic chronograms FLE females (n=12 and n=3 age and passage matched normal controls) enabled a prediction to be made that the PDP class of seed peptides and unaffected transmitting males (n=3 and n=3 age and passage is at least 18 million years old. The PawS1 genes that revealed the matched control males). We found that the expression of genes, which PDP family, were amplified by PCR, but in many cases PCR failed for normally show sex (male/female) biased expression in this cell type, species in which a PawS1 gene was expected based on phylogenies. was significantly altered (observed = 43/94 vs expected = 223/19223, The reason is, most likely, because the heterologous primers used p=1.09 x 10-55, two-tail Fisher’s exact test). Followup studies (including do not match the target sequence and the more distantly related the additional skin fibroblast cell lines) validated at least ~60% of selected species is, the less likely primers will amplify a PawS1 product. With the genes. From among several plausible biological candidates we focused development of lower cost next-generation sequencing technologies our attention on the aldo-keto reductase family 1, member C1-3 de novo transcriptomics has become a more viable method for (AKR1C1-3) genes, which play crucial role in neurosteroid hormone analysing non-model organisms. Therefore we intend to create de metabolism (which skin is endowed with). Additional support for steroid novo transcriptomes from mature seed RNA covering a wide range of and neurosteroid hormone role in the pathology of PCDH19-FLE came daisies to study the evolution of the PawS1 gene. De novo assemblies from the age of onset (mean ~10 months) and offset (mean ~12.5 years) made using CLC Genomics workbench (computation time ~1 day per of epilepsy (n=100 patients), both of which coincide with dramatically species) in 20 daisy species allowed us to identify several new PawS1 varying sex hormone levels (onset - after ‘minipuberty’ and offset - genes and some of the PawS-derived peptides were confirmed at the with the advent of puberty). This led us to postulate the neurosteroid protein level using LC-MS/MS. Therefore, de novo transcriptomics hypothesis, which may explain PCDH19-FLE and opens realistic seems to be a promising and cost effective approach for studying the opportunities for targeted therapeutic interventions. evolution of a single gene.

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SYM-04-02 SYM-O4-03 Sponsored by RAPID MOLECULAR DIAGNOSIS OF JAK2V617F The Australian Society for Biochemistry and Molecular Biology NEGATIVE MPN BY TARGETED DEEP SEQUENCING USING THE ION TORRENT PGM A SINGLE MOLECULE SEQUENCING EXPRESSION Mager G.1, Klose N.1, Tallack M.R.1, Mollee P.2 and Perkins A.C.1, 2 ATLAS 1Mater Research, Translational Research Institute, University of Queensland, Brisbane, Queensland, Australia 4101. 2The Princess Alexandra Hospital, Forrest A.R.R.1, 2, Kawaji H.1, 3, Rehli M.4, Baillie J.K.5, Lassmann T.1,2, Brisbane, Queensland, 4102, Australia. Itoh M.1, 3, Suzuki H.1, 2, Carninci P.1, 2, Hayashizaki Y.2, 3 and The Fantom5 Myeloproliferative neoplasms (MPN) are a heterogeneous group of blood disorders Consortium A.2 1 characterized by excess production of mature blood cells, increased risk of thrombotic RIKEN Centre for Life Science Technology, Division of Genomic complications and slow progression to myelofibrosis or, less often, leukemia. Activation Technology, Yokohama, Japan. 2RIKEN Omics Science Centre, 3 4 of the JAK-STAT signaling pathway is a common underlying feature of these diseases Yokohama, Japan. RIKEN PMI, Yokohama, Japan. Department of and JAK kinase inhibitors are efficacious in the more advanced forms of disease. Most Hematology and internal Oncology, University Hospital Regensburg, cases of polycythemia vera (PV) and approximately 60% of essential thrombocythemia Germany. 5The Roslin Institute, University of Edinburgh, UK. (ET) and primary myelofibrosis (MF) harbor a point mutation in JAK2 (V617F) which leads to constitutive JAK-STAT signaling and factor independent cell growth. The Using Cap Analysis of Gene Expression (CAGE) we have a generated remaining 40% of cases of MF and ET harbor a broad range of mutations in many a promoter level expression atlas based on single molecule sequencing genes including those involved in cytokine receptor signaling, other components or the across a broad panel of tissues, cell lines and primary cells from human JAK-STAT pathway or epigenetic regulators. This poses a challenge for rapid molecular and mouse. The first phase of the project is complete and represents diagnosis. Also, since ET is essentially a diagnosis of exclusion of reactive causes of the first single molecule sequencing atlas to date. Using this data we thrombocytosis, many cases of chronic ‘ET’ may not be clonal hematological neoplasms have obtained an exquisite snapshot of the expression patterns of but reactive conditions. We have developed a rapid deep sequencing pipeline to detect mammalian transcripts, including long non coding RNAs and novel mutations in 65 genes which have been implicated in MPN through previous reports of transcripts. I will present our progress to date, tools developed to human mutations, mouse models of MPN, or other known components of hematopoietic explore the data and the future directions of the project. cytokine receptor signaling. We used 10ng of DNA from blood to amplify and sequence all the exons of these 65 genes using Ampliseq and Ion Torrent PGM. Using 318 PGM chips and 8-fold multiplexing we achieved on average 200 fold coverage of the target exome. The bioinformatics of SNP validation and rapid generation of reports will be presented. From a pilot study of 30 cases referred for molecular diagnosis, we have detected the likely causative mutation in approximately 80% of ET and MF where JAK2 is wild type. Many of these mutations are known to be causative in MPN, including those in MPL, ASXL1, SET2, SH2B3 (LNK), EZH2, CBL, DNMT3A and other genes. We have identified a novel inherited mutation in a family with MPN and validated it in BAF3 factor-independency assays. We have identified further novel mutations in JAK3, EED, DNMT3A, APC and two phosphatases involved in silencing activated JAK-STAT pathway components. The biological significance of these is under investigation and progress will be reported. In many cases we find evidence for clonal evolution involving secondary mutations in epigenetic modifying proteins on top of driver mutations in the JAK-STAT pathway. In short, targeted exome re-sequencing using Ampliseq and Ion Torrent PGM provides a rapid and relatively cheap method for molecular diagnosis and characterization of most cases of MPN.

SYM-04-04 SYM-04-05 REPRESSION OF CHIMERIC TRANSCRIPTS A GENE IMPLICATED IN THE NEUROBEHAVIOURAL EMANATING FROM ENDOGENOUS ABNORMALITIES OF WILLIAMS-BEUREN SYNDROME, RETROTRANSPOSONS BY THE SEQUENCE-SPECIFIC GTF2IRD1, ENCODES A NOVEL EPIGENETIC TRANSCRIPTION FACTOR KLF3 REGULATOR

Funnell A.P.W., Mak K., Burdach J., Norton L.J., Pearson R.C.M. and Carmona-Mora P.1, Widagdo J.1, Tomasetig F.1, Taylor K.M.1, Cha Y.1, Crossley M. Pang R.T.-W.1, Twine N.A.2, Wilkins M.R.2, Gunning P.W.1, Hardeman School of Biotechnology and Biomolecular Sciences, University of E.C.1 and Palmer S.J.1 NSW, NSW, 2052. 1School of Medical Sciences, Cellular and Genetic Medicine Unit, University of New South Wales, Sydney, NSW. 2School of Whilst the expansion and integration of retroelements promotes genetic Biotechnology and Biomolecular Sciences, The New South Wales variation and drives evolution, it can also be causative of disease. This Systems Biology Initiative, University of New South Wales, NSW. is because retroelements, which comprise over 40% of the human genome, can disrupt or aberrantly activate the genes in which they Williams-Beuren syndrome (WBS) is caused by a microdeletion of 28 reside or to which they are proximal. Accordingly, host organisms genes on chromosome 7q11.23, occurring in 1:7,500 live births. WBS have evolved means of silencing retroelements, which typically involve has a broad presentation including craniofacial abnormalities and a epigenetic modification of histones and/or DNA. However, little is known distinctive neurocognitive profile. Genotype/phenotype correlations about the underlying mechanisms of this repression, specifically, which in patients with atypical deletions implicate a gene discovered in our DNA-binding proteins are involved and how silencing of distinct classes laboratory, GTF2IRD1, as responsible for these features. Gtf2ird1 of retrotransposons is achieved. KLF3 is a transcriptional repressor knockout (KO) mice recapitulate many of the defects of WBS, including that silences gene expression via the co-repressor CtBP, a protein hyperactivity and ataxia. To reveal GTF2IRD1 normal function, we that recruits multiple histone-modifying enzymes. Microarray and have combined protein interaction studies with gene expression RNA-Seq analysis of Klf3-/- foetal erythroid cells revealed upregulated analyses in Gtf2ird1 KO mice. The first approach comprised a expression of a number of genes only at their downstream exons. 5’ yeast two-hybrid screen in which we identified a panel of interacting RACE revealed that for one of these targets, Pu.1/Sfpi-1, a specific LTR partners that fall into functional groups; DNA binding proteins, post- retrotransposon functions as an internal transcriptional promoter in the translational modification machinery and chromatin modifying factors absence of KLF3. The retrotransposon generates a truncated protein involved in histone methylation, deacetylation and ubiquitination. isoform of PU.1 that displays dominant negative activity. Moreover, we Immunofluorescence and co-immunoprecipitation in mammalian cell found that a substantial number of KLF3 target genes have chimeric lines support the validity of these interactions. To assess the effect of transcripts driven by this class of retrotransposon. Lastly, we have GTF2IRD1 on gene expression, we conducted microarray analyses in also shown that the transcriptional activator KLF1, a master regulator corpus striatum tissue from Gtf2ird1 KO mice followed by qRT-PCR. of erythropoiesis, also drives expression from these LTRs in erythroid We found overexpression of genes involved in neuronal development cells, suggesting regulatory competition between these two KLFs. In and a cluster of immediate-early response genes that have previously summary, we have identified a novel role for KLF3 as a transcriptional been linked with hyperactivity. Our data are consistent with a role repressor that binds particular LTR promoter sequences to prevent for GTF2IRD1 as an epigenetic regulator of gene repression that aberrant, chimeric transcription of endogenous genes. coordinates interactions with transcription factors, DNA binding proteins and components of the chromatin modification machinery.

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SYM-05-01 SYM-O5-02 VENOMS TO DRUGS: TRANSLATING VENOM INNATE-LIKE T CELL RECEPTORS AND ANTIGEN PEPTIDES INTO THERAPEUTICS FOR TREATING RECOGNITION CHRONIC PAIN Patel O.1, Kjer-Nielsen L.2, Le Nours J.1, Birkinshaw R.1, Beddoe T.1, King G.F. Liu L.3, Sandoval-Romero M.L.1, Fairlie D.P.3, Mccluskey J.2 and Institute for Molecular Bioscience, The University of Queensland, Rossjohn J.1, 4, 5 Brisbane, Australia. 1Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia. 2Department of Microbiology Chronic pain is one of the most poorly managed medical problems in and Immunology, University of Melbourne, Melbourne, Australia. the world. The economic burden of chronic pain in the USA is currently 3Division of Chemistry and Structural Biology, Institute for Molecular $600 billion a year, which is more than the combined cost of cancer, Bioscience, University of Queensland, Brisbane, Australia. 4Australian heart disease and diabetes. Current-generation pain killers have Research Council Centre of Excellence in Structural and Functional significant problems such as dose-limiting side-effects, tolerance, Microbial Genomics, Monash University, Melbourne, Australia. addiction, and the potential for abuse, and hence there is much interest 5Institute of Infection and Immunity, Cardiff University, School of in developing new analgesics with greater potency and a better side- Medicine, Cardiff, UK. effect profile. The human voltage-gated sodium channel Nav1.7 is highly expressed in specialized primary sensory neurons (nociceptors) Mucosal associated invariant T cells (MAIT) are innate-like T cells that that are responsible for detection of noxious stimuli. Loss-of-function are preferentially located in the gut lamina propria. MAIT cells express mutations in the human Nav1.7 channel cause a congenital indifference alpha-beta T cell receptors (TCR) on their surface that recognize to all forms of pain, and therefore selective blockers of Nav1.7 are likely antigens bound to monomorphic non-classical Major Histocompatibility to be powerful analgesics. However, Nav1.7 is only one of nine human Complex (MHC) related molecule, MR1. It is well documented that T NaV subtypes, and improper function of certain members of this ion cells can be activated by peptide-based and lipid-based antigens channel family can cause debilitating or even lethal channelopathies. presented by classical MHC and Cluster of differentiation 1 (CD1) Thus, therapeutics designed to target Nav1.7 must have exquisite molecules respectively. However, to date the identity of the antigen that selectivity. The venoms of predators such as spiders and centipedes can bind to MR1 and activate MAIT cells was unknown. Here we present are dominated by disulfide-rich peptides that evolved to target a crystal structure of MR1 bound to 6-formyl pterin, a folic acid (vitamin neuronal ion channels in their prey. We took advantage of this natural B9) metabolite. The structure of MR1 has revealed how its binding cleft combinatorial library of ion channel modulators to uncover venom is ideally suited to present small organic compounds. Additionally, we peptides that potently and specifically target Nav1.7. I will describe the provide further insight into the recognition of vitamin metabolites by discovery and biophysical characterization of these peptides, including the MAIT TCR. This study provides a first example of how T cells can their subtype selectivity, 3D structure, channel binding site, and mode recognize antigens that are not peptide-based or lipid-based in nature. of action, and demonstrate that some of these peptides are even more Nature 2012 Nov 29; 491:717-23. Nature Communications 2013 June effective than morphine in rodent pain models. 13; Accepted.

SYM-05-03 SYM-05-04 THE ATRAZINE BREAKDOWN PATHWAY: EVOLUTION PROBING THE STRUCTURE AND FUNCTION OF A IN ACTION 1-MDA CHROMATIN REMODELING COMPLEX

Peat T.S.1, Balotra S.1, Wilding M.1, French N.G.1, Briggs L.J.1, Panjikar Webb S.1, Shepherd N.1, Silva A.1, Alqarni S.1, Saathoff H.1, Low J.1, S.2, Cowieson N.2, Newman J.1 and Scott C.1 Laue E.2, Blobel G.3, Matthews J.1 and Mackay J.1 1CSIRO. 2Australian Synchrotron. 1School of Molecular Bioscience, University of Sydney, NSW, Australia. 2Department of Biochemistry, University of Cambridge, UK. Atrazine is a commonly used herbicide that has been banned by the 3Children’s Hospital of Philadelphia, PA, USA. European Union but is still in common use in most of the world. The atrazine breakdown pathway found in Pseudomonas sp ADP consists The fact that DNA-binding transcription factors bind to specific sites in of six enzymes, AtzA through AtzF. The first three of these remove gene promoters and thereby regulate the expression of target genes various moieties from the ring structure leaving cyanuric acid. The is well established. However, at a molecular level, the steps between cyanuric acid hydrolase, AtzD, is the founding member of a newly DNA binding and changes in the expression levels of target genes are identified family of ring-opening amidases. We report the first X-ray only partly understood, and the goal of our work is understand at a structure for this family solved using SIRAS with a Xe derivative. The mechanistic level the changes in chromatin structure and occupancy structure is a novel fold (termed the ‘Toblerone’ fold) that likely evolved and the protein interactions that are made to drive these changes in via the concatenation of monomers of the trimeric YjgF-superfamily and gene expression. We are using a range of biochemical and structural the acquisition of a metal binding site. Structures of AtzD with bound approaches, including x-ray crystallography, NMR spectroscopy, substrate and inhibitors, along with mutagenesis studies, allowed the mass spectrometry and electron microscopy, to examine the structure identification of the active site. The AtzD monomer, active site and of the Nucleosome Remodeling and Deacetylase (NuRD) complex substrate all possess three-fold rotational symmetry, which extends to and the mechanism by which it is targeted to gene promoters, an three potential Ser-Lys catalytic dyads in the active site. Biochemical event that is essential for normal growth and development across all and crystallographic evidence suggests that only one of these dyads complex organisms. Combined with data that explore the molecular is catalytically active and a mechanism is proposed. The quaternary makeup of the NuRD complex, these results provide a glimpse into structure of AtzD was determined to be a D2 tetramer using SAXS and the mechanisms through which complex coregulator complexes are crystallographic data. The evolution of this pathway and this enzyme in recruited to target genes and help to map out the molecular events that particular will be discussed. drive gene regulation.

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SYM-05-05 SYM-O6-01 STRUCTURAL AND BINDING STUDIES OF THE CAV1.1 TARGETING THE VASCULAR NICHE: EVOLVING β1A SUBUNIT FEEDBACK MECHANISMS OF RESISTANCE

Casarotto M.G.1, Karunasekara Y.1, Aditya S.1, Capello J.1, Dulhunty A.F.1, Lu K., Rivera L., Chang J. and Bergers G. Board P.G.1, Oakley A.J.2 and Norris N.C.1 Department of Neurological Surgery, Brain Tumor Research Center and 1JCSMR, Australian National University, Canberra ACT Australia. UCSF Comprehensive Cancer Center, University of California, Helen 2Dept Chemistry, University of Wollongong, NSW Australia. Diller Family Cancer Research Center, San Francisco, CA 94143, USA.

Excitation-contraction (EC) coupling in skeletal muscle requires a A recent milestone in cancer research has been the revelation that physical coupling between the voltage gated calcium channel (Cav1.1) therapies targeting other cell types besides tumor cells; i.e., the tumor in the surface membrane and the ryanodine receptor (RyR) Ca2+ release vasculature can convey substantial improvements in radiographic channel in the sarcoplasmic reticulum Ca2+ store. Although the exact response, progression-free survival, and quality of life. This led to molecular mechanism of this interaction is unresolved, both the α1s and the accelerated FDA (US Food and Drug Administration) approval of β1a subunits of Cav1.1 are essential for EC coupling. The β1a subunit has bevacizumab (Avastin; Genentech/Roche), a humanized monoclonal a modular structure consisting of SH3/guanylate kinase (GK) domains antibody directed against VEGF-A, and several receptor tyrosine separated by a variable hook region. The GK domain binds with high kinase inhibitors blocking the VEGF/VEGFR pathway (e.g. Sunitinib, affinity to the α1 subunit, but the functional significance of the SH3 sorafenib) for use in various tumor types in the US. Despite these domain remains unclear. The structure of the Cav1.1 β1a subunit has not encouraging beneficial effects, patients inevitably develop resistance been experimentally determined. However, the SH3 polyproline-binding and frequently fail to demonstrate significantly better overall survival. site is occluded in all available Cav β-isoform structures by the α2 helix Unlike chemotherapies, however, in which tumors exhibit resistance due and the RT loop. Hence, it was predicted by others that none of the β to genetic mutation of drug targets, emerging evidence suggests that isoforms, including β1a, would bind polyproline motifs. This prediction is tumors bypass anti-angiogenic therapy while VEGF signaling remains at odds with our findings which shows binding of ~ 2 µM between the inhibited through a variety of mechanisms that are just beginning to

Cav1.1 β1a subunit and the α1s subunit II-III loop. We clearly show that be recognized. Because of the indirect nature of resistance to VEGF this interaction takes place through the SH3 domain of the β1a subunit inhibitors there is promise that strategies combining angiogenesis and a proline-rich region of the α1s II-III loop which has previously been inhibitors with drugs targeting such evasive resistance pathways will lead shown to be critical for skeletal type EC-coupling (1). Here we report to more durable efficacy and improved patient outcome. Leveraging from the first structure of the Cav1.1 1aβ subunit using X-ray crystallography the results obtained during the course of antiangiogenic therapy in brain, and through chimeric studies of different β-isoforms demonstrate that pancreatic and brain tumor mouse models and patient tumor samples, key amino acid residues in the RT loop facilitate the interaction with we found that relapsing tumors exposed cellular signals associated with proline-rich binding motifs. Based on these findings we propose a novel resistance that were rather transiently regulated in response to contextual model where the β1a subunit acts as conduit for the transmission of the signals that tumor cells received from their microenvironment and EC-coupling signal from the skeletal voltage-sensing Cav1.1 α1 subunit responded to during the course of treatment. Comparison of responding to RyR1. 1. Kugler, G. et al (2004). J Biol Chem 279(6): 4721-4728. and relapsing tumors during treatment has led to the discovery of a novel VEGFR2:cMet heterocomplex and its function in tumor cell invasion, and enabled the detection of an oscillating pattern of distinct innate immune cells within tumors in response to therapy modifications that quickly adapted to growth restrictions and provided resistance.

SYM-06-02 SYM-06-03 GENERATION OF IMMUNE-PRIVILEGED RESTORING ANTI-TUMOUR IMMUNE RESPONSES TO PREMETASTATIC NICHES BY PRIMARY TUMOUR PREVENT BREAST CANCER METASTASIS HYPOXIA Rautela J.1, 2 Slaney, C.1 and Parker B.S.1 Sceneay J.1, Wen S.W.1, Smyth M.J.2 and Möller A.1 1La Trobe Institute for Molecular Science, La Trobe University, 1Tumour Microenvironment Laboratory,. 2Immunology in Cancer and Melbourne, Victoria. 2Sir Peter MacCallum Department of Oncology, Infection Laboratory, Queensland Institute of Medical Research, The University of Melbourne, Parkville, Victoria. Herston, QLD. Breast cancer is the second most common cause of cancer-related Metastatic breast cancers are more difficult to treat than primary deaths in women. Patient mortality occurs due to metastatic spread, lesions and often do not respond to chemotherapeutics, causing with bone one of the most common metastatic sites. Therefore, in order mortality and morbidity in patients. The mechanisms underlying to improve patient survival, therapies aimed at preventing or treating this treatment resistance are largely unknown, but could be partially metastases are essential. Our recent data suggest that a key requirement due to the different stromal environments at metastatic sites. Pro- for breast cancer metastasis is evading anti-tumour immunity. We have tumourigenic properties of future metastatic sites are determined by the shown that breast cancer cells suppress innate type-1 interferon (IFN) interactions between factors secreted by the primary tumour and bone signalling to successfully grow in the bone environment. Restored IFN marrow-derived immune cells, which drives formation of permissive signalling in mice bearing 4T1. 2 breast tumour cells significantly reduced environments, called premetastatic niches, before the arrival of tumour bone metastasis and this anti-metastatic effect was entirely dependent cells. Hypoxia is a common feature and poor prognostic factor in many on the host cytotoxic lymphocyte compartment (NK and CD8+ T cells). solid cancers including breast cancer and acts as a strong selective Our current investigations are aimed at testing the therapeutic potential pressure that promotes angiogenesis, invasion and metastatic of IFN agonists in metastatic breast cancer models. Administration of spread of cancer cells. Here, we demonstrate in different immune IFNα or the viral mimetic poly(I:C) to mice bearing the bone metastatic competent, syngeneic breast cancer models, that primary tumour 4T1.2 cells significantly reduced metastasis to lung and bone. This was hypoxia promotes premetastatic niche formation. Cell-free supernatant associated with enhanced anti-tumour immune response. Our studies derived from hypoxic breast tumour cells results in increased bone are now focused on evaluating the value of such therapies in preventing marrow-derived cell infiltration into the lungs and leads to increased metastatic outgrowth and/or targeting established metastases, the metastatic burden in experimental metastasis models, suggesting potency of combining IFN-based therapies with commonly used reduced immune surveillance in premetastatic niches. We define bone chemotherapeutics such as doxorubicin, and determining patients that marrow-derived CD11b+/Ly6Cmed/Ly6G+ granulocytic myeloid-derived are most likely to benefit from these therapeutic approaches. suppressor cells and immature NK cells with reduced cytotoxic activity as main constituents of the premetastatic niche. Furthermore, tumour hypoxia causes increased secretion of exosome which contain different proteins and RNA compared to exosomes from normoxic cells. These exosomes are capable of establishing premetastic niches, and their unique protein and RNA content can be developed as novel diagnostic signatures and therapeutic targets for the prevention of metastasis.

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SYM-06-04 SYM-O6-05 REMODELLING THE IMMUNE MICROENVIRONMENT DEVELOPMENT OF NOVEL MOLECULES THAT TO ENHANCE IMMUNE THERAPY CONTROL CELL SURVIVAL BY REGULATING THE 14- 3-3 DIMER INTERFACE Johansson A.J., Hamzah J. and Ganss R. Western Australian Institute for Medical Research, University of Dhagat U.1, Holien J.K.1, Goodwin K.2, Carver J.2, Coolen C.2, Doughty L.1, Western Australia, Centre for Medical Research, Perth, Australia. Broughton S.E.1, Lopez A.2, Parker M.W.1 and Woodcock J.M.2 1Biota Structural Biology, St. Vincent’s Institute of Medical Research, Solid tumours maintain a barrier that renders them resistant to immune Fitzroy, Victoria. 2Department of Biochemistry and Molecular Biology rejection. In humans, systemic anti-cancer vaccines or adoptive Division of Human Immunology, Centre for Cancer Biology, Adelaide, transfer of anti-cancer immune cells often fail to induce a strong anti- South Australia. tumour response. Our work demonstrates that the stromal immune- microenvironment, including the abnormal tumour vasculature, is 14-3-3 proteins belong to a highly conserved family of dimeric phospho- an important component of this barrier. Importantly however, the serine binding proteins that interact and modulate the functions of key microenvironment can be remodelled to support cytotoxic immune cellular proteins involved in signalling. Many binding partners of 14-3- cells and consequently tumour rejection. To specifically manipulate 3 proteins are associated with cell survival and consequently 14-3-3 the tumour vasculature and stroma, we targeted low dose tumour proteins provide an important regulatory function in protecting cells necrosis factor alpha (TNFα) into murine endocrine pancreatic tumours against death through negative regulation of pro-apoptotic mediators. and investigated its effects on the immune microenvironment. We We have previously found that cytokine receptors can directly associate found that low dose TNFα effectively improves vascular perfusion and with 14-3-3 following cytokine stimulation and this mediates a survival CD8+ influx into tumours. Moreover, TNFα acts as a potent adjuvant signal. Given the above and the frequently observed overexpression for immunotherapy and substantially improves efficacy of anti-tumour of 14-3-3 proteins in many cancers we suggest that these proteins vaccination and adoptive transfer of cytotoxic T cells. Importantly, represent an attractive target for directed anti-cancer drug development. TNFα therapy also re-programmes intra-tumoral macrophages to We recently showed that the function of 14-3-3 proteins is regulated support anti-tumour immunity through secretion of immune- and through a mechanism that controls their dimeric state. This is critical vascular mediators such as monocyte chemoattractant protein 1 as disruption of 14-3-3 dimers drastically impairs the 14-3-3 protein’s (MCP1), inducible nitric oxide synthase (iNOS), and Angiopoietin 2 anti-apoptotic activity. From extensive site-directed mutagenesis (Ang2). In particular, macrophage derived Ang2 sensitizes endothelial studies we have gained a unique insight into the molecular basis of cells to TNFα treatment and enhances TNFα-induced up-regulation 14-3-3 dimerisation. We have established that dimers are held together of vascular cell adhesion molecule 1 (VCAM1) which in turn facilitates by critical inter-molecular salt bridges between conserved residues. leukocyte access into tumors. Thus, TNFα targets multiple components Taking advantage of the three dimensional structure of the dimer of the tumour microenvironment including vessels and macrophages, interface we have now screened a library of two million compounds improves vessel function and immunotherapy. Our findings strongly and identified new candidate compounds that target this pathway and suggest that intratumoral cytokine delivery is a promising strategy to reduce cell viability in vitro and tumour growth in vivo. These candidates change the inflammatory tumour milieu and break its resistance to show proof of principle and have the potential to be developed into immune therapy. novel therapies that selectively target cancer cells.

SYM-06A-01 SYM-06A-02 ADAPTING TO CHANGE: FLEXIBLE LEARNING IN THE DRIVING CURRICULUM REFORM THROUGH NEW MILLENNIUM NATIONAL GRADUATE LEARNING OUTCOMES

Poronnik P. Johnson E. School of Medical Sciences, The University of Sydney, NSW 2006 La Trobe University, Melbourne, Australia. Australia. The publication of Threshold Learning Outcomes for science graduates There has been significant activity for some years regarding on-line in 2011 marked a national change in the standards expected for science and flexible learning. Most of the tools that we need to help students graduates. Recent work by discipline education networks seeks to learn have also been around for quite some time. While the technology translate the description of graduate outcomes into teaching practice to enable many of these tools continues to improve, are we making within disciplines. Two of these networks (VIBEnet (sites.google.com/ the most of these opportunities, or are we seeing minor tweaks to the site/vibenet101) and CUBEnet (www.cubenet.org.au) are developing routines of yesterday? It is widely held that the most disruptive intrusion practice directly for molecular life sciences. Parallel work in chemistry into the tertiary curriculum space is the recent advent of the Massive (Chemnet), Physics (Physnet) and mathematics (AMSLaT) links Open Online Course (MOOC). This has put flexible learning at the foundation disciplines together through common objectives. Specific forefront of many debates and with it, an exponential rise in interest and good practice guides are also being developed for implementation of anxiety amongst the academic community. There is a strong case to be each of the five learning outcomes. Together these projects will provide made that MOOCs are a sign of the times, the catalyst for change that rich resources for science academics leading curriculum renewal. At has been some time in coming. The MOOCs are seen by some as a the same time, the Australian Council of Deans of Science (ACDS) mechanism to dramatically increase the quality and impact of campus has established its ACDS Teaching and Learning Centre (www.acds. learning and the chances for students to unbundle coursework potentially edu.au/tlcentre/) which aims to help Faculties of Science provide a increases mobility between institutions. MOOCs are therefore part of a supportive institutional context for curriculum reform. The Centre will suite of changes that the higher education sector faces such as mass be a hub for information on learning and teaching in university science participation, resource constraints, increased competition for research and aims to link science disciplines. It will focus the energies of learning funding and an increased regulatory environment. How do we capture & teaching leaders into change at Faculty and discipline level by the excitement and opportunities to lead to an adaptation and sustained constructing good practice guides providing distilled advice for teaching improvement to our tertiary teaching in this new millennium? Here we and learning. Through the Centre, the ACDS will develop effective will consider what this means for the curriculum, the staff, the students models for organizational change and curriculum development. The and our institutions. Centre aims to work collaboratively with science faculties, discipline groups & associations and learning & teaching leaders to develop and offer authoritative advice to Universities and regulators.

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SYM-06A-03 SYM-O6A-04 SCAFFOLDING STUDENT WRITING IN SCIENCE: LEARNING ABOUT RESEARCH: SUPPORTING INITIATIVES, IMPLEMENTATION AND INDICATORS OF STUDENT LEARNING IN AN UNDERGRADUATE SUCCESS RESEARCH PROJECT

Gleadow R.M.1, Rayner G.1 and Abbott K.1 Howitt S.M.1, Rivers J.1, Longbottom C.1, Mills R.1, Higgins D.1 and 1School of Biological Sciences, Monash University. 2School of Wilson A.2 Biological Sciences, Monash University. 3School of Biological 1Australian National University. 2Oxford Learning Institute. Sciences, Monash University. Research skills and experience are increasingly seen as desirable Graduates of universities are expected to gained competency in outcomes from an undergraduate degree but providing authentic critical thinking, data analysis, and communication. Despite this, many research experiences in large classes remains a challenge. We university undergraduates struggle to write coherently and are unable to describe a full semester research project undertaken by a second extract and summarise relevant information from the scientific literature. year class of 200 students. Students mutagenize E. coli, screen for We have developed a system of scaffolding student work to motivate lactose utilization mutants and then analyse their mutants to identify students to begin writing assignments earlier in the semester, and which component of the lac operon is defective. While lab sessions then step them through the process. The vehicle for this is a scientific are highly structured, with all procedures provided, the outcome literature review. The tasks form about one third of the assessment of is unknown so students must apply logic and link different strands a compulsory unit on Scientific Practice and Communication. Students of inquiry with lecture material to identify the mutations. Since the are introduced to databases, strategies for identifying key information project runs for the whole semester, students also need to keep good and curating tools for keeping track of their reading. The three parts records and understand the connections between different parts of the of the written assignment are (1) conceptualising and refining their experimental strategy. After running the project in 2012, it was clear that research question; (2) a summary of a single primary peer reviewed some students needed greater support to deal with these challenges. article; (3) An annotated bibliography of 5 articles relevant to their In 2013, optional peer assisted learning sessions were implemented, topic with an introductory paragraph and formatted reference list; (4) run by two undergraduate mentors who had previously completed the A draft that is reviewed by their peers in class; and (5) the final review. course. Specific activities that targeted difficulties encountered in 2012 Feedback is given at each step using variously rubrics, verbal feedback were developed by the peer mentors. The sessions aimed to provide and detailed written comments on one section. Plagiarism is minimised a supportive environment in which students could discuss results, by requiring students to check their own work using plagiarism detection experimental strategy and their concerns. Students were surveyed software, and appending the report to their assignment. There has throughout the semester, allowing concerns to be addressed in labs, been an apparent improvement in student writing and critical thinking peer assisted learning sessions and lectures. The surveys also asked in third year and Honours, although this has been hard to quantify. about learning throughout the semester and many students showed Students’ perceptions of their scientific writing skills increases over development from a focus on technical skills and short term outcomes each semester but does not necessarily translate into higher marks. to a more sophisticated understanding of the nature of scientific The challenge academics is to convert perceived improvement ability research with its inherent uncertainty. into a genuine improvement in writing skills that can be quantified.

SYM-06A-05 SYM-07-01 ENHANCING STUDENT MOTIVATION AND Sponsored by PARTICIPATION IN LARGE BIOLOGY COURSES The University of Western Australia THROUGH PEER ASSESSMENT, GROUP WORK AND NOVEL INCENTIVE-DRIVEN ASSESSMENT STRATEGIES REGULATION OF CELL ADHESION, ACTOMYOSIN CYTOSKELETON, AND MECHANICAL FORCES Galea A.M., Delaney S. and Wilson J.E. DURING APOPTOSIS IN DROSOPHILA DEVELOPMENT School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia. Toyama Y.1, 2, 3 1Department of Biological Sciences, National University of Singapore. 2 3 First and second year university students can face a number of Mechanobiology Institute, Singapore. Temasek Life Sciences educational disadvantages associated with their enrolment in large Laboratory. courses unless these matters are carefully addressed and managed. For example, the larger the cohort, the more difficult it becomes for Understanding tissue dynamics during embryo development requires course coordinators to ensure that all students receive consistent knowledge of active mechanical forces exerted by cells and passive formative assessment and thorough qualitative feedback on their response by the neighboring cells through cell-cell adhesion. Apoptosis, performance throughout the semester. In this study, we describe three or programmed cell death, is the process that remodels tissue by different learning and assessment strategies developed for large first eliminating cells that are no longer biologically necessary. During and second year biology courses that address these challenges. the apoptotic process among a tissue, 1) the dying cell is squeezed Online learning management systems are employed to streamline the out from the original tissue by the contraction of actomyosin cables deployment and administration of these large course activities and form within the dying cell and within the neighboring non-apoptotic also to enable a variety of student-student interactions, all of which are cells formed upon apoptosis and 2) the cell adhesion between dyting aimed at promoting student participation, motivation and, subsequently, and non-dying cells need to be well regulated so that the apoptotic the successful achievement of learning outcomes. The first approach cell is able to escape from the tissue wituout compromising the involves a learning and assessment strategy that integrates a reflective tissue integrity. The neighboring cells are deformed during the cell essay task with a comprehensive online peer assessment element. elimination, indicating that the mechanical stress in the surrounding This activity aims to introduce students to peer-reviewed scientific tissue is increased and that apoptotic process could mechanically literature whilst simultaneously assisting them to develop their written contribute to tissue dynamics. We study tissue replacement process communication skills. The second is a group project developed to know as Histoblast expansion during Drosophila Metamorphosis. enrich students’ critical and analytical thinking skills through the design, Abdominal histoblasts nests (precursor of adult epidermal cells) form execution and documentation of a simple experiment. The third task is among the cells of larval abdomen. During the expansion, histoblasts an activity designed to enhance students’ understanding and mastery undergo a rapid proliferation and the preexisting larval epidermal cells of individual learning objectives through an online, incentive-driven (LECs) undergo apoptosis. Eventually, the tissue replacement from framework that invites students to devise and share questions for their larval tissue to adult tissue completes. In this presentation, the spatial upcoming mid-term exams. All three strategies will be discussed with and temporal regulation of Caspase activation, cell adhesion, actin respect to their implementation and effectiveness in enhancing student and myosin dynamics, and cell deformation during apoptosis will be motivation, participation and achievement of biology learning outcomes. discussed.

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SYM-07-02 SYM-07-03 THE ROLE OF MYOSIN VI AND MUNC18-1 IN NOVEL ROLE OF TROPOMYOSIN ISOFORMS IN SECRETORY GRANULE TETHERING AND MOTILITY IN MODULATING ACTIN CYTOSKELETON DURING NEUROSECRETORY CELLS EXOCYTOSIS OF THE SECRETORY GRANULES IN RODENT MODELS OF EXOCRINE SECRETION Martin S.1, Tomatis V.M.1, Papadopulos A.1, Malintan N.T.1, Gormal R.S.1, Masedunskas A.1, Appaduray M.1, Hardeman E.2 and Gunning P.1 Kendrick-Jones J.2, Buss F.3, Martin J.L.4, Collins B.M.4 and Meunier F.A.1 1Oncology Research Unit, School of Medical Sciences, University of New South 1Queensland Brain Institute, The University of Queensland, Brisbane, Wales, Sydney, Australia. 2Neuromuscular and Regenerative Medicine Unit, QLD 4072. 2MRC Laboratory of Molecular Biology, Cambridge, CB2 School of Medical Sciences, University of New South Wales, Sydney, Australia. 0XY, UK. 3Cambridge Institute for Medical Research, University Regulated exocytosis of secretory granules (SGs) in exocrine glands has been of Cambridge, Cambridge CB2 0XY, UK. 4Institute for Molecular traditionally studied in ex vivo acinar preparations that may not recapitulate this Bioscience, The University of Queensland, Brisbane, QLD 4072. process as it happens in vivo. Recently we established an intravital microscopy based experimental system for studying SG dynamics and exocytosis in salivary During stimulated exocytosis in neurosecretory cells, secretory granules glands of living mice and rats. Using transgenic mice models expressing selected are recruited to the cortical actin network underlying the cell surface, fluorescently labeled molecules, we have previously described major differences prior to docking with the plasma membrane and undergoing SNARE- between in vivo and ex-vivo models in terms of the secretion initiation signal, as well as the kinetics and the modality of exocytosis. Furthermore, an actomyosin mediated membrane fusion. Despite the importance of these processes scaffold was found to be recruited around fused SGs which facilitated the in regulating the timing, amplitude and longevity of neurotransmitter completion of cargo and membrane delivery to the apical plasma membrane and hormone release, the precise molecular mechanisms underpinning (APM). In the current study we sought to explore how tropomyosins (Tms), these pre-fusion events are not yet fully understood. In recent studies which form co-polymers along the length of actin filaments, may be modulating we have analysed the role of two proteins, myosin VI and Munc18-1, the actomyosin scaffold, and hence controlling exocytosis. There are about in different aspects of secretory granule regulation. We identified the 40 different Tm isoforms and they are known as master regulators of the actin small insert isoform of myosin VI as critical to sustain neuroexocytosis cytoskeleton that specify the characteristics of actin filaments in time and space by recruiting secretory granules to the cortical actin network during either via either direct effect on filaments or by recruiting other actin binding sustained stimulation, an activity controlled by c-Src phosphorylation. proteins. First, we stained salivary gland sections with a battery of antibodies Loss of myosin VI small insert activity significantly impairs the specific to various Tm isoforms. We found that high molecular weight alpha- recruitment and tethering of secretory granules, and sustained tropomyosins, Tm4 and Tm5NM1 isoforms line the apical canaliculi of the acinar neurosecretion. We have additionally identified a role for the SNARE structures. Out of those Tms, the Tm5NM1 as well as Tm4 were most dramatically complex accessory protein Munc18-1 in regulating the movement enriched at the actin meshwork of the APM. Interestingly, the fine distribution of of secretory granules adjacent to the plasma membrane. Through this Tms did not overlap completely with the phalloidin staining suggesting that mutational analysis we identified a region in domain 3a of Munc18-1 these isoforms preferentially bind a subset of actin filaments. Upon stimulation that is important for secretory granule fusion in cells independent of with isoproteranol Tm5NM1 and Tm4 were recruited onto the fused SGs together with F-actin, but with different kinetics that bulk filaments. Intravital microscopy its binary binding to the plasma membrane SNARE protein syntaxin of transfected rat salivary glands revealed that Tm5NM1 and Tm4 are recruited 1a. Deletion of a region within this domain directly affects the ability after fusion of the SGs to the APM and after F-actin recruitment to the granules. of Munc18-1 to restrict the movement of cortical secretory granules at Genetic ablation of Tm5NM1 or its inhibition with TR100 compound altered the the onset of secretagogue stimulation. Our results highlight the diverse kinetics of granule exocytosis but did not prevent the completion of granule mechanisms acting on secretory granules on their way to the plasma exocytosis. In conclusion, using our in vivo experimental model system, we found membrane to undergo fusion. that tropomyosin isoforms are present at the APM and, in case of Tm5NM1 and Tm4 isoforms, directly participate in actin scaffolding and gradual delivery of the SG contents post-fusion.

SYM-07-04 SYM-07-05 ACTIN DYNAMICS AND ISOFORM SPECIFICITY IN REGULATION OF NEURITE BRANCHING BY ENDOTHELIAL VESICULATION DIFFERENT ACTIN FILAMENT POPULATIONS

Latham S.L.1, Chaponnier C.2, Dugina V.2, Goldsbury C.3, Couraud P.O.4, Curthoys N., Freittag H., Brettle M., Hall A., Desouza M., Schevzov G., Grau G.E.R.1 and Combes V.1 Hardeman E., Gunning P. and Fath T. 1Vascular Immunology Unit, Sydney Medical School, University of School of Medical Sciences, University of New South Wales, Sydney, Australia. 2Dept of Pathology and Immunology, Faculty of Kensington. Medicine, University of Geneva, CMU, Geneva, Switzerland. 3Alzheimer’s Disease Cell Biology Group, Sydney Medical School, University of The actin cytoskeleton has a pivotal role in regulating the morphology Sydney, Australia. 4Inserm U1016, Institut Cochin, Paris, France. and function of eukaryotic cells. Morphological changes, such as the formation and growth of cellular extensions in neuronal cells Elevated circulating endothelial microparticles (MP) are observed (neuritogenesis), are driven by different actin filament populations in numerous diseases, increasingly demonstrating them as disease with distinct structural and dynamic properties. These properties are progression promoters and vascular dysfunction markers. Vesiculation determined by tropomyosins, a family of actin-associated proteins. In requires modifications to the organisation and integrity of cellular neuronal cells, products from three tropomyosin genes (TPM1, TPM3 components including the actin cytoskeleton and its associated proteins. and TPM4) are found. We investigated the tropomyosin isoform- This study examines β- and γ-cytoplasmic actins during MP release in specific role of TPM1 (TmBr1, TmBr2, TmBr3) and TPM4 (Tm4) gene human cerebral microvascular endothelial cells. Specific subcellular products during neurite formation in B35 neuroepithelial cells. The localisations of both isoforms were defined by confocal microscopy using overexpression of different tropomyosins in these cells results in altered tumour necrosis factor (TNF) activation which significantly increases MP neuritogenesis. While overexpression of products from any of the TPM release at 18 h. Stimulation induced basal β-actin stress fibres which genes induces the formation of neurites in undifferentiated B35 cells, were increasingly associated with apical particulate structures rich in tropomyosins differentially impact on the extension and branching of both isoforms and cofilin. Correlative light and electron microscopy show neurites formed by differentiating B35 cells. TmBr2 is the only product a direct congruency between the particulate structures distributions and to promote neurite extension, whereas TmBr3 and Tm4 selectively electron-lucent membrane protrusions. Western blot showed variations lead to a pronounced increase in neurite branching. This increase in in the cytoskeletal composition of MP from the mother cell, with 3D branching is associated with an increase in the number of filopodia structured illumination microscopy indicating unique distributions for formed along these neurites. Live imaging of neurite branch formation these proteins within MP. Pharmacological Y-27632 Rho-kinase inhibition revealed a shift in the mode of branching. Whereas the majority of abolished basal β-actin stress fibres, minimised the apical association branches in control B35 cells are formed by growth cone bifurcation, of particulate structures and significantly reduced the number of both TmBr3 and Tm4 overexpression promotes interstitial branching along membrane protrusions and MP released to near basal levels. Functional the neurite shaft. Interestingly, the overexpression of TmBr3 and Tm4 siRNA experiments demonstrated that selective β-actin, but not γ-actin also resulted in an increased expression of the actin filament-bundling silencing, reduces TNF induced MP levels. Our data strongly suggest that protein fascin, which has previously been shown to promote filopodia the actin cytoskeleton plays more than a contractile role in vesiculation, formation. These findings suggest a fundamental role for different actin with novel cytoskeletal-rich structures increasing plasma membrane filament populations in regulating neurite outgrowth. disruption following activation due to their mechanical interaction with basally localised β-actin.

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SYM-08-01 SYM-08-02 MOLECULAR GENETICS, GENOMICS AND GENETICS THERMAL IMAGING AS A TOOL TO INVESTIGATE THE OF THE LEGUME TREE PONGAMIA (MILLETTIA) MECHANISMS OF THE RED-LIGHT RESPONSE OF PINNATA BIOFUEL PRODUCTION STOMATAL MOVEMENT

Gresshoff P.M., Indrasumunar A., Biswas B., Samuels S., Jiang Q., Busch F.A.1, Sirault X.2 and Von Caemmerer S.1 Kazakoff S. and Scott P.T. 1Research School of Biology, Australian National University, ARC Centre of Excellence for Integrative Legume Research; and Canberra, Australia. 2CSIRO Plant Industry, High Resolution Plant SAFS, The University of Queensland, St Lucia, Brisbane, QLD, 4072. Phenomics Centre, Canberra, Australia.

There is an essential need for knowledge and insights into the biology, Stomata control the uptake of CO2 and the loss of water in leaves of genetics and biotechnology of the legume tree Pongamia pinnata higher plants. To minimize water loss while maximizing CO2 uptake caused by global energy needs and associated biofuel industry stomatal movement is tightly regulated in response to environmental advancement. The key properties of Pongamia leading to global factors, such as light intensity or CO2 availability. While the response acceptance are: large seeds, high seed oil content, numerous seeds of stomata to CO2 and blue light is well described, the underlying per tree, flexible growth conditions, fast growth, salinity and drought mechanism of the response to red light is still unknown. The role tolerance, annual cropping without plant destruction and replanting, of photosynthesis as a trigger for the stomatal red-light response and symbiotic nitrogen fixation. Our research straddles the industrial- has been disputed for decades. We examined the hypothesis that applied aspects (oil composition, protein content), field studies (life- red light is sensed by the redox state of the photosynthetic electron cycle analysis) and productivity testing (geoclimatic control of yield transport chain, in particular by plastoquinone (PQ). PQ is a molecule and growth) and the here-described modern plant science. We have that shuttles electrons between photosystem II and the cytochrome developed technology for gene identification by deep DNA sequencing, b6f complex and is involved in balancing the supply of energy from DNA fingerprinting, diversity testing, chloroplast and mitochondrial absorbed light and the utilization of energy by the Calvin-Benson genomics, and gene transfer. Specifically we have characterised the cycle. Experiments in Arabidopsis and tobacco, in which we varied function of genes critical for fatty acid biosynthesis, seed storage the CO2 and O2 concentrations, red-light intensity and the redox proteins, and symbiotic nitrogen fixation. These advances will aid future state of PQ independently and in combination show that stomatal crop improvement, establishment of clonal elite lines, and genetic conductance does not correlate with these parameters per se, but can response to environmental variation and stresses. Jiang, Q., et al (2012) be well described by one parameter that all of these factors and their Genetic, biochemical, and morphological diversity of the legume biofuel interactions influence: the redox state of PQ. Attempting to identify the tree Pongamia pinnata. J. Plant Genome Science 1: 54-68. Kazakoff, molecular components involved in the stomatal red-light response we S.H., et al (2012) Capturing the biofuel wellhead and powerhouse: the designed a high-resolution Arabidopsis thermal imaging screen. Our chloroplast and mitochondrial genomes of the leguminous feedstock setup can detect small differences in leaf temperature resulting from tree Pongamia pinnata. PloS One 7: 12 | e51687 Scott, P.T., et al (2008) evaporative cooling, enabling the identification of mutants with low Pongamia pinnata: an untapped resource for the biofuels industry of stomatal conductance under red light. First results from this screen will the future. BioEnergy Research 1:2-11. Murphy, H.T., et al (2012) A be presented. common view of the opportunities, challenges and research actions for Pongamia in Australia. BioEnergy Research 5: 778-799. Samuel, S., Scott, P.T., and Gresshoff, P.M. (2013) Nodulation in the legume biofuel feedstock tree Pongamia pinnata. Agronomy Research (in press).

