The 2011 Molecular Biology Student Symposium

Brought to you by the PGSS, BGSA, MP Biomedicals, Conviron, Fisher Scientific and NEB Canada

Friday, August 19th, 2011

Thomson House Ballroom 3650 Rue McTavish Montreal, Quebec, H3A 1Y2

Event Schedule
MORNING 9:45 A.M. Registration and Coffee in Thomson House Ballroom 9:55 A.M. Opening Remarks by Professor Richard Roy 10:00 A.M. Graduate Student Talk: Sofia Ibarraran 10:25 A.M. Graduate Student Talk: Deena Gendoo 11:50 A.M. Graduate Student Talk: Jimin Guo 11:15 A.M. Coffee Break 11:45 A.M. Graduate Student Talk: Aileen Wang 12:10 P.M. Graduate Student Talk: Rajesh Ranjan 12:35 P.M. Graduate Student Talk: Olga Skorobogata AFTERNOON 1:00 P.M. Lunch 2:00 P.M. Undergraduate Student Talk: Stephanie Bourque 2:25 P.M. Undergraduate Student Talk: Kevan Lu 2:50 P.M. Graduate Student Talk: Katrina Gusev 3:15 P.M. Graduate Student Talk: Patrick Janukavicius 3:40 P.M. Coffee Break 4:00 P.M. Poster Session 4:45 P.M. Closing Remarks and Presentation of Awards EVENING 5:00 P.M. Closing Reception at Thompson House

Abstracts- Talks
PCA Analysis of Prion Protein Structures to Predict Amyloidogenic Segments Deena M.A. Gendoo, Paul Harrison The extraordinary conformational change witnessed between the normal, non-pathological prion protein, PrPC, and its virulent pathological form, PrPSC, in which the latter acquires almost a 50% increase in -sheet content, is a significant contributor to the role this protein plays as an agent of many Transmission Spongiform Encephalopathies (TSEs). Such diseases, of which include human Creutzfeld-Jakob Disease (CJD) and Bovine Spongiform Encephalopathy (BSE), are caused by the misfolding and subsequent aggregation of PrPSC to produce amyloid fibrils, highly ordered and distinct -sheet-rich molecular aggregates. The presented work attempts to identify segments of prions (PrPC) which are likely to undergo a conformational change during fibril formation, via an analysis of the tertiary structures of the globular domains of prion proteins. Using Principal Components Analysis (PCA) on the backbone of prion structures, a lower-dimensional representation of the structural dataset is generated which can allow for the identification and characterization of conformational differences in the proteins. Residues which contribute the most to the variation along the principal components (PCs), and ultimately separate structures based on their conformational differences are considered conformationally variable. Following the identification of the conformationally-variable segments, these segments are cross-referenced with the literature to determine their contribution to amyloidogenicity. Breast Cancer Anti-estrogen Resistant 3 regulates TGF-beta-induced cell signalling and invasion in breast cancer cells. Jimin Guo and Jean-Jacques Lebrun, McGill University Health Centre, Montreal QC Transforming Growth Factor-beta (TGF-beta) regulates mammary gland development and homeostasis through inducing differentiation, morphogenesis, and growth inhibition. During breast cancer progression, TGF-betas growth inhibitory effects are lost, while TGF-beta acts as a pro-invasive factor. In cultured breast cancer cells, insensitivity to growth inhibition and gain of cell invasion in response to TGF-beta correlate to a mesenchymal and estrogen receptor (ER) negative phenotype. Therefore, we hypothesize that specific mechanism allowing for ERindependent cell survival may also regulate TGF-beta signalling. Others have identified a list of Breast Cancer Anti-estrogen Resistance (BCAR) genes whose individual over-expression induces ER-independent cell survival. BCAR3 is a signalling adaptor protein that regulates various kinase activities in response to cell adhesion. BCAR3 directly interacts with p130Cas/BCAR1, a Tyrosine and Serine-rich protein, mediating its phosphorylation and thereby creating binding sites for signalling transduction. Notably, p130Cas/BCAR1 simultaneously regulates phosphorylation of two major TGF-beta signalling effectors, the SMAD3 transcription factor and the p38 stress activated protein kinases. We show here that high expression of BCAR3, but not p130Cas/BCAR1, correlates with a triple negative (ER-, PR-, Her2-) phenotype among breast cancer cells. We also found that knocking down BCAR3 in such cells potentiates TGF-beta signalling, leading to increases in SMAD3 activity and cell invasion in response to TGF-beta. Our results highlight a novel regulatory mechanism by which TGF-beta promotes breast cancer metastasis. Regulation of the Wnt pathway: analysis of b-catenin phosphorylation by the endogenous Axin-based complexes. Katrina Gusev, McGill Univerisity Wnt signaling has been known for its critical role in regulation of cell proliferation, differentiation, adult tissue homeostasis, tissue regeneration and stem cells maintenance through its canonical signaling - defined by the involvement of -catenin as its downstream effector. Inappropriate