SYM-08-03 SYM-08-04 LEGUME CROP AND PATHOGEN GENOMICS A GENOME-WIDE SURVEY OF DNA METHYLATION IN RICE ENDOSPERM REVEALS ITS ROLES IN GENOMIC Hane J.K.1, Anderson J.P.1, 2, Kamphuis L.G.1, 2, Williams A.H.1, IMPRINTING 1 2 1, 2 Sperschneider J. , Nelson M. and Singh K.B. 1 1 2 1 1 3 3 1 Luo M. , Hua Y. , Du M. , Taylor J. , Spriggs A. , Zhang H. , Wu Z. , Molecular Plant Pathology and Crop Genomics Laboratory, Plant 4 5 2 Cao X. and Koltunow A. Industry, CSIRO. The UWA Institute of Agriculture, The University of 1 Western Australia. CSIRO Plant Industry, PO Box 1600, Black Mountain Laboratories, ACT 2601, Australia. 2Potato Research Centre of Inner Mongolia University, Chengdu, Sichuan 611130, P.R. China. 3Rice Research Institute of The CSIRO and UWA Molecular Plant Pathology and Crop Genomics Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China. Laboratory researches various crop species, their pathogens and 4State Key Laboratory of Plant Genomics and National Centre for Plant their host-microbe interactions. This presentation focuses on recent Gene Research, Institute of Genetics and Developmental Biology, studies of Lupinus angustifolius, otherwise known as narrow-leaf lupin Chinese Academy of Sciences, Beijing 100101, China. 5CSIRO Plant (NLL), and Rhizoctonia solani, subspecies AG8. NLL is an important Industry, PO Box 350, Glen Osmond, South Australia 5064, Australia. grain legume crop which can be used in rotation with other crops for soil improvement, nitrogen fixation and disease breakage and also Genomic imprinting is an epigenetic phenomenon where the expression of an allele depends on its parental origin. In flowering plants, imprinting has health benefits useful for the management of obesity, high blood predominantly occurs in the endosperm, the tissue which nourishes the pressure and diabetes. Using various genomic and transcriptomic embryo and young seedling; it plays a critical role in seed development. resources for NLL, we have produced a stringent set of over 40,000 Hundreds of imprinted genes have been identified in the endosperm of rice, gene-based markers that are polymorphic across a panel of 22 but the mechanisms regulating imprinting in rice endosperm are currently cultivars and wild accessions. This represents a significant advance unknown. To analyse the role of DNA methylation in imprinting, we performed on existing marker resources and will be useful for genome assembly, MeDIP (Methylated DNA immunoprecipitation) on endosperm DNA of genetic resource management and for identifying marker-trait seeds obtained from reciprocal crosses between two subspecies, Japonica associations. On the pathogen side, Rhizoctonia solani AG8 causes (Nipponbare) and Indica (93-11) at 6 days after fertilization. Sequenced reads the disease “bare patch” in cereal and legume crops, including wheat of the MeDIP DNA were aligned to their respective genomes using single and lupin. To understand its pathogenicity mechanisms, we overcame nucleotide polymorphisms (SNPs). We found ~1000 differentially methylated significant bioinformatic challenges associated with the multinuclearity regions (DMRs) defined by methylated peaks spanning SNPs between the (autopolyploidy) and high heterozygosity of its genome. Novel genome maternal and paternal genomes. Over 95% of these regions are maternally assembly techniques have been developed which are appropriate for hypomethylated as in Arabidopsis, suggesting a conserved demethylation heterozygous polyploid genomes. We then applied various comparative program to regulate endosperm development across species. A subset of genomics analyses with related pathogens to identify the pathogenicity maternally or paternally expressed imprinted genes is associated with DMRs, genes and infer disease strategies of R. solani AG8. suggesting that they are potentially regulated by DNA methylation. DNA hypomethylation of the maternal genome was associated with activation of the maternal alleles in non-genic regions or alternative-splicing of transcripts. We have also sequenced the small RNA population in the endosperm of reciprocal crosses between these two rice subspecies. In contrast to Arabidopsis, the rice siRNAs are not all maternally derived. A large fraction of rice endosperm siRNAs are not imprinted as their expression is biallelic. In rice only a small number of imprinted siRNAs were found and these exhibit maternal or paternal expression, which may be mediated by DNA methylation.

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SYM-08-05 SYM-09-01 TRANSCRIPTOME AND CO-EXPRESSION ANALYSIS IDENTIFICATION AND FIELD TESTING OF NOVEL IN THE DISCOVERY AND CHARACTERISATION OF TRAITS FOR SALINITY TOLERANCE IN WHEAT AND BIOSYNTHESIS PATHWAYS ACTIVE DURING GRAPE BARLEY BERRY DEVELOPMENT Tilbrook J.1, Schilling R.1, Trittermann C.1, Zafar Z.U.1, 2, Ehsanzadeh P.1, 3, 1, 4 1 Drew D.P.1, 2, Sweetman C.1, Wong D.C.J.1 and Ford C.M.1 Tester M. and Roy S.J. 1School of Agriculture, Food and Wine, University of Adelaide, 1Australian Centre for Plant Functional Genomics and the University Australia. 2Department of Plant and Environmental Sciences, Faculty of Adelaide, PMB 1, Glen Osmond, SA 5064, Australia. 2Institute of of Sciences, University of Copenhagen, Denmark. Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan. 3Dept of Agronomy & Plant Breeding, Isfahan University of 4 The physiological and biochemical composition of grape berries Technology, Isfahan, Iran. Division of Biological and Environmental at harvest has a profound impact on the characteristics of the wine Sciences and Engineering, 4700 King Abdullah University of Science produced from those berries. The organoleptic properties of ripe and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia. berries are determined inter alia by the presence and concentration of primary metabolites, including sugars and tartaric and malic acids, and Salinity is a major abiotic stress which affects crop plants in Australia, secondary metabolites such as stilbenes, terpenes, norisoprenoids resulting in substantial loss of yield and millions of dollars of lost and a wide range of flavonoids. There is therefore great interest in revenue. Exposure to saline soils exposes the plant to a number of characterising the molecular and biophysical changes occurring specific stresses including shoot ion independent stresses, such as during berry development, including the coordination and temporal osmotic stress, and shoot ionic stress. Using destructive harvesting regulation of metabolic gene pathways. In order to investigate the and non-destructive imaging technology, we have identified a number global expression of transcripts in grape berries, we used an Illumina of quantitative trait loci (QTL) and candidate genes involved in a variety HiSeq2000 to conduct transcriptome sequencing at four developmental of salt tolerance mechanisms. We have identified QTL linked to osmotic stages: immature green berries, berries at the beginning and end stress tolerance and a several ionic stress tolerance mechanisms, such + - of veraison, and berries at harvest. This transcriptomic data has as the exclusion of Na and Cl from the shoot, and show there is great allowed us to identify genes and gene pathways active during berry potential for future gene discovery and intorgession of beneficial alleles development. We have used gene ontology assignment and statistical into crops. In addition, candidate genes for salinity tolerance which have clustering to describe transcriptional patterns, and have uncovered been identified in other species, such as from Arabidopsis thaliana, many examples of coordinated and highly regulated gene expression. have been transformed into both wheat and barley and assessed for We have also designed a grapevine co-expression database, VTCdb salinity tolerance in greenhouse and field conditions. Field trials of our (vtcdb.adelaide.edu.au/Home.aspx), which allows the identification most advanced GM barley lines indicate we have been able to increase of co-expressed genes using networks assembled from over 400 yield considerably on saline soils with no negative impact on growth in Nimblegen and 400 Affymetrix microarray datasets available in the non-saline soils. public domain. In situations where changes in metabolite levels through berry development have been described, but the genes involved in metabolism have not been characterised, the data from these investigations provide a valuable resource for candidate gene selection.

SYM-09-02 SYM-09-03 HOW DOES LAKE EUTROPHICATION LEAD NO CUSHION FOR CLIMATE EXTREMES: HYDRAULIC TO IMPAIRED AERATION AND ROOT OXYGEN ATTRIBUTES INDICATE VULNERABILITY OF ICONIC DEPRIVATION IN THE SUBMERGED AQUATIC SUB-ANTARCTIC ALPINE SPECIES TO INCREASING MACROPHYTE LAGAROSIPHON MAJOR? DROUGHT STRESS IN A CHANGING CLIMATE

Sorrell B.K.1 and Hawes I.2 Ball M.C.1, Rolland V.1, Lenne T.1, Egerton J.J.G.1, Stanton D.E.1, Choat 1University of Aarhus, Denmark. 2University of Canterbury, New B.1, Sack L.2, Bryant G.3, Holbrook N.M.4 and Bergstrom D.M.5 Zealand. 1Australian National University, Canberra ACT. 2University of California, Los Angeles, California, USA. 3RMIT, Melbourne VIC. Submerged aquatic plants are susceptible to root oxygen deprivation 4Harvard University, Cambridge, Massachusetts, USA. 5Australian because they cannot access atmospheric oxygen, and in the dark Antarctic Division, Kingston TAS. must rely solely on internal oxygen diffusion from the water column to maintain root aeration. This stress may be enhanced in eutrophicated The cushion plant, Azorella macquariensis, is endemic to Macquarie waters, where oxygen transport and plant aeration may be impaired by Island, where it occurs on a high elevation, alpine plateau that extends (i) higher epiphyte loads, (ii) greater self-shading, and (iii) accumulation the island. Within three years, the status of this iconic species changed of more organic matter and development of more reducing conditions from the dominant species to critically endangered. We examined in the sediment. We compared canopy structure, epiphyte loads, root hydraulic attributes of Azorella to assess its vulnerability to water morphology and anatomy, and alcohol dehydrogenase (ADH) activity in stress associated with subtle changes in climatic conditions. While a freshwater plant (Lagarosiphon major) in five lakes differing in degree rudimentary for a vascular plant, the hydraulic attributes were consistent of eutrophication. There was significant root ADH activity in four of the with adaptation to a cold, wet and low light environment. Water transport five lakes, suggesting that nocturnal root hypoxia is a normal feature was extremely limited by a poorly developed xylem with relatively few, for this plant even in mesotrophic (intermediate nutrient) lakes where it minute primary xylem vessels. The physical characteristics of stomata grows best. The lake with no root ADH activity had sandy sediments with limited conductance to 5 mmol m-2 s-1 and transpiration rates to 0.1 very high redox potentials, indicative of the presence of free oxygen. mmol m-2 s-1. Stems cavitated at 0.2 MPa, but in vivo visualisation The most eutrophic lake had sediments that were more reducing than of refilling showed that capillary forces could repair embolised vessels the others and correspondingly enhanced ADH activity, and much within minutes. These results provide novel evidence of a hydraulic shorter roots. Plants did alter canopy structure in the different lakes, basis for the cushion plant growth form, with the particulars of Azorella but there was no evidence of greater self-shading in more eutrophic (ie a strong reliance on direct, non-vascular water uptake by the shoots lakes, and the epiphyte loads were not higher on plants from eutrophic combined with extreme vulnerability to drought-induced cavitation) vs mesotrophic conditions. Roots of L. major have no external barrier contributing to an apparent extreme sensitivity to drying conditions that to radial oxygen loss or ability to enhance their cortical porosity, and are becoming more frequent and more severe with climate change. this presumably accounts for the particular importance of sediment conditions in determining the degree of hypoxia they experience. Our study demonstrates that root oxygen deprivation can be an important contributor to the growth reduction, collapse and disappearance of submerged aquatic plants caused by lake eutrophication.

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SYM-09-04 SYM-09-05 UTILIZATION OF THE IWGSC WHEAT SURVEY UNDERSTANDING THE MECHANISM OF ION TRANSPORT SEQUENCE FOR RNASEQ ANALYSIS OF WATER IN OXYGEN-DEFICIENT ROOTS OF CEREALS STRESS EFFECTS IN DEVELOPING WHEAT HEADS 1, Striker G.G.2, Pedersen O.1, 3, Clode P.L.4, LäUchli A.1, 5 and 1, 2 3 3 4 5 Kotula L. Webster H. , Tibbits J. , Hayden M. , Dell B. , FosuNyarko J. , Colmer T.D.1 1, 2 6 Keeble G. , Bellgard M. , InternationalWheatGenomeSequencing 1School of Plant Biology, The University of Western Australia, 35 Consortium I.W.G.S.C.7 and Appels R.1, 2 Stirling Highway, Crawley, 6009 WA, Australia. 2School of Plant 1Australia-China Centre for Wheat Improvement, Murdoch University, Physiology, IFEVA-CONICET, Faculty of Agronomy, University of Perth. 2Australian Export Grains Innovation Centre, Murdoch University, Buenos Aires, Buenos Aires, Argentina. 3The Freshwater Biological Perth. 3Department of Primary Industries Victoria, Victorian Agri Laboratory, University of Copenhagen, Helsingørsgade 51, 3400 Biosciences Centre, La Trobe University, Melbourne. 4Centre for Hillerød, Denmark. 4Centre for Microscopy, Characterisation and Phytophthora Science and Management, School of Biological Sciences Analysis, The University of Western Australia, 35 Stirling Highway, and Biotechnology, Murdoch University, Perth. 5Western Australian State Crawley, 6009 WA, Australia. 5Department of Land, Air & Water 6 Agricultural Biotechnology Centre, Murdoch University, Perth. Centre for Recourses, University of California, Davis, CA 95616-8627, USA. Comparative Genomics, Murdoch University, Perth. 7International Wheat Genome Sequencing Consortium. O2 deficiency during soil waterlogging adversely affects root growth and The genes/enzymes involved in supplying carbon to the developing head functioning, with subsequent consequences for shoots. For roots in O2 in wheat are not well characterized. The activities of cell wall invertases deficient media, large gradients in 2O occur across root tissues. Hypoxia (IVR1s) are known to be important in supplying sugars to pollen during the or even anoxia can develop in the stele, while the cortex and epidermis early stages of head development. Recently multiple IVR1 isoforms were receive sufficient 2O for respiration. O2 deficiency in stellar cells causes identified on the wheat genome (Webster et al 2012; Funct Plant Biol 39: energy deficits, as a result the activity of H+-ATPases is restricted by 569 - 579; GRDC-GRS10028) and were found to be located on a number low ATP. The present study aims to elucidate the relationship between of chromosome arms. These findings in addition to the existence of IVR1 root O2 supply and ion transport in roots affected by hypoxia, and the inhibitors, provides evidence to suggest a complex regulatory network combination of hypoxia and salinity. Two species, barley and wheat, exists, which controls the flow of sugars to maturing pollen. A reduction which differ in their tolerance to waterlogging and salinity were used. in supply of sugars to pollen in response to water stress during the early O2 status of roots was determined on plants grown in stagnant and stages of maturation leads to sterility. A large water stress experiment was stagnant+saline conditions and compared with plants grown in aerated carried out to analyse the transcriptome using RNASeq and RNA from and aerated+saline conditions. The O2 microelectrode profiles showed developing wheat heads. RNASeq performed on tissues extracted from that for roots in stagnant conditions, O2 deficiency first occurs in the developing heads, extending from immature floret formation through to stele whereas at the same time the cortex can contain some O2. This anthesis, enabled temporal profiling of transcript expression in response condition was more severe towards the root apex. Measurements of to water stress and non-limited water conditions. The IWGSC wheat Na+, K+ and Cl- across adventitious roots (epidermis, cortex, xylem genome survey sequence data was used as the reference to assign parenchyma, and xylem) from aerated, aerated+saline, stagnant, and chromosome arm locations of those transcripts showing expression in stagnant+saline treatments were determined using quantitative X-ray the RNASeq data. Mapping individual transcripts to an IWGSC survey microanalysis of freeze-substituted material. These measurements sequence contig facilitated downstream functional and biological showed that K+ concentration in xylem parenchyma cells was lower in + characterisation to identify key genes involved in the supply of carbon to O2 deficient roots, whereas the Na concentration in these roots was developing wheat heads. higher. Moreover, cells of roots subjected to salt stress produced Na+- binding starch granules.

SYM-10-01 SYM-10-02 RE-WIRING THE CANCER GENOME WITH FUNCTION OF PRC2 ACCESSORY FACTORS IN ENGINEERED TRANSCRIPTION FACTORS HAEMATOPOIETIC STEM CELLS

Blancafort P. and Stolzenburg S. Kinkel S.K.1, Flensberg C.2, Gearing L.J.1, Willson T.1, Taoudi S.1, Cancer Epigenetics group. School of Anatomy, Physiology and human Alexander W.S.1, Hilton D.J.1, Oshlack A.2, Majewski I.J.1 and 1 Biology. The University of Western Australia. Crawley WA. Australia. Blewitt M.E. 1Walter and Eliza Hall Institute of Medical Research. 2Murdoch Childrens Poorly differentiated breast and ovarian cancers are un-curable forms Research Institute. of tumors driven by a discrete number of newly identified genes. Many cancer drivers (such as Transcription Factors) are impossible to target We are interested in epigenetic control in haematopoietic stem cells with current drugs, and patients afflicted have only chemotherapy options. (HSCs). Our focus is on Polycomb Repressive Complex 2 (PRC2), Disease recurrence, resistance to chemotherapy and residual metastatic that has been studied extensively in ES cells, where it represses key disease often develops, resulting in patient death. In addition, because of developmental regulators. In addition to the core PRC2 members the lack of target specificity, chemotherapy drugs can cause severe side (Ezh1/2, Suz12 and Eed), accessory factors appear to modulate effects in the patients. The sequencing of the breast cancer genomes PRC2 activity and target its binding. We have previously shown that and the comprehensive characterization of the cancer epigenomes and reduction in the levels of core PRC2 members results in enhanced HSC transcriptomes have delineated an array of molecular aberrations in repopulating capacity, but the role of PRC2 accessory factors in HSCs cancer cells. Our objective is to create novel targeted protein-engineering has not been described. Using shRNA knockdown, we examined the approaches to revert and correct the aberrant epigenetic and transcriptomal function of six PRC2 accessory factors in HSCs by testing the capacity state of aggressive breast cancers. We describe the generation of novel of transduced murine foetal liver cells to competitively reconstitute engineered DNA-binding proteins able to recognize selectively sequences irradiated recipients. Depletion of the enzymatically inactive histone of the genome that are affected in the cancerous state and not in normal methyltransferase Jarid2 enhanced contribution to all blood cell cells. The module that reads the genomic sequence, for example, lineages compared to cells containing control constructs, similar to the engineered zinc finger modules, are linked to a writer or a module that carries a biochemical activity to modify the target gene. Novel effector domains phenotype observed upon depletion of core PRC2 components. Our incorporate epigenetic modifications or covalent modifications in the DNA data suggest that the enhanced activity of Jarid2 depleted cells is due and associated histones, that ultimately sculpt the chromatin structure to an increase in HSC number post Jarid2 knockdown, but that this and modulate gene expression. By incorporating specific combinations of role for Jarid2 is restricted to foetal HSCs. Foetal HSCs are dependent epigenetic modifications or marks, we can selectively modify the epigenetic on Ezh2-PRC2 rather than Ezh1-PRC2, so our data suggest Jarid2 is code and the resulting transcriptional state in targeted genes: For example, the accessory factor required in Ezh2-containing PRC2 complexes. by re-activating tumor suppressor genes that are otherwise silenced in Interestingly, mutations have been identified in PRC2 core components cancer, or by permanently silencing the oncogenes overexpressed or in haematopoietic malignancy, and recently mutations and deletions in overactive in cancer. Because epigenetic modifications are inherited, the JARID2 have also been reported. ultimate goal is to induce long-lasting phenotypic alterations of the targeted cells. The capacity of our engineered factors to selectively modulate a wide-range of cancer drivers is exemplified with the targeting of the tumor suppressor mammary serine protease inhibitor (maspin) and the oncogene SOX2 in breast cancer. In sum, we present novel engineering approaches to specifically target major “undruggable” cancer drivers, such as oncogenic transcription factors for the therapeutic treatment of recalcitrant cancers.

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SYM-10-03 SYM-10-04 LONG-RANGE GENE REGULATION: A COMMON ROAD RULES FOR TRAFFIC ON DNA: GENE MECHANISM UNDERLYING GWAS ASSOCIATIONS REGULATION BY ENCOUNTERS BETWEEN Edwards S.L.1, 5, Hillman K.1, Kaufmann S.1, Ghoussaini M.2, Meyer K.B.3, TRANSCRIBING RNA POLYMERASES AND DNA- Cloonan N.1, Korbie D.4, Chenevix-Trench G.1, Dunning A.2 and BOUND PROTEINS French J.D.1, 5 Hao N., Dodd I.B. and 1Queensland Institute of Medical Research, 300 Herston Road, Brisbane, Shearwin K.E. Queensland 4006, Australia. 2Department of Oncology, University of School of Molecular and Biomedical Science, The University of Cambridge, Strangeways Research Laboratory, Cambridge, United Adelaide, South Australia 5005. Kingdom. 3Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Cambridge CB2 0RE, UK. 4Australian Institute for Textbook depictions of the transcription of a gene show RNA polymerase Bioengineering and Nanotechnology, University of Queensland, Brisbane, binding to the DNA at the promoter, initiating transcription and then St. Lucia, Queensland 4072, Australia. 5School of Chemistry and transcribing uneventfully across the gene until reaching a terminator Molecular Biosciences, University of Queensland, St. Lucia, Queensland sequence and falling off the DNA. However, this picture of the DNA 4072, Australia. as a freeway for RNA polymerase does not apply within a cell, where the DNA is more like a crowded one-lane two-way street. As an RNA More than 80 breast cancer and/or ovarian cancer risk loci have now been discovered via Genome Wide Association Studies (GWAS) but until recently polymerase makes its way along the DNA, its progress can be affected it has not been possible to identify the causal variants that are directly by a myriad of proteins bound to the DNA, by difficult DNA sequences responsible for the association. In collaboration with geneticists at QIMR that make it pause, and also by other traffic - RNA polymerases moving and the University of Cambridge we have fine mapped the association at either in the opposite direction or more slowly in the same direction. several breast and ovarian cancer GWAS loci. Importantly, the majority The outcomes of these collisions are crucial in gene regulation, yet the of variants fall in non-coding regions of the genome such as introns and factors that determine the ‘winner’ – whether the DNA-bound protein intergenic regions. Using data made publically available by projects such blocks transcription by RNA polymerase or whether the polymerase as ENCODE, we have annotated non-coding variants at breast and ovarian dislodges the protein and interferes with its function – are unknown. cancer loci and show that many lie in regulatory elements that are scattered Here we integrate in vivo experiments in E. coli, using well-defined throughout the genome. Using chromosome conformation capture (3C)- genetic components, with mathematical modelling to study the factors based techniques we show that these regulatory elements can act as which influence the outcome of these interactions. Construction of a long-range enhancers and silencers of genes, physically located outside modular, chromosomally integrated single-copy chassis allows us to these regions, through long-range chromatin interactions. For example, we systematically vary parameters such as RNA polymerase density, fine-mapped the breast cancer risk at 11q13 and 2q35 and showed that promoter-roadblock spacing, and roadblock binding affinity. This the strongest risk SNPs at both loci mapped to transcriptional enhancers combined approach allows us to evaluate the significance of RNA that distally regulate genes (CCND1 and IGFBP5) located more than 120 polymerase - roadblock encounters, the factors that affect them and kb and 350 kb away, respectively. The risk SNPs at 11q13 were shown to may provide new tools for the manipulation of gene expression. affect the transactivation of the CCND1 promoter by affecting transcription factor binding. Whereas, the likely causative SNP at 2q35, increased the frequency of the chromatin interaction between the enhancer and the IGFBP5 promoter. Data using a genome wide 3C approach (4C-seq) will also be presented showing that the target gene of regulatory elements may also reside on different chromosomes. These results have significant implications for the functional follow-up of GWAS loci.

SYM-10-05 SYM-11-01 IDENTIFYING THE RNA TARGETS OF DBHS MEMBRANE TRAFFICKING IN THE ENDOSOMAL PROTEINS TO GIVE INSIGHTS INTO THE INTERNAL SYSTEM – A STRUCTURAL BIOLOGIST’S ORGANISATION AND FUNCTION OF A NUCLEAR PERSPECTIVE RNA:PROTEIN COMPLEX Ghai R., Norwood S.J., Bugarcic A., Teasdale R.D. and 1 2 1 Collins B.M. Fortini E. , Bond C. and Fox A. The University of Queensland, Institute for Molecular Bioscience. 1Centre for Medical Research, Western Australian Institute for Medical Research, University of Western Australia, Perth, WA, 6009, 2 Eukaryotic cells are highly compartmentalised, and have evolved highly Australia. School of Chemistry and Biochemistry, University of sophisticated protein machineries to control the flow of transmembrane Western Australia, Perth, WA, 6009, Australia. molecules and membrane lipids between organelles. Disruption Proteins of the DBHS (Drosophila Behaviour Human Splicing) of these processes are linked to numerous diseases including family are multifunctional and abundant nuclear proteins capable of neurodegenerative disorders, pathogen invasion and cancer. We are binding many different types of nucleic acids and influencing gene determining how these trafficking machineries are assembled and regulation via multiple mechanisms. One of their functions is to bind regulated at the molecular level through a combination of structural a long noncoding RNA (lncRNA), NEAT1, to form subnuclear bodies biology, biophysical, and cell biology approaches. This talk will discuss termed paraspeckles. LncRNAs are rapidly gaining interest as their our most recent work on a large family of molecules called the PX fundamental roles in the regulation of gene expression are beginning proteins or sorting nexins, that play critical roles in trafficking and to be unravelled. Paraspeckles therefore represent a valuable model signalling within the endocytic membrane system. system for studying lncRNA:protein interactions. Paraspeckles are also thought to play a role in controlling gene expression by sequestering specific mRNAs and proteins. Here I will present progress towards the first detailed analysis of the interactions between the DBHS core paraspeckle proteins, NONO and SFPQ, and the RNAs they bind. I have optimised a technique known as PAR-CLIP (Photoactivatable ribonucleoside enhanced crosslinking and immunoprecipitation) coupled with next generation RNA sequencing. Bioinformatic analysis of the deep sequencing data and various biological validations of the identified RNA:Protein interactions have allowed me to identify, to single nucleotide resolution, the RNA fragments bound by the paraspeckle proteins. This data will provide insights into the internal organisation of a nuclear RNA:Protein complex by revealing binding hotspots for the DBHS proteins in NEAT1. Further, my work will extend our knowledge of paraspeckle function by identifying RNAs that are bound by the DBHS proteins as a mechanism of regulating their expression.

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SYM-11-02 SYM-11-03 CRYSTAL STRUCTURES OF BAX REVEAL COMPLEXITIES OF TRANSCRIPTIONAL COMPLEXES MOLECULAR EVENTS INITIATING APOPTOSIS Matthews J.M. Czabotar P.E.1, 2 School of Molecular Bioscience, The University of Sydney NSW 2006. 1The Walter and Eliza Hall Institute of Medical Reasearch, Parkville, Victoria. 2Department of Medical Biology, The University of The transcriptional regulation of gene expression relies on the complex Melbourne, Parkville, Victoria. interplay of many different proteins and their target nucleic acids. We have been working to understand a set of cell-specific transcriptional A key event in apoptosis is the conversion of Bax from an inert monomer complexes that have key roles in blood cell development. These into a cytotoxic mitochondrial membrane perforating oligomer. Certain complexes have a core complex comprising one each of a GATA BH3-only relatives can initiate this step through direct interactions, protein, and LIM-only protein (LMO) families, a pair of bHLH proteins, yet the means by which conformational changes are invoked, and the and the LIM-domain binding (Ldb1) cofactor protein. Alternate members nature of the conformational changes themselves, has largely remained of the protein families are substituted in different cell types. We use a a mystery. Here we present crystal structures of Bax in complex with combination of structural biology and interaction studies to understand BH3 domains, providing the molecular details for these interactions. The how these protein complexes assemble, how substitution of different structures, and subsequent mutagenesis studies, define the sequence components affect protein- and DNA-binding specificity and how these signature of activator BH3 domains and reveal structural transitions complexes contribute to long range chromatin interactions that are required of Bax for death activity, in particular, the dissociation of Bax essential for biological activity. into previously unrecognized “core” and “latch” sub-domains. A further structure of the Bax “core” domain reveals a symmetric BH3-in-groove homodimer. This dimer, which is consistent with previous cross-linking studies and biophysical measurements, is believed to be the unit on which the pore-forming oligomer is assembled. These structures provide the first molecular insights into Bax activation and define key Bax transitions towards the initiation of apoptosis.

SYM-11-04 SYM-11-05 THE STRUCTURE OF SULFITE DEHYDROGENASE IN REGULATORY DETERMINANTS OF OREXIN COMPLEX WITH ITS ELECTRON ACCEPTOR RECEPTOR-ARRESTIN-UBIQUITIN COMPLEX FORMATION Maher M.J.1, Mcgrath A.P.2, Laming E.M.3, Guss J.M.3 and Kappler U.4 1Department of Biochemistry, La Trobe University, Bundoora, Vic Jaeger W.C., Seeber R.M., Eidne K.A. and Pfleger K.D.G. 3086, Australia. 2Centenary Institute, Locked Bag No 6, NSW 2042, Laboratory for Molecular Endocrinology-GPCRs, Western Australian Australia. 3School of Molecular Bioscience, University of Sydney, NSW Institute for Medical Research and Centre for Medical Research, 2006, Australia. 4School of Chemistry and Molecular Biosciences, University of Western Australia, Nedlands, Perth, Australia. University of Queensland, St Lucia, QLD 4072, Australia. The orexin system is involved in the fundamental orchestration between Sulfite-oxidizing enzymes are essential for many living cells. sleep-wake, energy metabolism and the promotion of behaviour Sulfite occurs naturally in the environment, and through metabolic that stimulates psychochemical reward. This system mediates these pathways such as the degradation of sulfur-containing amino acids, activities through the action of endogenous orexin neuropeptides, orexin organosulfonate metabolism and sulfur oxidation pathways in A and orexin B, on two G protein-coupled receptors, orexin receptors chemolithoautotrophic bacteria. Due to its highly reactive nature, the 1 and 2 (OX1/OX2). In an agonist dependent fashion, these receptors sulfite anion can react with vital cell components such as DNA and interact with β-arrestins; a set of ubiquitously expressed intracellular proteins. Therefore, cells need to be able to detoxify sulfite efficiently by multi-adaptor proteins that are critically involved in regulatory and oxidation to sulfate, facilitated by the sulfite oxidizing enzymes (SOE’s). signalling properties of GPCRs. A significant difference between Here, we present the structure of the sulfite dehydrogenase enzyme the OX receptors is the temporal stability of OX-arrestin proximity (SorT) from Sinorhizobium meliloti both in isolation and in complex over sustained periods of stimulation as observed through the use of with its cognate electron acceptor SorU (a cytochrome c). The three- bioluminescence resonance energy transfer (BRET), a biophysical dimensional structure of the complex defines a pathway for electron assay that measures protein proximity in live cells and in real-time. transfer between the SorT active site and the heme c cofactor of SorU, Specifically, OX2 displays more sustained proximity with β-arrestins with conformational changes in the structure of SorU acting to facilitate than OX1. The molecular mechanism of this difference in stability was inter-protein electron transfer. Structural comparisons between the investigated by evaluating the contribution of the C-terminus of OX ‘transient’ SorT/SorU protein-protein complex and related ‘permanent’ receptors, a key binding region for β-arrestins using BRET. These data heterodimeric complexes, reveal a number of structural features, which suggest that unlike OX1, two phosphorylation clusters are involved in are unique to the electron-transfer complex. These presumably play a sustaining stable OX2-β-arrestin proximity. This difference in temporal role in the fine-tuning of the complex to achieve both specificity and fast OX receptor arrestin-bound complex association has vital implications association/dissociation, which are required for rapid electron transfer. in regulatory processes including desensitization and internalization, as well as cellular signalling. These data indicate that secondary and perhaps tertiary structure is important for β-arrestin-receptor binding in addition to the requirement of primary determinants. This study provides insights into the molecular mechanism of this dynamic interaction that is likely to contribute to subtype-specific signalling and regulatory functions, a better understanding of which may help expose the therapeutic potential of these receptors.

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SYM-12-01 SYM-12-02 DEFINING THE AT1 RECEPTOR SIGNALLING EFFECTS OF CHEMOKINE RECEPTOR SULFATION ON NETWORK: A GENOME WIDE RNAI SCREEN CHEMOKINE BINDING AND SIGNALLING

Thomas W.G. Stone M.J.1, Ludeman J.P.1, Millard C.J.1, Tan J.H.Y.1, Bridgford J.L.1, School of Biomedical Sciences, The University of Queensland. Canals M.1, Christopoulos A.1 and Payne R.J.2 1Monash University. 2University of Sydney. G protein-coupled receptors (GPCRs) hijack epidermal growth factor receptors (EGFR) to drive cell growth in a process termed EGFR Chemokine receptors CCR2 and CCR3 play central roles in regulating transactivation. Our interest is in investigating the mechanism whereby the trafficking of monocytes and eosinophils in response to pro- the activated angiotensin type I receptor (AT1R) transactivates the inflammatory CC chemokines. The N-terminal regions of these receptors EGFR. While candidates, such as matrix metalloproteases and each contain two tyrosine residues in an appropriate sequence EGF ligands have been identified, the molecular events underlying environment to be post-translational sulfated. We have investigated this process remain unresolved. To study this mechanism, we the role of tyrosine sulfation in regulating the interactions of cognate have developed a stable cellular model of EGFR transactivation by chemokines with these receptors. Growth of receptor-expressing cells introducing the AT1 receptor (AT1R) into human mammary epithelial in the presence of the sulfation inhibitor chlorate substantially reduced cells using retroviral delivery, and determined that the cells were stably the binding and activation of expressed receptors by chemokines, expressing a functional form of the receptor. After stimulation of these indicating that the receptors are sulfated and that sulfation contributes cells with angiotensin II (AngII), we observe a robust activation of the to binding affinity. Moreover, synthetic peptides containing sulfated EGFR, which is blocked by the EGFR antagonist tyrphostin AG1478. tyrosine at specific positions exhibit dramatically enhanced chemokine We have used this model to optimise a high-throughput short interfering binding affinity relative to the corresponding non-sulfated peptides. RNA (siRNA) screening approach to identify genes that are involved We have used NMR spectroscopy to investigate the energetic and in AT1R/EGFR transactivation, and have subsequently performed structural basis of chemokine binding by receptor sulfotyrosine a primary screen with the Dharmacon siGENOME kinome siRNA residues. Although CCR3-derived peptides utilise a conserved surface library before embarking on a genome-wide screen. From our kinome for binding to receptor-derived sulfopeptides, the energetic effect of analysis, we have identified a number of genes of interest (TRIO, BMX, sulfation at multiple positions can be either additive or cooperative for CHKA), which we have tested in secondary and tertiary siRNA screens different chemokines. Consequently, different sulfation states of the to further validate these candidates. We are in the process of further receptor may differ in their selectivities among chemokine ligands. For characterising the role of these hits in AT1R/EGFR transactivation. CCR2, we found that sulfated peptides can bind to both the monomeric The outcomes from these studies will provide us with more information and dimeric forms of the chemokine MCP-1, suggesting that both forms about the specific factors involved in AT1R/EGFR transactivation, of MCP-1 may be capable of binding to CCR2. These results have and to discover potential targets for the prognosis and treatment of important implications for the sequence of molecular events leading to cardiovascular disease. leukocyte activation by CC chemokines.

SYM-12-03 SYM-12-04 N-TERMINAL PROCESSING OF THE ORPHAN GPCR, EFFECTS OF THE CB1 CANNABINOID RECEPTOR GPR37L1, AND ITS LINK TO CARDIOVASCULAR ALLOSTERIC MODULATOR, ORG 27569, ON AGONIST- DISEASE INDUCED ERK PHOSPHORYLATION

Smith N.J.1, 2 Khajehali E., Malone D.T. and Leach K. 1Victor Chang Cardiac Research Institute. 2University of New South Drug Discovery Biology, Monash Institute of Pharmaceutical Wales. Sciences, VIC 3052.

Despite great advances in our understanding of the pharmacology and Background: The majority of central effects of cannabis and other physiology of G protein-coupled receptors (GPCRs), a large number of cannabinoid compounds are mediated through CB1 cannabinoid receptors are yet to even be matched with their endogenous ligand, let receptors, which are the most abundant GPCRs in the brain (Devane alone ascribed a function in the body. Based upon unique expression et al., Mol Pharmacol. 1988). Therapeutic applications of cannabinoid patterns, homology to other GPCRs with known physiology, or knock- compounds are limited mainly due to their psychotropic effects. out mouse phenotypes, a number of orphan GPCRs are promising Selective allosterism at CB1 receptors may provide an approach to new therapeutic targets. In this talk I will outline our studies with increasing the therapeutic effects of cannabinoid compounds while GPR37L1, an orphan GPCR found in the heart and brain. Recently, reducing their unwanted side effects. Three novel compounds, named GPR37L1 knock-out mice were reported to develop profoundly Org 27569, Org 27759 and Org 29647 have been identified that act elevated blood pressure and concomitant left ventricular hypertrophy. as allosteric inhibitors of agonist function, but as enhancers of agonist Given the potential therapeutic relevance of GPR37L1, we sought to binding at the CB1 receptor (Price et al., Mol Pharmacol. 2005). The gain insight into the pharmacology of GPR37L1 by generating stable, aim of the present study was to investigate the effects of Org 27569 doxycycline-inducible, FlpIN T-REx HEK293 cells expressing human on CB1 agonist-mediated signalling pathways. Methods: Flp-In CHO GPR37L1 tagged with C-terminal enhanced Yellow Fluorescent Protein cells stably expressing human CB1 receptors were generated using (GPR37L1-eYFP). Confocal microscopy and cell-surface biotinylation Gateway technology and pERK1/2 phosphorylation was determined experiments showed wild type (WT) GPR37L1-eYFP reached the cell using the AlphaScreen surefire p-ERK1/2 kit from Perkin Elmer. surface but resided predominantly in intracellular vesicles, suggestive Results: Cannabinoid agonists WIN55212, CP55940, HU-210, Δ9- of constitutive activation and internalisation. Interestingly, Western blot THC, methanadamide, anandamide and 2-AG increased ERK1/2 analysis revealed that GPR37L1-eYFP is cleaved at the N-terminus in a phosphorylation in a dose-dependent manner. Org 27569 had no manner entirely blocked by broad-spectrum matrix metalloprotease/A effect by itself, and did not alter WIN55212- and Δ9-THC- induced Disintegrin and Metalloprotease inhibitors. The N-terminally processed ERK phosphorylation. However it decreased the response to CP55940 fragment of WT GPR37L1-eYFP ran at the same apparent molecular and HU-210. Conclusion: Our results indicate that Org 27569 displays weight as a truncated version of GPR37L1-eYFP lacking the first 122 probe-dependence, a characteristic of allosteric interactions. amino acids and was also insensitive to enzymatic deglycosylation, allowing us to identify a region of 17 amino acids within which N-terminal cleavage must occur. Given that GPR37L1 is down- regulated in cardiovascular diseases known to also be associated with elevated matrix metalloprotease levels, we are actively investigating the functional consequences of this protease-dependent regulation of GPR37L1.

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SYM-12-05 INVESTIGATING THE PHARMACOLOGICAL PROFILES OF ANGIOTENSIN II RECEPTOR HETEROMERS

Johnstone E.K.M.1 and Pfleger K.D.G.1, 2 1Laboratory for Molecular Endocrinology - GPCRs, Western Australian Institute for Medical Research and Centre for Medical Research, The University of Western Australia. 2Dimerix Bioscience Pty Ltd.

The renin-angiotensin system (RAS) plays a vital role in cardiovascular and renal physiology through the maintenance of blood pressure, as well as salt and water homeostasis. The primary effector of the RAS is angiotensin II, which acts upon two different G protein coupled receptors (GPCRs), the angiotensin II type 1 receptor (AT1R) and the angiotensin II type 2 receptor (AT2R). Almost all of the well characterised biological actions of angiotensin II are mediated by the AT1R, including vasoconstriction and antinatriuresis. In contrast, the molecular and physiological functions of the AT2R are poorly characterised, although it is often believed to counteract many AT1R-mediated effects. The establishment of the concept of GPCR heteromerisation has revealed an increased level of complexity within GPCR signalling systems. GPCR heteromers are complexes formed between different receptor units and have biochemical properties that are demonstrably different from those of their component receptors. This unique pharmacological profile may eventually enable heteromers to be targeted by heteromer- selective drugs with improved specificity and reduced side effects. The aim of this work was to identify and characterise novel angiotensin II receptor heteromers. Various cell-based approaches have been used to investigate signalling and regulation of these receptor complexes compared to the monomers/homomers of the component receptors, including utilising bioluminescence resonance energy transfer.

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SYM-13-01 SYM-13-02 ROLES OF CELL ADHESION IN ENTERIC NEGOTIATING 3D ENVIRONMENTS DURING CELL GANGLIOGENESIS: BIOLOGICAL AND CELLULAR INVASION: MORE THAN JUST SHAPE CHANGE AUTOMATA MODELS Bach C.1, Zhong J.1, 2, Shum M.1 and O’Neill G.M.1, 2 Newgreen D.F.1, Hackett-Jones E.J.2, Landman K.A.2, Zhang, D.C.1, 1Children’s Cancer Research Unit, Kids Research Institute, Children’s Rollo B.N.1, Howard M.J.3 and Dufour S.4 Hospital at Westmead. 2Discipline of Paediatrics and Child Health, 1Murdoch Childrens Research Institute, Parkville, Victoria 3052, Australia. University of Sydney. 2Department of Mathematics and Statistics, University of Melbourne, Victoria 3010, Australia. 3Department of Neurosciences University of Metastasizing tumour cells must transmigrate the dense extra-cellular Toledo Health Sciences Campus, Toledo, Ohio, USA. 4Compartimentation matrix that surrounds most organs. The use of 3-dimensional (3D) et Dynamique Cellulaires, Institut Curie, Paris, France. collagen gels has revealed that many cancer cells can switch between different modes of invasion that are characterized by distinctive Enteric neural crest cells (ENCCs) form small closely-spaced enteric morphologies (rounded versus elongated). The adhesion protein nervous system (ENS) ganglia while differentiating into neurons and glia NEDD9 can regulate the switch between elongated and rounded within the growing gut. Ganglionation is adhesion-dependent so cell- invasion in melanoma cells and thus we questioned the role of cell adhesion molecules were labelled. N-cadherin+/NCAM+ neurons NEDD9 in the invasion switch of other neuroectodermal tumour types, became centrally placed in nascent enteric ganglia and N-cadherin+/ namely glioblastoma and neuroblastoma. Notably, assessment of NCAM- glial/ENCCs became located to the periphery. Embryonic publicly available expression data suggests that there is a significant guts were dissociated and neural cells selected by FACS. These were correlation between levels of NEDD9 expression with poor prognosis in allowed to aggregate in vitro, forming spherical aggregates, dependent both glioma and neuroblastoma. siRNA mediated depletion of NEDD9 on cadherin and NCAM function, with central neurons and peripheral failed to induce cell rounding in these tumour cells, contrasting the glia/ENCCs. NC-specific KO of HAND2 in the mouse resulted effects that have been described by others in melanoma and by our in disorganization of ENS ganglionation, and molecular profiling team in fibroblasts. Given that Rac GTPase has been described to suggested alterations of polysialylation of NCAM may participate in mediate the switch between elongated and rounded invasion we tested this. NC-specific KO ofβ1-integrin in mouse caused larger, rounder and whether the Rac morphology switch is functional in the glioblastoma sparser ENS ganglia, and in vitro tests confirmed that this resulted from and neuroblastoma cells. Using both dominant negative Rac and Rac1- impaired ECM adhesion by ENS cells. Simultaneous KO of N-cadherin specific siRNA we confirm the presence of this morphological switch. In partially rescued this ENS phenotype, strongly suggesting that the the absence of a morphological change following NEDD9 depletion we ENS pattern relies on a balance of cell-cell and cell-ECM adhesion. instead quantified cell migration speed and demonstrated a significant Mathematical models of aggregation were made using cellular decrease in migration rate. Our data therefore reveal that NEDD9 can automaton (CA) algorithms encoding differentiation (ENCC to neuron), regulate 3D migration speed independently of the Rac1 morphology cell-cell adhesive strength (neurons≥ENCC), movement, proliferation switch. Our previous work showing that NEDD9 determines the (neurons≤ENCC), and gut growth. The CA model evolved multiple strength of adhesion to the extra-cellular matrix suggests that NEDD9 small, spaced clusters which became relatively stable, with a balance may modulate mechanocoupling of the cell to the extra-cellular matrix between the number of central neurons and peripheral ENCCs. These to regulate the rate of forward movement. results indicate that simple trends in only a few interacting adhesion components can drive resilient and seemingly complex morphogenetic events.

SYM-13-03 SYM-13-04 CLASP-MEDIATED LOCALIZED EXOCYTOSIS PROTEOGLYCANS WITHIN THE MESENCHYMAL STEM CONTROLS EXTRACELLULAR MATRIX CELL NICHE MEDIATE NEURONAL DIFFERENTIATION DEGRADATION AND FOCAL ADHESION TURNOVER POTENTIAL

Stehbens S.J.1, 3, Paszek M.J.1, 2, Pemble H.1, Gierke S.1 and Okolicsanyi R.K., Griffiths L.R. and Haupt L.M. Wittmann T.1 Genomics Research Centre, Griffith Health Institute, Griffith 1University of California San Francisco. 2Cornell University. 3Institute University. of Health and Biomedical Innovation, QUT. Multiple factors influence the lineage fate of hMSCs including the Turnover of integrin-based focal adhesions (FAs) with the extracellular extracellular environment or niche. The niche includes the extracellular matrix is essential for coordinated cell movement during tissue matrix (ECM), with the structural composition of the ECM and its remodelling and wound healing. In collectively migrating human associated components, including the proteoglycans, collectively keratinocytes, FAs assemble near the leading edge, mature as a result influencing hMSC stemness and differentiation. Direct cell-cell or of contractile forces, and subsequently disassemble underneath the cell-matrix interactions, as well as mediation of signalling pathways advancing cell body. We report that clustering of the microtubule- trigger downstream events, affecting the differentiation of these stem associated proteins CLASP1 and CLASP2 around FAs is temporally cells down specific lineages. Proteoglycans (PGs) are important correlated with FA turnover. CLASPs are recruited to FAs by the components of the cellular niche as they are ubiquitous and can be adaptor protein LL5β, and both CLASPs and LL5beta are required localised to the cell surface or embedded within the ECM. Heparan for FA disassembly. CLASP-mediated microtubule-tethering at sulfate (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) are FAs establishes a Rab6A-positive vesicle transport pathway for two families of proteoglycans known to influence cellular fate through delivery, docking and fusion of exocytic vesicles near FAs, thereby interactions with a number of signalling pathways and extracellular facilitating FA-associated, localized degradation of the extracellular components including FGFs, Wnts and BMPs. With evidence matrix. We propose that CLASPs function as key molecules coupling supporting a role for HSPGs and CSPGs in the specification of microtubule organization, vesicle transport and cell interactions with hMSCs down the osteogenic, chondrogenic and adipogenic pathways, the extracellular matrix, and that local matrix metalloprotease secretion it is conceivable that these important proteins also play a role in the may initiate FA turnover by severing cell-matrix connections. Targeted differentiation of hMSCs toward the neuronal lineage. In this study, we protease secretion is a mechanism by which tumour cells moderate expanded hMSC populations through ≥15 passages and ≥60 days in their microenvironment, allowing them to become highly invasive culture to examine neural potential and stemness following addition during tumour progression. Thus, understanding how cells regulate of heparin (heparan sulfate analogue) or chlorate (sulfation inhibitor). interactions with their surrounding matrix at a fundamental level, is We observed significant changes in the core protein (syndecans and crucial for the development of novel targeted therapies to combat glypicans) gene expression profiles at different growth phases. We also tumour progression. observed that these cells maintain both their stemness and their neural potential into late growth phases, and confirmed this through ICC of MSC markers including CD44 and CD29 and NSCs markers including Nestin and Sox2.

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SYM-13-05 SYM-14-01 LIPID REPLACEMENT OF SARCOPLASM IN UNCOVERING THE ROLES OF SECONDARY DYSFERLIN-DEFICIENT MUSCLES METABOLITES PRODUCED BY THE PLANT PATHOGENIC FUNGUS, LEPTOSPHAERIA MACULANS Grounds M.D.1, Radley-Crabb H.G.2, Terrill J.R.1, Robertson T.3, Papadimitriou J.3, Spuler S.4 and Shavlakadze T.1 Elliott C.E.1, Callahan D.L.2, Schwenk D.3, Hoffmeister D.3 and 1school of Anatomy, Physiology and Human Biology, the University of Howlett B.J.1 Western Australia. 2School of Biomedical Science, Curtin University, 1School of Botany, The University of Melbourne, Victoria 3010, Western Australia. 3School of Pathology and Laboratory Medicine, Australia. 2Metabolomics Australia, School of Botany, The University University of Western Australia. 4Max Delbrück Center for Molecular of Melbourne, Victoria 3010, Australia. 3Friedrich-Schiller-Universität, Medicine, Berlin, Germany. Department Pharmaceutical Biology at the Hans-Knöll-Institute, Beutenbergstrasse 11a, 07745 Jena, Germany. Dysferlin is a membrane associated protein involved in protein vesicle trafficking and fusion. Dysferlin is present in many cell types and defects Filamentous fungi produce a diverse array of secondary metabolites result in dysferlinopathies that manifest as human myopathies, including that have a tremendous impact on society; some are exploited for their Limb Girdle Muscular Dystrophy type 2B and Miyoshi Myopathy, and antibiotic and pharmaceutical activities, others are implicated in disease in the A/J and BLAJ dysferlin-deficient (null) mouse models of these interactions with plants or animals. Such metabolites are synthesized diseases. The precise basis for the adult-onset disease pathology is by gene clusters that include key genes such as non-ribosomal peptide not understood. Our studies show massive lipid accumulation that synthases and polyketide synthases. Epipolythiodioxopiperazines, a replaces myofibres in muscle of dysferlin-deficient mice and humans. class of toxins produced by a range of ascomycetes, confer toxicity due Using histological techniques. Oil red O staining (to identify lipids) and to a disulphide bridge, which can inactivate proteins via reaction with transmission electron microscopy, we show that in the most severely thiol groups, or generate reactive oxygen species by redox cycling. One affected psoas and quadriceps muscles of A/J and BLAJ dysferlin- such molecule is sirodesmin, produced by Leptosphaeria maculans, null mice, around 20-40% of the myofibers are replaced by fat by 8 the blackleg pathogen of canola, whilst another is gliotoxin, which is months of age; this appears to be due to the striking accumulation made by Aspergillus spp. that infect immunocompromised people. We of lipid droplets within myofibers and expansion of adipocytes. This are determining the role of two secondary metabolites of L.maculans lipid is also conspicuous in human muscle biopsies from patients with - sirodesmin and the polyketide-derived phomenoic acid, in blackleg dysferlinopathies. In the dysferlin-null mice, there is little evidence disease of canola. When secondary metabolites were first discovered of myonecrosis but severe disturbance of sarcoplasmic structures. in pathogenic fungi it was assumed that they had a key role in disease. These striking morphological changes in dysferlin-null mouse muscles However, now it is apparent that many secondary metabolites play are suggestive of altered gene expression within the myofibres. In only a minor role in plant or animal disease. Since synthesizing and quadriceps muscles of young (3 months) adult dysferlin-null mice maintaining these molecules is metabolically costly, it is likely that they there is no marked pathology, yet there is a striking increase (30-60% have an advantageous role to the producing organism. An increasingly compared with normal controls)) in gene expression associated with popular hypothesis is that secondary metabolite toxins enable induction of lipogenesis. These novel observations indicate severe microorganisms that produce them to survive in the environment. biochemical and metabolic disturbances in dysferlin-deficient muscles, that can account for the loss of muscle tissue and function.

SYM-14-02 SYM-14-03 DEPLOYING CHEMICAL DEFENCES IN EUCALYPT NITROGEN METABOLISM IN ACYANOGENIC MUTANTS SECRETORY CAVITIES OF SORGHUM BICOLOR

Woodrow I.E., Heskes A.M. and Goodger J.Q.D. Blomstedt C., Rosati V., Fromhold S., Edwards A., Neilson E. and The University of Melbourne. Gleadow R. School of Biological Sciences, Monash University, Clayton, Victoria One of the most distinctive features of eucalypts is the presence of Australia. essential oil secretory cavities which occur at a particularly high density in leaves. In some leaves, the secretory cavities can occupy Sorghum produces the secondary metabolite, dhurrin, a cyanogenic up to 25% of leaf volume and thus contribute significantly to defence glucoside with roles in defence and nitrogen storage and turnover. We against herbivores. We characterized the complex chemical cocktail have identified mutant sorghum lines with altered levels of dhurrin. found within the lumen of the secretory cavities of a range of eucalypts The totally cyanide deficient tcd1( ) line, has a mutation in the coding and mapped its organized spatial arrangement. Contrary to the long- region of the biosynthetic gene, CYP79A1, resulting in a non-functional held view that the secretory cavities simply contain a mixture of mono- protein, whilst adult cyanide deficient acdc1-3( ) mutants contain high and sesquiterpenes (essential oil) we found them to be rich in non- levels of dhurrin in the leaves of young seedlings but have negligible volatile compounds, especially monoterpene acid glucose esters. All leaf dhurrin levels when older. No alterations in the coding sequence of the characterized esters were found to be based on oleuropeic acid of dhurrin biosynthetic genes were identified in acdc mutants, which or methiafolic acid, or both, and many contained phenolic aglycones. are as tall or taller and leafier than non-mutated lines. We postulate All of the esters were shown to contain at least one α,β-unsaturated that these lines contain mutation(s) affecting growth regulation that carbonyl, which affords them various biological activities. We found might be due to alterations in nitrogen metabolism. In sorghum it has that the non-volatile component of the secretory cavities is spatially been shown that dhurrin can be turned over without the release of separate from the essential oil, partitioning to the periphery of the HCN, supporting the idea that plants may use cyanogenic glycosides spherical lumen. Our work shows that eucalypt secretory cavities as transportable, remobilisable nitrogenous storage compounds. To are a dynamic, highly organized, chemically complex domain for the investigate resource allocation in sorghum we are characterising the deployment of biologically active chemicals, which likely moderate tcd1 and acdc1 mutants compared to non-mutated lines under different interactions between leaves and herbivores. nitrogen levels during development. To monitor nitrogen use efficiency - (NUE) and the allocation of nitrogen we have assayed for HCNp, NO3 , total carbon/nitrogen, as well as morphological characteristics, such as growth rate, root/shoot ratio and biomass yield. Expression of key genes associated with dhurrin mobilization and catabolism are also being examined to investigate links between cyanogenic glucoside metabolism and the regulation of plant growth and development. A relatively large proportion (20-30%) of total nitrogen in sorghum is sequestered as dhurrin, making the recovery of nitrogen vital if NUE is to be improved, an important requirement in agriculture today.

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SYM-14-04 SYM-14-05 TRIGGERING THE PLANT DEFENCE RESPONSE USING EARLY STRESS-RESPONSIVE MARKER INDUCES ESSENTIAL OIL PRODUCTION IN INDIAN GENES TO SCREEN FOR ARABIDOPSIS MUTANTS SANDALWOOD WITH ALTERED PATHOGEN RESISTANCE

Tungngoen K.1, Norris L.4, Flematti G.2, Burgess T.5, Finnegan P.1, 3, Thatcher L.F.1, Foley R.C.1 and Singh K.B.1, 2 Ghisalberti E.2, Barbour L.3 and Plummer J.1 1CSIRO Plant Industry, Centre for Environment and Life Sciences, 1School of Plant Biology, University of Western Australia, Western Wembley, WA, 6913, Australia. 2University of Western Australia Australia. 2School of Chemistry and Biochemistry, University Institute of Agriculture, University of Western Australia, Crawley, WA, of Western Australia, Western Australia. 3Faculty of Science, 6009, Australia. University of Western Australia, Western Australia. 4Forest Products Commission, Kensington, Western Australia. 5School of Veterinary To identify genes regulating a plants first induced response to pathogen and Life Sciences, Murdoch University, Western Australia. attack we conducted forward genetic screens in Arabidopsis for mutants with altered stress-responsive promoter activity. We used promoters Indian sandalwood (Santalum album) produces sesquiterpene-rich from members of the Glutathione S-transferase (GST) family which essential oil inside the mature aromatic heartwood. This wood is dark perform key roles in protecting tissues from oxidative damage or toxic in colour and is mainly located at the base of the trunk, decreasing in products. In plants, transcriptional activation of GSTs can be induced by extent inversely with trunk height. Our previous work suggested that the a range of signals including hormones, herbicides and pathogen attack, defence response of S. album trees against fungal attack likely included with our interest on AtGSTF7 and AtGSTF8 which are predominantly the production of essential oil. In this project, two fungi that were expressed in roots. Characterising the transcriptional response of associated with rot in S. album were inoculated back into the branches AtGSTF7 and AtGSTF8 we identified several novel mutants with of 12-year-old trees. Branches were also inoculated separately with two loss-of- or gain-of-function promoter activity and which confer altered plant hormones that prime trees against fungal attack. All treatments resistance to diverse pathogens and pests. One mutant, disrupted in caused wood staining within 2 months. The essential oil extracted from stress response1 (dsr1), showed a loss of stress inducible AtGSTF8 the stained wood contained the sesquiterpenes α-santalol, β-santalol expression and increased susceptibility to specific bacterial and fungal and (Z)-lanceol. The composition of the oil changed with time, pathogens. The dsr1 mutation was mapped to the substrate binding resembling the oil in the trunk of a mature tree within eight months. The site of mitochondrial complex II succinate dehyrogenase subunit amount of oil produced differed among the treatments. Importantly, this SDH1-1 and caused reduced production of mitochondrial derived is the first clear demonstration that the branches of S. album can be reactive oxygen species (Gleason et al. 2011). A constitutive AtGSTF8 induced to produce oil. The promoter sequences for genes encoding promoter mutant, enhanced stress response1 (esr1), exhibits increased santalene synthase, monoterpene synthase and sesquiterpene resistance to a root-infecting fungal pathogen and altered gene synthase, enzymes involved in sesquiterpene synthesis in S. album, expression in cell recognition, stress and lipid/jasmonate signalling contain numerous sequence elements potentially responsive to networks, while another, esr3, exhibits increased resistance to aphid wounding, fungal infection or plant hormones, providing a promising attack and to specific root or leaf infecting fungal pathogens. Cloning molecular explanation for our results. of mutants is underway using NGS-mapping techniques with the goal of manipulating early stress responses in plants to breed for broad spectrum pathogen resistance in agricultural crops.

SYM-15-01 SYM-15-02 CLIMATE CHANGE IMPACTS ON YIELD AND THE EFFECTS OF LARGE-SCALE EXPERIMENTAL NUTRITIONAL VALUE OF CYANOGENIC TROPICAL RAINFALL EXCLUSION ON THE CARBON CYCLE OF CROPS TROPICAL RAIN FOREST

Gleadow R.M. Meir P.1, 2, Costa A.C.L.3, Galbraith D.R.4, Rowland L.2, Metcalfe D.E.B.5, School of Biological Sciences, Monash University. Vasconcelos S.6, Powell T.7, Carvalho C.6, Ferreira L.8 and Almeida S.8 1Australian National University, Canberra. 2University of Edinburgh, UK. Tropical crops will become increasing important for food production 3Federal University of Para, Brazil. 4University of Leeds, UK. 5Swedish as the planet warms and traditional food growing regions in temperate University of Agricultural Sciences, Sweden. 6Empresa Brasileira de latitudes become progressively drier. With the dwindling amount of Pesquisa Agropecuária, Brazil. 7Harvard University, USA. 8Museu arable land and the increasing cost of fertilisers, research to date Paraense Emilio Goeldi, Brazil. has largely focused on increasing yields, but food quality also needs to be considered. Sorghum, cassava and taro are among dozens of We report on results from a long term (10 yr) field-scale (1 ha) rainfall crops that produce cyanogenic glucosides that break down to release exclusion experiment in the eastern Amazon. Measurements were toxic cyanide gas and can be detrimental to human and animal health. made of canopy structure, tree growth and gas exchange by leaves, Cassava can cause acute cyanide intoxication, paralysis and even trees and soil in undisturbed forest and droughted forest (ie forest death of humans. Forage sorghum can cause similar symptoms in experiencing experimentally imposed soil moisture deficit). The data grazing animals. The concentration of the cyanogenic glycosides were used to improve understanding and then model the impact of depends on environmental conditions as well as genotype, ontogeny extended drought on GPP, autotrophic and heterotrophic respiration, and phenology. An on-going series of factorial experiments, conducted tree growth and mortality. Results indicate reductions in NEP and a in collaboration with the Monash Ecophysiology Group, has been delayed but substantial increase in tree mortality. Long term drought testing the effects of temperature, low soil moisture, nutrient supply has a large impact on the standing biomass and physiology of this and elevated CO2 on the growth and nutritional value of these species. tropical rain forest, with large trees of some genera more vulnerable Changes in secondary metabolite concentration are intricately linked to increased mortality than others. Dynamic vegetation models of to the primary metabolism and may act to mitigate oxidative stress. drought impacts on forest functioning are limited by our capacity to These crops appear to be well adapted to cope with global warming, represent mortality during drought and we discuss some ways forward with likely increases in yield, but care must to be taken to ensure food for improved understanding and subsequent simulation of mortality in safety, especially when soil moisture is low. Contributions by Rebecca rain forest trees. Miller, Tim Cavagnaro, Cecilia Blomstedt, Bruce Webber Steven Crimp, Rebecca Vandegeer, Alicia Brown, Natalie O’Donnell, Nikolai Macnee and Birger L. Moller are gratefully acknowledged.

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SYM-15-03 SYM-15-04 EVALUATING WHEAT TRAITS FOR ADAPTATION TO CALCIUM-DEPENDENT STOMATAL SIGNALLING, A FUTURE CLIMATES LIABILITY UNDER RISING ATMOSPHERIC CO2?

Palta J.A.1, 2, Dias De Oliveira E.1, 2, Bramley H.3 and Siddique K.M.H.3 Brodribb T.J. and McAdam S.A.M. 1CSIRO Plant Industry, Private Bag No 5, Wembley, WA 6913 School of Plant Science, University of Tasmania. Australia. 2School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Calcium signalling in plants regulates the dynamic opening and closing Crawley WA 6009, Australia. 3The UWA Institute of Agriculture of stomatal pores on the surfaces of leaves in response to changing (M082), The University of Western Australia, 35 Stirling Highway, 2+ concentrations of CO2 and water stress. The Ca -dependent signalling Crawley WA 6009, Australia. pathway in plants has only been characterised in model angiosperms, but an insensitivity of non-angiosperm stomata to elevated CO2 Australia Climatic conditions across the wheat growing regions of suggests that angiosperm stomatal signalling may differ from other Australia are predicted to change with increasing atmospheric CO2 plant groups. One possible explanation for the different behaviour of concentration and ambient temperatures and reducing rainfall. These angiosperm stomata is that Ca2+-dependent-signalling in stomata only changes poses a challenge for wheat production as the next generation evolved after the divergence of angiosperms from gymnosperms. We of wheat cultivars capable of keeping productive under future climates find strong support for this evolutionary hypothesis, showing that the will rely on genetic interventions based on process understanding stomata of lycophytes, ferns and gymnosperms lack characteristic 2+ and target traits more than ever before. The evaluation of how traits Ca -dependent responses to CO2 and the phytohormone abscisic respond to the current and future climate changes is an important step acid (ABA). In addition we observe that much of the diversity in Ca2+- in quantify the impact of climate change and hence to identify targets dependent signalling genes, and the Ca2+-activated stomatal anion for improvement. Studies into the impact of future climates have so channel in angiosperms, are absent in non-angiosperm genomes. This far focused on the effects of an increased average temperatures or evidence suggests that the unusual responsiveness of angiosperm CO2 concentration independently. However, most climate models stomata among vascular plants is partially due to the evolution of predict that increases in CO2 concentration, ambient temperature the Ca2+ signalling pathway in their guard cells. The benefit of this and incidence of the end-of–season drought (terminal drought) will evolutionary innovation appears to be faster stomatal movements, but occur simultaneously and over the season. Currently, information on we also identify a potential cost in terms of lowered productivity relative the physiology and genetics of the processes underpinning improved to non-angiosperms during rapidly rising atmospheric CO2. performance under these simultaneous changes is limited. This paper reports the evaluation of wheat traits for adaptation to drought and high temperature future climate scenarios by growing contrasting genotypes under elevated CO2 concentration and a range of ambient temperatures with and without terminal drought. Field tunnel houses in which CO2 concentration, temperature and terminal drought are controlled were specially designed and are a key facility for this research.

SYM-15-05 SYM-16-01 EVALUATING THE EFFECTS OF ELEVATED CARBON SMALL NONCODING RNAS AS MEDIATORS OF DIOXIDE ON IRON CONCENTRATIONS IN RICE TUMOUR ANGIOGENESIS

Riegert C.B.1, Tausz M.2 and Johnson A.A.T.1 Plummer P.N.1, Mittal V.M.2 and Mellick A.S.1 1School of Botany, The University of Melbourne, Victoria 3010, 1Host Response to Cancer Lab, School of Medical Science, Griffith Australia. 2Department of Forest and Ecosystem Science, Melbourne University, Gold Coast, QLD, Australia. 2Lung Cancer Centre, Weill School of Land and Environment, The University of Melbourne, Water Cornell Medical College, NY, United States. Street, Creswick, Victoria 3363, Australia. Driven by the push to develop improved anti-cancer strategies, there

Increased concentrations of carbon dioxide ([CO2]) change plant has been a recent increased interest in the mechanisms underlying physiology and morphology. Under elevated [CO2] (>550 ppm) C3 tumour vascularisation. Several therapies such as Avastin, inhibit plants increase biomass on average 13-28% and photosynthesis angiogenesis and target vascular endothelial growth factor (VEGF) rate by 13-46%, but show simultaneous decreases in protein and signaling directly; but become ineffective over time, in a process referred micronutrient concentrations. Soybean, barley, rice, radish, potato, to as evasive or adaptive resistance. Recent studies have shown that sorghum and wheat all decrease in micronutrient concentration many of the pathways regulating angiogenesis are modulated by under elevated [CO2]. The physiological mechanisms responsible for small noncoding RNAs (miRNAs). In cancer, miRNAs changes reflect decreased micronutrient concentrations are unresolved. Rice is the changes in pathology; as well as the proliferation, drug resistance, most important crop for humans as it is a staple for approximately half of motility and differentiation of tumour cells. Recent data suggest that the global population. Under current [CO2] rice consumption has been many of these same miRNAs play analogous roles in endothelial cell correlated with micronutrient deficiencies in humans, particularly iron biology, by regulating endothelial cell responsiveness to growth factor (Fe). Fe deficiency has many negative health consequences including signaling, differentiation, mobilization, and proliferation. The following anemia and increased mortality rates. A number of Fe-enriched rice presentation will outline the work of our lab in identifying key miRNA cultivars have been produced to combat human Fe deficiency due in pathways regulating EPC/EC tumour biology as targets for next rice based diets. Future [CO2] concentrations may further increase generation RNA-based anticancer therapy. the incidence of global malnutrition, and also raise questions about the efficacy of Fe-enriched rice. This study seeks to determine the direction and intensity of the [Fe] trend in rice under elevated [CO2] (~700 ppm). Tropical (Indica cv. IR64), temperate (Japonica cv. Nipponbare) and Fe-enriched rice plants were grown under ambient

(~400 ppm) and elevated [CO2] in glasshouse conditions. Selected data will be presented regarding measurements of Fe concentrations at tillering, anthesis and maturity as well as plant height, tiller number and stomatal conductance. The experiment will be repeated in the

2013-2014 growing season, with [CO2] swapped between chambers to eliminate the possibility of chamber effects.

Page 46 ComBio2013 s Perth, Western Australia s 29 September - 3 October, 2013 symposia TUESDAY

SYM-16-02 SYM-16-03 THE ROLE OF NON-CODING RNAS IN SEX FUNCTIONAL SIGNIFICANCE OF NON-CANONICAL DETERMINATION AND GONAD DEVELOPMENT MICRORNAS AND MICRORNA VARIATION

Wainwright E.N.1, Rakoczy J.1, Fernandez-Valverde S.L.1, Dinger M.E.1, Bracken C.P., Neilsen C.T., Thomson D.W., Anderson M.A., Lawrence D.L., Koopman P.1, Mattick J.S.2, Taft R.J.1 and Wilhelm D.1, 3 Pillman K. and Goodall G.J. 1Institute for Molecular Bioscience, The University of Queensland, Centre for Cancer Biology, SA Pathology, Adelaide. Brisbane, QLD 4072, Australia. 2Garvan Institute for Medical Research and St. Vincent;s Clinical School, University of New South Wales, The advent of mass-sequencing has revealed an extraordinary Darlinghurst, NSW 2010, Australia. 3Department of Anatomy and complexity of microRNAs, both in terms of naturally-occurring Developmental Biology, Monash University, VIC 3800, Australia. sequence variation (isomiRs) and microRNA-like molecules derived from larger non-coding RNAs. Using RNA-Seq to probe the small Disorders of sexual development are surprisingly common. They range RNA content of breast cancer cells, we find an extensive array of these from mild genital abnormalities to complete sex reversal. The cause variant and novel microRNA-like molecules are bound to Argonaute, of these problems is often a failure of the complex networks of gene the microRNA-binding component of the microRNA:protein effector regulation that regulate the differentiation of testes and ovaries. There complex, RISC. We extensively investigate whether these microRNA- is an increasing amount of evidence that gene activity is regulated post- sized molecules from diverse sources do indeed serve microRNA-like transcriptionally by non-coding RNAs (ncRNAs). We have performed functions. We also present evidence showing differential functions of high-throughput sequencing for small RNAs, as well as microarray endogenous microRNA sequence variants. In the case of miR-222 analysis for long ncRNAs, for mouse embryonic gonads from 11.5 for example, ~98% of all miR-222 in breast cancer cells possess a 3’ days post coitum (dpc), the time of sex determination, to 14.5 dpc, extension of between 1 and 5 nucleotides compared to the accepted when testes and ovaries are well differentiated. A number of small and “canonical” sequence. This has dramatic implications on cell cycle long ncRNAs were significantly differentially expressed and by using progression and apotosis for cells expressing these different forms. As gain- and loss-of-function analysis ex vivo and in vivo we are analyzing the majority of microRNAs exhibit sequence variation, the repertoire the functional relevance of these RNAs in the developing gonads. In of microRNA function may therefore be even more extensive than addition, we found that not only the expression but also the processing previously assumed. of microRNAs was highly controlled in a tissue-specific manner at the level of Drosha, Dicer and RISC, providing an ideal model system to identify and characterize regulatory factors.

SYM-16-04 SYM-16-05 RNA CAPTURE SEQUENCING FOR GENE DISCOVERY CONSTRUCTING THE SCAFFOLD OF THE PROTEIN- AND QUANTIFICATION BUILDING MACHINERY: IDENTIFICATION OF A PENTATRICOPEPTIDE REPEAT PROTEIN INVOLVED IN Clark M.B.1, 2, Mercer T.R.1, 2, Crawford J.1, Mattick J.S.2 and Dinger M.E.2 CHLOROPLAST RIBOSOMAL RNA BIOGENESIS 1The University of Queensland, St Lucia, QLD, Australia. 2Garvan Institute of Medical Research, Darlinghurst, NSW, Australia. Liu S., Small I. and Howell K.A. ARC Centre of Excellence in Plant Energy Biology, The University of Due to the large size and dynamic range of the human transcriptome, Western Australia. standard RNA sequencing (RNAseq) is an inefficient method for the assembly and quantification of a significant proportion of genes. We As chloroplasts are derived from a prokaryotic ancestor, they contain show that RNA Capture Sequencing (CaptureSeq), an exquisitely eubacterial 70S-type ribosomes. The ribosomal RNA component sensitive method for transcript detection, is superior in performance to is encoded by an operon in the chloroplast genome which shows RNAseq for the detection and quantification of lowly expressed genes, absolute conservation amongst higher plant chloroplasts. Maturation of which comprise 37% of the transcriptome and are overrepresented the rRNA precursor transcript is carried out by ribonucleases, but the in genes involved in human disease. We have utilised CaptureSeq precise mechanisms involved in chloroplast rRNA biogenesis remain to profile lowly expressed transcripts including ~30 000 human long to be discovered. One likely feature is the involvement of RNA-binding noncoding RNAs (lncRNAs) in cell lines and tissues. We find that many proteins, such as pentatricopeptide repeat (PPR) proteins, in defining currently annotated lncRNAs actually comprise fragments of much precursor ends by blocking nucleolytic activity. We have identified a larger transcripts. We also detect a massive increase in the complexity PPR protein, SOT1, which is involved in the biogenesis of the 23S rRNA and alternative splicing of lncRNA loci, discovering thousands of precursor. Analysis of rRNA species in the sot1 mutant by Northern novel lncRNA exons. Additionally, we have utilised CaptureSeq to blotting indicates a disruption in chloroplast 23S rRNA biogenesis. interrogate regions of the human genome identified as functionally Examination of the 5’ region of the 23S rRNA precursor, using 5’ RACE significant in various traits and diseases by genome-wide association (rapid amplification of cDNA ends), indicates that the 23S rRNA is studies (GWAS), but that appeared intergenic or “empty” of genes. We missing its 5’ extension which correlates with the reduced size of the find extensive transcription within and across these “empty” GWAS largest precursor identified in the 23S RNA blots. Using the “PPR code” regions, identifying ~1500 transcripts, ~60% of which are completely (1) to predict the binding site for SOT1 resulted in the discovery that the novel, while ~40% are novel isoforms of known Gencode transcripts sequence predicted aligns with the 5’ region of the 23S rRNA precursor that extend into the GWAS regions. We also identify many new coding identified by RACE. Moreover, the predicted binding site aligns with transcripts and at least 650 novel lncRNAs. These results demonstrate a putative RNA-binding protein footprint recently published (2). Taken the potential and the wide application of CaptureSeq for gene discovery together, these observations strongly suggest that SOT1 binds to the and profiling. 5’ region of the 23S rRNA precursor. Experiments to confirm direct binding of SOT1 to this specific sequence are currently underway. (1) Barkan et al, 2012, PLoS Genet, 8:e1002910 (2) Ruwe & Schmitz- Linneweber, 2012, Nucleic Acids Res, 7:3106.

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SYM-17-01 SYM-17-02 Sponsored by ENGINEERING STABILISED G PROTEIN-COUPLED The Australian Society for Biochemistry and Molecular Biology RECEPTORS AND OTHER INTEGRAL MEMBRANE PROTEINS FOR BIOCHEMICAL AND STRUCTURAL STRUCTURE OF THE AGONIST-BOUND STUDIES NEUROTENSIN RECEPTOR NTS1 1, 2 1, 2 1 1 3 White J.F.1, Noinaj N.1, Shibata Y.2, Love J.3, Kloss B.3, Xu F.1, Shah P.1, Scott D.J.1, 2, Bumbak F. , Yong K. , Lam N. , Gunn N. , Kummer L. , 3 1, 2 2 3 Shiloach J.1, Tate C.G.2 and Grisshammer R.1 Eglof f P. , Bathgate R.A.D. , Gooley P.R. and Plueckthun A. 1National Institutes of Health NINDS and NIDDK, USA. 2MRC Laboratory 1Florey Institute of Neuroscience and Mental Health, Parkville, Victoria of Molecular Biology Cambridge, UK. 3New York Consortium on 3010, Australia. 2The Department of Biochemistry and Molecular Membrane Protein Structure, USA. Biology, The University of Melbourne, Parkville, Victoria 3010, Australia. 3Department of Biochemistry, The University of Zurich, Neurotensin (NT) is a 13 amino acid peptide that functions as both a Switzerland. neurotransmitter and a hormone through activation of the neurotensin receptor NTS1, a G protein-coupled receptor (GPCR) signaling preferentially through Gq. We have solved the structure at 2.8 A resolution The G protein-coupled receptor (GPCR) gene family is the largest in the of NTS1 in an active-like state, bound to NT8-13, the C terminal portion human genome, yet only approximately 1% of GPCR family members of NT responsible for agonist-induced activation of the receptor. Because have been structurally characterised. This discrepancy reflects the wild-type NTS1 is unstable and thus not amenable to crystallization, we level of difficulty associated with manipulating and studying GPCRs used alanine-scanning mutagenesis to stabilize NTS1 and to select for in vitro. Integral membrane proteins (IMPs), such as GPCRs, need to an active-like conformation in the presence of agonist, which combined be extracted from cell membranes using detergents for purification. A with the bacteriophage T4 lysozyme fusion protein strategy and the lipidic major hindrance to biochemical studies of IMPs however, is that they mesophase crystallization method, resulted in diffracting crystals. The are typically unstable in detergent micelles. We have developed a agonist binding pocket is located at the extracellular receptor surface. directed evolution technique, termed CHESS (Cellular High-throughput The peptide agonist binds to NTS1 in an extended conformation nearly Encapsulation, Solubilisation and Screening), that allows the direct perpendicular to the membrane plane with the C-terminus oriented towards selection of detergent-stable IMP mutants from large gene libraries the receptor core. The NTS1 structure bears many hallmark features of an (>108 individual variants). CHESS has been applied to several class active-like receptor conformation such as an outward-tilted transmembrane A GPCRs including neurotensin receptor 1, α1A- and α1B-adrenoceptor, helix 6 at the cytoplasmic surface and key conserved residues in positions with the evolved variants able to be purified and stored for days in characteristic for active but not for inactive GPCRs. Our findings provided for harsh detergents. Stabilised receptors are valuable tools, enabling the first time insight into the binding mode of a peptide agonist to a GPCR. us to perform experiments that would be impossible to perform with This research was supported by the NIH Intramural Research Program and unmodified receptors including, automated in vitro binding assays, a joint grant from Pfizer Global Research and Development and the MRCT surface plasmon resonance binding experiments, X-ray crystallography, Development Gap Fund and core funding from the UK Medical Research Council. The New York Consortium on Membrane Protein Structure was small angle X-ray scattering measurements and nuclear magnetic supported by the NIH grant U54GM075026. The GM/CA-CAT beam line at resonance spectroscopy. Because the development of new drugs the Advanced Photon Source, Argonne National Laboratory was supported targeting IMPs is encumbered by a lack of information about their by the US Department of Energy, Basic Energy Sciences, Office of Science, molecular structure, it is hoped that studies such as these will aid in the under contract DE-AC02-06CH11357. identification of new lead compounds for drug development.

SYM-17-03 SYM-17-04 CRYSTAL STRUCTURE OF THE INTEGRAL ASSEMBLY OF PROTEIN SECRETION MACHINES IN MEMBRANE DIACYLGLYCEROL KINASE BACTERIAL PATHOGENS

Li D.1, Lyons J.A.1, Pye V.E.1, Vogeley L.1, Aragao D.2, Kenyon C.P.3, Lithgow T. Shah S.T.A.1, Doherty C.1, Aherne M.1 and Caffrey M.1 School of Biomedical Sciences, Monash University. 1School of Biochemistry and Immunology & School of Medicine, Trinity College Dublin, Dublin 2, Ireland. 2Australian Synchrotron, 800 Protein targeting and translocation across membranes is a fundamental Blackburn Road, Clayton, VIC 3168, Australia. 3CSIR, Biosciences, means by which all organisms deal with the issue of segregating their Meiring-Naude Road, Pretoria 0184, Gauteng, South Africa. proteome into distinct sub-cellular compartments, to effect specific metabolic and cellular functions. Pathogenic bacteria have subverted Diacylglycerol kinase catalyses the ATP-dependent phosphorylation the principles of protein targeting pathways, making insidious use of the of diacylglycerol to phosphatidic acid for use in shuttling water- process to send interfering and toxic proteins into specific sub-cellular soluble components to membrane-derived oligosaccharide and compartments of their host’s cells. We work on the mechanism by lipopolysaccharide in the cell envelope of Gram-negative bacteria. which protein secretion machines are assembled in membranes, and For half a century, this 121 residue kinase has served as a model for are particularly interested in how pathogenic bacteria assemble the investigating membrane protein enzymology, folding, assembly and protein secretion machines that they use against hosts. An essential stability. Here we present crystal structures for three functional forms “master machine” located in the bacterial outer membrane is the key of this unique and paradigmatic kinase, one of which is wild type. to assembling proteins into the outer membrane of bacteria. Both These reveal a homo-trimeric enzyme with three transmembrane physically and conceptually, this BAM complex is a classic example of a helices and an amino-terminal amphiphilic helix per monomer. Bound modular molecular machine, as are so many protein transport systems lipid substrate and docked ATP identify the putative active site that is [Lithgow & Waksman, 2013. EMBO Reports]. Recently we showed of the composite, shared site type. The crystal structures rationalize that the TAM is an additional module of this machinery, necessary extensive biochemical and biophysical data on the enzyme. They are, for the assembly of protein secretion machines [Selkrig et al 2012. however, at variance with a published solution NMR model in that Nature Struct. Mol. Biol. ]. Bacteria from which the TAM is deleted are domain swapping, a key feature of the solution form, is not observed in avirulent, because they fail to efficiently assemble their protein secretion the crystal structures. machines. We have solved structures of the components of the TAM and the BAM complex that are leading us to an understanding of how these sophisticated machines function, and whether they represent the next generation targets for therapeutic intervention against infectious diseases.

Page 48 ComBio2013 s Perth, Western Australia s 29 September - 3 October, 2013 symposia TUESDAY

SYM-17-05 SYM-18-01 WHAT IS THE MOLECULAR SWITCH FOR RELAXIN Sponsored by RECEPTOR SIGNALING? The Australian Society for Biochemistry and Molecular Biology

Petrie E.J.1, Diepenhorst N.1, 2, Kong R.1, 2, Lagadia S.1, 2, Bathgate R.A.D.2, 1 INTRACELLULAR COMPARTMENTALIZATION and Gooley P.R.1 OF GLUTATHIONE: REGULATING THE REDOX 1Department of Biochemistry & Molecular Biology, The University of GATEKEEPER Melbourne. 2Florey Institute of Neuroscience and Mental Health and Florey Department of Neuroscience and Mental Health. Foyer C.H. Centre for Plant Sciences, University of Leeds, School of Biology, The hormone Relaxin demonstrated beneficial effects and reduction of Leeds, UK. mortality rates in patients with acute heart failure in a recent Phase III clinical trial. While modulation of relaxin effects has clear therapeutic potential, its Oxidative stress signalling is pivotal in regulating plant responses to insulin-like two-chain structure has limited bioavailability, creating desire the environment and in phytohormone-dependent regulation of organ for small molecule relaxin-mimetics. Relaxin activates unique G-protein growth and development. The cellular reduction-oxidation (redox) hub, coupled receptor (GPCR), RXFP1. RXFP1 has a classic transmembrane which is comprised of oxidants such as reactive oxygen species and (TM) region, but complex ecto-region consisting of Leucine-rich repeats antioxidants such as reduced glutathione (GSH), integrates information (LRRs) and Low Density Lipoprotein Class-A (LDLa) module tethered by from metabolism and the environment to mediate these responses and linkers between the domains. Circulating relaxin is captured by the LRRs regulate gene expression. Regulation of the glutathione redox potential with high affinity. Removal of the N-terminal LDLa does not disrupt relaxin has important consequences for cellular functions, which are regulated binding; however signal activation is abolished. We hypothesize ligand by t hi o l - di sul fi de st atus. A p p r o p r i ate c o m par t m e nt aliz at i o n of t hi s c e nt r al binding induces the receptor confirmation necessary to initiate signaling redox gatekeeper is crucial to the maintenance of redox homeostasis and that the LDLa is essential to this process by making ‘ligand-like’ and oxidative signalling processes that regulate gene expression and contacts with the TM. Since RXFP1 and the closely related INSL3 receptor, determine the outcome of plant responses to stress. Mutants that RXFP2, are the only GPCRs to contain a LDLa, its role in signaling is lack chloroplast GSH transporters show depleted cytosolic GSH and unique. Interestingly, we can inhibit relaxin induced signaling of RXFP1 decreased pathogen responses. Similarly, GSH recruitment into the with recombinant LDLa. This brings promise that understanding where the nucleus early in the cell cycle causes oxidation of the cytosol. Oxidative LDLa docks and which residues are involved could lead to the design of stress drives oxidation of glutathione, and necessitates reconfiguration small molecules RXFP1 modulators. To this end we have determined the of sub-cellular distribution of GSH and glutathione disulphide (GSSG), structures of both the RXFP1 and RXFP2 LDLa to design a comprehensive which is sequestered in the vacuole. This talk will consider the impact of study using chimeric receptors and single point mutations and mapped the the intracellular partitioning of GSH and GSSG on the glutathione redox surface of the module involved in signal activation. To test our hypothesis potential of the cystosol, and describe the effect of GSH availability on that the LDLa makes ‘ligand-like’ interactions with the TM exo-loops (ELs) gene expression. we grafted RXFP1 ELs 1&2 onto the backbone of the protein GB1. This soluble scaffold presenting the ELs has shown interactions with both relaxin and LDLa in pull-down assays and NMR studies. Our comprehensive study is providing first insights into how the LDLa acts as the switch that leads to signal activation.

SYM-18-02 SYM-18-03 DETERMINATION OF THE FATE OF THE INNATE MEASURING ROS PRODUCTION AND OXIDATIVE IMMUNE RESPONSE TO RESPIRATORY VIRUSES: DAMAGE REDOX-DEPENDENT MECHANISMS Croft K.D. Grandvaux N. University of Western Australia. Départment de Biochimie, Université de Montréal, Montréal Québec 35292, CANADA. Atherosclerosis is a complex multifactorial disease, believed to be initiated by the uptake of modified low density lipoprotein (LDL) into Beside their reputed damaging role, Reactive Oxygen Species (ROS) the arterial wall. The pathogenesis of atherosclerosis is likely to involve have recently been characterized as modulators of signaling pathways, oxidative stress and damage to lipoproteins. Oxidised LDL can be taken mainly through reversible post-translational modifications of thiol up by the scavenger receptors of macrophages to form foam cells and residues in proteins. Viruses have long been reported to induce ROS products resulting from oxidation of LDL have been detected in human production in the host cells, but the origin and function of these ROS atherosclerotic plaque. Oxidants can also damage the endothelium of has long been ignored. Using respiratory ssRNA viruses as a model, blood vessels, leading to a loss of function, which can be an early sign our work in progress has clearly demonstrated that ROS produced by of cardiovascular disease. A number of methods have been developed two members of the ROS-generating NADPH oxidase enzyme family, to measure production of ROS and ‘oxidative stress’ including use NOX2 and DUOX2, positively contribute to the antiviral host response of fluorescent probes, measurement of ex vivo LDL oxidation, or the mounted by the host to restrict virus replication. The signaling cascades presence of oxidised LDL in plasma by ELISA. The level of antioxidants leading to the activation of two major transcription factors, NF-κB and and antioxidant enzyme activity can also be measured as well as the IRF-3, which control the expression of antiviral and proinflammatory so called ‘antioxidant capacity’ of plasma. However, these assays have cytokine genes are under the control of NOX2-dependent superoxides. limitations and we have taken the approach of measuring specific On the other hand, DUOX2-dependent hydrogen peroxide production biomarkers of oxidative damage in the circulation. F2-isoprostanes is induced by a novel delayed antiviral pathway and is currently are formed by free radical oxidation of arachidonic acid and represent thought to be required to sustain the antiviral response. The molecular specific markers of in vivo lipid oxidation. F2-Isoprostanes have been mechanisms, and more specifically the proteins that undergo oxidation detected in plaque tissue. Both free and esterified isopostanes are during this response, are currently being investigated. present in plasma and free isoprostanes are excreted in urine. Most circulating isoprostanes are associated with HDL. The usefulness of these and related measures to determine oxidative stress in humans will be discussed including results from recent human intervention studies.

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SYM-18-04 SYM-18-05 CHLOROPLASTS: ENVIRONMENTAL SENSING AND A STRATEGY TO MAP THIOL OXIDATION AT A NUCLEAR SIGNALING RESIDUE SPECIFIC LEVEL IN MUSCLE TISSUE

Pogson B.J. Boyatzis A.E.1, Bringans S.2, Joyce R.D.1, Piggott M.J.1, Lipscombe R.2, Research School of Biology, Australian National University. Fournier P.A.3 and Arthur P.G.1 1School of Chemistry and Biochemistry, University of Western Australia. Compartmentation of the cell, which has been critical to the success of Crawley, Western Australia. 2Proteomics International. Crawley, Western Eukaryotes, requires a complex set of subcellular messages including Australia. 3School of Sports Science, Exercise and Health, University of retrograde signals from the chloroplast to the nucleus to regulate gene Western Australia. Crawley, Western Australia. expression. Although some of the proteins that participate in different signalling cascades in higher plants have been identified, the actual Protein thiols are susceptible to oxidation by reactive oxygen and mobile signals and their mechanism of action are often debated. In nitrogen species (RONS). Reversible protein thiol oxidation has been recent years progress has been made by a number of groups, including shown to alter the function of proteins, and is implicated in a range ours, towards identifying specific signals and their pathways. This of pathological and physiological conditions. To understand the effect presentation will present insights into the mechanisms by which the of oxidation on protein function, elucidation of the identity of oxidised chloroplast perceives and responds to environmental stimuli resulting residues and the extent of oxidation is necessary. We have developed in signals that alter nuclear gene expression, chloroplast function and a technique to identify and quantify thiol oxidation at a residue-specific plant development as a whole. level. The differential alkylation of reduced and oxidised thiols using two novel isotopomeric biotinylated maleimides (“tags”) allows the mass differentiation of reduced and oxidised forms of peptides. The ratio of areas under resulting mass spectra peaks then gives the ratio of oxidised to reduced peptide. Proteins are separated by SDS-PAGE, excised, and digested with proteases. The tagged peptides are purified using a streptavidin affinity plate, then subjected to mass spectrometry analysis. The system has proven effective: all expected tagged peptides of purified lysozyme within the analysed mass range were detected. The proportion of oxidation of each residue was reflected in the resulting mass spectra. This technique has advantages over similar techniques for determining thiol oxidation status of proteins: it can be used to analyse tissue samples obtained from in vivo experimentation, and its ratiometric approach allows for accurate comparison between samples even where protein composition may vary. This novel technique therefore provides greater accuracy and flexibility when compared with alternative techniques.

SYM-18A-01 SYM-18A-02 USING LABGURU TO MANAGE LAB ACTIVITIES LABARCHIVES: ORGANISE, DOCUMENT, SHARE RESEARCH DATA Hatters D.M. School of Biochemistry and Molecular Biology, University of Hoy A.J.1, 2, 3 Melbourne, VIC 3010. 1Discipline of Physiology, School of Medical Sciences, University of Sydney, NSW 2006, Australia. 2Bosch Institute for Medical Research, Labguru is an online lab management system we have used for the last University of Sydney, NSW 2006, Australia. 3Boden Institute of 6 months. I will give a brief overview of the features available, and those Obesity, Nutrition, Exercise & Eating Disorders, University of Sydney, which we use. I will also discuss what we have found works well and Sydney, NSW 2006, Australia. what works less well. Finally I will provide my thoughts as to what extent it has changed our lab operations and other implications. LabArchives provides an easy to use platform for recording and sharing research data through its web-interface and Cloud storage structure. It also provides opportunities to have a central store of all laboratory information. What is achieved through electronic lab notebooks such as LabArchives, is that there is only a single version of every file generate in the lab, accessible to those who need it, but also retains all previous versions with time and date stamps. Finally, with LabArchives deep integration into popular software packages such as Prism GraphPad, FlowJo and MS Office, it makes it easy to retain active links between client software and the saved location in LabArchives.

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SYM-18A-03 SYM-19-01 USING AGILENT’S OPENLAB ELN SYSTEM FOR LAB LATE ENDOSOMAL AND NPC1-MEDIATED MANAGEMENT, EXPERIMENT DOCUMENTATION AND CHOLESTEROL TRANSPORT REGULATE SYNTAXIN COLLABORATION 6-DEPENDENT INTEGRIN RECYCLING AND CELL MIGRATION Millar H., Fenske R. and Taylor N. ARC CoE Plant Energy Biology, University of Western Australia, Grewal T. Crawley WA 6009. Faculty of Pharmacy, University of Sydney, Sydney, NSW 2006.

Data management is an increasing burden that needs to be solved in De-regulated cholesterol uptake and metabolism is the basis for our lab. We extensively use mass spectrometry for proteomics and several human diseases. We recently demonstrated that inhibition metabolomics which generates many large data files and we need of cholesterol export from late endosomes (LE) caused cholesterol to keep track of many different types of experiments on a range of depletion in the trans-Golgi-network (TGN). We now provide evidence transgenic lines. We also want to consider going paperless in day-to- for a completely novel role for LE-cholesterol (LE-chol) in cell migration. day operations (with tablet technology for note-taking and photography), Using Chinese Hamster Ovary (CHO) Niemann Pick Type C1 (NPC1) centralise storage and updating of lab protocols and primary data files mutant cell lines, pharmacological U18666A treatment, Annexin A6 from machinery, and allow text-searching of files inside the lab to build overexpression and human NPC1 mutant fibroblasts as models, we new collaborations. In the mid-term we want to provide post-docs demonstrate that LE-chol accumulation strongly reduces cell migration and students, once they had left the lab, with access to files as we and invasion. Mechanistic studies reveal that NPC1-dependent LE- prepare datasets for analysis and write papers. We are working with the chol accumulation perturbs post-Golgi membrane trafficking to trigger OpenLab ELN system to find solutions to these issues. Syntaxin 6 (Stx6) accumulation in Rab11-positive recycling endosomes. This interferes with Stx6-dependent recycling of αVβ3 and α5β1 integrins, key players in cell migration, through TGN compartments to reduce their cell surface delivery. Thus, our results provide insights into the mechanisms of LE-chol mediated cell migration and invasion.

SYM-19-02 SYM-19-03 STRESSING OUT - C-JUN N-TERMINAL KINASE 1 A NOVEL REGULATOR OF THE SECRETOME (JNK1) NUCLEAR TRAFFICKING BREAKS A STRESS- MODULATES CANCER METASTASIS ACTIVATED SIGNAL TRANSDUCTION PARADIGM Khew-Goodall Y. Bogoyevitch M.A. Centre for Cancer Biology, SA Pathology, Adelaide SA 5000. Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne. It is now becoming clear that cancer cells can secrete factors that modulate the pre-metastatic niche to favour their colonisation of Abiotic environmental stresses, including oxidative, genotoxic, or these niches long before they arrive at these sites. Comparisons of hyperosmotic stresses, are now known to impact on a diverse range the cancer cell secretome with that of its cell of origin often indicate of cell responses. Although stress can profoundly disrupt various significant differences in the composition of factors. However, the cellular parameters including intracellular ATP levels and the nuclear/ mechanisms behind global changes to the cellular secretome are not cytoplasmic gradients of Ran and importins, there is a limited well understood. Our earlier studies and subsequent studies by others appreciation of how these stress-induced changes impact on signal have suggested that the protein tyrosine phosphatase Pez (PTPN14) transduction and trafficking events. This is particularly surprising for has pleiotrophic effects but the mechanism by which it mediates cellular the c-Jun N-terminal Kinases (JNKs) that are traditionally considered functions remains unclear. Cancer-associated mutations in Pez have as members of the stress-activated protein kinases. We have used live been identified in breast and colorectal cancers, however the role of cell imaging including Fluorescence Recovery After Photobleaching such mutations in cancer progression remains undetermined. Our (FRAP) to define intracellular dynamics of JNK1 movement and its recent data suggests that Pez localises to vesicular compartments that entry into the nucleus. We have evaluated GFP-JNK1 trafficking into partially overlap with golgi / endosomal compartments in multiple cell the nucleus under both control and hyperosmotic stress situations, but types, modulates the secretion of multiple cytokines and influences also extended our study to define the nuclear entry of model cargoes the trafficking of various signalling receptors. In a xenograft mouse (GFP and GFP-Tantigen). Our studies showed a striking specific impact model of metastasis, reduced Pez expression leads to an increase in of hyperosmotic stress to slow GFP-JNK1 nuclear entry. This impact breast cancer metastasis. Furthermore, cell-free conditioned medium on GFP-JNK1 paralleled its slowed mobility in both the cytosol and the from Pez-low or Pez-high cells can modulate cancer metastasis. Data nucleus, suggesting its altered interactions in both the nucleus and will be presented on novel candidate Pez substrates and potential cytoplasm restrict its nucleocytoplasmic shuttling during hyperosmotic mechanism through which it regulates protein trafficking to alter the stress. Furthermore, we did not observe significant bulk accumulation cellular secretome and elaboration of cell surface receptors. of JNK1 during hyperosmotic stress. Our results thus break a paradigm of stress-induced nuclear trafficking of JNK1 and instead emphasize that JNK1 activated during hyperosmotic stress can continue to access its broad range of important substrates throughout the cell cytoplasm and nucleus. Thus, although stress-activated, JNK1 can continue to act beyond its archetypical role as a nuclear kinase targeting transcription factors such as c-Jun.

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SYM-19-04 SYM-19-05 REGULATION OF BREAST CANCER CELL MIGRATION REGULATION OF THE ERK/MAPK CASCADE BY THE AND INVASION BY CAMKII ACTIN CYTOSKELETON

Gilchrist J., Mayhew J., Hoffman A. and Skelding K.A. Schevzov G.1, Kee A.J.2, Coombes J.1, Wang B.1, Hook J.1, Lucas C.A.2, School of Biomedical Sciences and Pharmacy and Hunter Medical Stehn J.1, Sequeira V.1, Pleines I.3, Kile B. 3, Seger R.4, Hardeman E. 2 Research Institute, University of Newcastle, NSW, Australia. and Gunning P.1 1Oncology Research Unit. School of Medical Sciences, University Breast cancer is the most commonly diagnosed cancer among of NSW, NSW 2052. 2Neuromuscular and Regenerative Medicine Australian women, and once it has metastasized, it is thought to be Unit. School of Medical Sciences, University of NSW, NSW 2052. incurable. If we can better understand the mechanisms controlling 3Cancer and Hematology Division, The Walter and Eliza Hall Institute breast cancer metastasis, we can develop anti-cancer agents that of Medical Research, VIC 3052, 4Department of Biological Regulation, target these processes. Recently, the multi-functional serine/threonine Weizmann Institute of Science, Rehovot 76100, Israel (Thr) kinase, calcium/calmodulin-stimulated protein kinase II (CaMKII), was reported to be involved in osteosarcoma progression. CaMKII The actin cytoskeleton, a key regulator of cell proliferation, links functions are controlled by autophosphorylation at sites such as signalling pathways with cell cycle progression. Agents that disrupt Thr286 (increases CaMKII activity), and active CaMKII phosphorylates actin filaments lead to cell cycle arrest. Given that structurally and proteins involved in metastasis (e.g. Siva, Tiam-1). However, the role of functionally distinct populations of filaments co-exist within a cell, the autophosphorylated CaMKII in invasion/migration has not been directly lack of specificity of these agents impairs the ability to identify the examined. Therefore, we investigated whether CaMKII is involved specific filaments responsible for this process. Tropomyosin isoforms, in breast cancer cell migration and invasion in vitro. MDA-MB-231 which form co-polymers with actin, determine the functional capacity of cells were transfected with wild-type (WT) or Thr286 phosphomimic distinct actin filament populations. We show reduced cell proliferation in mutant (Thr286Asp) forms of αCaMKII. Cell proliferation (resazurin), tissues and primary mouse embryonic fibroblasts (MEFs) devoid of the clonogenicity (soft agar colony formation), migration (scratch) and tropomyosin isoform Tm5NM1, in contrast to Tm5NM1 overexpressing invasion (Transwell) were measured (n=3-4). Overexpression of WT- cells. Antibody microarray analysis revealed dysregulation of the MAPK/ CaMKII significantly increased cell proliferation, with Thr286Asp ERK pathway in cells devoid of Tm5NM1. Specific signalling pathway phosphomimic mutation producing an effect similar to that observed with inhibitors corroborate the array data and provide further evidence to WT-CaMKII (demonstrating that Thr286 phosphorylation has no effect support the isoform specific role of Tm5NM1 in regulating proliferation on cell proliferation). Furthermore, as shown in osteosarcoma cells, via the MAPK/ERK pathway. Moreover, we demonstrate that in the overexpression of WT-CaMKII significantly increased clonogenicity absence of Tm5NM1, nuclear translocation of pERK1/2 (essential for (p<0.001), migration (p<0.05) and invasion (p<0.05) when compared cell proliferation) is attenuated leading to altered expression of pERK1/2 to empty vector (EV) expressing cells, indicating that CaMKII may be and the downstream effector, cyclin D1. Finally, we show that Tm5NM1 involved in metastasis. Furthermore, Thr286Asp-CaMKII expression mediates its effects on ERK nuclear translocation via casein kinase 2, significantly increased the clonogenicity (p<0.0001), migration an activator of the nuclear translocation signal on ERK. We propose a (p<0.001) and invasion (p<0.01) of MDA-MB-231 when compared to novel function for actin filaments containing Tm5NM1 as a regulator of WT-CaMKII and EV cells. In conclusion, our results demonstrate that the MAPK/ERK pathway. CaMKII is involved in the invasion and migration of breast cancer, and that CaMKII autophosphorylation at Thr286 increases the migratory and invasive capability of breast cancer cells.

SYM-20-01 SYM-20-02 NOVEL FACTORS INVOLVED IN THE REGULATION OF INVESTIGATING THE ROLES FOR GIBBERELLIN, LEGUME NODULE DEVELOPMENT AUXIN AND STRIGOLACTONES IN LEGUME SYMBIOSES Ferguson B.J., Hastwell A.H., Li D., Mirzaei S., Reid D.E., Hayashi S., Batley J. and Gresshoff P.M. Foo E., Quittenden L.J., Hugill C. and Reid J.B. Center of Excellence for Integrative Legume Research, School of School of Plant Science, University of Tasmania, Hobart, 7001, Agriculture and Food Sciences, University of Queensland, St. Lucia, Australia. Brisbane, QLD, 4072, Australia. A major limit on food production is the availability of nutrients, Legume plants can enter into a symbiotic relationship with rhizobia especially the macronutrients nitrogen and phosphorous. Plants bacteria resulting in the formation of nitrogen-fixing root nodules. The employ strategies to maximise nutrient acquisition, including the host plant tightly regulates the number of nodules it forms following formation of specialised symbioses with soil microbes. We will present rhizobia-inoculation (autoregulation of nodulation) or nitrate-treatment new information on the central role of plant hormones in regulating the (nitrogen-regulation of nodulation). Both processes commence with two major symbioses formed by garden pea (Pisum sativum L.). The the production of a novel root-derived signal. We recently identified first is nodulation, the symbiosis between roots of mainly leguminous genes in soybean encoding CLAVATA3/ESR related (CLE) peptides plants and nitrogen-fixing rhizobial bacteria. The other is the much that exhibit increased expression following rhizobia inoculation more widespread symbiosis between plant roots and arbuscular (GmRIC1 and GmRIC2) or inhibitory nitrate treatment (GmNIC1). mycorrhizal fungi, which supplies previously inaccessible phosphate to Over-expression of these genes significantly reduces soybean the roots. We present evidence for novel roles for gibberellin, auxin and nodule numbers. The rhizobia-induced CLE peptides act systemically strigolactones in these symbioses from studies with hormone mutants, through the shoot, whereas the nitrate-induced CLE peptide acts direct hormone measurements and gene expression. Biologically locally in the root. Interestingly, all three CLE peptides, or a product active gibberellins suppress the formation of arbuscules, the nutrient of their action, are perceived by the same LRR receptor kinase, called exchange unit of the mycorrhizal symbiosis, and DELLA proteins are Nodulation Autoregulation Receptor Kinase (NARK). This perception essential for this response. We show that auxin promotes early events results in the production of a new factor that acts to inhibit further in mycorrhizal development, in part by interacting with strigolactones. nodule formation. Using comparative genomics, the orthologue of In addition to the well-known role of strigolactones as a stimulator of the GmNARK was identified in Phaseolus vulgaris (bean), making it the fungal partner, we present evidence for a new role for strigolactones first regulatory component of nodulation to be discovered in bean. A acting endogenously during mycorrhizal colonization and nodulation. novel mutant of Pvnark was also identified and used to demonstrate Finally, despite the widely discussed role for strigolactones in nutrient that the soybean CLE peptide GmRIC1 could function inter-specifically response, our studies with strigolactone-deficient pea mutants indicate via PvNARK in the autoregulatory pathway of bean. We also isolated that strigolactones are not essential for nutrient stress to regulate and phenotypically characterized a new soybean line mutated in the legume symbioses. paralogous gene of GmNARK, called GmCLAVATA1A. Comparisons between the genetic sequences and the genomic environments of GmCLAVATA1A and various orthologues of NARK revealed a number of interesting similarities. Findings regarding our progress in identifying and characterising the abovementioned nodulation factors will be presented.

Page 52 ComBio2013 s Perth, Western Australia s 29 September - 3 October, 2013 symposia TUESDAY

SYM-20-03 SYM-20-04 FINE ENDOPHYTES: IMPORTANT BUT UNDETECTED DROUGHT TOLERANCE IN ENDOPHYTE-INFECTED IN MOLECULAR ANALYSIS OF RESPONSE RYEGRASS - A TRANSCRIPTOMICS STUDY OF ARBUSCULAR MYCORRHIZAL FUNGAL Zhou Y.1, Schmid J.1, Johnson R.D.2, Hume D.E.2, Cox M.1, Dupont PY. 1 COMMUNITIES TO HOST PLANT SPECIES, SOIL TYPE and Bradshaw R.E.1 AND WATERLOGGING 1Bio-Protection Research Centre, Institute of Fundamental Sciences, College of Sciences, Massey University, Private Bag 11-222, Palmerston 2 Orchard S.1, Standish R.J.1, Nicol D.1, Vadakattu G.2 and Ryan M.H.1 North 4442, New Zealand. AgResearch, Grasslands Research Centre, 1School of Plant Biology, University of Western Australia, 35 Stirling Private Bag 11008, Palmerston North 4442, New Zealand. Highway, Crawley, WA6009. 2CSIRO, Ecosystem Sciences, PMB No Infection of the pasture ryegrass Lolium perenne by the endophytic fungus 2, Glen Osmond, South Australia, 5064. Neotyphodium lolii enhances grass performance, but its benefit on grass drought tolerance is influenced by host genotype. However, little is known Field studies highlight the influence of edaphic factors on both the regarding how the endophyte improves grass drought tolerance and why this community composition of arbuscular mycorrhizal (AM) fungi and effect varies among grass genotypes. Knowing this would help us to make their association with plants, yet few of these factors are tested under better use of endophytes, such as selecting and applying specific endophyte controlled conditions. We designed a glasshouse experiment to examine stains on grasses growing in arid areas; it would also increase our knowledge factors we identified in the field as influencing AM fungal colonisation: of this very important plant-microbe symbiosis. We selected a drought sensitive plant species, landscape zone and waterlogging. Ryegrass (Lolium (DS) and a tolerant (DT) N. lolii infected ryegrass genotype from the same rigidum) and lotus (Lotus subbiflorus) were grown in field soil from cultivar (Nine O One) in a glasshouse experiment. Endophyte-free grasses three zones. Waterlogging was imposed after 42 days. Percentage were generated from these and drought tolerance experiments involving root length colonised by AM fungi and their DNA concentration in soil genetically identical pairs of endophyte-infected (E+) and -free (E-) grasses were conducted in a controlled environment growth chamber, and both physiological were assessed 35 days later. AM fungal colonisation decreased with and transcriptome analyses were done. Physiological parameters (including waterlogging with the exception of that within lotus growing in lower relative water content, osmotic potential, Fv/Fm and biomass) were determined zone soils. DNA concentration was greatly reduced by waterlogging. and grass tissue (leaf, sheath, and root) was collected under two soil moisture Microscopic examination revealed a group of AM fungi probably conditions: 75% FC (field capacity) and 15% FC (maintained for one week to undetected by the DNA analysis, fine endophytes, in association with simulate drought conditions). The physiological results showed that endophyte lotus under waterlogged conditions. In summary, AM fungi can be enhanced drought tolerance in DT but not in DS plants. RNA was extracted from strongly influenced by small-scale spatial variation in plant species, grass sheath and duplicate cDNA libraries for each grass in each condition were soil type and waterlogging. Our study emphasises the importance of sequenced using Illumina next generation sequencing. More than 36 million 100 microscopic examination to complement molecular techniques to assist bp reads were obtained for each sample. Analysis of endophyte genes showed detection of fine and coarse endophytic AM fungi and so progress that 2,205 and 2,054 genes were differently expressed (> 2 fold) between towards improved understanding of their environmental niches. endophyte in DT and DS grass respectively, under drought conditions. Analysis of grass genes showed an increase in numbers of endophyte induced differently expressed genes in both DSE+ and DTE+ grasses under drought conditions (3,359 and 1,626 respectively) compared to well watered conditions (2,862 and 799 respectively). Initial functional analysis indicates that endophyte genes, involved in alkaloid production, antioxidant activity and sugar metabolism, and grass genes involved in photosynthesis, lipid biosynthesis and sucrose-starch metabolism were differently expressed. Further detailed analysis is in progress.

SYM-20-05 SYM-21-01 SYMBIOTICALLY EFFECTIVE N2-FIXATION ENHANCES IS MESOPHYLL CONDUCTANCE TO CO2 DIFFUSION 2-WAY NITROGEN-TRANSFER BETWEEN ROOT CO-REGULATED WITH LEAF WATER RELATIONS OR HEMIPARASITE SANTALUM ALBUM AND N2-FIXING PHOTOSYNTHETIC BIOCHEMISTRY? DALBERGIA ODORIFERA Barbour M.M. He X.H.1, 2, Lu J.K.3, Kang L.H.3 and Xu D.P.3 The University of Sydney. 1Institute of Agricultural Resources and Regional Planning, Beijing 2 100081, China. School of Plant Biology, UWA, WA 6009, Australia. C3 photosynthesis is significantly limited by the rate of diffusion of CO2 3Research Institute of Tropical Forestry, Guangdong 510520, China. between the leaf intercellular air spaces and chloroplasts. Mesophyll conductance (gm) has been shown to vary in response to environmental Little is known about nutrients movement between root hemiparasite conditions at both short and long time scales. Many of the reported responses mirror those of stomatal conductance (g ) leading to Santalum album and its N2-fixing host Dalbergia odorifera, both are s genuinely precious woody-trees but over-exploited globally. We show the suggestion that the two may be co-regulated. A comparison of the responses of g and g to changes in light, CO concentration, that N2-fixing D. odorifera is the best host to improve the S. album m s 2 growth, with higher tissue nutrition and photosynthetic rates, than temperature, vapour pressure deficit, water availability and leaf age across a wide range of species indicates that g is uncoupled N2-fixing Acacia confusa and non-N2-fixing Bischofia polycarpa or m 15 Dracontomelon duperreranum. With external N-labeling we then from gs in many cases, and is usually more closely correlated with photosynthetic rate. g decreases strongly as leaves age, and as water examine the role of N2-fixation in 2-way N-transfers between 7-month- m old Bradyrhizobium elkanii nodulated D. odorifera and its hemiparasitic availability and leaf temperature decrease. Interestingly, gm was more 15 S. album. Four potted-pairings were used, with N-labelling to either closely correlated to leaf hydraulic conductance (Kleaf) than to gs in host or hemiparasite and the host either nodulated or grown on Eucalyptus paniculata, when variability in Kleaf was caused by changes combined inorganic-N. Haustoria of S. album attached on D. odorifera in temperature and water availability. Significant genotypic variability in g is evident within wheat, and this extends to genotypic variability in roots and N2-fixation supplied 41-44% of total N inD. odorifera. Biomass, m 15 N and N were significantly greater in both nodulated D. odorifera and the degree of responsiveness of gm to light and CO2 concentration. A S. album grown with paired nodulated D. odorifera. Significantly higher new measurement technique demonstrates a strong decline in gm as 15 total plant N-recovery was in N-donor D. odorifera (68-72%) than leaves age in C4 plants, similar to C3 plants, and will allow quantification in N-donor S. album (42-44%) with regardless nodulation. Nitrogen of gm responses to environmental conditions in C4 plants. transfer to S. album was significantly greater (27.8-67.8 mg/plant) than to D. odorifera (2.0-8.9 mg/plant) and 2.4-4.5 times greater in nodulated than in non-nodulated pair. Irrespective of nodulation, S. album was the N sink plant. Amounts of 2-way N-transfer were increased by the presence of effective nodules, resulting in greater net N-transfers (22.6 mg/plant) from host D. odorifera to hemiparasite S. album. Our results may provide N management strategies for D. odorifera/S. album mixed plantations in the field.

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SYM-21-02 SYM-21-03 REGULATION OF WATER FLUX IN EXPANDING CELLS ACCLIMATION TO HUMIDITY MODIFIES THE LINK BY XYLEM DEVELOPMENT, SUCROSE METABOLISM BETWEEN LEAF SIZE AND THE DENSITY OF VEINS AND AQUAPORINS AND STOMATA

Ruan Y.-L. Carins Murphy M.R., Jordan G.J. and Brodribb T.J. School of Environmental & Life Sciences; University of Newcastle; School of Plant Science, University of Tasmania. NSW 2308. The coordination of veins and stomata during leaf acclimation to sun Cell expansion, essential for growth and development, is driven by influx and shade can be facilitated by differential epidermal cell expansion of water. Yet, the underlying mechanisms remain poorly understood. so large leaves with low vein and stomatal densities grow in shade, We use single-celled cotton fibre as a model to address this question. effectively balancing liquid- and vapour-phase conductances. As the Cotton fibre elongates to ~ 3 cm long in ~16 d after anthesis (DAA) difference in vapour pressure between leaf and atmosphere (VPD) and terminates elongation at ~20 DAA. Here we provide evidence determines transpiration at any given stomatal density, we predict that (i) despite water potential being more negative in leaves than that plants grown under high VPD will modify the balance between elongating fibres, backflow of water from expanding fibres is prevented veins and stomata to accommodate greater maximum transpiration. by a discontinuity in differentiating pedicel xylem strands of young Thus, we examined the developmental responses of these traits to fruit whilst phloem-derived water moves into fibres facilitated by local contrasting VPD in a woody angiosperm (Toona ciliata M. Roem.) and expression of plasma membrane intrinsic proteins (PIPs) and tonoplast tested whether the relationship between them was altered. High VPD intrinsic proteins (TIPs) and vacuolar invertase (VIN) that doubles the leaves were one-third the size of low VPD leaves with only marginally osmotic contribution of sucrose; (ii) xylem differentiation in the fruit greater vein and stomatal density. Transpirational homeostasis was pedicel correlates spatially and temporally with expression of sucrose thus maintained by reducing stomatal conductance. VPD acclimation synthase (Sus); (iii) xylem differentiation in the fruit pedicel is completed changed leaf size by modifying cell number. Hence, plasticity in vein by about 16 DAA, allowing backflow of water from fibres to leaves to and stomatal density appears to be generated by plasticity in cell size proceed thus terminating fibre cell elongation. Coincidentally, transcript rather than cell number. Thus, VPD affects cell number and leaf size and protein abundance of PIPs and TIPs in fibres were dramatically without changing the relationship between liquid- and vapour-phase reduced, which may help to impede the loss of cellular water that allows conductances. This results in inefficient acclimation to VPD as stomata completion of the major phase of fibre cellulose biosynthesis. Together, remain partially closed under high VPD. the data provide an example of systems regulation of water influx into and efflux from growing cells by coordinated actions of distal xylem development mediated by Sus and local expression of VIN and PIPs and TIPs.

SYM-21-04 SYM-21-05 LEAF HYDRAULIC CONDUCTANCE INCREASES VARIATION IN LEAF TEMPERATURE AND RATE WITH TRANSPIRATION RATE TO MINIMIZE WATER OF TRANSPIRATION AMONG WHEAT GENOTYPES POTENTIAL GRADIENTS GROWN UNDER TWO TEMPERATURE REGIMES

Simonin K.A.1, Choat B.2 and Franks P.J.1 Ranawana S.R.W.M.C.1, 2, Siddique K.H.M.2, Palta J.A.3 and Bramley H.2 1Faculty of Agriculture and Environment, University of Sydney, NSW 1School of Plant Biology, The University of Western Australia, 35 2570, Australia. 2Hawkesbury Institute for the Environment, University Stirling Hwy, Crawley, WA 6009, Australia,. 2The UWA Institute of of Western Sydney, Locked Bag 1797, Penrith 2751, NSW, Australia. Agriculture, The University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009. 3CSIRO Plant Industry, Centre for Environment Coordination between leaf gas-exchange parameters, hydraulic and Life Sciences, Underwood Avenue, Floreat, WA 6014. conductance and water potential has been observed across a wide range of plant species. Yet the mechanisms underlying this relationship High temperature is one of the most critical environmental variables remain uncertain. Here we tested the hypothesis that variation in that can lead to a drastic yield reduction in wheat. The canopy of a stomatal conductance (gs) and leaf hydraulic conductance (kleaf) are crop can be several degrees cooler than ambient air temperature due connected through the optimization of the water potential drawdown to evaporative cooling during transpiration. Therefore, transpiration across a leaf (ΔPsi(leaf)). Specifically, does short-term variation in kleaf, may be an important mode of temperature regulation in plants. Twenty when leaves are exposed to a variable environment, maximize level wheat genotypes with contrasting morpho-physiological characteristics gas exchange while minimizing ΔPsi(leaf). We evaluated the coordination and responses to heat and drought stress were assessed under between leaf and stem water potential, stomatal conductance, two temperature regimes to identify the genotypic variation in leaf transpiration rate and kleaf, over a diurnal cycle for three angiosperm and temperature and rate of transpiration. A pot experiment was carried out gymnosperm tree species growing in a common garden. Additionally, in two partially regulated glass-houses under well-watered conditions. the coordination between leaf gas-exchange parameters, kleaf and Instantaneous transpiration rate and stomatal conductance were ΔPsi(leaf) was evaluated as leaves desiccated. Over a diurnal cycle gs, measured along with leaf temperature. The results revealed significant transpiration and kleaf increased as leaf water potential decreased. A genotypic variation in leaf temperature and rate of transpiration. Some positive, non-linear relationship was observed between kleaf, gs and genotypes showed a higher leaf temperature depression compared to transpiration rate that ultimately minimized variation in ΔPsi(leaf). A others. Furthermore, a variation in leaf transpiration response to vapour similar relationship was observed as leaves desiccated. Traditionally, pressure deficit (VPD), which appears to be independent of stomatal increasing transpiration rate was thought mainly to lead to increased opening, was observed. The relationship between leaf temperature and probability of hydraulic failure, but our results suggest that within air temperature correlated well with the transpiration responses to VPD the zone of healthy xylem function, a positive dependence of kleaf in some genotypes, meaning that those genotypes whose transpiration on transpiration rate has several functional advantages. The strong rates increased more to VPD maintained lower leaf temperatures as air coupling between kleaf and transpiration rate suggests that leaves can temperature increased. However, in some genotypes, leaf temperature maximize gs for a given atmospheric demand by optimizing the water did not correlate with transpiration response. Those contrasting potential drawdown across a leaf. responses may be controlled by leaf physical attributes or water transport capacity of leaves and roots.

Page 54 ComBio2013 s Perth, Western Australia s 29 September - 3 October, 2013 symposia TUESDAY

SYM-22-01 SYM-22-02 THE ROLE OF GENE DUPLICATION AND ALLELIC ENVIRONMENTALLY INDUCED EPIGENETIC DIVERSITY IN ADAPTATION FOR HIGH SOIL BORON VARIATION AND INHERITANCE: PROGRAMMING THE RISK OF DISEASE Langridge P.1, Pallotta M.1, Schnurbusch T.1, 2, Hayes J.1, Hay A.1, Baumann U.1 and Sutton T.1 Cropley J.E.1, 2, Eaton S.A.1, 2, Li C.C.Y.1, Young P.E.1, Cowley M.J.3, 1Australian Centre for Plant Functional Genomics, School of Buckland M.E.4, Henstridge D.C.5, Cooney G.J.3, Febbraio M.A.5 and Agriculture, Food and Wine, University of Adelaide, Waite Campus, Suter C.M.1, 2 Urrbrae, South Australia 5064, Australia. 2Leibniz-Institute of Plant 1Victor Chang Cardiac Research Institute. 2Faculty of Medicine, Genetics and Crop Plant Research (IPK), Genebank Department, University of NSW. 3Garvan Institute of Medical Research. 4Discipline Corrensstr. 3, D-06466 Gatersleben, Germany. of Pathology, University of Sydney. 5Baker IDI Heart and Diabetes Institute. Boron has the narrowest range between deficient and toxic soil solution concentration of all plant nutrients, and boron deficiency and toxicity Exposure of parents to environmental factors can produce altered both severely limit crop production worldwide. In wheat, genetic phenotypes in their offspring, creating stable changes in physiology variation exists for tolerance to soil boron, and has been a long term that can have significant health consequences. A particularly pertinent priority for selection in wheat breeding programs in Australia. The exposure in the current health climate is overnutrition: an increasing major tolerance locus in wheat, Bo1, is located on 7BL while four QTL number of children are born to parents who are overweight or obese. for tolerance have been mapped in the barley genome. The wheat loci It is widely assumed that the mechanism underlying programmed and three of the four barley loci have been cloned and illustrate the phenotypes involves epigenetic changes to expression of metabolic processes that can now be used to identify and deploy variation. In genes, but evidence supporting this idea is limited. We use a mouse selecting for performance, early farmers around the Mediterranean, model of natural-onset obesity to examine the phenotypic and through the Middle East and into India and China developed wheat epigenomic consequences of parental obesity and diabetes. Our model landraces with varying levels of tolerance. A similar process was allows comparison of genetically identical mice whose parents were occurring in barley but the results were very different. Our data reveals either obese and diabetic, or lean with a normal metabolism. We have divergent evolution of B tolerance in wheat compared to barley. While found that the offspring both of obese mothers and of obese fathers in both species transcriptional regulation of B transporter genes is a carry a latent metabolic phenotype that is unmasked by exposure to common mechanism, tissue specificity differs and, in barley, tolerance a Western-style diet, resulting in glucose intolerance, defects in lipid is achieved by tandem gene duplication while in wheat gene duplication metabolism and hepatic steatosis. Remarkably, the altered glucose was combined with the generation of allelic diversity. It is curious that homeostasis is heritable into a second, unexposed, generation – the genetic basis for tolerance has developed so differently for these this heritability may be an important contributor to the current cycle two closely related species and the success of breeding for B tolerant of obesity. The first-generation offspring show changes in hepatic wheat versus barley may lie in the narrow allelic variation seen in barley gene expression and widespread but subtle alterations in cytosine compared to wheat. Matching B tolerance alleles to the level of B in methylation. These molecular changes do not point specifically to the environment appears to be critical but we now have germplasm, metabolic pathways but instead reside in broadly developmental markers and information on the mechanism of B tolerance. ontologies. This suggests that the changes may reflect generalised epigenomic damage that manifests most readily in metabolic disease, but may predispose to a variety of maladapted phenotypes.

SYM-22-03 SYM-22-04 REGULATION OF THE ITGA2 GENE IN PROSTATE IDENTIFICATION OF A NOVEL FAR-UPSTREAM CANCER ENHANCER OF SOX9 INVOLVED IN HUMAN DISORDERS OF SEX DEVELOPMENT (DSD) Chin S.P., Marthick J.R., Dickinson J.L. and Holloway A.F. Menzies Research InstituteTasmania, University of Tasmania, Hobart, Ohnesorg T.1, Tan J.1, Bowles J.2, Koopman P.2 and Sinclair A.1 Tasmania, 7000. 1Murdoch Childrens Research Institute, Parkville, VIC, Australia. 2Institute for Molecular Bioscience, Brisbane, QLD, Australia. A significant proportion of the morbidity and mortality associated with prostate cancer can be attributed to metastasis. Previous work SOX9 is a transcription factor essential for the formation of functional from our group has identified integrin alpha 2 (ITGA2) as a putative testes from bipotential gonads. Recent reports of 46,XX testicular DSD prostate cancer susceptibility gene, and there is increasing evidence (female-to-male sex reversal) patients carrying duplications and 46,XY suggesting it is involved in prostate cancer progression and bone gonadal dysgenesis patients carrying deletions ~500-600kb upstream metastasis. However, little is known about how ITGA2 gene expression of SOX9 indicate the presence of a gonad specific enhancer of SOX9 is controlled, and we are therefore investigating the transcriptional and in this region. Using a comprehensive luciferase tiling approach epigenetic regulation of ITGA2 in prostate cancer. A large CpG island we identified two novel putative enhancers within this region. The exists in the ITGA2 promoter and differential methylation is associated enhancer with the strongest activity in vitro was further analysed and with differences in gene expression in prostate cancer cell lines. High used to generate transgenic reporter mice. We show that this enhancer DNA methylation and low chromatin accessibility correlates with lower mediates SOX9 auto-regulation in vitro and leads to strong reporter gene expression in the non-invasive LNCaP compared to the highly gene expression in embryonic gonads at the time of sex determination metastatic PC3 cells. This is also consistent with the DNA methylation and gonad differentiation. Our results strongly suggest that copy pattern observed in prostate tumour samples compared to normal number variations of this enhancer lead to DSD. prostate tissue. Reporter gene assays confirmed that the ITGA2 promoter is susceptible to silencing by DNA methylation. Multiple Sp1 transcription factor binding sites are found across the ITGA2 promoter and Sp1 activates the promoter in reporter assays. However regulation of the promoter by Sp1 is unaffected by DNA methylation and does not explain the differential expression of the ITGA2 promoter in prostate cancer cell lines. In contrast, expression of ITGA2 in prostate cancer cell lines inversely correlates with expression of the EMT-associated Snail1 transcription factor and Snail1 overexpression represses ITGA2 promoter activity in reporter assays. A potential Snail binding site is found in the ITGA2 promoter in a region of differential methylation, suggesting that Snail and epigenetic factors may regulate the ITGA2 promoter in prostate cancer.

ComBio2013 s Perth, Western Australia s 29 September - 3 October, 2013 Page 55 symposia TUESDAY

SYM-22-05 SYM-23-01 IDENTIFICATION OF A NOVEL SMALL RNA PATHWAY TO AFFINITY AND BEYOND. A STRATEGY TO IN TUMOUR ANGIOGENESIS ELABORATE “FRAGMENTS” INTO MORE POTENT LIGANDS Plummer P.N.1, Balsara S.1, Umer B.1, Ferro V.2, Mittal V.3 and Mellick A.S.1 1Host Response to Cancer Laboratory, Griffith University, Parklands Adams L.1, Vazirani M.1, Mohanty B.1, Ilyichova I.1, Mulcair M.1, Dr, Gold Coast, QLD, Australia. 2School of Chemistry and Molecular Williams M.L.1, Heras B.2, Simpson J.S.1 and Scanlon M.J.1, 3 Biosciences, University of Queensland, St Lucia QLD, Australia. 1Monash Institute of Pharmaceutical Sciences, 318 Royal Parade, 3Neuberger Berman Lung Cancer Centre, Weill Cornell University Medical Parkville, VIC 3052. 2Department of Biochemistry, La Trobe College, 525E 68th St, 1300 York Ave, New York, NY, USA. University, Bundoora, VIC 3086. 3ARC Centre of Excellence for Coherent X-ray Science, Monash University, Parkville, VIC 3052. Tumour angiogenesis is an important process in the development, growth and spread of tumours. This is a process in which the host vasculature is Fragment-based screening (FBS) has emerged as a mainstream enrolled to the tumour, resulting in new vessel development in response to approach for the rapid and efficient identification of key building-blocks growth factors. There are several anti-angiogenic therapies currently used that can be used to develop high-affinity ligands against a specific for cancer treatment, however these only extend the life of patients for a protein target. One of the attractions of FBS is the relative ease and limited period of time due to the development of adaptive resistance and low cost of the primary screen to identify fragments that bind. However, serious side-effects. Bone marrow (BM)-derived endothelial progenitor cells fragments that emerge from primary screens often have low affinities (EPCs) play an important role in tumour angiogenesis and are mobilized (KD values in the high μM to mM ranges). Therefore a significant in response to growth factors. We have previously showed EPCs migrate challenge for FBS is to develop the initial fragments into more potent from the BM to sites throughout the body, including the BM compartment of ligands. To do so generally requires structures of the fragments the tumour-stroma1,2. Here we report the identification of a novel small RNA pathway required for regulation of EPC function and tumour angiogenesis. bound to their target proteins as well as the availability of a range of In this pathway microRNA (miR)-10b is up-regulated in EPCs, and tumour biophysical and biochemical assays to track potency and efficacy. The vasculature in response to tumour challenge. The expression of miR-10b low potency of the initial “hits” requires that these assays be conducted was also found to be in the vasculature of aggressive late stage invasive in the presence of high concentrations of the fragments, which in turn ductal carcinoma using a novel fluorescent in situ hybridization method often necessitates the use of co-solvents to maintain the fragment developed by our lab. We have also demonstrated that miR-10b can be solubility. These requirements render the assays susceptible to artifacts targeted in vivo using an anti-miR encapsulated in stealth liposomes and therefore a more potent ligand is desirable as a positive control. tagged with an RGD-peptide (to target tumour vasculature). This resulted However, for new targets, it is often the case that no suitable high- in a significant decrease in tumour growth and EPC mobilization1. We affinity ligand is available. In this presentation I will discuss some of the are currently altering this system so that the liposome will be tagged with pitfalls that can be encountered in developing ligands from FBS hits in a novel EPC-peptide, thus resulting in directed delivery of the anti-miR to such a situation and describe a strategy that we have implemented to EPCs. This method is believed to target tumour vasculature formed during enable weakly-binding fragment hits to be elaborated into more potent angiogenesis, as opposed to normally occurring vasculature within the body. ligands. It is proposed that this approach may lead to new anti-angiogenic therapies which significantly decrease the adverse side-effects associated with current anti-angiogenic treatments. 1. Plummer PN, Freeman R, et al. Cancer Research (2013) 73:341-52. 2. Mellick AS, Plummer PN, et al. Cancer Research (2010) 70:7273-82.

SYM-23-02 SYM-23-03 INHIBITOR DESIGN OF CARBOHYDRATE- Sponsored by RECOGNISING PROTEINS The University of Western Australia

Blanchard H. DEVELOPING TOOLS TO STUDY LIPID SIGNALLING Institute for Glycomics, Griffith University. MOLECULES

Carbohydrate-recognising proteins (Lectins) have significant roles in Flanagan J.U.1, 2, Smythe M.L.3, Sweet M.J.3, Kulis C.3, Kendall J.D.1, 2, disease. These include facilitating interactions between pathogens and Rewcastle G.W.1, 2, Denny W.A.1, 2 and Shepherd P.R.4, 2 host cells during infection and also promoting metastasis in cancer. 1Auckland Cancer Society Research Centre, The University of The design of inhibitors of lectins is important in reducing or eliminating Auckland, New Zealand. 2Maurice Wilkins Centre for Biodiscovery, serious disease but is often challenging due to the nature of their ligand/ The University of Auckland, New Zealand. 3Institute for Molecular inhibitor binding sites. This presentation focuses on our structure- Bioscience, The University of Queensland, Australia. 4School of based design of inhibitors of lectins, including the galectins that are Medical Sciences, The University of Auckland, New Zealand. involved in assisting cancer cell evasion of the immune response enabling increased tumour survival and metastasis. Crystallographic Lipid signalling molecules have key functions in a range of diseases structures of these lectins in complex with inhibitors will be presented from inflammation to cancer. We are interested in developing tools that giving insight into the progression of design of potent inhibitors. probe the function of these molecules by blocking the enzymes that synthesize them. The enzymes of interest include the hematopoietic prostaglandin D2 synthase (HPGDS) enzyme, the major source of prostaglandin D2 during allergic and asthmatic responses, and the oncogenic phosphatidylinositol-3-kinase (PI3K) enzyme p110α. New HPGDS enzyme inhibitors developed by fusing different compounds will be presented along with the use of protein structure in identifying interactions between p110α and its inhibitors that control potency and specificity.

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SYM-23-04 SYM-23-05 GIBBS FREE ENERGY FORMULA: THE FIRST INHIBITOR PEPTIDE DESIGN – IMPROVING AFFINITY PRINCIPLE OF AB INITIO PROTEIN STRUCTURE WITHOUT LOSING SPECIFICITY PREDICTION Gunzburg M.J.1, Kulkarni K.1, 2, Brandt R.1, Watson G.1, Lim R.1, Payne R.3, 2 1 Fang Y.1, 2 Perlmutter P. , Wilce M.C. and Wilce J.A.1 1Nanchang University. 2Australian National University. 1Department of Biochemistry and Molecular Biology, Monash University, VIC, Australia. 2School of Chemistry, Monash University, VIC, Australia. 3 Anfinsen’s thermodynamic principle of protein folding says that the School of Chemistry, The University of Sydney, NSW, Australia. native structure has the minimum Gibbs free energy which depends only on the protein’s peptide chain. There was no theoretically derived A challenge in the development of inhibitors of intracellular targets is to Gibbs free enery formula G(X), for any conformation X of the protein. achieve specificity and high affinity for those targets. Here peptides, that Some even deny there is such a function. Many developed emperical can mimic specific protein-protein interaction surfaces, can provide the formulas to substitute G(X). No wonder ab inito prediction has no answer, so long as cell permeability and stability issues are addressed. success, it lacks firm theoretical basis. We will show how to apply Here we describe progress in developing a specific inhibitor of the quantum statistics to derive G(X) which only depends on the peptide Grb7-SH2 domain involved in cancer progression. Grb7 is an adapter chain of the protein. Supprisingly G(X) does not include pairwise protein, aberrantly co-overexpressed with erbB-2 and identified as poteintials commonly used in all force fields, but levels of hydrophobicity an independent prognostic marker in breast cancer. Grb7 signals the play importantly roles. With theoretically established G(X), ab initio activation of erbB-2 and erbB-3, which play key roles in disregulated structure predicting not only becomes possible, but also simple, cell growth in cancer. Grb7 also mediates signalling pathways from seeking the minimizers of G(X). No need of searching landscapes, just another tyrosine kinase, focal adhesion kinase (FAK) exacerbating cell migration and the metastatic potential of cells. It is thus a prime following the -DG (the gradient of G) from any initial conformation X0 to X = X - sDG(X ), where s is a small positive number and i = 0, 1,..., target for the development of novel anti-cancer therapies. We have i+1 i i characterised a non-phosphorylated cyclic peptide (G7-18NATE) that the native structure will be reached eventually. The initial X0 determines the pathway. If a protein’s inital confomations varis wildly, one will is a specific inhibitor of Grb7 and inhibits cellular growth and migration observe multple pathways. The -DG is generalized force in physics, it in cancer cell lines. X-ray crystallographic structure determination of gives insight to the kinetic procedure of protein folding. The derivation the G7-18NATE/Grb7-SH2 domain complex and binding studies using of G(X) reveals that to obtain G(X) one has to take a single X and its surface plasmon resonance have revealed the basis of affinity and immediate environment to form the thermodynamic system, againist specificity of the peptide1-3. Here we describe how this information has the intuition that statistics only deals large quantities of molecules. After been used to successfully design second generation bicyclic peptides 20 years of single molecule experiments, this is the begining of a single that show enhanced binding without loss of specificity. These peptides molecule theory of protein folding. Current G(X) is only true for self- can be coupled to cell penetrating peptide sequences to be taken up folding single domain globular proteins, other proteins will have similar by cells, lowering cell growth and migration. We anticipate that these but more complicated G(X). Grb7 inhibitor peptides will form the basis of novel therapeutics that can be used in conjunction with existing therapies against breast cancer. References 1. Ambaye ND et al., (2011) J. Mol. Biol. 412, 397-411. 2. Gunzburg MJ et al., (2012) J. Mol. Recog. 25, 57-67. 3. Gunzburg et al., (2013) Biopolymers. Mar 15.[Epub ahead of print].

SYM-24-01 SYM-24-02 SENESCENT ENDOTHELIAL CELLS SHOW AN UNIQUE INFLAMMATION TRIGGERED BY THE NLRP1 AND ANTI-INFLAMMATORY PHENOTYPE NLRP3 INFLAMMASOMES

Gamble J.R.1, 2, Vadas M.A.1, 2, Powter E.1, Coleman P.1, Lay A.1 and Masters S.L. Hutas G.1, 2 The Walter and Eliza Hall Institute. 1Centenary Institute, Newtown, NSW. 2University of Sydney, Newtown, NSW. The NLRs (Nucleotide-binding domain and Leucine-rich repeat containing Receptors) constitute a large family of intracellular proteins Inflammation and senescence have been linked at both the clinical that regulate innate immune defense. Upon activation, NLRs form and molecular levels. In general, senescent cells have been described protein complexes called “inflammasomes” that dimerize caspase-1, as pro-inflammatory based on their senescence associated secretory resulting in proteolytic activation of the cytokines pro-IL-1β and pro- phenotype (SASP). However, endothelial cells when induced to IL-18, and causing a caspase-1-dependent form of cell death known become senescent by three of the chief signals associated with as pyroptosis. The activation of one family member, NLRP3, is well ageing; oxidative stress, disturbed flow and hypoxia display both studied, and we have shown that an amyloid present in the pancreas anti-inflammatory and pro-inflammatory senescent phenotypes. The (IAPP) may trigger this pathway during type 2 diabetes. We have also anti-inflammatory phenotype is also seen in senescence induced by identified negative regulation of NLRP3 by an endogenous microRNA overexpression of ARHGAP18 or SENEX (Blood Coleman et al 2010). (miR-223) and a viral microRNA (EBV-mir-BART15). In contrast, very The mosaic of inflammatory states is assessed at the individual cellular little is known about NLRP1 despite human genetic associations with level and is based on the classic criteria for inflammation in endothelial autoimmune disease and infection. We have now studied mice with cells- leucocyte adhesion and expression of the associated adhesion an activating mutation in NLRP1a, and made mice that are genetically molecules and cytokines. One population of senescent cells is deficient for all NLRP1 alleles. Activation of NLRP1a causes a lethal powerfully anti-inflammatory while the remainder of the senescent cells systemic inflammatory disease which is driven by Caspase-1 and behave like normal endothelial cells, are responsive to inflammatory IL-1β but was independent of ASC and negatively regulated by IL- stimuli, such as TNFα, and become pro-inflammatory. Senescence 18. Surprisingly, in the absence of IL-1β-driven inflammation, active is associated with an increase in cell size, an increase in number of NLRP1a triggered pyroptosis of progenitor cells resulting in leukopenia caveolae with an increase in expression of the caveolae proteins, at steady state. Furthermore, during periods of hematopoietic stress caveolin-1, cavin-1 and cavin-2 and a decrease in NFkB signaling, induced by chemotherapy or lymphocytic choriomeningitis virus consistent with caveolae being negative regulators of the inflammatory (LCMV) infection, active NLRP1a caused prolonged cytopenia, bone state. Depletion of the caveolae proteins by siRNA reduced the number marrow hypoplasia and immunosuppression. Conversely, NLRP1- of senescent cells but interestingly this was a preferential reduction in deficient mice showed enhanced recovery from chemotherapy and the anti-inflammatory senescent cells and a corresponding increase in LCMV infection. In summary we show for the first time in vivo the effect the pro-inflammatory population. Thus we hypothesize that the anti- of an activating mutation in NLRP1 as well as NLRP1 deficiency in inflammatory population of senescent endothelial cells may have an response to hematopoietic stress induced by infection. We demonstrate unique protective role, to inhibit uncontrolled proliferation and to limit that this inflammasome activity induces caspase-1 dependent the local inflammatory response. pyroptosis of hematopoietic progenitor cells, suggesting that NLRP1 acts as a cellular sentinel to alert caspase-1 to hematopoietic and infectious stress.

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SYM-24-03 SYM-24-04 A NOVEL ROLE FOR INTERLEUKIN 2 IN REGULATING THE RABIES VIRUS INTERFERON ANTAGONIST PULMONARY TYPE 2 INFLAMMATION P-PROTEIN INTERACTS WITH ACTIVATED STAT3 AND INHIBITS GP130 RECEPTOR SIGNALLING Roediger B.1, Tay S.S.1, Mitchell A.J.1, Bolton H.A.1, Guy T.V.1, Fazekas De St. Groth B.1, 2 and Weninger W.1, 2, 3 Lieu K.1, 2, Brice A.1, Wiltzer L.1, 2, Hirst B.1, Jans D.A.2, Blondel D.3 and 1The Centenary Institute, Newtown, NSW 2042, Australia. 2Discipline Moseley G.W.1 of Dermatology, University of Sydney, Camperdown, NSW 2006, 1Viral Pathogenesis Laboratory, Department of Biochemistry and Australia. 3Department of Dermatology, Royal Prince Alfred Hospital, Molecular Biology, Monash University, Victoria 3800, Australia. Camperdown, NSW 2050, Australia. 2Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia. Type 2 cytokine-dependent immune responses are essential for 3Laboratoire de Virologie Moleculaire et Structurale, CNRS, Gif sur protection against helminth infections but also underlie the pathology Yvette, France. of numerous allergic diseases such as eczema and asthma. While the central role of CD4+ type 2 helper T cells in allergic disorders has been The type I interferon (IFN)-mediated host immune response is the well established, the innate signalling pathways that contribute to type primary defense against viral infection through the production of IFNα/β, 2 inflammation remain incompletely understood. We have identified a which bind to receptors on infected and neighbouring cells to activate hitherto undescribed role for the cytokine IL-2 in type 2 inflammation intracellular signal transducers and activators of transcription STAT1 in the lung. IL-2 was required for the spontaneous development of and 2 proteins. Activated STAT1/2 translocate to the nucleus to activate eosinophilic crystalline pneumonia in immunodeficient mice, a condition gene expression leading to the establishment of a potent antiviral state. characterised by increased type 2 cytokines, alternatively activated Subversion of the IFN system is thus a key component of pathogenic macrophages, eosinophils and group 2 innate lymphoid cells (ILC2). viral infection and is mediated by virus-encoded IFN-antagonist proteins. Additionally, in vivo administration of IL-2 promoted ILC2 expansion Immune evasion by the lethal rabies virus (RABV) depends on specific and activation in the lung, which was associated with an increased targeting of activated STAT1/2 by the IFN-antagonist P-protein, but eosinophilic infiltrate. This response to IL-2 was the result of a direct targeting of other STATs has not been investigated. Using a combination effect on ILC2, which express the high affinity IL-2 receptor, CD25. of quantitative confocal microscopic analysis and immune signalling Strikingly, IL-2-driven activation of ILC2 promoted the development of assays, we investigated the potential role of P-protein in targeting STAT3, consolidated lung lesions within treated mice, demonstrating that ILC2 which is activated both by IFNs and by IL6-family cytokines such as can represent a potent effector population in type 2 cytokine-mediated oncostatin M (OSM) through Gp130-dependent signalling. P-protein pulmonary inflammation. expression was found to significantly inhibit cytokine-activated STAT3 nuclear translocation, and immunoprecipitation assays indicated that this correlates with a specific interaction between P-protein and activated STAT3 that appears to be unique to RABV. Critically, we found that RABV infection or P-protein expression significantly inhibits OSM/Gp130- dependent signalling, indicating that STAT3 antagonism may be important to RABV pathogenesis. Our current research is focused on understanding the mechanisms involved in P-protein-STAT3 targeting, which may aid in the development of new vaccines or therapeutic approaches to combat currently incurable rabies disease.

SYM-24-05 EFFECT OF UV RADIATION ON SIGNALLING PATHWAYS AND THE RELEASE OF TNFα FROM HUMAN KERATINOCYTE CELL LINES

Muthusamy V., Ravi R. and Piva T.J. School of Medical Sciences, RMIT University, Bundoora 3083, Victoria, Australia.

Ultraviolet (UV) light is main carcinogen involved in the formation of skin cancer. UV radiation is a potent inducer of TNFα and cytokine gene expression in human keratinocytes. TNFα plays an important pro- inflammatory role in the skin, and is cleaved from its precursor by the action of Tumor Necrosis Factor-α Converting Enzyme (TACE). TACE is cleaved from its preproform by furin, a proprotein convertase. The effect UV radiation has on the intracellular signalling pathways and that of furin’s expression and/or activity in keratinocytes is not known. The major signalling pathways activated by UV radiation are p38 MAPK, JNK and NF-κB. We investigated the effect 1 MED UV radiation [UVA (40 KJ/m2), UVB (2 KJ/m2) and UVAB (40+2 KJ/m2)] had on the expression of p-p38, p-JNK, NFκB as well as the activity of furin, and secretion of TNFα were examined using human primary keratinocytes (HEK), immortalised HaCaT cells as well as the squamous cell carcinoma Colo16 cell line. Colo 16 cells were more sensitive to high dose UVB and UVAB radiation than were HaCaT or HEK cells, though neither cell line was sensitive to UVA radiation. p38 MAPK was the major signalling pathway activated by UVA and/or UVB in all cell lines, though the level of activation differed. Only in the presence of IL1α did UVB and UVAB radiation induce maximal secretion of TNFα from HEK cells with lesser amounts secreted from the other cells. Of interest was that UV radiation increased furin expression in Colo16 and HaCaT cells which did not correspond to that of TNFα released from these cells. The differences observed between HaCaT cells and that of HEK cells raise questions about the use of these cells in studies on keratinocyte cell biology, the implications of which will be discussed.

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SYM-25-01 SYM-25-02 A DUAL ROLE FOR THE E3 LIGASE E6AP IN THE THE LOSS OF C-CBL E3 UBIQUITIN LIGASE ACTIVITY REGULATION OF CELLULAR SENESCENCE PROMOTES THE DEVELOPMENT OF ACUTE MYELOID LEUKAEMIA IN FLT3-ITD MUTANT MICE Wolyniec K., Levav-Cohen Y., Young R., Russell P., Wright G., Solomon B., Do H., Dobrovic A., Haupt S. and Haupt Y. Taylor S.J., Thien C.B.F., Dagger S.A., Duyvestyn J.M. and Langdon W.Y. Peter MacCallum Cancer Centre, Melbourne 3002, Victoria, Australia. University of Western Australia.

The E6-Associated Protein (E6AP) is known for its role in promoting FLT3 is a receptor tyrosine kinase (RTK) expressed on haematopoietic p53 for degradation in HPV-infected cells. We have recently revealed a progenitors and is mutated in approximately 30% of acute myeloid novel function of E6AP in the regulation of cellular senescence in mouse leukaemias (AML). The most common mutation involves internal tandem embryonic fibroblasts (MEFs) by modulating the p53 pathway. E6AP duplications (ITD) within the juxtamembrane domain of FLT3 causing deficient MEFs bypass oncogene- and stress-induced senescence and constitutive activation and mislocalization in the ER/Golgi network. display accelerated tumourigenesis in transplanted mice. In a search A key consequence of these perturbations is the aberrant activation for a molecular explanation for this impairment, we found that in E6AP of STAT5, a transcriptional regulator that is not activated by wild-type knockout MEFs the expression of the tumour suppressors encoded FLT3. c-Cbl is RING finger based E3 ubiquitin ligase that targets by the INK4/ARF locus is reduced. A mechanistic explanation for the ligand-activated wild-type FLT3 for degradation. c-Cbl is mutated in a regulation of the INK4/ARF locus by E6AP, which controls cellular range of haematopoietic neoplasms, and a c-Cbl RING finger knock- senescence via the p16-pRb and p53 pathways, will be presented. in mouse develops a severe myeloproliferative disease that involves To examine the relevance of our findings to human cancer we chose enhanced FLT3 signaling. We have recently crossed the c-Cbl RING non-small cell lung cancer (NSCLC) as a cancer model, in which finger knock-in mouse with a FLT3-ITD knock-in to determine if the two the p16 gene is frequently silenced. We found that the regulation of mutations cooperate to enhance leukaemia development. We find mice cellular senescence by E6AP occurs in a cohort of NSCLC patients and transplanted with fetal liver cells from double mutant embryos develop a correlates to poor survival. Overall, our study explores a new pathway rapidly fatal AML, a phenotype that is markedly more severe than either to the silencing of the INK4/ARF locus, which provides an explanation of the single mutations. The effects of the c-Cbl RING finger/FLT3-ITD for how E6AP controls cellular senescence via the p16-pRb and p53 double mutation on haematopoietic progenitors will be discussed. pathways.

SYM-25-03 SYM-25-04 CULLING BAD MITOCHONDRIA: THE MOLECULAR A BALANCING ACT BETWEEN THE CHAPERONE MECHANISMS OF PARKINSON’S DISEASE GENES NASP AND LYSOSOMAL DEGRADATION TO FINE- PINK1 AND PARKIN TUNE HISTONE SUPPLY AND MAINTAIN GENOME INTEGRITY Lazarou M., Jin S.M., Kane L.A., Narendra D.P. and Youle R.J. Biochemistry Section, Surgical Neurology Branch, National Institute Cook A.J.L.1, 2, 3, Gurard-Levin Z.1, Vassias I.1, Tremethick D.2 and of Neurological Disorders and Stroke, National Institutes of Health, Almouzni G.1 Bethesda, Maryland, United States. 1Institut Curie & CNRS UMR218, 75248 Paris cedex 05, FRANCE. 2John Curtin School of Medical Research, Australian National Mitochondrial dysfunction appears to be a central player in the University, Canberra ACT, AUSTRALIA. 3Centenary Institute of pathogenesis of sporadic Parkinson’s disease (PD). The ubiquitin Cancer Medicine & Cell Biology, Camperdown NSW, AUSTRALIA. E3 ligase Parkin and the mitochondrial kinase PINK1 are mutated in familial PD and were recently shown to function together to maintain Proper genome packaging and thus genome stability requires mitochondrial health by clearing defective mitochondria. PINK1 coordination of both DNA and histone metabolism. While histone gene accumulates on the outer membrane of damaged mitochondria transcription and RNA processing can respond to scheduled changes where it recruits Parkin from the cytosol and activates Parkin’s ligase in demand, how histone supply adjusts to unexpected changes in activity. Parkin conjugates ubiquitin to a variety of proteins on the demand remains is less clear. We recently revealed a role for the histone outer membrane to promote mitochondrial elimination by selective chaperone Nuclear Autoantigenic Sperm Protein (NASP) to protect a autophagy and preserve the health of the mitochondrial population. Our reservoir of soluble histones H3-H4 in mammalian somatic cells. The studies reveal how PINK1 senses mitochondrial damage and identify reservoir can be fine-tuned, increasing or decreasing depending on the a PINK1-TOM complex that down regulates PINK1 when damaged level of NASP. Our data suggest that NASP does so by balancing the mitochondria regain function. Generation of a cell free ubiquitination activity of the heat-shock proteins Hsc70 and Hsp90 to direct H3-H4 assay recapitulating PINK1 dependent activation of Parkin enabled us for degradation by chaperone-mediated autophagy. The importance of to study Parkin’s catalytic mechanism. We show that PINK1 activates NASP is revealed upon histone over-load, engagement of the reservoir the formation of a Parkin-ubiquitin thioester intermediate that is upon acute replication stress and when Asf1 activity is perturbed. These essential for Parkin activity and translocation to mitochondria. Prior insights into NASP function and the existence of a tunable reservoir in to formation of the thioester enzyme intermediate, PINK1 stimulates mammalian cells demonstrate that contingency is integrated into the Parkin to self-associate and form a complex upstream of mitochondrial histone supply chain to respond to unexpected changes in demand. binding shedding light on how Parkin is activated. Taken together Curiously, NASP isoform expression is misregulated in cancer cells our studies describe the mechanisms that regulate PINK1/Parkin and it is well-established that improper histone supply causes genome mitochondrial quality control paving the way to a better understanding instability, a hallmark of cancer, hinting that NASP plays a functional of PD pathogenesis. role in cancer. Our recent efforts to address this question and a putative role for NASP in responding to genome instability in cancer cells will be presented.

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SYM-25-05 SYM-26-01 DEFINING THE ROLE OF THE E3 LIGASE UBR5 DESIGNING A BIO-DESALINATOR IN CANCER: AN INTEGRATED APPROACH TO SYSTEMATIC IDENTIFICATION OF SUBSTRATES Honsbein A.1, Madsen M.A.1, Minas K.2, Karunakaran E.3, Bond T.4, Gandy C.5, Amezaga J.5, Templeton M.4, Biggs C.3, Lawton L.2 and 1 Iconomou M., Shearer R., Uhl F., Ali N., Daly R. and Saunders D.N. Amtmann A. 1Molecular, Cellular & Systems Biology, University of Glasgow, UK. Kinghorn Cancer Centre, Garvan Institute of Medical Research, 2 3 Sydney, Australia. Life Sciences, Robert Gordon University Aberdeen, UK. Chemical & Biological Engineering, University of Sheffield, UK.4 Civil & 5 Altered expression and somatic mutations of the HECT E3 ligase Environmental Engineering, Imperial College London, UK. Civil UBR5 (EDD) have been observed in numerous cancer types (e.g. Engineering & Geosciences, University of Newcastle, UK. breast, ovarian, lymphoma) and UBR5 modulates chemoresistance in ovarian cancer, likely through regulation of the DNA damage While water covers three quarters of the earth’s surface less than response. Components of the UPS, including E3 ligases, have 0.5% of it is freshwater. Increasing global population, industrialization become an attractive target for development of novel cancer therapies. and particularly agriculture exert significant pressures on this limited Identification of E3 ligase substrates is key to defining their biological resource. With the aim to unlock the vast water resource in the oceans, function and understanding their role in disease. However, even with there is increasing interest in desalination technologies. However, advances in proteomics and in vitro assays, substrate identification current technology, based on physicochemical processes, is a highly remains a significant challenge. To this end, we have developed an energy demanding process and its application is limited to fuel-rich integrated approach to systematically define high-confidence E3 and affluent countries. We have turned our attention to biological ligase substrates, combining novel cell models with proteomics-based mechanisms to remove NaCl from seawater (‘bio-desalination’). detection of ubiquitylated peptides and interacting proteins, and BiFC- Marine organisms employ energy-consuming transport processes to based detection of protein-protein interactions in situ. UBR5 interacting maintain low sodium concentrations inside their cells. The energy for proteins were isolated using GFP-Trap affinity purification followed by this natural desalination ultimately comes from sunlight harvested by tandem MS based identification and label free quantitation. We identified photo-autotrophic organisms at the bottom of the marine food chain. 198 putative UBR5-interacting proteins with good reproducibility. Initial Based on available information on ion flux rates through individual analysis of differentially ubiquitylated peptides following UBR5 depleted transport proteins and their abundance in cell membranes, and taking by shRNA in a breast cancer cell line using di-Gly affinity purification into account the total cell surface area and volume generated by high- identified approximately 400 individual ubiquitlyation sites. We are now density bacterial cultures, we propose that the energized low-sodium extending these studies using BiFC to characterize putative interactions internal volume of photosynthetic microbial cultures can be used as and ubiquitylation in in situ, ubiquitin ligase activity assays on high- an ion exchanger to remove NaCl from the surrounding seawater. This density protein microarrays, and investigating the role of various hypothesis is currently put to the test in a multi-pronged experimental functional domains in UBR5 using disease-specific mutants. These project carried out by an interdisciplinary team of biologists and orthogonal but complementary approaches are providing interesting engineers. We are generating the biological tools that enable us to new insights into the function of E3 ligases in cellular signaling, with control membrane transport in cyanobacteria, and we are designing a implications for understanding regulation of DNA damage response, simple and energy-efficient process for growth, exposure and removal metabolic reprogramming and other key pathways important in tumour of the cultures in/from the seawater. biology.

SYM-26-02 SYM-26-03 EXPRESSION OF TIBETAN WILD BARLEY HVHKT1 GABA-GATED ANION CHANNELS IN PLANTS REGULATES ROOT ION HOMEOSTASIS AND CONFERS SALT, OSMOTIC AND OXIDATIVE STRESS Ramesh S.A.1, 2, 3, Tyerman S.D.1, 2, 3, Ryan P.R.4 and Gilliham M.1, 2, 3 TOLERANCE IN ARABIDOPSIS 1ARC Centre of Excellence in Plant Energy Biology. 2School of Agriculture, Food and Wine, The University of Adelaide. 3Waite 4 Han Y.1, Chen Z.H.2, Yin S.1, Wu X.1, Liu X.H.2 and Zhang G.1 Research Institute. CSIRO Plant Industry, Canberra, ACT 2601. 1Agronomy Department, Key Laboratory of Crop Germplasm Resource of Zhejiang Province, Zhejiang University, Hangzhou Gamma-aminobutyric acid (GABA) is a neurotransmitter regulating 310058, China. 2School of Science and Health, Hawkesbury Campus, membrane potential in nerve cells. GABA rapidly accumulates in University of Western Sydney, Richmond, 2753 NSW, Australia. plant tissues in response to various stresses, and regulates important processes such as pollen tube growth, root and hypocotyl elongation, K+/Na+ homeostasis regulated by HKT transporters is one of the key and pathogen defence. There is much speculation that GABA signalling components of salinity tolerance in plants. ~70% of Tibetan wild barley occurs in plant cells but definitive proof has been lacking, and the (Hordeum spontaneum) lines are more tolerant to salt than a salt-tolerant molecular components have remained elusive. We have identified cultivar CM72. Under salt stress, the relative transcripts of HvHKT1 in GABA-binding sites within plant Aluminium-activated Malate Transporter the roots were up to 4-fold higher in Tibetan wild barley X16 than those (ALMT) proteins with homology to GABA-binding motifs of mammalian GABA receptors (GABA-gated anion channels). Furthermore, we in other genotypes. We, therefore, tested the hypothesis that HvHKT1 A will restore the salinity tolerance in salt sensitive Arabidopsis knockout demonstrate that anion flux through ALMTs can be regulated by GABA mutants. Overexpressing HvHKT1 improved the salt tolerance of hkt1- and its analogs with an IC50 in the low micromolar range. Site-directed 4 and sos1-12, which was evident by an increase in dry weight of the muatagenesis of this site alleviates GABA block but does not alter other transgenic plants grown in soil, in hydroponic solution and on Petri channel properties. Using wheat ALMT1 we show that endogenous dishes in salt treatment. Meanwhile, hkt1-4 and sos1-12 showed higher GABA, which accumulates in cells at low pH, closes the channel and Na+ accumulation in both shoots and roots than those in the HvHKT1 inhibits cellular malate efflux. At higher pH ALMT1, which is expressed overexpressing lines. Moreover, levels of reactive oxygen species in root apical cells, is open and at alkaline extracellular pH functions to − acidify the cell wall and maintain root growth. A survey of other ALMTs such as H2O2 and superoxide anion radical (•O2 ) and malondialdehyde (MDA) and proline were significantly lower in the overexpression lines showed variable GABA sensitivity related to binding site residues. This compared to hkt1-4 and sos1-12. Last but not least, the transgenic may reflect different roles for GABA signalling in the context of the wide plants exhibited remarkable capacity to regulate root K+, H+ and Ca2+ range of functions attributed to ALMTs. Our findings demonstrate that homeostasis in response to salinity, osmotic and oxidative stress. We most ALMT proteins act as GABA-gated ion channels in plants that propose that HvHKT1 may play an important role not only directly in parallel the role of GABAA receptors in animals. This means GABA regulating Na+ and K+ transport, but also indirectly in modulating Ca2+ can be considered a conserved messenger for cellular communication and H+ homeostasis. Therefore, genetic engineering of HvHKT1 could across multiple kingdoms. contribute to the improvement of salinity tolerance in other crops.

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SYM-26-04 SYM-26-05 DIVERSE PHOSPHATE ACQUISITION BIOTECH STRATEGIES TO PRODUCE AND ALLOCATION PATTERNS IN SEVEN MICRONUTRIENT-ENRICHED WHEAT PHYLOGENETICALLY CLOSELY-RELATED SPECIES OF HAKEA Johnson A.A.T. School of Botany, The University of Melbourne, Victoria 3010, Mirfakhraei B., Jost R. and Finnegan P.M. Australia. School of Plant Biology, The University of Western Australia, 35 Stirling Highway, Crawly WA 6009, Perth, Australia. Graminaceous plants such as wheat and rice employ a chelation- based strategy to absorb iron (Fe) from the rhizosphere - an Phosphorus (P) is essential for plant growth and development and approach commonly referred to as Strategy II Fe uptake. In rice, this is a non-renewable resource. Inorganic phosphate (Pi) is the form of system involves the synthesis of a small molecular weight mugineic P that is absorbed directly by root cells. Therefore, it is important to acid phytosiderophore called deoxymugineic acid (DMA) that is understand the mechanisms that are used by plants that are highly subsequently secreted from the root, in a diurnal pattern, via the efficient in Pi acquisition, as this information may assist in the breeding plasma membrane-localized phytosiderophore transporter TOM1. The released DMA phytosiderophore solubilizes ferric Fe (Fe3+) in the of more P-efficient crops. Hakea prostrata (Proteaceae) is a highly 3+ P-efficient plant. However, it has a decreased ability to down-regulate rhizosphere and the Fe -DMA complex is then absorbed into root cells Pi uptake, and suffers P toxicity symptoms at Pi supply rates that are not via yellow stripe 1-like (YSL) transporters such as OsYSL15. Once toxic to other species. We compared the patterns of P acquisition and inside the rice plant, Fe can remain complexed to DMA or transfer to a allocation in Hakea prostrata with those in six other phylogenetically number of additional chelating molecules, such as citrate, depending closely-related species (H. pritzelii, H. denticulata, H. amplexicaulis, H. on pH of the surrounding plant tissue. Central to the chelation-based megalosperma, H. ruscifolia and H. drupacea). H. prostrata had the Fe uptake strategy of graminaceous plants is the DMA precursor highest Pi concentration in the leaves, followed by its phylogenetically nicotianamine (NA), a non-protein amino acid that is a strong chelator closest relative, H. pritzelii. The relative inability of H. prostrata to of transition metals in all higher plants. Nicotianamine is formed by esterify Pi into organic compounds in the leaves, even compared to trimerization of three molecules of S-adenosyl methionine in a process H. prizelii, is likely to be a critical driver for the accumulation of Pi to catalyzed by nicotianamine synthase (NAS) enzymes. We are using toxic levels. The other species had lower and similar Pi concentrations OsNAS genes from rice to generate wheat varieties that load increased in the leaves, suggesting a stronger capacity to regulate Pi allocation. concentrations of Fe and Zn into the grain. Our preliminary results The distribution of Pi in roots and cluster roots was variable among indicate that OsNAS overexpression in wheat leads to 1.3- to 2-fold the species and was not dependent on phylogenetic relationships. more Fe, and 1.3- to 2.2 fold more Zn, in whole wheat grain compared Moreover, transcript sequences from thirteen H. prostrata PHOSPHATE to wild-type grain. Additionally, many OsNAS overexpressing wheat TRANSPORTER (PHT) genes have been determined from cDNAs plants have larger shoot systems and produce more grain than wild- and from next generation RNA sequencing. The relationships between type wheat in the glasshouse. The development of micronutrient- the abundance of these transcripts and Pi allocation patterns are now enriched wheat provides an inexpensive and sustainable method for being determined. increasing micronutrient intakes in many developing countries.

SYM-27-01 SYM-27-02 DECONSTRUCTING THE BREAST: THE GENETIC DEFINING MOLECULAR MECHANISMS OF GERMLINE PROGRAM FOR CELLULAR ASYMMETRY DIRECTS STEM CELL MAINTENANCE AND DEVELOPMENT CELL FATE, TISSUE ARCHITECTURE AND CANCER PROGRESSION IN THE MAMMARY GLAND Hobbs R.M.1, 2, De Seram M.1, Nishinakamura R.3, Chai L.4 and Pandolfi P.P. 2 Humbert P.O. 1Australian Regenerative Medicine Institute, Department of Anatomy Peter MacCallum Cancer Centre. and Developmental Biology, Monash University, Clayton, Australia. 2Beth Israel Deaconess Medical Center, Harvard Medical School, Cell polarity, the universal property of all cells to be asymmetrical, Boston, USA. 3Institute of Molecular Embryology and Genetics, coordinates cell movement, differentiation, proliferation and cell fate Kumamoto University, Japan. 4Brigham and Womans Hospital, decisions to build and maintain complex epithelial tissues such as Harvard Medical School, Boston, USA. the mammary gland. Loss of polarity and the deregulation of these processes are critical events in malignant progression but precisely how Maintenance of fertility in adult males is dependent on a pool of and at which stage cell polarity loss impacts on mammary development undifferentiated germline cells (spermatogonial stem and progenitor and tumourigenesis is unclear. To provide insight as to how cellular cells; SSPCs) within the testis that self-renews and generates asymmetry can regulate epithelial organ formation, we investigated differentiating germ cells for production of spermatozoa. We have Scrib and GPSM2/Pins, core mammalian members of two highly demonstrated that SSPC self-renewal in the mouse is critically conserved key cell polarity complexes that act as tumour suppressors dependent upon expression of Promyelocytic leukemia zinc finger and regulate asymmetry in Drosophila. Utilizing a conditional (Plzf), a transcription factor that acts as a defining marker for this cell mouse model, we report that Scrib is essential for mammary duct population. While the role of Plzf in maintenance of SSPC function morphogenesis and inhibits the initiation and progression of sporadic remains poorly understood, we have recently characterized key mammary tumors. Scrib-deficiency resulted in fully penetrant ductal downstream targets controlling SSPC fate. The transcription factor hyperplasia characterized by high cell turnover, MAPK hyperactivity, Spalt-like 4 (Sall4) is essential for early development and embryonic polarity loss, aberrant luminal cell spindle orientation and expansion stem cell function. Importantly, we have found Sall4 to be dynamically of atypical luminal cells. We recently identified GPSM2 in a polarity expressed in SSPCs and to physically and functionally interact with tumour suppressor RNAi screen. Similarly to Scrib, GPSM2 loss can Plzf. Through germline-specific deletion of Sall4 we demonstrate key cooperate with activated oncogenes to promote invasion and tumour roles for this factor in embryonic germ cell maintenance and SSPC growth in transplant model of breast cancer. Using a mouse model of differentiation; distinct functions to that of Plzf. We find that Sall4 and GPSM2 loss, we show that in contrast to Scrib, deregulation of GPSM2 Plzf antagonistically co-regulate Kit and Sall1, genes linked to SSPC inhibits the repopulating frequency of mammary stem cells and favours differentiation and development respectively. On-going studies are the ectopic expansion of luminal progenitor cells previously identified aimed at elucidating the complex functional interaction between these as the cell of origin for estrogen receptor negative breast cancers. two stem cell factors within the germline and respective gene targets. These studies identify distinct and essential roles for cell polarity Based on our data, we propose that a balance between Sall4 and regulators Scrib and GPSM2 in mammary gland development and Plzf activities regulates SSPC function and suggest a new model for highlight the importance of the proper regulation of cellular asymmetry formation of stable adult stem cell pools from embryonic precursors. in the suppression of breast cancer.

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SYM-27-03 SYM-27-04 LUNG REGENERATION AFTER INFLUENZA-MEDIATED STEM CELL AND GENE THERAPY APPROACHES FOR EPITHELIAL INJURY COMBINATORIAL PRECLINICAL THERAPIES FOR SPINAL CORD INJURY Filby C.E.1, Viitaniemi K.1, McQualter J.2, Belz G.1, Galvis L.1, Bertoncello I.2 and Asselin-Labat M.L.1 Hodgetts S. 1The Walter and Eliza Hall Institute of Medical Research, Parkville Anatomy, Physiology and Human Biology, University of Western 3052 Victoria, Australia. 2The University of Melbourne, Parkville 3052 Australia, 35 Stirling Highway, Crawley WA 6009. Victoria, Australia. Over the last decade, our laboratory has focussed on the potential of Seasonal and pandemic flu are responsible for a large number of purified (Stro-1+) adult human mesenchymal precursor cells (hMPCs) respiratory tract disorders and constitute a health burden worldwide. to repair the injured spinal cord after transplantation into T cell deficient Infections by H1N1 influenza A strain induces acute respiratory distress athymic nude rats following acute and chronic moderate contusive, and syndrome marked by a rapid onset of pneumonia, diffuse alveolar complete transection spinal cord injury (SCI). Isolated from the bone damage and elevation of inflammatory cytokines. Although the role marrow of SCI patients, these multipotent hMPCs have been used of immune and inflammatory cells during the early phase of Influenza in combinatorial approaches and been shown to markedly improve infection has been well studied, regeneration of the lung epithelium after morphological and functional outcomes in our rat models. This occurs influenza infection is largely underexplored. This study aims to evaluate despite the fact that the cells ultimately do not survive long term. mechanisms regulating regeneration of the lung epithelium after flu Combinatorial experiments have repeatedly highlighted these donor infection and to determine the cellular and molecular events driving cells as promising candidates for SCI therapies. Our laboratory is epithelial repair. Regeneration of the lung epithelium after flu injury was currently investigating the ability to prolong donor hMPC survival post evident two weeks after intranasal administration of H1N1 virus in mice transplantation via modulation of the host immune response and further with the appearance of keratin 5-positive, BrdU-positive regions. Using enhance these outcomes, as well as exploring the use of inducible flow cytometry and a combination of cell surface markers, we identified pluripotent stem cell (iPSC) technology, nanotechnology delivery and a novel discrete population of epithelial progenitor cells that is activated gene therapy techniques as preclinical strategies designed to promote after H1N1 infection and is responsible for lung repair after H1N1- regeneration after SCI. induced damage. These cells had high colony-formation capacity in vitro. Gene profiling of this population by RNAseq led to the identification of genes driving progenitor activity, including the transcriptional modifier LMO4 (Lim domain Only protein 4). LMO4 has known roles in maintaining progenitor populations in epithelial organs (ie. breast) and in oncogenesis. Our results showed that Shh-cre;LMO4fl/fl mice that lack LMO4 in the lung epithelium presented a higher sensitivity to flu infection and a delayed repair of the epithelium. The number of lung progenitor cells in Shh-cre;LMO4fl/fl mice was reduced, further demonstrating its role in repair after injury. In conclusion, we have identified a novel progenitor population of lung epithelial cells that is activated following flu-mediated injury. RNAseq studies identified pathways that are critical for lung progenitor activity and regeneration of the damaged lung.

SYM-27-05 SYM-28-01 THE LYSINE ACETYLTRANSFERASE HBO1 IS ROLE OF ATRX-H3.3 COMPLEX IN THE MAINTENANCE ESSENTIAL FOR MAINTAINING THE POISED OF CHROMATIN STATE INTEGRITY AT REPETITIVE CHROMATIN STATE OF NEURAL STEM CELLS GENOMIC REGIONS

Thomas T., Kueh A. and Voss A.K. Chang F.T.M.1, Udugama M.1, McGhie J.D.1, Chan F.L.1, Mann J.2 and Walter & Eliza Hall Institute of Medical Research, Melbourne. Wong L.H.1 1Monash University, Australia. 2Murdoch Children Research Institute, The five MYST proteins (KAT5-8) constitute one third of the Australia. mammalian genome’s capacity to regulate transcription and chromatin conformation at the level of histone acetylation. We have reported the Mammalian telomeres contain a specific protein complex that acts biological and molecular roles of the MYST histone acetyltransferases to protect telomeres from the DNA damage response. In addition, a in vivo in mice.1 MOZ (MYST3) and QKF (MYST4) are essential for proper maintenance of telomeric chromatin state is also essential the development and self-renewal of haematopoietic stem cells and for telomere protection, for example, the deposition of histone H3.3 neural stem cells, respectively.1 Interestingly, MYST family members by the ATRX/DAXX chaperone complex is essential for chromatin can act to regulate chromatin structure throughout the genome or at repression at the telomere. In mouse embryonic stem (ES) cells, specific loci. MOF (MYST1) is required genome-wide histone 4 lysine the assembly of telomeric chromatin state occurs within the PML 16 acetylation (H4K16ac) and in the absence of MOF chromatin promyelocytic leukemia (PML) bodies. Disassembly of these bodies, undergoes rapid condensation.1 In contrast, MOZ is unexpectedly following PML RNAi-knockdown, disrupts the binding of ATRX-H3.3 specific and regulates H3K9ac at Hox and Tbx loci, correspondingly, and proper establishment of histone methylation pattern, leading to Moz mutation leads to homeotic transformation of the axial skeleton2 DNA damage at the telomere. PML bodies act as platforms for the and DiGeorge-like defects in heart development.3 Surprisingly, HBO1 assembly of the telomeric chromatin to ensure a faithful inheritance appears to be required for genome wide acetylation of H3K14.4 Neural of epigenetic information at the telomere. The function of ATRX-H3.3 stem cells lacking HBO1 (MYST2) are devoid of H3K14ac, yet undergo in maintaining the telomeric chromatin state is reinforced by a strong self-renewing proliferation for months in culture. Wild type neural stem association of ATRX mutation with telomerase negative alternative cells are multipotent. In contrast, Hbo1 null neural stem cells are unable lengthening of telomeres (ALT) in human cancers. Importantly, recent differentiate into neurons, forming only astrocytes. In vivo, Hbo1 null studies also suggest the role of ATRX in directing DNA replication of cortical neurons fail to express key regulators of cortex development. complex secondary DNA structures, such as G-quadruplex (G4) DNA, Re-expression of HBO1 in neural stem cells restores H3K14ac and which are found at telomeres and other genomic sites. Given ATRX multipotency, when conducted 3 days, but not 5 weeks after decline binds a range of DNA repeats including ribosomal DNA, pericentric in HBO1 protein, suggesting irreversible changes to the chromatin satellite repeats and G4 repeats, we explore the function of ATRX-H3.3 after prolonged absence of HBO1. Our data suggest that HBO1 and in maintaining replication fidelity and chromatin marks at these DNA H3K14ac are essential for the maintenance of multipotency and the repeats. In human ALT cells, we observe severe DNA recombination poised chromatin state. 1Voss and Thomas (2009) Bioessays 31:1050; and damage at pericentric satellite repeats, accompanied by an 2Voss et al (2009) Developmental Cell 17:674; 3Voss et al (2012) aberrant pattern of H3.3 phosphorylated at serine residue 31 Developmental Cell 23:652; 4Kueh et al (2011) Molecular and Cellular (H3.3S31P) on the chromosome arm. These aberrant chromatin states Biology 31:845. may contribute to the overall genome instability in these human cancer cells. We will discuss the underlying mechanisms that are responsible for the abnormal H3.3S31P profile in ALT cancer cells.

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SYM-28-02 SYM-28-03 THE EVOLUTION OF A CONTAGIOUS CANCER, THE NEAT1 LONG NONCODING RNA SUPPRESSES TASMANIAN DEVIL FACIAL TUMOUR DISEASE TRANSCRIPTION VIA PROTEIN SEQUESTRATION WITHIN SUBNUCLEAR BODIES Ujvari B.1, Pearse A.-M.2, Madsen T.3 and Belov K.1 1Faculty of Veterinary Sciences, University of Sydney, NSW, 2006, Hirose T. 2, Virnicchi G.3, Tanigawa A.2, Naganuma T.2, Li R.1, Pierron G.3 Australia. 2Devil Facial Tumour Project, Diagnostic Services, Animal and Fox A.H.1 Health Laboratory, Department of Primary Industries, Water and 1WAIMR/Centre for Medical Research, UWA, WA, 6009, Australia. Environment, Launceston, Tasmania, 7250, Australia. 3School of 2Institute for Genetic Medicine, Hokkaido University, Sapporo 060- Biological Sciences, University of Wollongong, Wollongong, NSW, 0815, Japan. 3Centre National de la Recherche Scientifique, UMR- 2522, Australia. 8122, Institut Gustave Roussy, Villejuif 94805, France.

Transmissible animal tumours are informative models to study cancer Paraspeckles are sub-nuclear structures formed around NEAT1/ evolution at genetic as well as epigenetic levels. The Tasmanian devil MENε/β long noncoding RNA (lncRNA). We sought conditions where facial tumour disease (DFTD) is one of the two, clonally transmissible paraspeckles became altered, and found they become dramatically cancers. The first case of DFTD was observed in the east of Tasmania elongated following proteasome inhibition. This enlargement was in 1996 and since has led to the dramatic decline of Tasmania devil mainly caused by NEAT1 transcriptional up-regulation. We performed (Sarcophilus harrisii) populations. The cancer causes large ulcerating microarrays from NEAT1 knockdown cells and found that NEAT1 tumours primarily around the head of Tasmanian devils. Animals represses transcription of several genes including the RNA-specific generally die within six months of the first appearance of lesions due adenosine deaminase B2 (ADARB2) gene. In contrast, we found a to starvation or complications from metastases. Devil Facial Tumour paraspeckle protein, SFPQ that activates ADARB2 transcription. This (DFT) cells possess highly rearranged genomes, characterized by led us to hypothesize that ADARB2 expression is controlled by NEAT1- tumour specific chromosomal rearrangements. While the clonal nature dependent sequestration of SFPQ. Accordingly, we found SFPQ of DFTs is unequivocal, recently four distinct chromosomal strains have sequestration within the enlarged paraspeckles upon proteasome been described. In the present study we investigated the temporal and inhibition was enhanced and ADARB2 expression reduced, with a spatial evolution of DFTD at genetic and epigenetic levels. No spatial concomitant reduction in SFPQ-binding to the ADARB2 promoter and genetic or epigenetic variations were observed between strains, but depleted nucleoplasmic SFPQ. Finally, NEAT1-/- fibroblasts were more tumour methylation levels decreased over time. Temporal changes were sensitive to proteasome inhibition that triggered cell death, suggesting also observed in genes associated with methylation. The variations in that the paraspeckle-mediated regulatory mechanism attenuates the gene expression and methylation suggest that this cancer should not cell death pathway. These data provide a model whereby induced be treated like a static entity, but rather as an evolving pathogen. NEAT1 controls target gene transcription by protein sequestration into paraspeckles.

SYM-28-04 SYM-28-05 AN ENU MUTAGENESIS SCREEN IDENTIFIES THE POLYCOMB REPRESSIVE COMPLEX 2 (PRC2) FIRST MOUSE MUTANTS OF A NOVEL EPIGENETIC SUPPRESSES Eμ-myc LYMPHOMA DEVELOPMENT MODIFIER, REARRANGED L-MYC FUSION (RLF) Lee S.C.W.1, Phipson B.1, Hyland C.D.1, Blewitt M.E.1, 2, 4, Smyth G.K.1, 3, Harten S.K.1, Bourke L.1, Bharti V.1, Oey H.2, Whitelaw N.1 and Alexander W.S.1, 2 and Majewski I.J.1, 2 Whitelaw E.1, 2 1The Walter and Eliza Hall Institute of Medical Research. 2Department 1QLD Institute of Medical Research. 2La Trobe University. of Medical Biology. 3Department of Mathematics and Statistics. 4Department of Genetics, University of Melbourne. An ENU mutagenesis screen was designed to identify novel genes involved in establishing or maintaining the epigenetic state of the genome in the Polycomb group (PcG) proteins are transcriptional repressors that mouse. The mice used in the screen carry a multi-copy GFP transgene act at many loci throughout the genome. PcG proteins function in two array, which is expressed in a variegated manner in erythrocytes and distinct complexes called Polycomb Repressive Complex 1 (PRC1) is highly sensitive to epigenetic silencing1. A gene discovery pipeline and 2 (PRC2). Deregulation of PRC1 and PRC2 via mutation or altered involving SNP arrays and whole exome sequencing allowed the rapid expression is associated with diverse human cancers. Loss-of-function identification of causative mutations in ~40 mutant lines. The screen has mutations in EZH2, EED and SUZ12, members of PRC2, occur in T-ALL produced mouse mutants of both known modifiers of epigenetic state, such and in myeloid malignancies. In contrast, activating mutations in EZH2 as Dnmt1 and Smarca52, and novel modifiers, such as Re-arranged L-Myc are commonly observed in B-cell lymphoma, implying cancer-specific fusion (Rlf). Here we report three independent lines with mutations in Rlf. effects of individual mutations. To define the underlying mechanisms Each line shows a reduced percentage of GFP expressing cells compared via which altered PcG proteins contribute to disease, we compared to wild-types. Homozygous Rlf mutants show increased methylation at the and contrasted the effects of loss of PRC1 or PRC2 activity on Myc- transgene and haploinsufficiency for Rlf alters transcriptional silencing at driven lymphomagenesis in Eμ-myc mouse model. Unlike PRC1, Agouti Viable Yellow (Avy), an independent endogenous epiallele. Taken PRC2 functions as a tumor suppressor in Eμ-myc lymphomagenesis: together, these findings suggest that Rlf is a modifier of epigenetic state. lymphoma onset was accelerated by heterozygosity of a loss-of- Rlf encodes a protein containing multiple widely-spaced zinc finger function mutation in Suz12 or by shRNA-mediated knockdown of domains, about which very little is known. Homozygous Rlf null mutants Suz12 or Ezh2. PRC2-deficient lymphomagenesis was characterised weigh less than wild-types, and show postnatal lethality, indicating that Rlf by increased accumulation of B-lymphoid cells above that caused by is critical for proper development. Initial histology suggests the presence Eμ-myc alone in the absence of effects on apoptosis or cell cycling. of a heart defect, which is the subject of further investigation. Analysis of However, Suz12-deficient B-lymphoid progenitor cells exhibited RNA-Seq data, comparing RNA from wild-type and Rlf mutant fetal livers, enhanced serial clonogenicity. Our data suggest that PRC2 normally showed differential expression of genes involved in metabolism. Levels of restricts self-renewal of B-lymphoid progenitors, the disruption of 4-hydroxyphenylpyruvic acid dioxygenase (HPD), an enzyme involved in which contributes to lymphomagenesis. This finding provides new tyrosine catabolism, were markedly reduced. Genetic deficiency of HPD insight regarding the functional contribution of mutations in PRC2 that underlies Tyrosinemia type 3 in humans and is associated with mental contribute to a range of haematological malignancies. As the exciting retardation and ataxia. Genome-wide bisulphite sequencing studies are prospect of new cancer therapies that target epigenetic regulation via underway to examine the role of Rlf in the control of methylation both PRC2 emerges, our findings have important implications for how to within and outside of CpG islands. 1. Blewitt et al. PNAS, 2005. 2. Chong approach this complex therapeutically. et al. Nature Genetics, 2007.

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SYM-29-01 SYM-29-02 FUNCTIONAL DECORATION: POST-TRANSLATIONAL PROTEOME ANALYSIS OF THE NODULE MODIFICATIONS AND THEIR CROSSTALK IN DEVELOPMENT IN MEDICAGO TRUNCATULA MYOCARDIAL ISCHEMIA / REPERFUSION INJURY Van Noorden G.E.1, Jin J.1, Harris C.1, Costa S.2 and Mathesius U.1 Parker B.L.1, White M.Y.2 and Cordwell S.J.1, 2 1Australian National University, Canberra, Australia. 2University of 1Discipline of Pathology, School of Medical Sciences, The University Coimbra, Coimbra, Portugal. of Sydney, Australia 2006. 2School of Molecular Bioscience, The University of Sydney, Australia 2006. We are studying the control of symbiotic nodule development in legumes. To this end we have used proteome analysis to identify protein changes Myocardial ischemia and cardioprotection by ischemic pre-conditioning associated with the early stages of nodule development in the model (IPC) induce signal networks aimed at survival, or cell death if the legume Medicago truncatula. To identify proteins potentially involved in ischemic period is prolonged. These pathways are mediated by protein in the control of nodule numbers, we have examined changes in protein post-translational modifications (PTM). A temporal signal profile expression in response to carbon and nitrogen availability, as well generated by phosphoproteomics over a time-course of ischemia as changes between wild type plants and mutants unable to control in the rat heart model identified specific activation of kinases in an nodule numbers. We have used Difference In Gel Electrophoresis for ordered and time-dependent manner. Drug inhibition of such kinases protein differential display, DeCyder software for statistical analysis and resulted in significant changes to the functional recovery of ischemic MALDI-TOF/TOF based mass spectrometry for protein identification. hearts. Phosphopeptides and lysine acetylated (AcetylK) peptides The analysis demonstrated an important role of redox-related proteins were also quantified in isolated rat hearts subjected to ischemia or IPC, that could play a role in controlling defense responses and auxin with and without inhibition of sirtuin deacetylase 1 (SIRT1). We show oxidation during the control of nodule development. In addition, we SIRT1-dependent activation of AMPK, AKT and PKA kinases during found that auxin application appears to phenocopy many of the early ischemia. Phosphorylation and AcetylK sites mapped onto protein changes in protein expression during nodulation. The role of these tertiary structures revealed they were often proximal, yet were mutually proteins in context of nodulation and early infection of rhizobia will be exclusive in >20 proteins containing the KxxS phosphophilic protein discussed. kinase consensus motif. Modifications in this motif were modeled using the C-terminus of muscle-type creatine kinase. AcetylK increased proximal dephosphorylation by 10-fold. Structural analysis by 2D-NMR revealed stabilization through a lysine-phosphate salt bridge, which was disrupted by AcetylK resulting in backbone flexibility and increased dephosphorylation. This study determined novel inter- and intra- molecular PTM crosstalk, and an association with metabolic events, during myocardial ischemia and cardioprotection.

SYM-29-03 SYM-29-04 TARGETED PROTEOMICS OF PATHWAYS AND PLASMA BIOMARKERS FOR DIABETIC KIDNEY TURNOVER OF PROTEOMES IN PLANTS DISEASE: DISCOVERY TO VALIDATION

Millar A.H., Nelson C.J., Taylor N.L., Fenske R. and Li L. Lipscombe R.1, Bringans S.1, Winfield K.1, Ito J.1, Phillips M.1, Davis W.3, ARC Centre of Excellence in Plant Energy Biology, The University of Peters K.3 and Davis T.3 Western Australia. 1Proteomics International, Perth. 2WAIMR, Perth. 3Fremantle Hospital, Perth. Shotgun approaches dominate proteome studies as discovery tools to find changes in protein abundance. Key limitations, however, Diabetes is a metabolic time bomb that is becoming one of the greatest are they only provide a limited image of the proteome response health challenges of the 21st century. Globally there are ~370 million and they focus just on the proteins that are changing in abundance people with diabetes, and in combination with obesity the economic to find biological insights. I will show data from three alternative cost in Australia alone is $80 billion annually. Chronic kidney disease approaches: (1) targeting identification of changes in biochemical is a significant complication of diabetes that affects >35% of patients. pathways; (2) analysing protein synthesis and degradation rates with Diabetic kidney disease is a major cause of renal transplantation progressive stable isotope labelling; and (3) native complex analysis globally and 10-20% of diabetics will die of kidney failure. The current with isotope labelling to measure the age of protein complexes to study diagnostic test for diabetic kidney disease uses a single urine biomarker in vivo assembly of protein machinery. Through developing optimised that measures microalbumin levels which has limited utility. Clinicians, selected reaction monitoring (SRM) assays for a series of peptides, however, require better tools that can deliver more effective diagnosis, and multiplexing these SRM assays for whole biochemical pathways, prognosis and monitor treatment to notably improve health outcomes. we can begin to use peptide mass spectrometry to follow the dynamics Candidate biomarkers were identified using 2D chromatography and of complete biochemical pathways. I will show the example of the TCA mass spectrometry in a pilot study of 90 highly curated clinical samples. cycle. Through progressive 15N labelling of plant cells from nitrate and The simplicity of the study design combined with the power of the ammonia salts and modelling incorporation fits, we can calculate the biomarker platform used is highly efficient and provides strong data. rate at which proteins which are static in abundance in the proteome This attracted $1 million in funding from Commercialisation Australia are turning over, and thus provide an extra dimension to proteome to produce a diagnostic kit. The team is now analysing a diagnostic analysis. Through combining progressive 15N labelling with separation and prognostic study of 1,000+ individuals with samples collected at of protein complex and subcomplexes by native electrophoresis, we three key time points that are clinically significant (Years 0, 2, 4). The can observe the in vivo turnover rate of assembly intermediates of diagnostic study has identified 8 protein biomarkers at high stringency protein complexes in the mitochondrial respiratory chain. Combined using mass spectrometry, including proteins involved in metabolism, there approaches provide new avenues for peptide mass spectrometry inflammation and oxidative stress. Further work is on-going to cross- to provide answers to a wide range of questions in biology. validate these findings against FDA approved antibody-multiplexing approaches. The target outcome is a point-of-care test or companion diagnostic for diabetic kidney disease.

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SYM-29-05 SYM-30-01 STRUCTURE AND FUNCTION STUDIES ON HUMAN A CELL, TISSUE AND WHOLE ORGAN PROFILE OF PLASMINOGEN GLYCOFORMS KIDNEY MORPHOGENESIS

Law R.H.P.1, Caradoc-Davies T.2, Cowieson N.2, Lu B.3, Quek A.J.1, Short K.M.1, Combes A.N.2, Lefevre J.2, Ju A.L.2, Georgas K.2, Horvath A.J.3, Coughlin P.B.3 and Whisstock J.C.1 Cairncross O.2, Hamilton N.A.2, McMahon A.P.3, Little M.H.2 and 1Department of Biochemistry and Molecular Biology, Monash Smyth I.1 University, Clayton, Melbourne, VIC 3800 Australia. 2Australian 1Monash University, Melbourne. 2University of QLD, Brisbane. Synchrotron, 800 Blackburn Rd, Clayton, Melbourne, VIC 3168 3University of Southern California, Los Angeles. Australia. 3Australian Centre for Blood Diseases, Monash University, Prahran, Melbourne, VIC 3004 Australia. Epithelial branching morphogenesis drives the development of many organs. At its heart, this process relies on the complex interplay between Plasminogen is a 7-domain protein (with an N-terminal Pan-apple the morphogen gradients that exist between arborising epithelia and domain, five kringle domains and a C-terminal serine protease their intimately associated mesenchymal cells. We have recently domain) that adopts a closed, activation-resistant conformation in the developed a program which employs Optical Projection Tomography circulatory system. The recruitment of plasminogen to its target sites is imaging to quantify how branching occurs in three dimensions dependant on the lysine binding sites of the kringle domains. Binding during organ development. Using this approach we have profiled the to lysine residues on cell receptors and fibrin clots simultaneously elaboration of the ureteric bud, which is critical for establishing kidney triggers a conformational re-arrangement of the molecule to adopt an architecture and nephron number. By analysing 94,036 morphological open conformation. The open form is readily converted to plasmin by measures that describe nearly 10,000 branch segments developing tissue- and urokinase-type Plasminogen Activators (tPA and uPA). kidneys, we demonstrate a hitherto unappreciated dynamism in form Plasmin is a serine protease with a broad specificity that plays a key during the course of renal development. To relate these changes to the role in number of physiological and pathological processes including behaviour of the cells driving branching, we measured the flux in cell degradation of extracellular matrices, cell migration, tissue remodeling, proliferation, population and morphology in the progenitor niches of the wound healing, angiogenesis, inflammation, pathogen invasion and ureteric bud tips and their surrounding mesenchyme. Taken together, cancer migration. Recently, we have solved the X-ray crystal structure this multi-scale analysis provides one of the most comprehensive of human plasminogen in the closed conformation. Our results revealed profiles of the orchestrated changes in the cells and tissues involved that the N-terminal Pan-apple domain and the serine protease domain in the development of an organ. As well as delineating several key maintain the closed conformation via interactions made throughout the phases in kidney development, our study has generated a baseline kringle array, in particular, Kringles 2, 4 and 5. Our data suggests that resource essential for understanding the temporal and spatial impacts Kringle 1 governs proenzyme recruitment to target sites and binding to of individual morphogens on organogenesis. external lysine of Kringle 5 in the closed conformation may trigger the formation of the open conformation. Glycosylation affects the overall conformation of the protein and therefore its functions. There are two plasminogen glycoforms in the plasma; our structural data suggests that these glycoforms have distinct structural characteristics. This observation supports the notion that the two glycoforms have different target specificity and therefore physiological roles. Here we discuss our recent studies on the structure and function of these two glycoforms.

SYM-30-02 SYM-30-03 CARDIAC GENE REGULATORY NETWORKS IN DEFINING THE MECHANISMS BY WHICH THE DEVELOPMENT AND DISEASE UBIQUITIN LIGASE NEDD4 CONTROLS PATTERNING AND MORPHOGENESIS OF THE VASCULATURE Ramialison M.1, 2, Bouveret R.1, 2, Waardenberg A.1, Doan T.1, De Jong D.1, Schönrock N.1 and Harvey R.P.1, 2 Secker G.1, Sutton D.1, Kazenwadel J.1, Betterman K.1, Boase N.1, 1Victor Chang Cardiac Research Institute. 2St. Vincent’s Clinical Schwarz Q.2, Kumar S.1, 3 and Harvey N.1, 3 School, University of New South Wales. 1Division of Haematology, Centre for Cancer Biology, SA Pathology, PO Box 14 Rundle Mall, Adelaide, SA, 5000. 2Division of Human During embryogenesis the heart is the first organ to form and this Immunology, Centre for Cancer Biology, SA Pathology, PO Box 14 complex process is tightly orchestrated by a gene regulatory network Rundle Mall, Adelaide, SA, 5000. 3School of Medicine, University of (GRN) encompassing cardiac transcription factors (TFs) that engage Adelaide, Adelaide, SA, 5000. into concerted transcriptional regulation of shared target genes. To date, our knowledge of the topology of the heart GRN is incomplete Lymphatic vessels are vital for tissue fluid homeostasis, the absorption and furthermore, little is known about the intricate cooperativity of of dietary fats and immune cell trafficking. Despite the integral role the cardiac TFs embedded within the GRN. With the goal to deepen that lymphatic vessels play in homeostasis and human disease, little our knowledge of the cardiac GRN, we have undertaken a systems is known about the signals that direct construction of the lymphatic biology approach to systematically interrogate the genome-wide vasculature during development. Here, we present recent work targets of wild-type and mutant cardiac TFs, and decrypt the network dissecting the role of the ubiquitin ligase Nedd4 in embryonic vascular of interactions that they participate in. Bioinformatics analysis of our development. Ubiquitylation has been demonstrated to target proteins genome-wide data has discovered that the Elk family of TFs, which are for degradation, regulate receptor endocytosis and direct protein not cardiac specific, play an essential role during heart development. trafficking within the cell, thereby providing multiple levels of control First, we established that they are directly integrated in the cardiac over cell signalling pathways. Our work has revealed that Nedd4 GRN by interacting with cardiac specific TFs. Second, we determined is crucial for morphogenesis of the blood and lymphatic vascular that genome-wide targets of Elks significantly overlap with those of networks during mouse embryogenesis; Nedd4-/- embryos exhibit cardiac specific TFs. Finally, we demonstrated that loss of function of sparse, ruptured blood vessels and strikingly mispatterned lymphatic Elk1 in vivo in zebrafish embryos leads to a cardiac specific phenotype vessels. We have demonstrated that both the levels and the localisation supporting their involvement in early stages of cardiogenesis. Our of multiple components of the key pro-lymphangiogenic VEGF-C/ study highlights for the first time that Elks are directly embedded within VEGFR-3 signalling pathway are modified following Nedd4 knockdown the heart GRN and are essential for proper heart development. in primary embryonic lymphatic endothelial cells. Current work aims to define the mechanisms by which Nedd4 regulates activity of this key endothelial cell signalling pathway and to further dissect the endothelial cell autonomous versus non-autonomous roles of Nedd4 during vascular development.

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SYM-30-04 SYM-30-05 THE MIR17-92 MICRORNA CLUSTER AND THE EXTRA-EMBRYONIC YOLK SAC AND EMBRYONIC SPONTANEOUS MALFORMATION DORSAL AORTA: TRANSCRIPTOME ANALYSES OF TWO CRITICAL HEMATOPOIETIC NICHES Farlie P.G.1, Welfare M.F.1, Tan T.Y. 2, White S.M.2 and Kannu P.3 1Murdoch Children Research Institute Parkville Vic. 2Victorian Clinical Antas V.I.1 and Fraser S.T.1, 2 Genetics Services Parkville Vic. 3Sick Kids Hospital Toronto Canada. 1Discipline of Physiology, School of Medical Sciences, University of Sydney. 2Anatomy and Histology, School of Medical Sciences, A duplication of the miR17-92 miRNA cluster was detected in an University of Sydney. individual with pre-axial polydactyly and craniofacial dysmorphism. We used a chick embryo overexpression system to determine whether Mammalian embryonic hematopoiesis (or blood production) is a duplicated miR17-92 was responsible for these birth defects. We complex process that occurs in different organs as development found that a proportion of infected embryos exhibited additional digits progresses. Shortly after an initial (or ‘primitive’) wave of embryonic suggesting that this was the case. However, we also saw a range of more hematopoiesis, a definitive hematopoietic stage starts. The latter is complex limb malformations including duplications of entire limbs and characterized by the generation of multilineage hematopoietic stem and partial duplication of the limb with otherwise normal skeletal elements. progenitor cells that form the basis of the adult hematopoietic system. In In addition to limb phenotypes we also see a range of abnormalities the midgestational mouse conceptus, transient hematopoietic activity involving other tissues including exencephaly, gastroschisis, superficial occurs in the extraembryonic yolk sac (YS) followed by the dorsal aorta papillae extending from the body wall, adhesion of embryonic of the embryonic aorta-gonad-mesonephros (AGM) region. These two membranes to the flank/limbs and skin spurs. Most affected embryos distinct hematopoietic niches harbour a rare population of endothelial have only a single malformation and many embryos overexpressing cells (named hemogenic endothelial cells) that give rise to definitive miR17-92 appear to develop normally. This range of dysmorphology hematopoietic stem and progenitor cells. The stem cells generated involves diverse cell types that do not appear to follow any clear pattern. in these two distinct tissues show differences in transplantation and The broad range of phenotypes suggests that overexpression of these reconstitution activity. The molecular mechanisms that regulate miRNAs does not produce any one specific birth defect but creates a hematopoiesis mediated by hemogenic endothelial cell intermediate propensity for birth defects, leaving the embryo susceptible to diverse and the temporal regulation of hematopoietic activity in the YS and structural abnormalities. We hypothesise that miRNAs encoded by AGM are poorly defined. The present study aims to investigate the these miRNA clusters regulate a surveillance system which normally gene expression profiles of mouse YS and AGM through microarray suppresses spontaneous structural anomalies arising stochastically analyses, and to identify differentially expressed genes involved in throughout development. Further, we hypothesise that repression temporal and spatial regulation of hematopoietic activity in each niche. of this surveillance system through dysregulation of miR17-92 family Genes found to be up regulated in the YS (FDR≤0.05) include cubilin miRNAs is responsible for a range of apparently spontaneous human and folate receptor 1, which are critical for vasculature development birth defects. and possibly involved in hematopoietic regulation. In contrast to the YS, four members of the forkhead box (Fox) gene family were up regulated in the AGM (FDR≤0.05). Fox transcriptional regulators are crucial for cardiovascular development, including blood vessel specification and induction of endothelial and hematopoietic genes. The exact role of differentially expressed genes in hemogenic cell function and their expression in other hemogenic tissues have to be further investigated.

SYM-31-01 SYM-31-02 SUPER RESOLUTION OF CYTOKINESIS IN BACTERIA WHEN A PUSH COMES TO A SHOVE: RELEASE OF USING ULTRA-FAST 3D STRUCTURED-ILLUMINATION VACCINIA VIRUS MICROSCOPY Newsome T.P.1, Horsington J.1, Lynn H.1, Karupiah G.4, Whitchurch C.B.2, Strauss M., Liew A., Turnbull L., Whitchurch C., Monahan L. and Turnbull L.2, Cheng D.3 and Braet F.3 Harry E. 1School of Molecular Bioscience, University of Sydney. Cnr Maze University of Technology Sydney (UTS). Cres & Butlin Ave 2006 Sydney. 2The ithree institute, University of Technology Sydney, Sydney, New South Wales, Australia. 3Australian FtsZ is an essential tubulin-like bacterial cell division protein, which Centre for Microscopy & Microanalysis, University of Sydney, orchestrates division by self-assembling into the Z ring. Despite its Sydney, New South Wales, Australia. 4John Curtin School of Medical importance in division the mechanisms that allow assembly and Research, Australian National University, Canberra, Australian Capital subsequent constriction of the ring to allow cytokinesis are not clear. Territory, Australia. The spatial resolution afforded by conventional wide-field fluorescence microscopy has not been sufficient to clearly visualize these processes Vaccinia virus (VACV) is a large DNA virus that following replication in in live bacterial cells, that are as small as 1 μm in diameter. We have the cytoplasm generates two infectious forms: wrapped virus (WV) and therefore utilized 3D-structured illumination microscopy (3D-SIM) and intracellular mature virus (MV). Wrapped virus is released from infected a new form of fast live 3D-SIM, known as OMX Blaze, to generate cells by fusion of the outermost of two trans-Golgi derived membranes 3D super resolution images of FtsZ and other division proteins in with the host plasma membrane. Extracellular, cell-associated WVs the rod-shaped bacterium, Bacillus subtilis and in spherical cells of possess the capacity to activate actin polymerisation in the underlying the pathogen, Staphylococcus aureus. Unlike conventional wild-field cytoplasm, thereby promoting cell-to-cell spread. Here we report an fluorescence microscopy, which depicts the Z ring as a continuous belt, analysis of VACV during release using fluorescent recombinant viruses, 3D-SIM shows that FtsZ distribution within the ring is heterogeneous, achieving a resolution of ~20 nm with three-dimensional structured with a bead-like arrangement in both organisms. Despite this similarity, illumination microscopy (3D-SIM). We have imaged recombinant the Z ring in S. aureus does not appear to assemble via remodelling of viruses that differentially label the virus core and viral membranes, we helical FtsZ structures like B. subtilis. Furthermore, we use ultra-fast were able to resolve the trans-Golgi-derived membranes as spheres 3D-SIM (OMX Blaze) to uniquely show that the bead-like arrangement that encompass the viral core of intracellular WV. As WV fused with of FtsZ is dynamic throughout constriction. Our direct demonstration of the plasma membrane, the outer WV membrane redistributed to form FtsZ redistribution within the Z ring during constriction is consistent with a platform adjacent to extracellular WV. The viral protein responsible the dynamic behavior of FtsZ proposed in the iterative pinching model. for initiating actin polymerisation, the transmembrane protein A36, was Other core components of the division apparatus display a dynamic found to be tightly associated with de novo formed F-actin filaments at and heterogeneous localization pattern similar to FtsZ. Our data lead the onset of actin polymerisation. We describe a role for actin nucleation us to propose that FtsZ guides the division machinery to adopt a similar in the untethering of virus into the surrounding extracellular space. localization pattern to ensure Z ring constriction only proceeds following complete assembly of this machinery.

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SYM-31-03 SYM-31-04 IMAGING THE MOLECULAR AND CELLULAR TROPOMYOSIN BASED REGULATION OF THE ACTIN MECHANISMS CONTROLLING EARLY MAMMALIAN CYTOSKELETON DEVELOPMENT Vindin H.1, Macmillan A.2, Stehn J.1 and Gunning P.1 Plachta N.P.1 1School of Medical Sciences, University of New South Wales, Sydney, 1EMBL Australia. 2ARMI, Monash University. NSW, 2052, Australia. 2Biomedical Imaging Facility, University of New South Wales, Sydney, NSW, 2052, Australia. We have developed new in vivo single-cell imaging methods to understand how cells in the early mouse embryo start adopting The actin cytoskeleton is a dynamic and complex system which plays a distinct lineage identities, and how they form the first tissue-like central role in many essential cellular processes. It’s incorporation into structures of mammals. To obtain a systems-level view of the dynamic such a wide range of cellular processes is due to its interaction with a events patterning early embryos, we have combined single-molecule large number of actin binding proteins which gives it the ability to form methods based on fluorescence correlation spectroscopy (FCS) and multiple filament populations with different structural and functional light-based manipulations, to probe how transcription factors critical properties. One family of proteins which are of particular importance are for preimplantation development such as Oct4 and Sox2 search the the tropomyosins which play an essential role in regulating interactions DNA, and how they control the specification of the first pluripotent and between actin and other binding proteins. The mechanisms behind how extraembryonic lineages in the nuclei of cells deep in the embryo. We single tropomyosin isoforms sort to distinct subcellular locations and have also recently used live imaging to reveal how initially rounded cells bind to actin filaments creating filament systems with unique functional of the embryo become polarized and how they form the first epithelia- properties are still unknown. A greater understanding of the processes like structures during development. Our findings provide a new involved in the formation of a multifilament system in a common dynamic view of the molecular and cellular mechanisms underlying cytoplasm will not only lead to important insights into the regulation early mammalian embryogenesis. of the cytoskeleton, but may also provide clues as how the actin cytoskeleton is manipulated in disease. Research into this complex process has previously been difficult as many thin filament bundles are too small to be resolved using conventional light microscopy. The use of stimulated emission depletion (STED) and deconvolution microscopy has provided new insights into the regulation of the actin cytoskeleton. STED microscopy has allowed us to achieve a spatial resolution of up to 50nm, well below the resolving power seen using confocal microscopy. This improved resolution has allowed for a much greater appreciation of the diversity of actin filament populations found within a cell will allow for more sophisticated studies surrounding the regulation of the actin cytoskeleton.

SYM-31-05 SYM-32-01 IMAGING THE DYNAMICS OF CALCIUM COMPREHENSIVE ANALYSIS OF THE RICE MICRODOMAINS FOLLOWING NEUROTRAUMA USING TRANSCRIPTOME, METHYLOME AND SMALL NANOSIMS RNAOME IN RESPONSE TO PI STRESS USING DEEP SEQUENCING Lozic I.1, 2, 3, Bartlett C.A.1, Shaw J.A.3, Iyer K.S.2, Dunlop S.A.1, Kilburn M.R.3 and Fitzgerald M.R.1 Secco D.1, Walker H.1, Schultz M.2, Ecker J.R.2, Lister R.1 and Whelan J.1 1School of Animal Biology, UWA, 35 Stirling Hwy, Crawley WA 6009. 1Australian Research Council Centre of Excellence in Plant Energy 2School of Chemistry and Biochemistry, UWA, 35 Stirling Hwy, Biology, University of Western Australia, Crawley 6009, WA, Australia. Crawley WA 6009. 3Centre for Microscopy, Characterisation and 2Genomic Analysis Laboratory, The Salk Institute for Biological Analysis, UWA, 35 Stirling Hwy, Crawley WA 6009. Studies, La Jolla, CA 92037,USA.

Following neurotrauma, changes in Ca2+ concentration result in the Phosphate starvation is one of the most limiting factors for plant onset and spread of secondary degeneration mechanisms thought growth and development, thus greatly affecting crop productivity. A to result in oxidative stress in uninjured tissue. While fluctuations in time course experiment, covering both the early and late stages of Ca2+ form a normal and essential part of neural function, excess Ca2+ Pi starvation, as well as the early responses to Pi re-feeding in root influx into cells is a feature of CNS injury and is thought to contribute and shoot tissues was performed, using next-generation sequencing. to the death of neurons and glia vulnerable to secondary degeneration. Analyses of 126 RNAseq libraries, provided a comprehensive overview Ca2+ involved in signalling events is often found in highly localised of the responses of rice to Pi stress. Several genes showed expression increased areas of concentration, referred to as Ca2+ microdomains. of novel splice-isoform specifically under Pi stress, including the well- However, quantitative analyses of changes in size and distribution characterized regulator of phosphate homeostasis PHO2. Taken of Ca2+ microdomains in specific cell types in whole tissue samples together, the response to Pi starvation is dynamic over time, has has been limited by analytical resolution and reliance on indirect Ca2+ identified many novel Pi responsive genes, including those encoding indicator systems. Using nanoscale secondary ion mass spectrometry a variety of transcription factors and organ specific (root or shoot). (NanoSIMS) and immunohistochemistry on excised rat optic nerve Genome-wide DNA methylation profiling revealed the first report of the segments we have directly quantified changes in number, size and involvement of DNA methylation in transcript regulation upon abiotic intensity of Ca microdomains in glial and axonal tissue. We found that stress. Indeed several key regulators of Pi homeostasis showed altered Ca microdomains can be classified into two types, those that do or do methylation status upon long term Pi starvation, resulting in changes not colocalised with P enriched regions. There were significantly more in gene expression. Deep sequencing of smRNAs revealed a direct non-P colocalised Ca microdomains in glial than axonal regions in relationship between the location of smRNAs and DNA methylation as normal optic nerve. Both glial and axonal located non-P colocalised Ca well as the identification of the differentially regulated miRNA upon Pi microdomain density was observed to decrease significantly following stress. This study represents a comprehensive and integrated dataset injury, a change not observed with P colocalised Ca microdomains. of the molecular responses of the rice transcriptome, methylome and Our observed overall decrease in Ca microdomain numbers suggest smallRNAome to Pi stress, providing key elements for future research an efflux of Ca2+ from non-P colocalised microdomains, possibly as well as providing the first report of dynamic DNA methylation contributing to the structural and functional deficits observed in nerve changes upon nutrient stress, as a mean to regulate gene expression. vulnerable to spreading damage following neurotrauma.

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SYM-32-02 SYM-32-03 THE IMPACT OF ARBUSCULAR MYCORRHIZAS ON LEARNING FROM THE EXTREMISTS - THE PLANT P NUTRITION AND INTERACTIONS WITH PHOSPHORUS-EFFICIENT SOUTHWEST AUSTRALIAN EDAPHIC FACTORS NATIVE HARSH HAKEA

Cavagnaro T.R. and Watts-Williams S. Jost R.1, Hubberten H.-M.2, Giavalisco P.2, Sulpice R.2, Stitt M.2, Monash University. Hoefgen R.2, Scheible W.-R.2, Lambers H.1 and Finnegan P.M.1 1School of Plant Biology, University of Western Australia, 35 Stirling Most terrestrial plants form arbuscular mycorrhizas (AM). These Hwy, Crawley, WA 6009, Australia. 2Max-Planck Institute of Molecular associations play an important role in plant acquisition of nutrients, Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany. especially P, but also N, Zn and other nutrients. One of the challenges in the study of AM relates to the establishment of appropriate non- Proteaceae in Australia’s southwest are well adapted to the extremely mycorrhizal control treatments. For example, fumigation of soils can low phosphorus (P) content of the highly weathered soils, e.g., in eliminate other members of the soil biota as well as affect crop growth the Spearwood and Bassendean dunes of the Swan Coastal Plain and nutrition through other mechanisms. This in turn can affect the and the Peron slopes. They have evolved phosphate (Pi)-mining cycling of nutrients in the soil, and thence, plant nutrition. To overcome capabilities to access sparingly available P resources, producing this issue, we have used a novel experimental system to study the dense clusters of short tertiary lateral rootlets, so called proteoid or effects of arbuscular mycorrhizal fungi (AMF) on plant growth and cluster roots. These increase the surface area of the roots to boost nutrition, soil ecology and nutrient cycling. We grew a mycorrhizal local organic acid, nuclease and phosphatase secretion to liberate defective tomato mutant (rmc) and its mycorrhizal wildtype progenitor Pi from organic molecules or soil particles. Proteaceae species also (76RMYC+) in both the field and glasshouse to study the biology of AM. efficiently remobilise iP from senescing organs. Since these adaptive Here we present result from a number of experiments in which we have traits are costly, their production will require extensive reprogramming used our mutant based approach to study the interactions between AM, of primary metabolism. We have chosen harsh hakea (Hakea prostrata plant P nutrition and edaphic factors. In addition to presenting results R.Br.) as a model to study acclimation responses to varying Pi supply from individual experiments, we will also present a short synthesis of on a physiological and molecular level. Metabolite profiling revealed all studies published using this experimental system, in an attempt relatively high levels of essential P metabolites such as phospholipids in to identify larger scale patterns. We will also explore the potential for growing tissues which may help to sustain the formation of cluster roots AM to play a role in biologically regulated nutrient supply systems. and new leaves. Moreover, harsh hakea appears to adjust rates of N Taken together, the results of our recent and ongoing research will assimilation and protein synthesis during development and in response be discussed in the context of the role of AM in achieving agricultural to external Pi availability, most likely by tightly controlling nitrate uptake. sustainability. These and other metabolic adjustments will be discussed in the context of this species’ extraordinary P efficiency and opportunities for translational research into creating smarter crop plants better able to

acquire and efficiently use Pi.

SYM-32-04 SYM-32-05 CAN AUSTRALIAN FARMERS IMPROVE CROP DOES LOW SOIL PHOSPHORUS IN WESTERN PHOSPHORUS NUTRITION THROUGH MANAGING AUSTRALIA REDUCE THE THREAT OF INVASION BY A ARBUSCULAR MYCORRHIZAL FUNGI? NON-NATIVE TREE?

1, 2 1, 3 1 1 and Kirkegaard J.A.2 Trudgen M.S.1, 2, Webber B.L. , Scott J.K. and Lambers H. Ryan M.H. 1 1School of Plant Biology, UWA, Perth, WA. 2CSIRO Plant Industry, School of Plant Biology, The University of Western Australia, Crawley, 2 Canberra, ACT. WA 6009, Australia. CSIRO Ecosystem Sciences and Climate Adaptation Flagship, Wembley, WA 6913, Australia. 3School of Animal Phosphate (P) reserves are limited and therefore there is an urgent need Biology, The University of Western Australia, Crawley, WA 6009, to improve the P-use efficiency of agriculture. Arbuscular mycorrhizal Australia. fungi (AMF) colonise the roots of most crop species and are reported to enhance P uptake and growth of crops in many glasshouse studies Tipuana tipu (Fabaceae) is an invasive non-native tree, and is listed and some field studies. Thus, should Australian farmers prioritise as a national alert weed in Australia. It is an ornamental species management practices that favour AMF? To address this question we that is widely planted in urban gardens, as a street tree and in the identified all reports on field experiments focussed on AMF in Australian racehorse industry. It is an aggressive invader in subtropical southern extensive cropping systems. Wheat was the crop most often studied. Queensland and northern New South Wales. Despite high propagule Findings were reviewed in the context of the large body of Australian pressure from horticultural plantings and street trees, the species has agronomic literature. It was found that low root colonisation by AMF not naturalised in temperate Western Australia. While climate is likely occurs in crops following non-mycorrhizal break-crops (e.g. canola and to play a role, recent insight suggests that T. tipu is able to germinate lupin) and plant-free fallow. Low colonisation may also occur if soil P and survive across a wide range of climatic conditions. Other factors, levels are high (e.g. after P fertiliser is applied). In the northern cropping therefore, may play an important role in the ability of T. tipu to naturalise zone, low colonisation sometimes causes yield reductions in wheat and and invade. We hypothesised that low soil nutrient levels in Western other crops. In the larger southern cropping zone, low colonisation Australia inhibit invasion. We grew T. tipu in washed quartz river sand has no effect on, or even enhances, wheat yield, even on low P in a glasshouse trial, across a phosphorus gradient (0 – 640 mg/kg) soils. Information is limited for other crops, but there are no reports to represent natural, agricultural and phosphorus-enriched urban of poor growth when colonisation is low. Evidence for significant non- soils. We found that soil phosphorus had a significant effect on the nutritional benefits from high colonisation by AMF (e.g. in control of root establishment and growth rate of young T. tipu seedlings. It appears diseases or maintenance of soil structure) is lacking. It is concluded that that the naturally low phosphorus levels in Western Australian soil may farmers generally do not need to consider AMF when planning their be contributing to the lack of invasion success for T. tipu. crop management regime and that agronomic practices that underpin sustainable productivity in Australian cropping systems, such as non- mycorrhizal break-crops and P fertiliser, often reduce colonisation by AMF. These results highlight that potential benefits from manipulation of the soil biological community must be assessed in a whole-of-system agronomic context.

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SYM-33-01 SYM-33-02 CELLULAR CYTOKINE CROSS-TALK DURING ORGANISATION AND REGULATION OF LUNG CHRONIC LIVER DISEASE, FIBROGENESIS AND EPITHELIAL STEM CELLS REGENERATION Van Der Velden J., McCarthy R., Bertoncello I. and McQualter J. Ramm G.A. University of Melbourne. Queensland Institute of Medical Research, Brisbane. Regeneration of the adult lung epithelium is regulated by the intrinsic In chronic liver disease, myofibroblast precursors called hepatic stellate potential of resident stem/progenitor cells and extrinsic cues derived cells (HSCs) play a crucial role in wound healing via inflammation, from the local microenvironment. We have recently provided evidence extracellular matrix remodelling and fibrillar collagen deposition, which that the adult mouse lung contains a population of multipotent epithelial if left uncontrolled can lead to fibrosis. HSCs do not work in isolation stem/progenitor cells (EpiSPC). These cells are able to self-renew and and are impacted upon by local production of soluble mediators generate descendants which give rise to airway and alveolar epithelial produced in response to tissue injury and direct cell-cell contact with cell lineages in vitro. However, the lack of specific markers precludes liver progenitor cells (LPCs), inflammatory cells and other parenchymal lineage tracing analysis of these cells in vivo. In this study we have cells. LPC expansion, migration and differentiation occurs in an used our colony-forming assay to determine how changes in the attempt to replace lost ductular and parenchymal tissue when the stromal microenvironment affect the proliferation and differentiation of normal replicative capacity of hepatocytes is compromised in the EpiSPC in vitro. Using this approach we have identified an important injured liver. Chronic liver diseases, although diverse in aetiology, show role for resident lung mesenchymal stromal cells (MSC) in regulating a similar progression of pathologies, characterised by three major lung EpiSPC. We have shown that resident MSC (or factors thereof) cellular changes: inflammation, expansion of the LPC compartment are necessary and sufficient to support epithelial colony formation and fibrogenesis involving HSC activation. Each of these processes from primary isolated EpiSPC. Conversely, we have shown that has been extensively studied in isolation, however, few studies have myofibroblastic differentiation of MSC results in a loss of their epithelial investigated how these cellular responses are cross-regulated and how supportive capacity, which can be reversed by blockade of the TGF-β interaction between cell types may contribute to the regenerative and signalling pathway. This supports the concept that the regenerative fibrogenic response of the liver to chronic injury. Recent studies from potential of EpiSPC is regulated by their interaction with the our group and others support the concept that hepatic fibrosis is driven microenvironment and suggests that its inhibitory or permissive nature by expanding/migrating LPCs (including their biliary differentiation, i.e., is regulated by TGF-β1. These studies demonstrate that changes in “ductular reaction”), suggesting potential cross-talk between HSCs the cellular composition of the local microenvironment can significantly and LPCs. Key mediators in this process have now been identified alter the regenerative potential of adult lung EpiSPC. and include the macrophage/NK cell-derived LPC mitogen TNF-like weak inducer of apoptosis (TWEAK), acting via its receptor Fn14; LPC surface-bound lymphotoxin-β (LT-β) acting via the LT-β Receptor (LT- βR) on activated HSCs to induce RANTES-induced chemotaxis of CD45+ lympohocytes, HSCs and LPCs; and the demonstration that myofibroblast expression of the Notch ligand, Jagged 1, promotes Notch signaling in LPCs driving their biliary differentiation (“ductular reaction”).

SYM-33-03 SYM-33-04 Sponsored by A SYSTEMATIC APPROACH TO IDENTIFY NOVEL The University of Western Australia COOPERATIVE TUMOUR SUPPRESSOR GENES IN RAS-DRIVEN TUMOURIGENESIS LIVE AND LEAVE ALIVE: MATRIX COMPONENTS IMPROVE MSC SURVIVAL Manent J.1, Banerjee S.1, Willoughby L.F.1, Penninger J.M.3, Simpson K.J.1, 2, Humbert P.O.1 and Richardson H.E.1 Wells A. 1Peter MacCallum Cancer Centre; Sir Peter MacCallum Department University of Pittsburgh, PA, USA. of Oncology, University of Melbourne, Melbourne, Australia. 2Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, Multipotential stromal cells / mesenchymal stem cells (MSCs) are ideal Melbourne, Australia. 3Institute of Molecular Biotechnology of the candidates for regenerative therapy as these cells differentiate into Austrian Academy of Science, Vienna, Austria. multiple lineages and positively influence neighboring cells. However on implantation, these cells are faced with nutrient deprivation and The development of a tumour is a multistep process in which non-specific inflammation, resulting in cell loss. Surface-restricted neoplastic cells accumulate mutations and reactivate developmental epidermal growth factor receptor (EGFR) signaling by a tethered pathways to maintain a progenitor-like state, thereby sustaining self- form of EGF (tEGF) enhances MSC survival in these challenging replicative potential, survival and migratory properties. The Ras small environments. Interestingly, during wound repair an ECM component G proteins are key regulators of these cellular processes, and Ras- Tenascin C (TNC) containing EGF-like repeats (EGFL) is upregulated. activating mutations are found in ~30% of human cancers. However, The EGFL bind wtih low affinity / high avidity causing sustained surface- Ras alone is not sufficient for the development of tumours. Therefore, restricted EGFR signaling. TNC is attractive as a naturally occurring it is of utmost importance to decipher the networks driving neoplastic molecule that would not be immunogenic. MSCs grown on TNC and behaviours in cells in which the Ras oncogene is constitutionally Collagen I displayed a survival advantage in the presence of FasL. active. To identify genes and processes that act in concert with Ras TNC neither sequestered nor neutralized FasL; rather the effects were to promote proliferation, survival and invasion, we have undertaken a via cell signaling of TNC activating EGFR and downstream pathways systems level approach combining genetic screens in vivo and in vitro. of Erk and Akt through EGFL. MSCs deposit TNC in proliferative media In vivo, we have found novel tumour suppressor genes that cooperate in culture. Importantly, TNC, like tEGF, neither impelled or impeded with oncogenic Ras to confer increased hyperplasia of the Drosophila MSC differentiation into osteoblasts or adipocytes, enabling this to be developing eye epithelia. In vitro, we have developed a shRNA used in cell therapies. Going in vivo, both tEGF and TNC protected screening platform to translate the fly screen to human epithelial cells MSC when implanted into mice in collagen plugs. tEGF has been grown in 3D, and validate Ras-cooperative genes and processes attached to tricalcium phosphate particles and improves bone healing conserved across species. Our study has identified clusters of genes in a canine model. These results suggest that providing MSC with a involved in the regulation of diverse biological processes such as cell non-immunogenic ECM moiety like TNC enhances their survival in the polarity, chromatin remodelling or autophagy. The effect of these novel presence of death factors via signaling of EGFR. This matrix is being tumour suppressor genes on differentiation, proliferation, survival and used to supplement MSC delivery on scaffolds to provide a survival invasion is being examined. advantage against non-specific inflammation in vivo and thus provide a novel avenue for supporting stem cell incorporation into regenerating tissue.

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SYM-33-05 SYM-34-01 SIGNALLING PATHWAYS CONTROLLING INTESTINAL BUILDING THE INTRACELLULAR METHYLPROTEOME STEM CELL BIOLOGY AND TUMOUR FORMATION NETWORK

Germann M., Xu H., Pereira L., Malaterre J. and Ramsay R.G. Wilkins M.R. Peter MacCallum Cancer Centre, Melbourne, VIC, Australia. School of Biotechnology and Biomolecular Sciences, University of New South Wales NSW 2052 Australia. The regenerative capacity and activity of stem cells is tightly regulated by signalling pathways, many of which are aberrantly activated in Methylation of proteins occurs predominantly on arginine and lysine cancer. Our lab is interested in how these pathways interact to control residues in the eukaryotic cell. Until recently, its predominance was self-renewal, proliferation, differentiation, as well as tumour formation. unknown and its role obscure. This presentation outlines our efforts Among other tissues we mainly focus on the intestinal epithelium as to construct the first ‘methylproteome network’ for a eukaryotic cell a highly self-renewing and tumour-prone tissue. To do this we are and provides evidence that arginine methylation modulates protein- concurrently inhibiting and activating different pathways in intestinal protein interactions in this network. We analysed the S. cerevisiae cells using various crosses of transgenic mice. Looking at the intestine methylproteome to identify methylated proteins and precise in vivo and in organotypic cultures in vitro we follow homeostatic and modification sites. Immonium ion-based scanning and targeted data tumorigenic processes in real-time. This approach further helps us to acquisition - electron transfer dissociation MS/MS was used, as understand how stem cells interact with their microenvironment during were yeast proteome arrays (containing 4,400 chips spotted onto these processes. Thereby we show how signalling pathways such as microscope slides). This showed that protein methylation is widespread Wnt, Notch and Myb act together to control stem cells and that aberrant in the eukaryotic cell. To build the intracellular methylation network, activation of single pathways determine or essentially contribute to all known and putative methyltransferases in yeast were knocked out different steps of tumorigenesis. Activity of these pathways depends on and the methylproteome re-analysed to determine which enzyme was extrinsic signals, which derive either from the stem cell niche or from responsible for which methylation event. This led to the discovery of the surrounding stroma, such as inflammatory signals. In return, the a new lysine methyltransferase, we named Efm2. Enzyme-substrate stem cell signalling pathways themselves control generation of stem links were further investigated by the analysis of recombinant substrate cell niche cells as well activation of surrounding stroma, which in theory proteins methylated by recombinant enzymes, by in vivo methylation again may feed back on aberrant stem cell activity. This demonstrates assays and/or the incubation of proteome arrays with recombinant how easily aberrant activity of stem cell signalling pathways can lead enzymes. Validated enzyme-substrate links were integrated with to neoplasia and how tightly they need to be regulated to guarantee the yeast protein-protein interaction network to generate the first proper homeostasis. methylproteome network. Interestingly, this suggested that many protein-protein interactions could be controlled by protein methylation. To test this, we constructed a new conditional two-hybrid (C2H) system. Interactions of proteins were tested in the presence of a methyltransferase or in the presence of the same enzyme with active site knocked out. Of the protein-protein pairs involving arginine methylated proteins, half of those tested to date have shown increases in interaction in association with methylation. The implications of this will be discussed.

SYM-34-02 SYM-34-03 DECODING CALCIUM SIGNALS IN THE HEART TRANSCRIPTOME RESPONSES TO ANAEROBOSIS IN RICE AND ARABIDOPSIS Crampin E.J.1, 2 1The University of Melbourne. 2NICTA. Narsai R.1, 2 and Whelan J.1, 3 1ARC CoE PEB, University of Western Australia. 2Centre for The dynamics of calcium signalling in heart cells presents many puzzles Computational Systems Biology, University of Western Australia. for biomedical researchers. Calcium signals coordinate contraction 3Department of Botany, School of Life Sciences, La Trobe University. of the heart muscle following electrical stimulation. Calcium signals arising from hormonal stimulation may also regulate gene expression, Plants face a variety of environmental stresses and have evolved for example during development and in cardiac hypertrophy. Given that molecular mechanisms to survive these challenges. One of these these two types of calcium signals coincide in the cell, how do they not stresses is low-oxygen conditions, which can occur under flooding interfere with one another? A quantitative modelling analysis of these stress. Rice (Oryza sativa) is somewhat unique for its ability to tolerate signaling pathways is required to address this issue. We demonstrate and even germinate under low to no oxygen conditions. In this study that a small, localised hypertrophy-related calcium signal can be we examined global transcriptomic responses over the course of detected by key calcium-sensing proteins against a background of germination and in response to low-oxygen and abiotic stresses the large beat-to-beat cytosolic calcium transients that coordinate cell in rice and Arabidopsis (Arabidopsis thaliana). Analysis of genes contraction with each beat of the heart. This forms part of a broader that responded differently in rice compared to Arabidopsis revealed initiative in cardiac systems biology to model interactions between responses specific to anaerobic germination in rice, including the signaling, mechanical and metabolic pathways in the heart in health down-regulation of genes encoding redox functions and up-regulation and disease. of receptor kinases. Comparison of a range of hypoxia/anoxia studies within and across Arabidopsis and rice revealed conserved and species specific changes in gene expression (e.g. Arabidopsis specific up-regulation of WRKYs and rice specific down-regulation of heme), unveiling unique transcriptomic signatures of the low-oxygen response. A comparison of the low-oxygen response with cold, salt, drought and heat stress revealed some similarity with the response to heat stress in Arabidopsis, which was not seen in rice. Comparison of these heat- responsive, abiotic stress marker genes in Arabidopsis with their rice orthologues revealed that while low-oxygen may be perceived as an abiotic stress in Arabidopsis, this is not the case in rice. Finally, changes in genes encoding protein involved in epigenetic modification of DNA under anoxia in rice, not observed in other abiotic stresses, suggest a role for epigenetic regulation in response to anoxia in rice.

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SYM-34-04 SYM-34-05 A TALE OF TWO TERMINI: PROFILING MRNA 5’-3’ GENOME-WIDE INVESTIGATION OF THE HOST INTERACTIONS AND TRANSLATIONAL REGULATION microRNA RESPONSE TO COXIELLA BURNETII INFECTION IN HUMAN MACROPHAGE CELLS Archer S.K.1, Shirokikh N.E.1, Hallwirth C.3, Yuan J.3, Beilharz T.2, Sobti M.3 and Preiss T.1 Lisanby M., Beer M., Unsworth N. and Liu C.Q. 1John Curtin School of Medical Research, Australian National DSTO Land Division, 506 Lorimer Street, Fishermans Bend, University. 2Monash University. 3Victor Chang Cardiac Research Melbourne VIC 3207, Australia. Institute. Coxiella burnetii is a Gram negative obligate intracellular pathogen that Interactions between the 5’ and 3’ termini of mRNAs have long been is the causative agent of Q fever. Humans normally become infected postulated to occur in the cell. This “closed-loop” model of mRNA can by exposure to contaminated aerosolized animal body fluids. The initial explain the ability of 3’ UTR features and the poly(A) tail to modulate step of Q fever pathogenesis is the phagocytic uptake of C. burnetii small translation initiation and other events at the 5’ end. Here we introduce cell variants by alveolar macrophages. In order to survive within these a novel assay to detect the closed-loop conformation of specific professional phagocytic cells, C. burnetii has to subvert the standard mRNAs in vivo for the first time. Using Saccharomyces cerevisiae, we host immune response to foreign antigen and establish an intracellular demonstrate that the closed-loop is the predominant conformation of niche that will support bacterial replication. C. burnetii accomplishes this the subset of mRNAs stabley bound by the cap-binding complex eIF4F. by hijacking the autophagic pathway and undermining phagolysosomal However, the bulk of mRNAs are not in a stable closed-loop conformation maturation using a Type IV Secretion System. Little is known about during normal growth, most likely due to the limiting concentrations of how the bacterium modifies these pathways or which host factors are eIF4F components in the cytoplasm. Various examples are known involved in host-pathogen interactions. In this study, we focussed on where interactions between eIF4F and PABP, which are required for microRNAs because of their well-established roles in eukaryotic host closed-loop formation, come under regulatory intervention, resulting responses to various viruses and extracellular bacterial pathogens. To in reduced translational efficiency. Thus, detecting altered closed- investigate the possible involvement of microRNAs in the pathogenesis loop status represents a catch-all method of identifying mRNAs that of C. burnetii, we conducted the first microarray study to examine the are translationally regulated. We are currently investigating global and global cellular microRNA response to C. burnetii infection. Using an in transcript-specific regulation of the closed-loop conformation, which vitro human macrophage-like cell-line infection model, we identified 22 will identify regulatory mechanisms that enable organisms to rapidly miRNAs that were significantly, differentially regulated during the mid- alter gene expression in response to intrinsic and extrinsic signals. log phase of a C. burnetii infection compared to uninfected controls. These microRNAs were predicted to be involved in various host cellular functions, such as apoptosis, cell cycle and cholesterol metabolism. Our findings provide new insight into host mechanisms that are required for C. burnetii infection.

SYM-35-01 SYM-35-02 Sponsored by Sponsored by The University of Western Australia The University of Western Australia

ACTIVATION MECHANISM OF THE UNFOLDED LIPID MEMBRANES MODULATE PROTEIN PROTEIN RESPONSE DURING LIPID DISEQUILIBRIUM AGGREGATION RELATED TO NEURODEGENERATION

Wu H.1, 2, Xu C.2, 3, Ng D.T.W. 2, 3 and Thibault G.1, 2, 4 Legleiter J. 1School of Biological Sciences, Nanyang Technological University, The C. Eugene Bennett Department of Chemistry, West Virginia Singapore. 2Temasek Life Sciences Laboratory, National University of University, Morgantown, West Virginia 26505, United States. Singapore, Singapore. 3Department of Biological Sciences, National University of Singapore, Singapore. 4Lee Kong Chian School of The formation of nanoscale protein aggregates generally termed Medicine, Nanyang Technological University, Singapore. amyloids is the hallmark of several neurodegenerative diseases, including Alzheimer’s disease (AD) and Huntington’s disease (HD). While the precise The transmembrane protein Ire1p mediates the unfolded protein toxic mechanism associated with amyloidogenic protein aggregates response (UPR) by regulating HAC1 mRNA splicing. It is currently is yet to be determined, amyloid-forming proteins have been shown to accepted in the field that Ire1 lumen domain senses ER stress by directly interact with lipid membranes. Lipid membranes can mediate directly binding unfolded proteins accumulating in the lumen. In recent protein aggregation by promoting or stabilizing the formation of specific reports, it was demonstrated in yeast and mammalian cells that Ire1 is aggregate forms. Furthermore, lipid membranes may also be targeted by activated from perturbed ER membrane through its transmembrane or toxic protein aggregates, leading to dysfunction. A detailed understanding cytosolic domains. However, the cascade of events required to activate of the influence of cellular surfaces in driving protein aggregation and/ the UPR from perturbed membrane remain unclear. or stabilizing specific aggregate forms could provide new insights into toxic mechanisms associated with these diseases. Using a variety of scanning probe microscopic techniques, the interaction of the β-amyloid (Aβ) peptide associated with AD and mutant huntingtin (htt) proteins with expanded polyglutamine domains associated with HD with supported lipid membranes was explored. The interaction of Aβ and htt with supported lipid bilayers was highly dependent on bilayer composition and protein context. For example, certain domains of Aβ appeared to facilitate its interaction with lipid bilayers, and specific flanking sequences adjacent to a the polyglutamine domain in htt were required for the initial binding of the protein to membranes. We also demonstrated that lipid bilayer structure is disrupted by amyloid-forming proteins, leading to altered mechanical properties of the membrane that are consistent with a decreased packing efficiency of the lipids within the bilayer. The ability of these proteins to perturb membrane integrity could directly lead to membrane dysfunction, and it is an intriguing possibility that induced changes in lipid membranes may represent a common toxic mechanism for amyloid diseases.

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SYM-35-03 SYM-35-04 WHEN QUALITY CONTROL GOES WRONG: USING TDP-43, FUS AND SOD1 FORM PROTEIN NEUROPATHIC GAUCHER DISEASE TO DISSECT AGGREGATES VIA DISTINCT PATHWAYS IN CELLS PARKINSON’S DISEASE Farrawell N., Lambert-Smith I. and Yerbury J.J. Osellame L.D.1, 4, Rahim A.A.2, Waddington S.N.2, Schapira A.V.H.3 IHMRI, University of Wollongong. and Duchen M.R.1 1Department of Cell and Developmental Biology, University College ALS is characterised by the progressive and selective death of upper London, WC1E 6BT, UNITED KINGDOM. 2Gene Transfer Technology and lower motor neurones in the motor cortex and spinal cord. This Group, Institute for Women’s Health, University College London, WC1E leads to loss of muscle control, muscle atrophy and invariably death, 6BT, UNITED KINGDOM. 3Department of Clinical of Neurosciences, generally within 3-5 years of diagnosis. The actual cause(s) of most Royal Free Hospital and University College Medical School, London, cases of ALS (sporadic ALS; sALS) remain undefined, however, NW3 2PF, UNITED KINGDOM. 4Current Address: La Trobe Institute for approximately 5-10% of cases are inherited (familial ALS; fALS). ALS Molecular Sciences, La Trobe University, Melbourne, 3086, AUSTRALIA. pathology is characterised by a number of inclusions including Bunina bodies, basophilic inclusions, axonal spheroids and ubiquitinated round, Gaucher Disease (GD) is the most common Lysosomal Storage Disorder conglomerate or skein-like inclusions. The composition of ubiquitinated and results from mutations in the glucocerebrosidase (GBA) gene. Loss of inclusions varies considerably depending on whether the disease is functional GBA in lysosomes leads to accumulation of glucocerebroside, sporadic or familial and the genetics of the familial forms. Both FUS and resulting in non-functional organelles. Strong evidence has emerged SOD1 mutations cause ALS with FUS and SOD1 positive inclusions linking lysosomal dysfunction and Parkinson’s Disease (PD); individuals respectively. However, most other cases have inclusions positive for with a homozygous loss of GBA have up to a twenty fold increased risk TDP-43. Inclusions in sALS have been observed to contain the fALS of developing PD, thus loss of GBA activity is the highest genetic risk associated proteins FUS, optineurin, ubiquilin2 and p62. Collectively factor for PD. Using a murine model of type II neuropathic GD, gba-/- these data implicate protein aggregation as an underlying mechanism primary neurons and astrocytes have a defective autophagy pathway with important to ALS pathology. This study aimed to test whether fALS reductions in LC3-I/II and Atg5/12 levels. Defective autophagy impinges associated proteins aggregate via distinct mechanisms in the cellular on the UPS with increases in ubiquitinated proteins, p62/SQSTM1 context. Using ALS associated proteins tagged with fluorescent protein aggregates as well as α-synuclein deposits in the midbrain. As a result we show that TDP-43 will co-aggregate with FUS but not SOD1 in NSC- of defective quality control mechanisms dysfunctional mitochondria, that 34 cells. In addition, SOD1-GFP co-aggregates early with ubiquitin, are unable to recruit the E3 ubiquitin ligase Parkin, are not flagged for while ubiquitin appears co-localised with FUS and TDP-43 later in turnover and accumulate. gba-/- cells have reductions in CI, CII/III activity the aggregation process. Moreover, we find that SOD1 inclusions are and membrane potential and hydrolyse ATP in order to maintain it. Loss microtubule dependant, while TDP-43 and FUS aggregates do not diminish in number upon microtubule destabilisation. We also observed of membrane potential is rescued by the antioxidant MitoQ10, suggesting damage is mediated by ROS generation from CI. In addition treatment that while FUS will co-localise with dense Httex146Q-RFP inclusions TDP-43 and SOD1 do not. These data suggest that the properties of with MitoQ10 does not restore defects in autophagy, confirming a primary lysosomal defect affects cellular quality control. Taken together these TDP-43, FUS and SOD1 inclusions and the pathways through which findings suggest that cellular dysfunction observed in GD, like that of PD, they form are distinct in ALS and are thus unlikely to be a common is a consequence of defects in autophagy/mitophagy pathways, resulting underlying cause of ALS. in failed clearance of damaged mitochondria.

SYM-35-05 SYM-36-01 CHARACTERISING THE BINDING SITES OF A NOVEL FACTORS INFLUENCING THE SEX-SPECIFIC BETA-AMYLOID INTERACTING PEPTIDE: TOWARDS DIFFERENTIATION OF FETAL GERM CELLS PEPTIDOMIMETICS FOR ALZHEIMER’S DISEASE Bowles J.1, Spiller C.M.1, Feng C.-W.1, Gillis A.2, Looijenga L.H.J.2 and Barr R.K.1, 2, Verdile G.1, Taddei K.1 and Martins R.N.1, 2 Koopman P.1 1Centre of Excellence for Alzheimer’s Disease Research and Care, 1Institute for Molecular Bioscience, The University of Queensland, Edith Cowan University, 270 Joondalup Drive, Joondalup, WA 6027, Brisbane, Australia. 2Josephine Nefkens Institute, Erasmus University Australia. 2Alzhyme Pty Ltd. Medical Center, Rotterdam, The Netherlands.

According to the “amyloid hypothesis”, the development of Alzheimer’s Germ cells respond to molecular cues that regulate their sex-specific Disease pathology results from a shift in the production, metabolism and development, and the decision to withdraw from the cell cycle, in the clearance of the human beta amyloid 1-42 (Abeta42) peptide, causing fetal gonads. In fetal ovaries, germ cells enter meiosis whereas in fetal oxidative stress, neuronal loss and cognitive decline. This highlights testes, germ cells arrest mitotically. We report here on several signaling the potential utility of agents that target the toxic Abeta42 peptide. We systems that contribute to the control of fetal germ cell fate in vitro and have previously reported a novel 15mer Amyloid Neutralising Agent in vivo. These are : --- (1). Germ cells in female mouse fetal gonads (ANA-1) peptide that targeted Abeta42 and attenuated its neurotoxicity are triggered to enter meiosis by the signaling molecule retinoic acid (Taddei et al. 2010 Neurobiol Aging. 31:203-14). We have since (RA). RA induces germ cells to express Stra8, which is essential for demonstrated that in vitro, ANA-1 reduces the formation of neurotoxic initiation of meiosis. In the developing testis, germ cells avoid entering “oligomers” and concurrently promotes the formation of non-toxic, meiosis because RA is actively degraded by a cytochrome P450 insoluble aggregates. To better understand how the ANA-1-Abeta42 enzyme, CYP26B1. --- (2). FGF9 acts directly on testicular germ cells interaction results in reduced formation of toxic oligomers, we have to prevent up-regulation of Stra8 and hence their entry into meiosis. conducted complementary studies to ascertain the relative importance Thus, Stra8 expression, and hence meiosis, is regulated both positively of regions within both peptides. Using a PepSet of modified ANA-1 and negatively by RA and FGF9 respectively. --- (3). The TGFβ peptides, we have determined the minimal sequence required for Abeta morphogen Nodal and its co-receptor Cripto regulate the balance binding and also the relative importance of individual ANA-1 residues between continued germ cell proliferation and cell fate commitment in for the interaction. We have further conducted an “epitope-mapping” the developing testis. Compromised Nodal signaling in male germ cells experiment, where sequential 15mer peptides from the Abeta42 led to reduced pluripotency, and premature differentiation. Conversely, sequence were assayed for ANA-1 binding activity, to clarify the ANA-1 human testicular germ cell tumours showed abnormally high levels of binding site within Abeta42. Here we provide evidence that the binding NODAL and CRIPTO. Our results support the hypothesis that testicular between these peptides most likely occurs via both H-bonding and germ cell tumours and some forms of infertility have their origins in hydrophobic interactions, and involves regions of Abeta42 previously dysregulation of pathways controlling germ cell behaviour in the determined to contribute to its self-association and aggregation. Using embryo. this data, we hope to design novel peptidomimetics that mimic the action of ANA-1, but with improved pharmacokinetic profiles.

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SYM-36-02 SYM-36-03 THE UNIQUE ROLES OF RNA-BINDING MALE INFERTILITY - THE CANARY IN YOUR PROTEINS MUSASHI-1 AND MUSASHI-2 DURING COALMINE SPERMATOGENESIS: INSIGHTS FROM TRANSGENIC MOUSE MODELS O’Bryan M. Department of Anatomy and Developmental Biology, Monash Sutherland J.M.1, Koopman P.2, Hime G.R.3 and McLaughlin E.A.1 University, Melbourne. 1School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308 Australia. 2Institute for Molecular Bioscience, Male infertility affects 1 in 20 Australian men of reproductive University of Queensland, Brisbane, QLD 4072, Australia. 3Anatomy age and for at least half a precise diagnosis cannot be provided. and Cell Biology, University of Melbourne, Parkville, VIC 3010, Conversely, for the majority of other people additional contraceptive Australia. options, including those that target the male germ line, are desirable. Both deficits are in reality caused by the same problem, a lack of Mammalian spermatogenesis is a complex developmental process knowledge regarding the processes why which sperm are produced requiring the strict regulation of stage-specific gene expression to and how they function. Interestingly epidemiological data is suggesting produce physically mature spermatozoa from undifferentiated germ the human male infertility is associated with a decreased life cells. Crucial to this process are RNA binding proteins responsible expectancy. In an effort to address these knowledge gaps, we conduct for the regulated post-transcriptional control of essential mRNAs, a random mouse mutagenesis (forward genetic) screen to identify particularly important during the two periods of inactive transcription additional genes/proteins involved in male fertility. The screen was which occur during spermatogenesis. Here we focus on the Musashi conducted through the Australian Phenomics Network and was very family of RNA binding proteins, in particular the roles of mammalian successful. We identified numerous genes that had not previously Musashi-1 (MSI1) and Musashi-2 (MSI2), both previously identified been implicated in fertility, and often in any other biological process. as key translational regulators in various stem cell populations. In the We identified multiple genes involved in microtubule dynamics present study, the expression patterns of MSI1 and MSI2 throughout and protein transport within haploid germ cells; regulators of RNA all key stages of mammalian spermatogenesis are characterised metabolism and others involved in stem cell function. Each of these and described. Extensive examination of the role of Musashi in mouse lines is now revealing novel insights of relevance not only to spermatogenesis was achieved through the use of two transgenic male fertility, but often to diverse tissues throughout the body. mouse models with germ cell specific over-expression of full-length Msi1 or Msi2. These models demonstrated that regulation of Msi2 is essential for normal spermatogenesis, and the controlled expression of both Msi1 and Msi2 are crucial to fertility. Finally, novel evidence is presented for the stabilisation of Msi2 via direct interactions with MSI1 during early spermatogenesis, prior to the nuclear translocation of MSI1 by Importin-5 during the transition from mitosis to meiosis in pachytene spermatocytes. Overall this study suggests a crucial role for MSI2, unique to that of MSI1, during the process of germ cell development in the mammalian testis, and establishes the Musashi family proteins as key regulators of germ cell development during the process of spermatogenesis.

SYM-36-04 SYM-36-05 miRNA PROFILE OF DIVERGENT REPROGRAMMING REGULATION OF GENE EXPRESSION BY RNA ROUTES TO MULTIPLE STEM CELL STATES POLYMERASE II PROMOTER PAUSING DURING MOUSE EMBRYONIC DEVELOPMENT Clancy J.L.1, Patel H.R.1, Cloonan N.2, Corso A.3, Puri M.C.3, Tonge P.D. 3, Nagy A.3 and Preiss T.1 Weston M., Ng J. and Wilson M.J. 1JCSMR, Australian National University, Australia. 2Queensland Department of Anatomy, Otago School of Medical Sciences, Institute of Medical research, Australia. 3Samuel Lunenfeld Research University of Otago, Dunedin 9054, New Zealand. Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. Proximal-promoter pausing by RNA polymerase II is a key rate- The recent establishment of secondary fibroblast reprogramming limiting step in transcription initiation. Recent genome-wide studies systems allows observations of whole culture reprogramming. using chromatin-immunoprecipitation to detect stalled RNA-pol II Intriguingly, we have found that modifying Yamanaka factor levels can have shown that promoter-pausing occurs for a number of genes, produce multiple, distinct pluripotent cell states, including: traditional particularly developmental control genes. This stalling is believed to be iPSC states; two variant, alternative, transgene-dependent, teratoma- a mechanism of gene regulation, causing RNA-pol II to be paused near competent states; and a 3-factor derived state (OSK), all with distinct a promoter region, ready to respond to environmental or developmental miRNA profiles. The alternative L/C-class state displays low expression cues. Two transcription elongation factors DSIF and NELF control of many core miRNA-mediators of pluripotency, including the miR-302 promoter stalling by RNA-pol II. Our laboratory studies sex-specific cluster. Nevertheless miRNAs expressed higher in this alternative state differentiation of developing mouse embryo tissues and we have utilized target many core reprogramming pathways (e.g. cell cycle), while also this system to study RNA pol II stalling and its effect on gene expression displaying divergent targeting (e.g. MET) consistent with their differing over key developmental stages. Using ChIP-seq with antibodies morphologies. miRNA expression change is often rapid and extreme. against RNApol II, we identified many promoters that have RNA-pol II For example, almost 1/3 of all miRNAs change expression >4-fold in stalled in a sex-specific manor in both gonad and head tissue at 13.5 the first 48 hours. In contrast, while many of the core reprogramming dpc. This corresponded to differences in gene expression between the miRNAs have 5’ isomiRs (which alter their targeting spectrum) and/ sexes for the associated gene transcript (as assayed by qPCR). For or have multiple variant 3’ ends (due to non-templated addition or some transcripts, RNA pol II stalling marked them for future activation alternative processing), we find that the relative proportions of these at a later time point in development. In other cases, paused RNA pol II variants remains surprisingly stable during the reprogramming was associated with genes that were becoming down regulated. This process. Broader analysis of the dataset also shows the wider small data also reveals that promoter pausing can occur differently between RNA profile changes markedly after induction of the Yamanaka factors sexes during development. We also have preliminary evidence that and we describe a novel mitochondrial-derived small RNA that is indicates that components of NELF and DSIF complexes (that control massively expressed in the L/C-class state. This work challenges the RNA pol II pausing) are expressed differently between the sexes during assumed requirement of a single specific miRNA expression profile for embryogenesis. Together our data suggests that some genes are being pluripotency and, as part of a multi-omics, international collaborative poised to respond to a signal and then strongly upregulated, whereas effort, forms an integral resource for understanding divergent transcription at other genes is stalled but not activated, perhaps in the pluripotent states. absence of an appropriate signal.

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SYM-37-01 SYM-37-02 ROLES OF ASCIZ AND DYNEIN LIGHT CHAIN IN CREB-BINDING PROTEIN (CBP) REGULATES REGULATING BIM-DEPENDENT APOPTOSIS DURING β-ADRENOCEPTOR (β-AR)-MEDIATED APOPTOSIS B CELL DEVELOPMENT Lee Y.Y.1, Moujalled D.1, Doerflinger M.1, Weston R.1, Rahimi A.1, Heierhorst J.1, 2 De Albroran I.2, Herold M.3, Bouillet P.3, Du X.-J.4 and Puthalakath H.1 1St. Vincent’s Institute of Medical Research. 2Dept of Medicine SVH, 1La Trobe University, Bundoora, Melbourne. 2Centro Nacional de University of Melbourne. Biotecnología, Madrid, Spain. 3The Walter and Eliza Hall Institute for Medical Research, Melbourne. 4Baker IDI Heart and Diabetes Developing B lymphocytes expressing defective or auto-reactive pre-B Research Institute. or B-cell receptors (BCRs) are eliminated by programmed cell death, but how the balance between death and survival signals is regulated Catecholamines regulate the β-adrenoceptor/cyclic AMP-regulated to prevent immunodeficiency and auto-immunity remains incompletely protein kinase A (cAMP/PKA) pathway. Deregulation of this pathway understood. Here we show that absence of the essential ATM substrate can cause apoptotic cell death and is implicated in a range of human Chk2-interacting Zinc-finger protein (ASCIZ, also known as ATMIN/ diseases, such as neuronal loss during aging, cardiomyopathy and ZNF822), a protein with dual functions in the DNA damage response septic shock. The molecular mechanism of this process is, however, and as a transcription factor, leads to progressive cell loss from the only poorly understood. Here we demonstrate that the β-adrenoceptor/ pre-B stage onwards and severely diminished splenic B cell numbers cAMP/PKA pathway triggers apoptosis through the transcriptional in mice. This lymphopenia cannot be suppressed by deletion of p53 induction of the pro-apoptotic BH3-only Bcl-2 family member Bim or complementation with a pre-arranged BCR, indicating that it is in tissues such as the thymus and the heart. In these cell types, the not caused by impaired DNA damage responses or defective V(D)J catecholamine-mediated apoptosis is abrogated by loss of Bim. recombination. Instead, ASCIZ-deficient B cell precursors contain highly Induction of Bim is driven by the transcriptional co-activator CBP reduced levels of DYNLL1 (LC8), a recently identified transcriptional (CREB-binding protein) together with the proto-oncogene c-Myc. target of ASCIZ, and normal B cell development can be restored by Association of CBP with c-Myc leads to altered histone acetylation ectopic Dynll1 expression. Remarkably, the B cell lymphopenia in and methylation pattern at the Bim promoter site. Our findings have the absence of ASCIZ can also be fully suppressed by deletion of implications for understanding pathophysiology associated with the pro-apoptotic DYNLL1 target Bim. Our findings demonstrate that a deregulated neuroendocrine system and for developing novel ASCIZ plays a key role in regulating the survival of developing B cells therapeutic strategies for these diseases. by attenuating pre-BCR/BCR-initiated death signals through DYNLL1- dependent inhibition of Bim.

SYM-37-03 SYM-37-04 ELUCIDATING THE ROLE OF THE PRO-SURVIVAL THE PSEUDOKINASE MLKL MEDIATES NECROPTOSIS BCL-2 FAMILY MEMBER A1 IN MELANOMA AND VIA A NOVEL MOLECULAR SWITCH MECHANISM OTHER CANCERS Murphy J.M.1, Czabotar P.E.1, Hildebrand J.M.1, Lucet I.S.2, Webb A.I.1, Lee E.1, 2, Rohrbeck L.1, 2, Lang M.1, 2, Tai L.1, 2, Strasser A.1, 2, Fairlie D.1, 2 Alvarez-Diaz S.1, Strasser A.1, Babon J.J.1, Silke J.1 and Alexander W.S.1 and Herold M.J.1, 2 1Walter and Eliza Hall Institute. 2Monash University. 1The Walter and Eliza Hall Institute of Medical Research. 2Department of Medical Biology, The University of Melbourne, Victoria, Australia. Mixed lineage kinase domain-like (MLKL) was recently identified as a component of the ‘necrosome’, the multi-protein complex that The deregulated expression of pro-survival Bcl-2 family members triggers TNF-induced cell death by the process termed necroptosis. has been implicated in the sustained growth of diverse tumour types. To define the specific role and molecular mechanism of MLKL action, Extensive efforts have led to the development of a novel class of anti- we generated MLKL-deficient mice and solved the crystal structure cancer drugs, the so-called BH3-mimetics ABT-263, which allow of MLKL. While MLKL-deficient mice were viable and displayed no specific inhibition of pro-survival Bcl-2 proteins. However, none of the hematopoietic anomalies or other obvious pathology, cells derived from currently available BH3-mimetics inhibit the pro-survival A1. Importantly, these animals were resistant to TNF-induced necroptosis unless MLKL A1 has been implicated as a therapeutic resistance factor for many expression was restored. Structurally, we show that MLKL comprises different tumour types and has recently been shown to be amplified in a four-helical bundle tethered to the pseudokinase domain, which >30% of human melanoma samples. To test an involvement of A1 in the contains an unusual pseudoactive site. Although the pseudokinase sustained growth of melanoma lines, we generated inducible A1 shRNA domain binds ATP, it is catalytically inactive and we show that its constructs. While the mutant B-Raf inhibitor Plexxikon (PLX) could kill essential non-enzymatic role in necroptotic signaling is induced by the melanoma lines, its killing capacity was significantly enhanced when RIPK3-mediated phosphorylation. Remarkably, structure-guided combined with A1 specific knockdown. To mimic an A1 specific drug, we mutation of the MLKL pseudoactive site resulted in constitutive, RIPK3- developed an A1 peptide ligand, using as a guide the different binding independent necroptosis, demonstrating that modification of MLKL is specificities of the pro-apoptotic Bcl-2 proteins for the pro-survival essential for propagation of the necroptosis pathway downstream of Bcl-2-like proteins. Importantly, we could show that the inducible RIPK3. expression of the A1 ligand can efficiently kill several melanoma lines, even in the absence of PLX treatment. We are currently developing an A1 specific small molecule BH3-mimetic with the aim to translate these efforts into the clinic for the treatment of patients with melanoma. Since the expression of A1 is usually restricted to the hematopoietic system, we believe that an A1 targeting drug would spare most healthy tissues, thereby reducing the undesirable side effects associated with conventional chemotherapy.

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SYM-37-05 SYM-38-01 DETERMINING THE ROLE OF TROPOMYOSINS IN Sponsored by ACTIN-MEDIATED APOPTOSIS The Australian Society of Plant Scientists

Desouza M., Gunning P. and Stehn J. TRACE ELEMENT NUTRITION OF PLANTS AND Cellular and Genetic Medicine Unit, School of Medical Sciences, ANIMALS University of New South Wales, Sydney, Australia. White P.J.1 and Broadley M.R.2 The actin cytoskeleton has been implicated as both a sensor and mediator 1The James Hutton Institute, UK. 2University of Nottingham, UK. of apoptosis (programmed cell death). Tropomyosins are a family of proteins that define the functional properties of actin filaments in an Trace elements are defined by biologists, and nutritionists, as isoform specific manner. Tropomyosin-1 (Tm1) has been highlighted as a essential dietary minerals required in minute quantities for proper sensor of apoptosis induced by cell detachment. Further studies suggest growth, development and physiology of an organism. Trace elements that actin may mediate apoptosis via interaction with pro-apoptosis for both plants and animals include iron (Fe), zinc (Zn), copper (Cu), proteins such as Bmf. We aim to determine the role of specific tropomyosin manganese (Mn) and molybdenum (Mo), and animals also require filament populations in apoptosis and to elucidate the mechanism by cobalt (Co), selenium (Se), iodine (I), and several other elements in which specific tropomyosins alter sensitivity to various apoptotic stimuli. trace amounts[1]. The phytoavailability of trace elements for plants can Rat neuroblastoma cell models that over-express specific tropomyosins limit crop production. For example, the phytoavailability of Fe, Zn, Cu (vector only, Tm1, Tm3, Tm4 and Tm5NM1) were utilised to study the and Mn often limit crop production on alkaline and calcareous soils, isoform-specific role of tropomyosins in apoptosis. The tropomyosin and Mo deficiency can contribute to reduced yields on acid soils[2]. cell models were initially analysed for anchorage-independent survival In addition, when crops are grown on soils with low concentrations, using the chemical PolyHEMA which prevents cell adhesion. Tm1 over- or low phytoavailabilities, of trace elements, this is reflected in low expression specifically reduced cell viability in the presence of PolyHEMA concentrations of these elements in edible products. Indeed, human relative to control cells. Tropomyosin over-expressing cell models diets commonly lack sufficient quantities of trace elements for adequate were then treated with actin and microtubule targeting compounds and nutrition, and dietary deficiencies of, for example, Fe, Zn, Cu, Se and I apoptosis sensitivity was investigated via detection of caspase 3/7 activity. are prevalent worldwide[3]. One strategy to address mineral malnutrition Over-expression of Tm1 and Tm4 increased apoptosis sensitivity to the of animals is to increase the concentrations of mineral elements in actin stabilising compound jasplakinolide whereas Tm1, Tm4 and Tm5NM1 over-expression reduced sensitivity to the actin de-stabilising compound edible crops (biofortification) through the application of fertilizers and/or the development of genotypes with an increased ability to acquire and latrunculin A. Furthermore Tm3 over-expression increased apoptosis [3] sensitivity to microtubule stabilising and de-stabilising compounds accumulate mineral elements in edible portions . This presentation will (paclitaxel and vinblastine). Finally tropomyosin over-expressing cell consider the merits of agronomic and genetic strategies to improve the models were analysed for the expression of pro-apoptosis proteins Bax acquisition of trace elements by crops to increase both crop yields and and Bak, revealing a significant increase in Bak expression when Tm1 is the delivery of trace elements to human diets. References: [1] White over-expressed. To conclude, over-expression of specific tropomyosins and Brown (2010) Ann. Bot. 105, 1073-1080. [2] White et al. (2013) altered sensitivity to apoptosis induced by cell detachment and by a Frontiers Plant Sci. 4:193. [3] White and Broadley (2009) New Phytol. variety of cytoskeleton targeting compounds. Over-expression of specific 182, 49-84. tropomyosins altered the expression levels of Bak and future studies will determine whether specific tropomyosins interact with or activate pro- apoptosis proteins to mediate cell death.

SYM-38-02 SYM-38-03 DO COMPLEMENTARY PLANT NUTRIENT- TRANSPORT SYSTEMS THAT UNDERPIN NITROGEN ACQUISITION STRATEGIES PROMOTE GROWTH? EFFICIENT MAIZE

Teste F.P.1, Veneklaas E.1, Dixon K.2 and Lambers H.1 Dechorgnat J.1, Wen Z.1, Vandeleur R.1, Francis K.1, Wignes J.1, 1The University of Western Australia, School of Plant Biology. 2Botanic Dhugga K.2, Rafalski A.3, Tyerman S.D.1 and Kaiser B.N.1 Gardens and Park Authority, Kings Park and Botanic Garden. 1School of Agriculture Food and Wine, The University of Adelaide, Urrbrae, SA. 2Pioneer HiBred, Johnston, Iowa, 50131, USA. 3DuPont Better understanding what promotes positive plant interactions in the Crop Genetics, Wilmington, Delaware, 19803, USA. context of species interacting in nutrient-poor soils is a priority, particularly in phosphorus (P) limited ecosystems where plants with contrasting Nitrogen (N) is an essential nutrient required for plant growth where nutrient-acquiring strategies co-occur and in agroecosystems, since deficiencies reduce yield and/or quality of important agricultural food global P stocks are being depleted. In this study, we assess positive crops. With most high-input cropping systems (maize, wheat, rice and interactions between plants with contrasting nutrient-acquiring strategies canola), N fertilisers are used to overcome annual deficiencies in the that co-occur in a biodiversity hotspot in severely P-impoverished soils soil in order to achieve economically viable yields. Unfortunately, the of south-western Australia. Four plant species (Bm: Banksia menziesii, use of N fertilisers by most crop plants is inherently inefficient where Em: Eucalyptus marginata, Vn: Verticordia nitens, and Mp: Melaleuca combinations of agronomy, soil- and plant-dependent factors contribute preissiana) forming non-mycorrhizal cluster roots, ectomycorrhizal (EM), to poor nitrogen use efficiency (NUE). Soil-dependent factors include arbuscular (AM), or dual AM/EM roots, respectively, were grown together nitrate leaching and N volatilisation (NH3, N2O, NOx), while plant- in a novel ‘common garden’ microcosm experiment with nutrient-poor dependent factors include constraints at the root/soil interface that or fertilised soil and with or without root intermingling. We measured limit the uptake of N and internal constraints to N assimilation, storage growth, mycorrhizal colonisation, root intermingling and nutrient uptake capacity and remobilisation. In many cases, the activity of N transport to determine negative or positive patterns amongst the various plant assemblies. Growth of AM/EM host Mp was best when interacting with proteins are often correlated with N demand and supply, indicating both EM host Em and a nutrient-mining plant with cluster roots (Bm) in that N transport can be rate limiting. Our understanding of the plant N microcosms where root intermingling was not possible. This promoted transport network remains incomplete. This limits targeted approaches growth was only seen in pots with nutrient-poor soils, where the better to enhance the underlying transport systems responsible for N uptake growth of Mp coincided with higher shoot P, manganese, calcium, iron, and the subsequent remobilisation of N across cells and tissues over and boron content. Furthermore, the dual AM/EM Mp exhibited enhanced key developmental stages of plant growth. Our research program is EM colonisation and favoured EM over AM fungi when grown beside Em using maize as a model system to define the N transport network and Bm. We surmise that mycorrhizal networks were instrumental in the responsible for nitrate and ammonium uptake and redistribution. variation in both mycorrhizal type and colonisation levels. We conclude To do this we are currently using a combination of approaches that that complementarity between nutrient-acquisition strategies in plants include traditional cellular/tissue profiling of transport proteins in( situ can promote growth. The results suggest a synergistic effect between EM microscopy, promoter GUS fusions, mRNA expression), heterologous hyphal scavenging and mobilisation of limiting nutrients by cluster roots. expression of cloned transport proteins in Xenopus laevis oocytes and The positive and negative mechanisms that interact enable coexistence whole plant physiological analysis of inbred and corresponding hybrid to go far beyond the traditional view that plants interact mainly through maize populations. Our results are contributing to a defined N transport resource depletion. We provide a better understanding of how root network for maize that will aid in forward activities that target enhanced interactions can shape plant communities and promote diversity. N access and utilisation in line with plant growth and yield objectives.

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SYM-38-04 SYM-38-05 ANALYSIS OF THE NATURAL GENETIC VARIATION OF HOW DOES PHOSPHITE PROTECT PLANTS AGAINST PHOSPHITE SENSITIVITY IN ARABIDOPSIS THALIANA PHYTOPHTHORA?

Kollehn D.O.1, O’Brien P.A.1, Hardy G.E.St.J.1 and Berkowitz O.1, 2 Borges Osorio M., Jost R., Barbetti M. and Finnegan P. 1Murdoch University, 90 South Street, Murdoch WA 6150, Australia. School of Plant Biology, The University of Western Australia, 35 2University of Western Australia, 35 Stirling Hwy, Crawley WA 6009, Stirling Hwy, Crawley, WA 6009, Australia. Australia. Phytophthora cinnamomi, the causal agent of dieback disease, is a Phosphorus is one of the most critical macronutrients for plants significant threat to Australia’s biodiversity. As a measure to manage - and taken up from the soil in the form of phosphate (H2PO4 , Pi) by this destructive soil borne oomycete, the phosphate (Pi) analogue specific transporters. It is frequently a growth-limiting factor due to phosphite (Phi) has been applied by aerial spraying at high rates. While low availability in the soil. Hence plants have developed strategies to Phi cannot be directly metabolized by plants, it can act as a slow- adjust to Pi starvation with adaptations including the alteration of root release Pi fertilizer through oxidation by soil microbes. This fertilization architecture or the secretion of organic acids to raise Pi uptake capacity. might affect fragile Pi-sensitive flora such as that in Southwestern Pi depleted plants also increase the expression of genes involved Australia that is unable to cope with increased phosphorus (P) supplies. in Pi acquisition, e.g. Pi transporters and purple acid phosphatases. Phi is also known to disrupt normal plant growth, perhaps partially by - Phosphite (H2PO3 , Phi) is the reduced form of Pi and taken up by mimicking Pi and disrupting P homeostasis. How Phi protects plants plants through phosphate transporters. Although metabolically inert, against oomycetes is still unclear. Although the chemical is able to Phi is able to suppress Pi starvation responses, which exacerbates directly inhibit pathogen growth, it also seems to activate a variety of Pi depletion leading to an inhibition of plant growth. In addition, Phi signaling cascades in plants, some of which are associated with known induces plant defence responses and effectively inhibits colonisation by defense responses. Most progress on the mechanism of Phi action oomycete pathogens (e.g. Phytophthora spp.), which have devastating comes from the analysis of shoot pathosystems, while root responses effects in horticultural and native ecosystems. Although Phi is the only to soil borne pathogens are less well studied and are likely to differ reliable measure to control these pathogens its mode of action remains significantly in their molecular signature. Therefore, my overall aim unclear. To better understand the effects of Phi on plant growth and is to provide a further understanding of the mechanisms by which induced pathogen resistance we are analysing the genetic basis of Phi Phi protects plants against Phytophthora infection, with focus on the sensitivity in Arabidopsis thaliana. We have investigated the phenotypic interaction of the pathogen with the root. Reproducing the pathosystem responses of 18 Arabidopsis accessions and EMS mutagenized lines in Arabidopsis thaliana will give me access to this plant’s physiological to Phi treatment. This has led to the identification of a major QTL and and molecular responses to Phi and the pathogen. Unraveling these also a mutant line with increased phosphite tolerance. Findings from responses will contribute to the development of new strategies to this research will improve our knowledge of the mode of phosphite manage P. cinnamomi without affecting plant P homeostasis, which action in plant defence responses, and may have implications for the is the key long-term challenge for the current use of Phi in natural understanding of Pi signalling or metabolism. ecosystems.

SYM-39-01 SYM-39-02 IMMUNE REGULATION OF REGENERATION VASCULARISED CARDIAC ORGANOIDS FOR RECONSTRUCTIVE SURGERY Rosenthal N.1, 2 1Australian Regenerative Medicine Institute, Monash University. Dilley R.J.1, 2 and Morrison W.A.2, 3 2National Heart and Lung Institute, Imperial College London. 1School of Surgery, University of Western Australia, WA 6009. 2Department of Surgery, University of Melbourne, VIC 3010. 3The The adult mammalian body does retain the robust repair capacity of O’Brien Institute, Melbourne, VIC 3000. embryonic stages. In contrast to the effective regeneration of other vertebrates, the limited restorative capacity of many adult mammalian Cardiac tissue engineering is being pursued as an alternative tissues has been attributed to the loss of adequate cell replacement approach to transplantation for treating heart failure. Shortage of coupled with persistent inflammation. Using genetic manipulation we organ donors and complications of orthotopic heart transplant remain have investigated the role of growth factors and resident immune cells major challenges to the modern field of transplantation. Engineering in the resolution of tissue injury, in both mouse and axolotl, an efficiently functional myocardium de novo requires an abundant source of cells regenerating member of the urodele amphibian family. We have that can form cardiomyocytes, a biocompatible scaffold material uncovered a complex interaction between local repair mechanisms and and a functional vasculature to sustain the high metabolism of the innate immune cells, which participate in the removal of necrotic tissue, construct. Progress in cardiac cell biology, stem cells and biomaterials secrete growth factors that limit inflammation, and promote progenitor research is promising for cardiac tissue engineering however currently cell-mediated tissue replacement. We have recently discovered an employed strategies for vascularization continue to limit the volume unexpected connection between regenerative processes and adaptive of tissue formed. Over ten years we have developed an in vivo tissue immune tolerance. Our work supports the feasibility of improving engineering model to construct vascularised cardiac tissue from cardiac regenerative capacity by modulating key signaling pathways various cell and tissue sources. In order to increase the volume of this controlled by specific components of the immune system, which tissue we have also tested the hypothesis that angiogenic cytokines provide new targets for clinical intervention and improving prospects and growth factors secreted by mesenchymal stem cells may be used for molecular and cellular combination therapies. to promote vascularisation and tissue growth for tissue engineering in vivo. Human adipose-derived stem cells (ASCs) expressed mRNA for a range of angiogenic factors and secreted a proliferative activity for human microvascular endothelial cells. VEGF- and Angiogenin- blocking antibodies reduced the activity. MSCs implanted in vivo in a subcutaneous chamber with a femoral arteriovenous loop survived implantation and were incorporated into the newly formed tissue. The volume of newly generated tissue was significantly higher in chambers containing MSCs and they were enriched with vasculature. We conclude that human MSCs promote tissue growth and angiogenesis in the rat vascularized chamber, thereby showing promise for tissue- engineering applications for regenerative therapy.

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SYM-39-03 SYM-39-04 ENGINEERED MATRIX MICROENVIRONMENTS FOR BIODEGRADABLE POLYURETHANES FOR SPINAL- REGULATION OF STEM CELLS CORD TISSUE REPAIR

Choi Y.S. Filardo F.1, Dargaville B.1, Yu A.1, Micallef A.S.1, O’Shea M.S.2, Tsai E.C.3, Kolling Institute of Medical Research, University of Sydney, Royal Whittaker A.1 and Rasoul F.1 North Shore Hospital, ST LEONARDS NSW 2065. 1Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St. Lucia, Queensland, 4066, Australia. In recent years, ECM stiffness and resulting cell contractility 2CSIRO Materials Science and Engineering, CSIRO, Clayton, Victoria, have been identified as potent stem cell differentiation regulators. 3168, Australia. 3Ottawa Hospital Research Institute, University of Successful stem cell-based therapies will require acclimating cells to Ottawa, Canada. the abnormally stiff ECM of muscular dystrophy while inducing and/ or maintaining myogenesis, fusion, and dystrophin delivery. Here The personal, social and economic effects of spinal cord injury (SCI) we showed that adipose-derived stem cells (ASCs) more completely on the victims, their families and society at large are enormous. The undergo extracellular matrix (ECM)-directed myogenesis than their most common SCI, resulting in paraplegia or quadriplegia, is the bone marrow-derived counterparts (BMSCs) by expressing myogenic bruising of the myelin sheath of axons, followed by the progressive markers 40-fold higher and forming fused muscle in vitro; fusion rates, degeneration of neuronal tissue and glial scar and cyst formation. In however, still produce too few myotubes to constitute a clinically viable addition, axonal regeneration is repressed by inhibitory proteins such cell source (~2%). To encourage end-on cell fusion to form aligned as Nogo and myelin-associated glycoprotein (MAG). With advances skeletal muscle, a mechanically patterned substrate with alternating in the neurosciences and cell biology, there has been a major push neuronal- and muscle-like stiffness that mimicks innervated muscle to develop therapies that promote SCI repair. A successful strategy may be a more physically appropriate environment. This mechanically will most likely require a combination of biomolecular-, cellular- and patterned substrate or ‘Zebraxis’ matrix was fabricated with alternating biomaterial-based approaches. We have developed a range of regions of soft (neurogenic), firm (myogenic), and/or stiff matrix (fibrotic biocompatible and biodegradable polyesterurethanes based on the or osteogenic). ASCs, C2C12 myoblasts, and PC12 neuronal cells all well-defined PCL-PEG-PCL tri-block co-polymer building blocks and differentially sorted themselves based on their stiffness preference: lysine-based polyisocyanates that can be used to form a 3-dimensional cardiomyocytes, myoblasts, and ASCs all durotaxed to the myogenic scaffold to bridge the SCI. The aim of the scaffold is to promote axonal regions of the pattern whereas neurons had opposing behavior. With growth, allow realignment of nerve tracts and provide mechanical additional alignment, ASCs fused into multi-nucleated myotubes at a protection to prevent ingress of scar tissue. We will report results on rate almost twice that on static hydrogels. Moreover, a great fraction the biological compatibility including cytotoxicity and motor neuron of ASCs-derived myotubes underwent multiple rounds of fusion due (MN) cell adhesion and proliferation of both bulk and electrospun to alignment of their cadherin-rich ends. Most importantly, the multi- polyurethane samples. The effect of the biomaterial on cell viability and nucleated myotubes that form are resistant to transdifferentiation when proliferation of rat neural progenitors obtained from both the brain and plated onto a stiffer matrix mimicking a more fibrotic-like stiffness. spinal cord has also been investigated. Future experiments will involve Singly nucleated ASCs are not, suggesting that additional strategies in-vivo studies using the biomaterial in spinal cord injured rats. are necessary to achieve a pure myotube fraction. However differential sorting, enhanced fusion, and multiple fusion events supports using ECM to create spatially-patterned in vitro muscle for regenerative uses in fibrotic muscle diseases such as muscular dystrophy.

SYM-39-05 SYM-40-01 THE APPLICATION OF NOVEL BIOMATERIALS TO SYNTHETIC SENSING AND SIGNAL TRANSDUCTION OTOLOGY CASCADES BASED ON ARTIFICIAL AUTOINHIBITED PROTEASES Shen Y.1, 2, 3, Redmond S.L.1, 2, Teh B.M.1, 2, Yan S.4, Wang Y.5, Atlas M.D.5, Zheng M.H.1, 2, Marano R.J.1, 2 and Dilley R.J.1, 2 Alexandrov K. and Stein V. 1Ear Sciences Centre, School of Surgery, The University of Institute for Molecular Bioscience, The University of Queensland, Western Australia. 2Ear Science Institute Australia. 3Department Brisbane, QLD, Australia. of Otolaryngology, Head & Neck, Ningbo Lihuili Hospital (Ningbo 4 Medical Centre), Ningbo, Zhejiang, China. Key Laboratory of The central tenet of the emerging field of Synthetic Biology is that Combined Multi-organ Transplantation, Ministry of Public Health; biological components can be refined into a toolkit of plug-and-play Department of Surgery, The First Affiliated Hospital, School of building blocks. The experimental evidence supporting this idea has 5 Medicine, Zhejiang University, China. Centre for Orthopaedics so far been limited to relatively slow synthetic gene expression circuits. Research, School of Surgery, The University of Western Australia. Real-time events in biological process are mediated by protein-based signaling circuits that can operate up to the millisecond scale. Ability Tympanic membrane (TM) perforations lead to significant hearing loss to design protein-based circuits would enable us to rewire cellular and middle ear infection. Surgery to repair perforations uses various signalling cascades and make them respond to non-native queues in graft materials to promote TM regeneration, but these have limitations. non-native fashion. Further, the same set of principles is applicable to the We evaluated the safety, efficacy and feasibility of novel graft materials, design of analytical and diagnostic applications. To create orthogonal silk fibroin (SF) and acellular collagen (AC), compared to paper patch toolbox of protein-based signal detectors and amplifiers we combined and Gelfoam for TM regeneration. Scaffolds were implanted in rats structure-based engineering and directed evolution of proteins. This subcutaneously or in middle ear for safety study and via onlay myringoplasty approach allowed us to create artificial auto-inhibited proteases with for TM perforation models using both rats and guinea pigs. Surface non-overlapping substrate specificities. These molecules are inactive morphology, mechanical properties and biocompatibility of grafts were in their ground state but are activated by changes in their structure that evaluated before TM repair. The morphology of TM healing was assessed dislodge the inhibitor from the active site. Incorporation of a ligand- at various time points post-implantation using otoscopy, light and electron binding domain into such basic signaling unit allowed us to create microscopy, and functional outcomes by auditory brainstem responses. an allostercally regulated receptor protease. We demonstrate that a Tensile strength and elasticity of SF and AC were in the range for human complete signal detection and amplification cascade can be assembled TM. SF and AC were compatible in subcutaneous and middle ear implant from such synthetic protein units. The applications of the developed assays for up to 6 months. In rats and guinea pigs the healing of perforated technology will be discussed. TM was similar. SF and AC significantly accelerated TM perforation closure, obtained optimal TM thickness, and resulted in better trilaminar morphology with well-organized collagen fibers and early restoration of hearing. However, paper patch and Gelfoam lost their scaffold function in the early stages and showed an inflammatory response, which may have contributed to delayed healing. This study indicates that compared to paper patch and Gelfoam, SF and AC are safe and effective in promoting early TM regeneration and improved hearing, suggesting that these novel scaffolds may be potential substitutes for clinical use.

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SYM-40-02 SYM-40-03 METABOLIC AND REGULATORY MODELS FOR UNDERSTANDING HUMAN COMPLEX I BIOGENESIS SYNTHETIC BIOLOGY DESIGN THROUGH TALEN-MEDIATED GENE DISRUPTION

Nielsen L.K. Stroud D.A.1, 2, Formosa L.E.1, 2, Surgenor E.E.1 and Ryan M.T.1, 2 The University of Queensland. 1Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086. 2Centre of Excellence Detailed metabolic and regulatory models are critical to guide efficient for Coherent X-ray Science, La Trobe University, Melbourne 3086. microbial metabolic engineering design. First the potential of developing new pathways using heterologous or even de novo evolved enzyme Complex I deficiency is the most common mitochondrial disease and functions must be established. Next pathway integration into desired most often results in multi-system disorders with a fatal outcome. host organisms must be explored and potential indel gene targets Human complex I consists of 44 subunits, 14 of which constitute its identified. This talk will review the status of rational in silico strain enzymatic core and are conserved from bacteria to man, while the design with a particular focus on assumptions and limitations of current remaining 30 are termed accessory subunits. Despite one third of strategies. complex I accessory subunits having been identified as mutated in patients with mitochondrial disease, their roles in human complex I function and/or assembly remains largely unclear. As complex I is not present in genetically amendable model organisms such as yeast, research aimed at understanding the molecular defects of complex I has typically utilized patients fibroblasts. Patient cell lines however have limited life spans, are difficult to transfect, lack appropriate isogenic control cell lines, and in some cases were not collected at the time of patient presentation. Transcription activator-like effector nucleases (TALENs) are new tools that act as site-specific “molecular scissors” - generating small, targeted genomic deletions - resulting in permanent and complete disruption of gene expression in routinely cultured human cell lines. We have introduced TALENs in HEK293T cells to disrupt a number of genes involved in the biogenesis of human complex I. Analysis of these cell lines has revealed important new insights into how these gene products are important for complex I and cellular function.

SYM-40-04 SYM-40-05 BIOLOGY FROM THE BOTTOM UP: ARTIFICIAL DNA LOOPS THAT INHIBIT OR ENHANCE ONE SYNTHESIS OF THE BACTERIAL FLAGELLAR MOTOR ANOTHER’S FORMATION

Hynson R., Vernhes E. and Lee L.K. Priest D.G.1, Shearwin K.E.1, Dodd I.B.1, Kumar S.2 and Dunlap D.D.2 The Victor Chang Cardiac Research Institute. 1The University of Adelaide, Australia. 2Emory University, Atlanta, USA. Nanoscale rotary motors are everywhere in nature. These biological machines perform functions as diverse and as fundamental as DNA looping is of fundamental importance for gene regulation in synthesising ATP, mediating vesicular transport and bone remodelling. Prokaryotes and Eukaryotes. In Prokaryotes, full repression of the The largest of these, the bacterial flagellar motor (BFM), is an Lac operon requires DNA looping via the Lac repressor to distal ~11 mega Dalton protein superstructure consisting of hundreds of operators. In Eukaryotes, promoter activation often requires the subunits that powers the rotation of long, helical flagellar filaments, action of enhancers, which are DNA elements often located many which allows bacteria to swim through viscous media. Fuelled by an kilobases from the promoter. Enhancers are bound by transcription electrochemical gradient, it is equipped with an impressive molecular factors and function by looping to target promoters thus bringing engine that converts a flux of cations into mechanical rotation at speeds them into an environment rich in transcriptional activators. Although of up to 1700Hz, yet also allows the motor to shift into reverse in a specific proteins can mediate enhancer-promoter contacts, enhancers handful of milliseconds, tune its speed and stop either via a molecular also have general activity in reporter assays. Therefore mechanisms brake or clutch. Understanding the molecular structure and function must exist to insulate enhancers from off-target promoters. The Loop of the BFM promises enormous insight into dynamic macromolecular Domain model proposes that topological isolation of DNA elements into superstructures in general. For example, how do hundreds of different separate DNA loops can limit their interaction, therefore suggesting that proteins dance in unison to perform one function? But a highly dynamic genomes can be compartmentalised through subdivision into separate ~11 MDa transmembrane protein is no ‘ordinary’ protein structure. It ‘loop domains’. Providing solid evidence for the Loop Domain model falls into a category where high-resolution structural biology cannot in a Eukaryotic model is technically challenging, however the Loop reach. Here I will describe our progress towards elucidating a complete Domain model can be reduced to the hypothesis that; (1) alternating atomic-scale molecular picture of the BFM structure and dynamics, DNA loops interfere with one another’s formation, (2) nested DNA which includes a new initiative to artificially synthesise the BFM using loops will aid one another’s formation, and (3) DNA loops located side- DNA nanotechnology. by-side will have no effect on one another. We’ve employed two well- studied DNA looping proteins, Lac and lambda CI repressors, in E. coli, to quantitatively show these tenets of the Loop Domain model. By comparing our in vivo data with experiments in vitro and in silico, we’ll have a rich dataset on the biophysics of DNA looping, which will form the foundation for further experiments in Eukaryotes.

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SYM-41-01 SYM-41-02 SEEING PLANTS IN A NEW LIGHT - ENGAGING LARGE “TELL ME AND I’LL FORGET.”: STRATEGIES FOR FIRST-YEAR CLASSES IN EXPERIMENTAL PLANT SCIENCE ENHANCING STUDENT UNDERSTANDING

Liu D.Y.T. Lutze-Mann L.H. School of Biological Sciences, The University of Sydney. School of Biotechnology and Biomolecular Sciences, The University of NSW, Sydney, NSW, 2052. Teaching introductory biology classes presents a number of related challenges including low student engagement, large class sizes, When teaching undergraduate students, especially in large classes, and the diversity of student background knowledge and interests. one of the most challenging aspects is providing students with an For these reasons, first-year biology units need to instil in students a engaging experience that will foster their learning and increase their sense of scientific discovery instead of just delivering content, while understanding of complex material. As the classic Chinese proverb providing safe avenues for authentic inquiry-based learning to engage reminds us: “Tell me and I’ll forget; show me and I may remember; these students. A further challenge for plant scientists is students’ involve me and I will understand”. There are numerous studies in the relatively low innate attraction to this subject material compared to literature which document the advantages for students of participating the study of humans or furry animals. This presentation outlines a in ‘active learning’ where interacting with other people (students, number of new plant-based student-centred practical sessions that academic staff) in real or virtual worlds maintains their interest and were designed and implemented into two introductory biology courses hence enhances their learning, especially when they receive immediate at the University of Sydney. These courses have enrolments of feedback on their performance. However, it is not easy to design over 600 students each, and introduce a diverse range of biological learning tasks that encourage student interaction, are academically concepts. By applying various aspects of their biology, plants were rigorous and also able to engage the current generation of digitally integrated into inquiry-based practicals that included digital imaging, native students. Three strategies for enhancing student understanding enzyme assays, fluorescence microscopy, isolation and analysis of live using active learning will be presented: the formative assessment organelles, preparation of live tissue for histology, and investigation of lecture using mobile response devices; virtual laboratory activities using whole-organism physiology. Although the practicals covered a range an adaptive feedback platform and student-led game design. The value of concepts and competencies, they were structured to provide a of each these strategies, apart from increasing student engagement, safe environment for students to build their laboratory and scientific is that student misconceptions are frequently revealed allowing direct thinking skills which were then applied to the design and analysis of remediation, which further enhances student learning and provides their own open-ended experiments through which they also developed instructors with feedback on troublesome areas of the curriculum. a strong collaborative ethic. Because students were invested in their investigations, they were more engaged with the subject material and, notably, better understood and appreciated the relevance of plants. Operationally, these practicals were designed to be cost-effective, easy to implement, and adaptable, and they addressed the educational principles of scaffolding, engaged enquiry, and research-enriched teaching. Students frequently commented that learning and then independently applying scientific skillsets, as well as collaborating with their peers to investigate an authentic question, produced a much more fruitful and engaging learning experience.

SYM-41-03 SYM-41-04 INCREASING ENVIRONMENTAL ENGAGEMENT FEEDBACK-INFORMED PROBLEM-BASED LEARNING THROUGH THE USE OF CITIZEN SCIENCE AND A TO PREPARE STUDENTS FOR THE REAL WORLD STUDENT JOURNAL IN UNDERGRADUATE BIOLOGY

1 2 3 2 1 Schaeffer P.M. Mitchell N. , Triska M. , Weatherill R. , Barker S.J. and Longnecker N. Comparative Genomics Centre, School of Pharmacy and Molecular 1School of Animal Biology M092, The University of Western Australia, 2 Sciences, James Cook University, DB 21, James Cook Drive, 35 Stirling Hwy, Crawley WA 6009 AUSTRALIA. School of Plant Biology Townsville, QLD 4811, Australia. M090, The University of Western Australia, 35 Stirling Hwy, Crawley WA 6009 AUSTRALIA. 3Earthwatch Institute Australia, 126 Bank Street South Melbourne Victoria 3205 Australia. In the undergraduate Biotechnology/Biochemistry/Molecular Biology program the School and Faculty need to ascertain that the students Inquiry based learning can increase student engagement and understanding who progress into Honours/postgraduate studies or leave to join the beyond the active learning techniques that are typically used in science industry have the appropriate theoretical knowledge and practical laboratories. In our study, a citizen science exercise using a national skills to be “ready” for their next educational/professional steps. The program known as Climate Watch was introduced to UWA undergraduate Biotechnology subject is an essential third year subject for students biology units in 2011, 2012 and 2013. In 2011, students collected and majoring in Biochemistry and Molecular Biology in the Faculty of entered phenological observations of plants and animals into a website, Medicine, Health, and Molecular Sciences. This subject is notoriously and in 2012 and 2013, students both entered data for Climate Watch and demanding due to its multidisciplinary and technical nature. Although wrote scientific articles based on their analysis of Climate Watch datasets. student satisfaction for this subject is generally high as demonstrated Surveys of students showed that their interest and engagement with the by previous SFT/SFS results, this is not reflected in the pass rate at environment increased after the assignment(s) in all years. The addition of the final exam. To enable early identification of students who may have the inquiry based learning (analysis and article writing) further challenged problems applying theoretical knowledge and skills in a laboratory students to think in depth about the species observed, about the value relevant manner I developed a framework that drives learning in a of phenological data, and about climate change, but it did not decrease way that enables students to apply their knowledge and skills with their overall interest or level of engagement. However, students were much confidence in the laboratory. The subject has been redeveloped to more concerned with the reliability of data collected by citizen scientists include feedback-informed problem-based learning strategies based than they were in 2011, when they only collected data. This is likely to on periodical surveying of students. The feedback from the survey be because the students were deliberately provided with unvalidated (and (through measurement of skills and performances) was used to often inaccurate) data, much of which had been submitted by previous identify knowledge and skills gaps and to develop specific problem- UWA students. A further learning outcome of this project has been an based learning tools and strategies to enhance learning of students. A introduction to the practice of scientific publication, because we mimicked dramatic improvement in pass rate at the exam was achieved last year the online submission and peer review process of real journals using but interestingly at the expense of the overall satisfaction of students. UWA’s Learning Management System. Further, we ‘published’ the best articles in an open-access Wiki: cygnus-biologystudentjournal.wikispaces. This framework is now available as an online tool on Learn-JCU to com/Journal+Home. Overall, the Climate Watch and journal projects have provide a flexible and custom-made learning-experience to all students. been a challenging but valuable addition to first year biology teaching at UWA, as they have enhanced students’ environmental awareness and potentially increased the likelihood that students will participate in citizen science programs.

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SYM-41-05 SYM-42-01 SHOW ME THE NUMBERS - QUANTITATIVE ROLES OF RND PROTEINS IN NEURONAL ASSESSMENT OF LABORATORY TEACHING AND DEVELOPMENT LEARNING PRACTICES Pacary E. Ar thur P.G., Attwood P.V. and Ludwig M. Neurocentre Magendie, INSERM U862, 33077 Bordeaux, France. The University of Western Australia. The complex functions of the central nervous system rely on the proper Undergraduate science laboratories provide an opportunity to acquire development of several neuronal populations, in an orchestrated and apply a range of generic and scientific skills. Measuring the success and multistep process which can be divided as follows; proliferation, of students in obtaining and employing particular skills would assist in migration, differentiation and integration into neural circuits. These recognizing weaknesses in teaching delivery for ongoing improvement different developmental steps have an essential role in the functional of laboratories and identifying students needing additional assistance. organization of the brain and failure of one of this aspect during Student marks can be used to provide feedback on student learning development impacts on subsequent brain structure and function. The achievements. However, in many laboratory courses there is only a understanding of these critical processes is therefore of considerable grade for a particular laboratory or group of laboratories, which does not importance to ultimately provide further insights into the pathogenesis provide sufficient information about student acquisition and application of many neurodevelopmental disorders. The Rnd proteins, which form of skills. We have developed a skills-based practical program in our a distinct sub-group of the Rho family of small GTP-binding proteins, Level 2 Biochemistry and Molecular Biology units that focuses on have been shown to regulate the actin cytoskeleton. In a previous sustained learning and critical thinking. It is also designed to be an work, we showed that Rnd2 and Rnd3 control distinct steps of the evidence-based approach to improving student learning. It provides migratory process in the developing cerebral cortex, multipolar-bipolar quantitative information on the student learning experience by including transition and locomotion respectively, by inhibiting RhoA in different quizzes to probe different aspects of student learning (51 assessable cellular compartments. More recently, we have demonstrated that items for each student over the course of the semester). To effectively Rnd3, in radial glia cells, regulates interkinetic nuclear migration, the use these data, they need to be readily interpretable by academics, orientation of mitotic spindles and is required for the maintenance demonstrators and, where appropriate, students. We found the usual of adherens junctions through its action on the actin cytoskeleton practice of providing averages with standard deviation, or percentage whereas it limits the divisions of basal progenitors via the suppression of students achieving a particular grade, was not particularly useful of cyclin D1 translation. In line with these previous studies, I am now in identifying relationships that could be used to improve a course investigating the role of Rnd in the regulation of the last steps of or student learning. Instead, we have developed an approach using neuronal development, i.e. dendrite, spine and synapse formation and numerical and graphical representations of the marks for the top and finally synaptic integration. Understanding how Rnd proteins contribute bottom 25% of the student cohort. We propose this approach enables to neuron development is of fundamental importance both from a basic ready identification of weaknesses in teaching delivery and/or student scientific perspective, with the goal of providing mechanistic insights understanding, and provides quantitative evidence to guide reflective into the critical aspect of neuronal development, and from a clinical teaching and learning practices. perspective, with the goal of providing effective therapies for a range of brain disorders.

SYM-42-02 SYM-42-03 AXONAL FUSION IN REGENERATING AXONS SHARES PEERING INTO NEURAL STEM CELLS IN THE MOLECULAR COMPONENTS WITH THE APOPTOTIC DEVELOPING BRAIN: A TALE OF NEUROGENESIS, PATHWAY MORPHOGENESIS, NEURONAL MIGRATION AND THE SMOOTH BRAIN Neumann B.1, Coakley S.1, Giordano-Santini R.1, Linton C.1, Yang H.2, Xue D.2 and Hilliard M.A.1 Tsai J.W.1, 2, 3 1Queensland Brain Institute, The University of Queensland, Brisbane, 1Institute of Brain Science, School of Medicine. 2Brain Research Australia. 2Dept of MCD Biology, University of Colorado, Boulder CO, Center. 3Biophotonics and Molecular Imaging Research Center, USA. National Yang-Ming University, Taipei, Taiwan.

Understanding the molecular mechanisms regulating axonal The vertebrate CNS originates from a neuroepithelium composed of regeneration is essential for the development of effective therapies for highly organized neural stem cells, which give rise to virtually all of nerve injuries. Despite a substantial knowledge being gained into how the neurons and glia in the brain. During development, these neural axonal re-growth is initiated, our understanding of the mechanisms stem cells and their progeny go through a series of motile events, which needed to achieve target reconnection remains very poor. We and are tightly regulated by numerous genes. Any small perturbation in others have shown that precise reconnection of severed axons can these processes can cause neural developmental disorders, such as occur in C. elegans and in other species through a process of axonal lissencephaly, microcephaly, double cortex, schizophrenia, and even fusion, whereby the proximal regrowing fragment recognises and autism. In order to study the etiology of these developmental disorders, re-establishes membrane and cytoplasmic continuity with its own we established animal models to elucidate the functions of their causal separated distal fragment, preventing it from undergoing degeneration. genes in vivo. For example, we knocked down the expression of the We have now characterised the axonal fusion process at the molecular lissencephaly gene, LIS1, in the rat embryo using RNA interference level, uncovering a critical role for molecules previously shown to (RNAi) and in utero electroporation techniques and found that mediate the recognition of apoptotic cells by neighbouring phagocytes. knockdown of LIS1 completely blocks the interkinetic nuclear migration PSR-1 has been shown to bind exposed phosphatidylserine (PS) on the (INM), as well as the subsequent radial migration of committed surface of apoptotic cells, an “eat-me” signal necessary for recognition neuronal precursors. We further developed a culture system to observe and engulfment by phagocytic cells. In animals carrying mutations neural cells in brain slices using high resolution light microscopy. in the psr-1 gene, the proximal axon regenerates and contacts the Live imaging of coexpressed histone, centrosome, and microtubule distal fragment, but is unable to fuse, as a result of which the distal plus-end markers revealed that LIS1 is required for both nuclear and fragment degenerates. We have extended our analyses and have centrosome movement in the radially migrating cells. We have also identified similar axonal fusion defects in animals lacking the secreted applied these approaches to the behavior of neural stem cells and transthyretin-like protein TTR-52, which also binds PS, the phagocyte found that INM involve a cell cycle-dependent switch between dynein- receptor CED-1 that TTR-52 directly interacts with, and the adaptor and nonconventional kinesin-driven nuclear transport. More recently, phosphotyrosine-binding protein CED-6, which acts downstream we found a population of novel neural stem cells in the developing of CED-1 to transduce the engulfment signal. We propose that the brain in both human and rodents, termed OSVZ radial glial (ORG) cells. processes of recognition between two separated regenerating axonal Using these approaches, we have elucidated the mechanism of normal fragments and recognition between an apoptotic cell and phagocytes brain development and shed light on how various genes cause many share molecular components and mechanisms. neural developmental disorders.

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SYM-42-04 SYM-42-05 THE HISTONE ACETYLTRANSFERASE, MOZ, IS ROLE OF SECRETED WNT SIGNALLING INHIBITORS REQUIRED FOR MULTIPLE GENETIC PATHWAYS IN CROSS-TALK BETWEEN TROPHOBLAST AND DURING PALATE DEVELOPMENT DECIDUAL CELLS IN CO-CULTURE

Vanyai H.K., Sheikh B.N., Phipson B., Smyth G., Thomas T. and Voss A.K. Nayeem S.B.1, 2, Dharmarajan A.M.2, 3 and Keelan J.A.1 The Walter and Eliza Hall Institute of Medical Research, IG Royal 1School of Womens and Infants Health. 2School of Anatomy, Parade, Parkville, Melbourne, Victoria 3052, Australia. Physiology and Human Biology, University of Western Australia, Perth. 3School of Biomedical Sciences, Curtin University, Perth. Monocytic leukaemia zinc finger protein (MOZ/MYST3/KAT6A) is a member of the MYST family of histone acetyltransferases. The Implantation of the blastocyst and early development of the placenta molecular role of MOZ is to acetylate lysine 9 of histone 3 (H3K9), a are crucial for successful fetal growth and development. Wnt signalling chromatin mark associated with activation of gene transcription. MOZ pathways play an important role in implantation and placentation. has multiple roles in development, including the activation of Hox genes To explore the role of secreted Wnt inhibitors in blastocyst-uterine and in the formation of haematopoietic stem cells. Furthermore, loss of signalling, human placental trophoblast and decidual cells were MOZ in mice phenocopies the human DiGeorge syndrome, a congenital extracted and cultured in a Transwell model. Gene expression profiles disorder characterised by palatal, facial, thymic and cardiovascular and secretion of Wnt inhibitors was compared in monolayered and abnormalities. DiGeorge syndrome is caused by a 22q11 deletion or, co-cultured cells (trophoblasts grown on Transwell inserts above in rare cases, by heterozygous mutation of the TBX1 gene, which is decidual monolayers). Analysis of Wnt component expression by PCR central to the 22q11 deletion interval. In the absence of MOZ in mice, array (Qiagen) showed that the majority of the 84 Wnt-related genes Tbx1 expression, H3K9 acetylation and occupancy of the Tbx1 locus on the array were detectable in both cell types; most genes were not by ING5, a MOZ complex protein, are halved, suggesting that the Tbx1 differentially expressed between cell types or in co-culture. However, gene is a direct target of MOZ. However, Moz homozygous mutants expression of Wnt inhibitors sFRP4 and Wif1 were up-regulated and have a higher penetrance of DiGeorge-like abnormalities including cleft Wnt5A and Dkk1 were down-regulated in trophoblast with decidual palate than Tbx1 heterozygous mutant mice, indicating that Tbx1 is not co-culture respectively. The release of sFRP4 (which inhibits both the only MOZ target in palate development. Using a candidate gene canonical/non-canonical Wnt signalling) from trophoblast was approach and expression profiling by RNA sequencing, we found that increased by >400% in co-culture media; conversely, release of Dkk-1 expression of the distal-less gene family requires MOZ. In the absence (canonical pathway inhibitor) was reduced by ~50%. Msh homeobox 1 of MOZ, H3K9 acetylation and expression of Dlx genes and their and cyclooxygenase-1, two implantation/placentation-related proteins downstream targets is reduced. Single and compound mutations of the shown interact with the Wnt signalling components, were up-regulated Dlx1, Dlx2 and Dlx5 genes are known to cause cleft palate, suggesting in trophoblasts with co-culture, consistent with an increased inhibition that disruption of the distal-less genetic pathway in the absence of MOZ of non-canonical Wnt signalling in response to increased sFRP4/Wif- may contribute to palate defects in Moz mutant mice. 1. Our studies suggest that differences in secreted Wnt antagonists reflecting paracrine regulation of Wnt signalling are involved in fetal- maternal communication during successful implantation. Opposing changes in Dkk1 expression and other components may reflect a shift from non-canonical to canonical signalling.

SYM-43-01 SYM-43-02 MALARIA PARASITE ORGANELLES: UNDER THE PROTEIN QUALITY CONTROL IN THE MICROSCOPE MITOCHONDRIAL INTERMEMBRANE SPACE

Millet C.1, 2, 3, Dixon M.1, 2, 3, Hanssen E.2, 3, Dearnley M.1, 2, 3, Mcmillan P.2, 3, Baker M.J.1, Knight K.J.1, Mooga V.P.2, Ryan M.T. 2 and Stojanovski D.1 Hliscs M.1, 2, 3 and Tilley L.1, 2, 3 1The Department of Biochemistry and Molecular Biology & Bio21 1Department of Biochemistry & Molecular Biology, The University of Molecular Science and Biotechnology Institute, The University of Melbourne. 2ARC Centre of Excellence for Coherent X-ray Science. Melbourne, Parkville, 3010, Australia. 2Department of Biochemistry, La 3Bio21 Institute, The University of Melbourne. Trobe Institute for Molecular Science, La Trobe University, Melbourne, 3086, Australia. New microscopy techniques are providing amazing views of the cellular landscape. We have used 3-D structured illumination microscopy (SIM) The majority of mitochondrial proteins are encoded in the nucleus and and 3D-Electron Tomography to explore the sub-cellular topography of must be imported into one of the four mitochondrial sub-compartments: the malaria parasite, Plasmodium falciparum. As the parasite develops the outer membrane, intermembrane space, inner membrane and matrix. within human erythrocytes it establishes membrane organelles outside The MIA (Mitochondrial Intermembrane Space Assembly) pathway its own limiting membrane in the cytoplasm of its host cell that are used is the most recently described import pathway into mitochondria and to traffic virulence proteins to the erythrocyte surface. The pathway is dedicated to the biogenesis of intermembrane space proteins that followed and vehicles used to trafficking proteins across multiple possess twin cysteine motifs (CXnC, n is typically 3 or 9 amino acids). membranes are only partly defined. We have used 3D-SIM to probe the These cysteine residues are rapidly oxidized to disulfide bonds once traf ficking pathway for the major virulence proteins, the knob -associated the precursor is imported into mitochondrial intermembrane space histidine-rich protein (KAHRP) and P. falciparum erythrocyte-binding and this is mediated by the oxidoreductase Mia40 and the sulphydryl protein-1 (PfEMP1)(1). We have also explored changes in the cellular oxidase Erv1. The trafficking and biogenesis of intermembrane space structure of the parasite as it undergoes the remarkable transformation precursors can produce mislocalised, unfolded and unassembled in preparation for transmission to a mosquito and sexual reproduction proteins, especially under situations of cellular stress. Such protein (2). We have characterized the membrane organelle that helps co- species can form aggregates and be deleterious for mitochondrial ordinate gametocyte elongation and hypothesize that elongation function. Mitochondria have in place quality control systems equipped enables circulating gametocytes to pass through the sinusoidal slits with chaperones and proteases to aid in protein folding and removal of in the spleen, thereby avoiding host surveillance mechanisms. We misfolded or damaged proteins. Quality control mechanisms within the have probed the parasite’s digestive processes (3) and show that mitochondrial matrix are well established, however our understanding endocytosis and haemoglobin digestion occur via an orchestrated of the mechanisms and machineries that govern protein biogenesis process that is initiated earlier than previously recognized. This has and quality control in the intermembrane space is limited and we have important implications for the mechanism of action of artemisinin (4). no information into the cellular consequences that ensue when these References (1) P. J. McMillan et al., Cell Microbiol (in press), (2013). (2) events fail. The mitochondrial intermembrane space is fundamental to M. K. Dearnley et al., J Cell Sci 125, 2053 (2012). (3) N. A. Abu Bakar, mitochondrial function since many intermembrane space proteins are N. Klonis, E. Hanssen, C. Chan, L. Tilley, J Cell Sci 123, 441 (2010). (4) involved in key cellular pathways, including apoptosis, mitochondrial N. Klonis et al., Proc Natl Acad Sci U S A 110, 5157 (2013). respiration and transport of proteins and lipids. We are investigating the mechanisms that govern protein quality control within this vital compartment upon conditions of incomplete protein oxidation and protein misfolding.

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SYM-43-03 SYM-43-04 INSIGHTS INTO CILIARY TRAFFICKING THROUGH NUCLEAR TRAFFICKING OF PROTEINS FROM RNA GENE DISCOVERY IN SKELETAL CILIOPATHIES VIRUSES: POTENTIAL TARGET FOR ANTI-VIRALS?

Cortes C.R.1, McInerney-Leo A.M.2, Schmidts M.3, Leo P.J.2, Beales P.L.3, Jans D.A., Caly L., Dean J.E. and Wagstaff K.W. Brown M.A.2, Zankl A.4, Mitchison H.3, Duncan E.L.2, 5 and Wicking C.1 Monash University, Monash Vic. 3800. 1Institute for Molecular Bioscience, The University of Queensland, Qld, Australia. 2The University of Queensland Diamantina Institute, A key aspect of the infectious cycle of many viruses is the transport of Translational Research Institute, Qld, Australia. 3Molecular Medicine Unit specific viral proteins into the host cell nucleus to perturb the antiviral and Birth Defects Research Centre, Institute of Child Health, University response. Examples include a number of RNA viruses that are significant College London, London WC1N 1EH, UK. 4The University of Queensland, human pathogens, such as human immunodeficiency virus (HIV)-1, UQ Centre for Clinical Research, Qld, Australia. 5Department of influenza A, dengue, respiratory syncytial virus, and rabies. Inhibiting Endocrinology, Royal Brisbane and Women’s Hospital, Qld, Australia. the nuclear trafficking of viral proteins as a therapeutic strategy offers an attractive possibility, with important recent progress having been Ciliopathies form an expanding class of congenital disease resulting made with respect to HIV-1 and dengue. The results validate nuclear from dysfunction of the primary cilium. Genes mutated in ciliopathies protein import as an antiviral target, and suggest the identification and commonly encode proteins implicated in ciliary assembly, function development of nuclear transport inhibitors as a viable therapeutic and/or associated protein trafficking pathways. In particular, a approach for a range of human and zoonotic pathogenic viruses. subset of ciliopathies characterised by skeletal defects, are due to mutations encoding genes that affect intraflagellar transport (IFT), the process responsible for trafficking cargo along the ciliary axoneme. These skeletal ciliopathies include short rib polydactyly, Jeune and Sensenbrenner syndromes. Using whole exome sequencing to analyse a cohort of SRP and Jeune syndrome individuals, we have uncovered mutations in genes with a previously uncharacterized role in these disorders. For one of these, WDR60, we have analysed fibroblasts derived from affected individuals and show a severe reduction in cilia number relative to control cell lines. Immunofluorescence analysis reveals localization of this protein at the base of the cilium and further analysis suggests a role in regulating ciliary trafficking. We are further exploring protein interactions to more thoroughly understand the function of WDR60 in ciliogenesis. These studies expand the repertoire of proteins regulating the function of the primary cilium, and provide insights into the role of this dynamic signalling and sensory organelle in health and disease.

SYM-43-05 SYM-44-01 MOUSE XENOCYBRID MODELS OF HUMAN MTDNA Sponsored by DISEASE The Australian Society of Plant Scientists

McKenzie M.1, Carey K.T.1, Van Bergen N.2 and Trounce I.A.2 LIGHT AND TEMPERATURE CONVERGENCE IN THE 1Centre for Reproduction and Development, Monash Institute of CONTROL OF PLANT DEVELOPMENT Medical Research, Melbourne, Australia. 2Centre for Eye Research Australia, Royal Victorian Eye & Ear Hospital, Melbourne, Australia. Casal J.J.1 1IFEVA-Facultad de Agronomía, Universidad de Buenos Aires and The creation of live mtDNA mutant mouse models has been hampered CONICET, 1417- Buenos Aires, Argentina. 2Fundación Instituto Leloir, by the lack of suitable mouse mtDNA mutations in culture and the 1405-Buenos Aires, Argentina. unique features of mtDNA which make site-directed mutagenesis impractical. To overcome these barriers we have created mouse The light environment provides major cues in the control of plant growth ‘xenocybrids’ by introducing mitochondria from different mouse species and development. Although light signals can be specifically manipulated into Mus musculus domesticus (Mm) mtDNA-less (rho 0) fibroblasts. As under laboratory conditions to rule out confounding effects, the natural the genetic divergence of our mtDNA donors increases mitochondrial environment is more complex. Light signals can overlap with changes oxidative phosphorylation (OXPHOS) defects of increasing severity in temperature or occur under extreme temperatures that could result. Using blue native (BN)-PAGE analyses, in conjunction with distort the light signalling mechanisms. We have observed that while pulse-chase labeling of mtDNA-encoded subunits, we have found that constant elevated warm temperatures tend to oppose light effects on the introduction of distantly related species results in the disruption of the control of plant development, short periods of high temperatures OXPHOS complex biogenesis and the reduction of mature complex (heat shocks) enhance the effect of light on seedling morphology during steady-state levels. This is associated with deficiencies in OXPHOS the transition from skotomorphogenesis to photomorphogenesis. This enzyme activities, increased generation of reactive oxygen species and synergism involves heat shock effects on two major branches of the alterations in sensitivity to cell-death inducers. We have transferred one light signalling network, i.e. the COP1-HY5 pathway and the PRR7/ of our in vitro xenocybrid models (which uses Mus dunni (Md) mtDNA) PRR9-LHY/CCA1-PIF4/PIF5 pathway. Via the second pathway, heat to an in vivo setting by introducing Md mitochondria into rhodamine- shocks generate a circadian rhythm of sensitivity to light. However, 6G-treated Mm mouse embryonic stem (ES) cells. The resultant Md the action of the COP1-HY5 does not obviously involve the circadian xenocybrid ES cells remain pluripotent and can contribute to the mouse clock. In addition, heat shocks modify the subcellular distribution germ line, generating homoplasmic Md xenomice. Although these of the light receptor phytochrome B but this effect bears no obvious xenomice develop normally we have observed an accelerated age- relation with the physiological output. These observations indicate that related decline in brain mitochondrial respiration and mild OXPHOS heat shocks impact on key components of the light signalling network; defects in the heart. These results highlight the feasibility of creating while some of these effects can be regarded as noise others (which live mouse models of mtDNA disease using a xenomitochondrial are dominant) reinforce light signalling and contain informative value approach and we are currently generating different xenocybrid ES cell for plant development. lines for the creation of mice with other OXPHOS disorders.

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SYM-44-02 SYM-44-03 LATERAL BRANCHING OXIDASE - A NOVEL GENE IN CARLACTONE-INDEPENDENT SEEDLING THE STRIGOLACTONE PATHWAY MORPHOGENESIS IN ARABIDOPSIS

Brewer P.B. and Beveridge C.A. Scaffidi A.1, Waters M.T.2, Ghisalberti E.L.1, Dixon K.W.3, 4, Flematti G.R.1 The University of Queensland, School of Biological Sciences, St Lucia and Smith S.M.1, 2 Qld 4072. 1School of Chemistry and Biochemistry, The University of Western Australia, Crawley WA 6009. 2Plant Energy Biology, The University Strigolactones are a class plant hormone with diverse signalling roles of Western Australia, Crawley WA 6009. 3Kings Park and Botanic in plant development. Generally they act as a barometer in plants to Garden, West Perth, WA 6005. 4School of Plant Biology, The adjust growth and development in response to resource availability. University of Western Australia, Crawley WA 6009. They also act as stimulants for microbe symbiosis and weed parasitism in the rhizosphere. Strigolactones are produced in shoot and root Strigolactones are carotenoid-derived plant hormones that regulate and can move upwards over graft junctions. They are carotenoid- shoot and root architecture, and promote the colonisation of roots derived, with the initial biosynthetic steps occurring in plastids whereby by arbuscular-mychorrhizal fungi. Structurally, strigolactones are the intermediate, carlactone, is produced. Biosynthetic steps after characterised by a methyl-butenolide ring linked via an enol-ether carlactone are predicted but not yet elucidated. Using our knowledge bridge to a tricyclic lactone. Biosynthesis of strigolactones requires the of patterns of auxin and feedback signalling on strigolactone gene sequential action of a carotenoid isomerase (D27) and two carotenoid expression in Arabidopsis, combined with microarray analysis, we cleavage dioxygenases (MAX3 and MAX4) to generate the precursor identified a novel co-expressed gene that we named LATERAL carlactone. Mature strigolactones are perceived through the combined BRANCHING OXIDASE (LBO). LBO encodes an oxidoreductase- activity of the α/β-hydrolase D14 and the F-box protein MAX2. While like enzyme of the 2-oxoglutarate and Fe(II)-dependent dioxygenase MAX2 is also necessary for normal seedling development, D14 and the super-family. Based on grafting studies measuring shoot branching, known strigolactone biosynthesis genes are not, raising the question of this protein functions in shoot and root to control a mobile signal and is whether endogenous, canonical strigolactones derived from carlactone required downstream of previously reported strigolactone biosynthesis have a role in seedling morphogenesis. We report the chemical enzymes. The synthetic strigolactone GR24 can restore branching synthesis of carlactone and show that it represses Arabidopsis shoot inhibition in lbo mutants shoots, but unlike previously reported branching and influences leaf morphogenesis via a mechanism strigolactone biosynthesis mutants, carlactone fails to inhibit branching dependent on the cytochrome P450 MAX1. In contrast, carlactone has in lbo mutant shoots. This indicates that LBO acts downstream of limited weak activity in seedlings, and predominantly signals through carlactone to produce the active strigolactone(s). LBO will be useful D14 rather than its paralogue KAI2, in a MAX2-dependent but MAX1- to identify strigolactones involved in receptor binding and signalling as independent manner. KAI2 is necessary for the perception of karrikins, a well as in understanding strigolactone diversity and function. class of butenolide germination stimulants derived from wildfire smoke. Crucially, KAI2 is also essential for normal seedling morphogenesis. Hence, we infer that early stages of plant development employ as yet unidentified butenolide morphogens that are generated independently of carlactone, but for which karrikins represent specific surrogates.

SYM-44-04 SYM-44-05 MOONLIGHTING CATALYTIC FUNCTIONS TREHALOSE-6-PHOSPHATE – A SUGAR SIGNAL MODULATING SIGNALLING PATHWAYS IN A FAMILY THAT REGULATES PLANT METABOLISM AND OF RECEPTOR LIKE KINASES DEVELOPMENT

Wheeler J.I.1, Kwezi L.2, Muleya V.1, Freihat L.1, Ruzvidzo O.2, Lunn J.E. Manallack D.1, Gehring C.3 and Irving H.R.1 Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 1Monash Institute of Pharmaceutical Sciences, Monash University 14476 Potsdam-Golm, Germany. Parkville VIC 3052. 2North-West University, Mmabatho, 2735, South Africa. 3King Abdullah University of Science and Technology, 23955- Trehalose metabolism was once thought to be unimportant or even 6900 Thuwal, Kingdom of Saudi Arabia. absent from higher plants. This view was completely overturned by the discovery of genes encoding enzymes of trehalose biosynthesis in Cyclic GMP is an important signalling molecule involved in regulating Arabidopsis thaliana and many other higher plants, and from analysis a wide variety of physiological effects ranging from plant hormone of trehalose metabolic mutants, which display an extraordinary range of dependent responses to induction of plant defence responses. As the developmental phenotypes: arrested embryo development, altered root guanylate cyclase (GC) enzymes responsible for cGMP synthesis were and leaf growth, delayed senescence, early/late flowering and changes elusive, we designed a search motif using consensus residues present in inflorescence architecture. Many of these developmental defects in the catalytic domain of GCs from lower eukaryotes and animals have been linked to changes in the level of trehalose 6-phosphate to identify putative GCs in Arabidopsis. The search returned over 40 (Tre6P), the phosphorylated intermediate of trehalose biosynthesis, putative GCs, the majority of which are annotated as receptor-like rather than trehalose itself. There is now compelling evidence that kinases (RLKs) where the GC catalytic centre is embedded in the kinase Tre6P acts as a signal metabolite in plants, reflecting the availability domain which is distinct from animal GCs where the two domains are of sugars, particularly sucrose. We are investigating how sucrose separated. The large family of RLKs with both GC and kinase domains determines the level of Tre6P, and how this signal metabolite influences implies that these dual functionalities have coevolved due to the flowering and other aspects of plant development. In addition to its importance of both enzyme activities in plant development and stress role in controlling plant development, we have also found evidence response where the GC is a moonlighting activity. We have shown that that Tre6P regulates transitory starch metabolism in leaves, helping to several RLKs such as BRASSINOSTEROID INSENSITIVE 1 (BRI1), balance the rate of starch breakdown at night with demand for sucrose WALL ASSOCIATED KINASE LIKE 10 (WAKL10), PHYTOSULFOKINE from sink organs. A number of other potential targets of Tre6P signalling RECEPTOR 1 (PSKR1) and PEP1 RECEPTOR 1 (PEPR1) contain both have been identified, along with a role for trehalose in regulation of kinase and GC activity in vitro and that the natural ligands of BRI1 and stomatal opening. PSKR1 stimulate increases in cGMP in protoplasts. Hence it is likely that the GC kinases switch between downstream cGMP-mediated or kinase-mediated signalling cascades to elicit desired outputs to particular stimuli. The current challenge lies in understanding the interaction between the kinase and GC domains at the molecular level and how these receptors capitalize on their dual functionality. To that end we have established that calcium, phosphorylation and cGMP itself influence the different enzymatic activities of these molecules and that this phenomenon extends beyond the plant kingdom.

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SYM-45-01 SYM-45-02 CELL CHIPS: NANOSTRUCTURED MATERIALS NANOPARTICLES: A NEW TOOL IN BIOLOGICAL AND THAT CAN REPORT ON THE RELEASE OF MATRIX MEDICAL RESEARCH METALLOPROTEASES FROM JUST A FEW CELLS Barnard A.S. Zhu Y.1, 2, Gupta B.1, 2, Soeriyadi A.1, 2, Guan B.1, 2, Kilian K.A.1, Le Saux G.1, CSIRO Materials Science and Engineering. Boecking T.3, 2, Reece P.J.4, Gaus K.3, 2 and Gooding J.J.1, 2 1School of Chemistry, UNSW Australia, Sydney 2052. 2Australian Nanoparticles are being proposed for new applications in biology, Centre for NanoMedicine, UNSW Australia, Sydney 2052. 3Centre biotechnology and medicine more and more every year. In many cases, for Vascular Research, UNSW Australia, Sydney 2052. 4School of they are targeted to well known and well established applications, and Physics, UNSW Australia, Sydney 2052. aim to replace existing molecular technologies. In other cases they are targeted to new and novel areas of research, where current methods Out long term goal is to design surfaces that can monitor the release fail to provide sufficient stability, specificity or sensitivity. However, of biomarkers from arrays of single cells. Such surfaces could then be irrespective of the application area, there are a number of challenges used as cell based diagnostic devices, or cell chips (for applications associated with engineering the right nanoparticle for the job, and as diverse as drug testing, personalized medicine and nanotoxicology) dealing with the complexity associated with collections of particles that as well as provide fundamental information on the variation of cellular are not all identical, and cannot be truly purified. Small nanoparticles responses to stimuli. This talk will outline our progress towards this (smaller than cells) have many advantages in penetrating tissues, but goal using photonic crystals that can monitor the release of matrix this also means they have a high surface area, and are very reactive to metalloproteases (MMPs) from macrophage cells. As cells sense their surroundings. In this presentation we will discuss some of these their environment with nanoscale precision, how cells interact with a issues, using an exemplary system: diamond nanoparticles. These tiny surface can influence cell phenotypes and responses. Therefore how a ~4 nm fluorescent particles show no cytotoxicity, and so are finding use manmade surface is designed, with regards to its density of cell surface in a range of advanced diagnostic and therapeutic applications. ligands and topography, will be important for cell based diagnostic devices and biomaterials. As a consequence this talk will first discuss the design of surfaces with molecular level control that influence the outside-in signaling and migration of bovine aortic endothelial cells. We show how both the density of ligands and the topography influence cell function and show how the expression of cell adhesive ligands can be switched on demand. We next show similar trends can be observed for macrophage cells before moving to surfaces that can monitor the release of enzymes from human macrophage derived monocytes (HMDMs). Upon stimulation of the macrophage cells with lipopolysaccharide the expected upregulation in MMPs is observed from just 1500 cells. The convergence of this technology to detecting the MMP release from just a few cells is discussed as is how to apply the devices in vivo. Finally a demonstration of how to make the photonic crystals selective for a specific MMP is also presented.

SYM-45-03 SYM-45-04 TARGETED NANOPARTICLES FOR THE DELIVERY OF LAYERED DOUBLE HYDROXIDE NANOPARTICLES THERAPEUTICS IN PREGNANCY FOR HEPARIN AND SIRNA DELIVERY

Keelan J.A.1, 4, Waddell B.J.2, 4 and Iyer K.S.3, 4 Xu Z .P. and Gu Z. 1School of Women’s and Infants’ Health. 2School of Anatomy, AIBN, The University of Queensland. Physiology and Human Biology. 3School of Chemistry and Biochemistry. 4University of Western Australia. Layered double hydroxides (LDHs), also known as anionic clays and exemplified by hydrotalcite (Mg6Al2(OH)16CO3.4H2O), find a high Around 20% of all pregnancies are complicated by conditions potential as the drug/gene delivery vehicle. In this talk, I will report our that involve some form of placental pathology or dysfunction, with findings that LDH NPs can be quickly taken up by various cell lines, in potentially serious fetal and/or maternal consequences. However, the a clathrin-mediated dose-dependent and time-dependent endocytosis treatment of feto-placental conditions during pregnancy poses some pathway. More excitedly, we have for the first time discovered that the unique therapeutic challenges due to the presence of three discrete rod-like LDH NPs are mostly located in the nucleus while plate-like LDH compartments: mother, baby and placenta. Each of these possesses NPs in the perinuclear area, which may imply a strategy that can be different pharmacokinetic characteristics and access to maternally- used to target the subcellular compartments by controlling the shape/ administered drugs. Modalities for delivering therapeutics are needed size of delivery vehicles. I will then present two practical examples that allow the selective targeting of maternal, placental or fetal tissues to demonstrate the feasibility using LDH NPs as clinical vehicles. while avoiding unwanted off-target effects and minimising risk to the One example is anti-restenotic drug delivery. Low molecular weight fetus. In this context, drug delivery via nanoparticles offers enormous heparin (LMWH) is a good and commonly used anti-restenotic drug advantages over conventional drug delivery strategies. In particular, to intervene the biological activity of vascular smooth muscle cells nanoparticles can be functionalised to target specific cell types or (SMCs). Intercalation of LMWH into LDH enables the release to sustain tissues and thus restrict their actions to those tissues that would benefit for over 5 days. More significantly, LMWH-LDH nanohybrids enhance from the therapy. The placenta, with unfettered access to substances the inhibition to SMC proliferation and migration by ~60% compared circulating in the maternal blood stream, offers an excellent therapeutic with the control, e.g. promoting the biological functions of LMWH on target as well as a potential conduit for fetal drug administration. We SMCs, which is actively pursued for anti-restenosis treatment. The and others have shown that the uptake and passage of nanomaterials other example is delivery of functional small interfering RNA (siRNA) across the placenta can be controlled by altering their size, structure and its DNA mimic to various cell lines, with the efficiency superior to and charge. We have explored the placental uptake and biodistribution the commercial polymeric vehicle. of PEI-coated polymeric nanoparticles using human (ex-vivo) and animal (in-vivo) pregnancy models. We are now exploring the use of nanoparticles decorated with targeting ligands (polypeptides with high binding specificity to placental surface proteins or receptors) to selectively deliver pharmaceuticals to human and rodent placental tissue. Preliminary findings to date suggest that these approaches are likely to be effective, albeit data from animal models are still pending.

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SYM-45-05 SYM-46-01 Sponsored by CLICKABLE NANOFORMULATION: The Australian Society for Biochemistry and Molecular Biology MULTIFUNCTIONAL FORMULATION FOR FOLATE RECEPTOR TARGETED CHEMOTHERAPY DEFECTIVE POLYADENYLATION IS A MOLECULAR

2 1 1 PATHOLOGY IN HUMAN MITOCHONDRIAL MRNA Singh R.1, 2, Lim L.Y. , Iyer K.S. and Smith N.M. MATURATION 1School of Chemistry and Biochemistry, The University of Western Australia, Crawley, WA 6009, Australia. 2Pharmacy, School of Wilson W.C.1, Jourdain A.2, Spahr H.3, Chang J.H.4, Tong L.4, Bruni F.1, Medicine and Pharmacology,The University of Western Australia, Larsson N.-G.3, Crosby A.H.5, Chrzanowska-Lightowlers Z.M.A.1 and Crawley, WA 6009, Australia. Lightowlers R.N.1 1Wellcome Trust Centre for Mitochondrial Research, Medical School, 2 One of the current challenges in cancer treatment is enhancement Newcastle University, Newcastle Upon Tyne, NE2 4HH, UK. University of of tumor specific targeting of both the imaging probe and the Geneva, Departement of Cell Biology, 30 quai Ernest Ansermet 1211 Geneva 4 Switzerland. 3Max Planck Institute for Biology of Ageing, Gleueler Strasse chemotherapeutic agent. In this presentation we demonstrate the 4 ability to deliver both imaging modality and therapeutic in one single 50a, D-50931 Cologne, Germany. Department of Biological Sciences, Columbia University, New York, NY 10027. 5Centre for Medical Genetics, St. dose using an in-vitro model to target the folate receptor. This receptor George’s University, Cranmer Terrace, London SW17 0RE, UK. is highly overexpressed in many human tumors including ovarian, lung, head, neck, renal cells and breast cancer whereas in normal tissue its Polyadenylation of transcripts is a common phenomenon but one that has expression is significantly lower. Using the facile click chemistry we different consequences depending on the organism and the subcellular will report the synthesis of the polymeric (polyglycidyl methacrylate) compartment. It is a post-transcriptional modification that plays a crucial carriers with folic acid moieties on the surface. The nanoparticles role in mammalian mitochondrial gene expression. In human mitochondria, consists of Docetaxel (chemotherapeutic agent) and Rhodamine the one certain role that polyadenylation plays is the generation of UAA stop (single cellular imaging). Using confocal imaging, flow cytometry, and codons that would otherwise remain incomplete in seven transcripts following electron microscopy we demonstrate the ability of the nanoparticles to their excision from a polycistronic RNA species. Using cell lines derived from selectively deliver payloads in folate positive ovarian cancer (SKOV- patients harboring a 1432A>G mutation in the PAPD1 gene, which encodes the 3), folate negative lung adenocarcinoma (A549) and normal fibroblast mitochondrial poly(A) polymerase, we have been able to confirm that defects in mtPAP cause a form of autosomal-recessive spastic ataxia and optic atrophy. (3T3) cells. These mutant cells show truncated poly(A) tails on mt-mRNAs. Analysis of mitochondrial transcript steady-state levels, de novo protein synthesis, protein steady-state levels, and respiratory complex assembly show non-uniform dysregulation of gene expression. To confirm the pathological nature of the mutation, a complementation experiment was performed, and showed that lentiviral expression of WT PAPD1 gene the rescued the mutant phenotype. In vitro activity assays with WT and N478D recombinant mtPAP found that the mutation appears to compromise enzymatic activity. As part of these studies, we investigated the effects on in vitro polyadenylation of LRPPRC, a putative coordinator of mitochondrial translation, with and without SLIRP, a protein with which LRPPRC is know to complex. Finally, to identify potential interacting factors with mtPAP, co-immunoprecipitation and sucrose gradient experiments were undertaken. Our results give a comprehensive understanding of the impact of mitochondrial polyadenylation in vivo.

SYM-46-02 SYM-46-03 Sponsored by The Australian Society for Biochemistry and Molecular Biology RBP ATLAS: AN EXPLORATION OF INTERACTIONS BETWEEN MRNA AND PROTEINS AND THEIR IMPACT ON CARDIOMYOCYTE BIOLOGY RBFA, A MITOCHONDRIAL RIBOSOME ASSEMBLY 1 2 2 2 1 2 FACTOR ? Liao Y. , Castello A. , Foehr S. , Leicht S. , Yang H. , Fischer B. , Horos R.2, Krijgsveld J.2, Hentze M.W.2 and Preiss T.1 1 1 1 2 John Curtin School of Medical Research, Australian National University, Chrzanowska-Lightowlers Z.M.A. , Rozanska A. , Rorbach J. , 2 3 4 Australia. European Molecular Biology Laboratory (EMBL), Heidelberg, Richter R. and Lightowlers R.N. Germany. 1The Wellcome Trust Centre for Mitochondrial Research, Institute for Ageing and Health, Newcastle University, The Medical School, RNA-binding proteins (RBPs) control all aspects of RNA fate, often by Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. 2current organizing multiple functionally related RNAs into post-transcriptional address, MRC MBU Hills Road Cambridge CB2 0XY,UK. 3current operons. Defects in RBP function furthermore underlie a broad spectrum of address, Institute of Genetics, University of Cologne, Cologne, human pathologies (1). How such RBP networks operate in cardiomyocytes Germany. 4The Wellcome Trust Centre for Mitochondrial Research, and respond to (patho-) physiological cues in the heart is poorly understood. Institute for Cell and Molecular Biosciences, Newcastle University, We chose to investigate this in murine HL-1 cardiomyocytes, a cell line that The Medical School, Framlington Place, Newcastle upon Tyne, NE2 can be propagated in culture while maintaining the ability to contract and 4HH, UK. other differentiated cardiac morphological and functional properties (2). Deploying a recently developed combination of UV-crosslinking of proteins to RNA in living cells with identification of proteins co-purifying with poly(A)+ We have identified that human mitochondria have an orthologue of a RNA by mass spectrometry (3), we have now identified ~1000 proteins bacterial protein, RbfA (ribosome assembly factor A) that as its name as the mRNA interactome of cardiomyocytes. Domain features and gene implies is involved in the synthesis of 70S ribosomes. In bacteria this ontology enrichment broadly validate the sensitivity and specificity of the protein is responsible for final processing step of the 5’ end of the 17S cardiomyocyte RBP capture. About half of this RBP set overlaps with our rRNA removing a short RNA sequence to generate the 16S rRNA. The previously published human HeLa cell mRNA interactome, while many equivalent rRNA species that is incorporated into the small subunit of of the remainder represent cardiomyocyte-specific RBPs. ~370 of the the human mitochondrial ribosome is a 12S rRNA and is encoded by identified proteins have no RNA-related annotations and ~65 of these were mitochondrial DNA as part of a large polycistron. In contrast to the case identified as RBP candidates for the first time. Notably, ~50 of the identified in bacteria, the final 12S rRNA is generated by a single cleavage at RBPs have established functions as enzymes of intermediary metabolism. the 5’ terminus and a further single cleavage at the 3’ end. Neither Of direct relevance to cardiac pathology, ~180 of cardiomyocyte RBP genes cleavage event is dependent on RbfA begging the question of what are associated with heart disease (based on genecards.org), and only ~50 RbfA’s function in human mitochondria may be. of these have established links to RNA biology. Ongoing work is focused on probing changes to the RBP network in cardiomyocytes responding to metabolic challenges, as well as identifying the RNA targets of selected RBPs, with particular emphasis on probing the potential interplay between cellular metabolism and gene regulation through RNA-binding by metabolic enzymes (4). 1. Castello A et al. 2013. RNA-binding proteins in Mendelian disease. Trends Genet. 29:318-27. 2. Claycomb, W. C. et al. (1998). HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci USA, 95(6), 2979–2984. 3. Castello, A. et al. (2012). Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell, 149(6), 1393–1406. 4. Hentze MW, Preiss T. 2010. The REM phase of gene regulation. Trends Biochem Sci. 35:423-6.. ComBio2013 s Perth, Western Australia s 29 September - 3 October, 2013 Page 85 symposia THURSDAY

SYM-46-04 SYM-46-05 TOWARDS UNDERSTANDING TRANSLATIONAL THE ROLE OF AN UNCONVENTIONAL RIBOSOMAL CONTROL IN GERM CELLS PROTEIN IN MITOCHONDRIAL FUNCTION

Sengupta M.S.1, Raymant G.1, Bär I.2, Sarov M.2 and Boag P.R.1 Richman T.R., Davies S.M.K., Shearwood A., Ermer J., Scott L., 1Department of Biochemistry and Molecular Biology, Monash Rackham O. and Filipovska A. University. 2Max Planck Institute of Molecular Cell Biology and Western Australian Institute for Medical Research, The University of Genetics, Germany. Western Australia.

During oogenesis many germ cell mRNAs are produced and stored in a Mitochondria are ubiquitous organelles of eukaryotic cells that translationally repressed state and are subsequently activated at specific contain a circular double-stranded genome encoding 13 polypeptide times during oocyte and early embryonic development. Some of these subunits of the mitochondrial respiratory chain. The genes for these translationally repressed mRNAs are stored in perinuclear cytoplasmic polypeptides are transcribed and translated in the mitochondrial matrix, granules called germ granules, which are key sites of mRNA storage using 22 tRNAs and 2 rRNAs encoded by the compact mitochondrial and post-transcriptional gene regulation. To further understand how DNA. Consequently, mtDNA is dependent on nuclear encoded proteins maternal mRNAs are post-transcriptionally regulated, we are studying for replication, repair, transcription and translation. Mammalian the eIF4E-binding protein IFET-1, in the model organism C. elegans. mitochondrial mRNAs generally begin at the start codon and lack We have found that IFET-1 is required for translational repression of conventional 5’ untranslated regions or Shine-Dalgarno sequences. several maternal mRNAs, and is required for normal oogenesis but not In addition mitochondrial encoded proteins are hydrophobic and are spermatogenesis. IFET-1 functions in conjunction with other broad- likely to be co-translationally inserted into the membrane-embedded scale translational regulators (CGH-1 and CAR-1) to control germ complexes providing unique constraints for their translation. cell sex determination, presumably through regulation of networks Mitochondrial ribosomes must have evolved an alternative way to of maternal mRNAs. Interestingly, IFET-1 is required for the normal regulate translation initiation and elongation to ensure accurate start ultrastructure of germ granules and for the localization of CGH-1 and codon recognition and facilitate protein complex assembly. Mammalian CAR-1 to germ granules. Our findings, together with others, suggests mitochondrial ribosomes are unique from bacterial and cytoplasmic that IFET-1 is a key evolutionarily conserved node in germ granule ribosomes of eukaryotes because their ribosomal RNA has been formation and post-transcriptional gene regulation during oogenesis. reduced considerably and has been replaced by additional proteins. These proteins may have new functions in mitochondrial translation and recognition of mitochondrial mRNAs. We have identified an additional protein and found that it localizes to mitochondria, associates with mitochondrial ribosomes specifically, and has important roles in mitochondrial gene expression and consequently cellular energy metabolism.

SYM-47-01 SYM-47-02 MEMBRANE RIGIDITY, VESICLE FORMATION AND SEC14 OF CRYPTOCOCCUS NEOFORMANS IS THE PROTEIN QUALITY CONTROL CHECKPOINT REQUIRED FOR EXPORT OF CELL WALL-MODIFYING ENZYMES AND CELL SEPARATION Miller E.A. Biological Sciences, Columbia University, New York, NY 10027, USA. Lev S.1, Crossett B.2, Wilson C.F.1, Desmarini D.1, Li C.1, Chayakulkeeree M.3, Williamson P.4, Sorrell T.C.1, 5 and Djordjevic J.T.1 Eukaryotic secretory proteins exit the endoplasmic reticulum (ER) via 1Centre for Infectious Diseases & Microbiology, Westmead Millennium transport vesicles generated by the essential COPII coat proteins. This Institute & Sydney Medical School, U. of Sydney at Westmead Hospital step represents a critical quality control checkpoint in that misfolded Westmead NSW. 2School of Molecular Bioscience, University of Sydney, proteins are generally excluded from COPII vesicles. In a search for NSW. 3Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok yeast mutants that have defective ER retention of misfolded proteins, Thailand. 4Laboratory of Clinical Infectious Diseases, National Institute we identified several bypass-of-sec-thirteen (bst) mutants, where the of Allergy & Infectious Diseases, National Institutes of Health, Bethesda 5 otherwise essential COPII protein, Sec13, becomes dispensable. A Maryland USA. Sydney Emerging Infections & Biosecurity Institute, U. of genome-wide screen for the full complement of bst mutants identified Sydney at Westmead Hospital Westmead NSW. nine “core” proteins that generally contribute to the packaging of Secreted proteins contribute to the pathogenesis of the opportunistic asymmetrically distributed cargo proteins, including the particularly fungus, Cryptococcus neoformans. We previously established that the abundant GPI-anchored proteins. Our genetic and biochemical cryptococcal phosphatidylinositol/phosphatidylcholine transfer protein, analyses suggest that within the outer coat scaffold formed by Sec13 and Sec14, regulates secretion of the fungal invasin, phospholipase B1 (Plb1), Sec31, Sec13 acts to rigidify the COPII cage, increasing its membrane- and is essential for cell wall integrity and virulence. To identify additional bending capacity. This structural integrity is not required when a bst Sec14-regulated proteins that potentially contribute to the pathogenesis mutation renders the membrane more amenable to deformation by of C. neoformans, we analysed the secretomes of WT (strain H99) and a depleting cargoes. We propose that the lapse in ER quality control that SEC14 deletion mutant (Δsec14) using mass spectrometry. We identified occurs in bst mutants reflects the altered biophysical properties of the 105 proteins in WT secretions: 27 contained a signal peptide, implying membrane and coat itself to create structures that are more permissive that they are canonically secreted via the ER/Golgi. The abundance of to the inclusion of aberrant proteins. 25 proteins was reduced in Δsec14 secretions, 7 of which were cell wall- associated/modifying enzymes, including the virulence-related enzymes, Plb1, laccase (Lac1), acid phosphatase Aph1 and α-1,3-glucan synthase (Ags1). Comparison of the subcellular localization of fluorescent-labelled Plb1and Aph1 revealed that Plb1 was transported directly to the cell periphery from the ER/Golgi, while Aph1 accumulated in endosome-like structures en route to the plasma membrane and vacuoles. Both proteins were enriched in bud necks, implicating them in bud formation and/or septation. Microscopic examination of Δsec14 revealed that the cells were enlarged, and that mother and daughter cells often remained connected via the septum following mitosis. Taken together, our findings demonstrate that Sec14 regulates the traffic of proteins involved in cell wall remodelling via endosome-dependent and -independent secretion routes to ensure cell wall integrity and timely dissolution of the septum.

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SYM-47-03 SYM-47-04 PURINE METABOLISM AS AN ANTIFUNGAL TARGET GENE EXPRESSION IN 3D: HIDDEN GENOMIC FUNCTIONALITY IS REVEALED IN THE MATURING Fraser J.A. YEAST COLONY University of Queensland, St Lucia, 4072. Heinz E.1, 2, Harrison P.2, 3, Powell D.2, 3, Lee M.1, Traven A.1 and We have investigated the potential of the purine biosynthesis pathway Beilharz T.1 as a chemotherapeutic targets in the human pathogen Cryptococcus 1Department of Biochemistry and Molecular biology; Monash neoformans, a common cause of fatal fungal meningoencephalitis. University. 2Victorian Bioinformatics Consortium. 3VLSCI: Life Science We find that de novo GTP biosynthesis, but not the alternate salvage Computation Centre. pathway, is critical to cryptococcal dissemination and survival in vivo. Loss of inosine monophosphate dehydrogenase (IMPDH) in the de Human development is a clear example of how multicellular communities of novo pathway results in slow growth and virulence factor defects, while genetically identical cells can generate an astounding level of complexity. loss of the cognate phosphoribosyltransferase in the salvage pathway We recently showed that the individual cells of the simple model eukaryote, yielded no phenotypes. Structural and functional characterization of Saccharomyces cerevisiae, display different gene-expression patterns IMPDH from Cryptococcus has revealed potential strategies for the depending on their position within a mature colony. Whereas cells of the development of fungal-specific inhibitors; the crystal structure reveals colony exterior are metabolically active, the cells of the colony interior the position of the IMPDH moveable flap and catalytic arginine in the are starving. To our surprise, a disproportionately large number of genes open conformation for the first time, plus unique, exploitable differences that were differentially expressed between the inner and outer layers of in the highly conserved active site. Treatment with mycophenolic acid the colony have no known function, encoding uncharacterised proteins led to significantly increased survival times in a nematode model, or are non-coding. To better understand how the growth environment of validating de novo GTP biosynthesis as an antifungal target in yeast shapes gene-expression, we used a custom RNA-seq approach Cryptococcus. to transcriptionally profile yeast cells growing in either the standard laboratory conditions (liquid culture with high glucose and high aeration) and compared these to maturing (2, 3 and 4 day) yeast colonies grown on nutritionally identical solid media. These experiments reveal that the mature yeast colony expresses a large proportion of its genome that is silent under standard conditions. Among genes specifically unregulated in the colony are non-coding RNA, transcription factors, regulatory RNA- binding proteins, and a suite of enzymes with catabolic functions that are normally expressed at low liquid culture population. Of particular interest to us is Ngl3, a poorly characterised deadenylase whose expression is unregulated ~50 fold in cells growing as colonies and whose expression is limited to the colony interior. We will present data showing that Ngl3 is a homolog of the metazoan protein Nocturnin, a deadenylase essential for normal fat metabolism. We will report on our current work in search of the targets of deadenylation by Ngl3 in the mature yeast colony as part of our ongoing interest in understanding the role Nocturnin mediated deadenylation in energy homeostasis.

SYM-47-05 SYM-48-01 BOVINE PANCREATIC TRYPSIN INHIBITOR (BPTI) MUSCLE PROGENITOR CELL BIOLOGY IN MUSCLE INHIBITS THE GROWTH OF YEAST BY A NOVEL GROWTH AND REGENERATION IN THE ZEBRAFISH MECHANISM THAT INVOLVES PERTURBATION OF CELLULAR MAGNESIUM HOMEOSTASIS Gurevich D.B.1, 2, Phan J.M.1, Hall T.E.1, Verkade H.2 and Currie P.D.1 1Australian Regenerative Medicine Institute, Monash University, Bleackley M.R.1, Hayes B.M.1, Potter I.D.1, Traven A.2, Clayton, VIC, Australia. 2School of Biological Sciences, Monash Van Der Weerden N.L.1 and Anderson M.A.1 University, Clayton, VIC, Australia. 1La Trobe Institute for Molecular Science, Bundoora, VIC. 2Monash Univeristy, Melbourne, VIC. We have recently investigated the developmental origin of muscle progenitor and stem cells in the zebrafish myotome. Using a combination Antimicrobial peptides (AMPs) are a diverse set of molecules expressed in of lineage analysis, whole somite imaging and gene specific loss of all kingdoms of life to protect against microbial pathogens. Their activities function approaches, we have determined the developmental origin of against a broad spectrum of species have made them a promising set of specific muscle populations during primary and secondary myogenesis molecules for the control of infections. Traditionally AMPs were thought in the zebrafish emebryo. We have defined a specific layer of cells to act via membrane disruption. However, recent studies have revealed functionally equivalent to the amniote dermomyotome, termed the more complex mechanisms. Many AMPs that are active against fungi are external cell layer which generates distinct muscle populations during small, cationic and stabilized by disulphide bonds. We recognised that embryogeneis. Despite these analyses the cellular basis for the many Kunitz type protease inhibitors also fulfilled these requirements and prodigious growth evident in the post embryonic zebrafish myotome thus assayed the prototypical member of this family, the bovine pancreatic have remained undescribed. The work presented here focuses on trypsin inhibitor (BPTI), for antifungal activity. A novel antifungal activity for identifying the source and mechanism of post-embryonic muscle BPTI against the model yeast Saccharomyces cerevisiae and the human growth in zebrafish. We show that zebrafish possess a compartment pathogen Candida albicans was identified. BPTI does not kill yeast but of satellite cell-like muscle precursor cells that perjure throughout instead inhibits cellular replication. FACS analysis of the DNA content the post-embryonic development of the fish. Using fluorescent of BPTI retarded yeast revealed a decrease in the proportion of cells transgene expression and morphometric analysis, we identify the actively replicating DNA. Screening of S. cerevisiae deletion collections zones of new growth throughout the myotome for larval to adult revealed the plasma membrane magnesium transporter ALR1 as a stages of fish development. Additional fluorescent transgenic lines potential target for BPTI. Resistance to BPTI in ESCRT mutants indicates allow us to investigate the myotomal location and cellular behaviour a role for the ESCRT pathway in cellular magnesium homeostasis, likely involved in the transition from an undifferentiated mononuclear MPC in the recycling of Alr1p from the membrane. That BPTI was interfering into a multinucleate muscle fibre within larval/adult skeletal muscle with cellular magnesium homeostasis was confirmed through growth in vivo. Finally, we use cellular injury and ablation of the mutant and assays and atomic absorption spectroscopy. Magnesium has long been transgenic fish to visualise the role of MPCs in post-embryonic muscle linked to a number of processes essential for cell division thus perturbing regeneration, and to investigate the genetic programs used. magnesium homeostasis would cause cell cycle check points not to be met and prevent division. This study has revealed a novel function for the well-studied protein BPTI in fungal growth inhibition and to our knowledge this is the first report of a protein inhibitor of a cellular magnesium transporter.

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SYM-48-02 SYM-48-03 DEFINING THE MOLECULAR BASIS OF SARCOPENIA, YAP CONTROLS STEM/PROGENITOR CELL AND IDENTIFYING BIOMARKERS TO MONITOR THE PROLIFERATION IN THE MOUSE POSTNATAL IMPACT OF INTERVENTIONS EPIDERMIS

1, 2 2 3, 4 2 3, 4 Shavlakadze T.1, Chai J.1, Barns M.E.1, Soffe Z.R.2, Mcmahon C.3, Beverdam A. , Claxton C. , Zhang X. , James G. , Harvey K.F. Sayer A.3 and Grounds M.D.1 and Key B.2 1School of Anatomy, Physiology and Human Biology, the University 1Department of Anatomy, The University of New South Wales. 2School of Western Australia. 2Agresearch Ltd, New Zealand. 3University of of Biomedical Sciences, The University of Queensland. 3Sir Peter Southampton, United Kingdom. MacCallum Department of Oncology. 4Department of Pathology, University of Melbourne. With ageing, the progressive loss of muscle mass and function, sarcopenia, results in loss of independence and quality of life, frailty Tissue renewal is an ongoing process in the epithelium of the skin. and rising health costs. The mechanisms of sarcopenia are unclear. We have begun to examine the genetic mechanisms that control stem/ We have described the time course of sarcopenia in C57Bl/6J mice progenitor cell activation in the postnatal epidermis. The conserved with significant loss of muscle mass by 24 months (m) of age and Hippo pathway regulates stem cell turn over in arthropods through further loss by 28m. This mouse model provides a powerful tool to to vertebrates. We show here that its downstream effector Yes- dissect the molecular basis of sarcopenia. Our intensive histological, associated protein (YAP) is active in the stem/progenitor cells of the immunohistochemical and molecular studies have identified striking postnatal epidermis. Overexpression of a C-terminally truncated changes during the transition phase between 15 and 24m of age in YAP protein mutant in the basal epidermis of transgenic mice caused mice: these include changes in metabolic pathways (protein synthesis, dramatic expansion of epidermal stem/progenitor cell populations. degradation and autophagy) and loss of muscle innervation. Other Our data suggest that the C-terminus of YAP controls the balance studies with exercised mice, show that life-long exercise (for 24m) between stem/progenitor cell proliferation and differentiation in the without resistance and short term (3m) exercise with resistance postnatal interfollicular epidermis. We conclude that YAP functions as reduces sarcopenia. Molecular analyses of muscle samples from both a molecular switch of stem/progenitor cell activation in the epidermis. of these intensive exercise regimes, combined with fully described Moreover, our results highlight YAP as a possible therapeutic target for phenotypes, links the profile of the biomarkers with the benefits of diseases such as skin cancer and psoriasis. exercise on sarcopenia. Based on these molecular changes in ageing mouse muscles, selected targeted analyses are being carried out at UWA on 120 muscle biopsies from elderly humans (aged>70 years) from the Hertfordshire study in the UK. This translational study (with full phenotype data available for human subjects) aims to identify molecular biomarkers to monitor future interventions to reduce sarcopenia in humans.

SYM-48-04 SYM-48-05 TWO NOVEL MOUSE MODELS OF HUMAN EYA AND SIX FAMILY MEMBERS REGULATE CRANIOFACIAL DYSMORPHOLOGY MYOGENIC STEM CELL FATE

Miller K.1, Tan T.2, Welfare M.1 and Farlie P.1, 3 White R.B.1, 2, Lee J.3, Davies J.3, Thomas M.G.1, 2, Fear D.4 and 1Craniofacial Development, Murdoch Childrens Research Institute. Zammit P.3 2Department of Paediatrics, Victorian Clinical Genetics Services. 1Parkinson’s Centre, School of Medical Sciences, Edith Cowan 3Department of Pediatrics, University of Melbourne. University. 2Experimental and Regenerative Neuroscience, School of Animal Biology, University of Western Australia. 3Randall Division, Approximately one third of all congenital abnormalities involve the King’s College London. 4MRC Centre in Allergic Mechanisms of craniofacial structures, where they are frequently associated with other Asthma, Guy’s Hospital, UK. clinical characteristics such as defects in the limbs and/or other organ systems. Thus delineating the molecular control of normal development Purpose: Satellite cells are the primary stem cell responsible for in any individual structure will impact on our understanding of craniofacial growth, maintenance and repair of skeletal muscle, and as such dysmorphologies. Our current knowledge of the developmental are a viable cell source for the therapeutic treatment of muscular processes governing anomalous development of the craniofacial dystrophies. Satellite cells live a complex life: they lie dormant in stable complex is poor due to our deficits in our understanding of normal muscle, however when repair is required, satellite cells activate and development of these structures. We have identified two novel ENU produce sufficient progeny to differentiate effecting repair, and then mouse models of two distinct human craniofacial dysmorphologies. return themselves to quiescence, poised for next time. This requires Mutant snoopy embryos display a unilateral facial hypoplasia phenotype an exemplary capacity to respond appropriately to signalling. During that involves the mandible, mid-face and ears. These characteristics development, myoblasts express a suite of transcription factors that are very similar in appearance to the human condition Goldenhar are critical in deciding their fate – when to proliferate, differentiate, syndrome. The developmental origins of Goldenhar syndrome are not migrate, quiescence, and die. The exquisite, fine regulation of cell fate well documented and no genetic lesion has yet been associated with this decisions in muscle satellite cells requires a highly adaptive platform condition. Kanyon embryos have a mid-facial cleft, ocular anomolies of gene expression. We have explored the function of some major (micropthalmia) and a variable mid-brain exencephaly, phenotypes that players responsible for modifying the satellite cell transcriptional mimic human fronotonasal dysplasia. The facial cleft often varies in landscape during activation, proliferation and myogenic differentiation. severity, from a disastrous lesion completely disrupting the face to a Methods: We have investigated the role of Pax, Six, and Eya family discrete cleft lip and palate phenotype. Thus, kanyon in its mildest form members in controlling the transcriptional landscape of satellite cells may also be a model for cleft lip and palate. Characterisation of these using retroviral expression, dominant negative constructs, siRNA- ENU mouse mutant strains will highlight the fundamental mechanisms mediated knockdown, immunocytochemistry, RT-qPCR, chromatin- responsible for normal development of the craniofacial structures. This immunoprecipitation, microarray and next-generation sequencing. data will facilitate the identification of underlying mutations in correlating Results: We have defined members from each gene family that human conditions. participate in regulating critical functions of satellite cells during in vitro myogenesis. Conclusion: This research provides an in-depth analysis of transcription factor function in a model stem cell. This approach has uncovered interesting new players in satellite cell biology, and revealed some new tricks for an old dog.

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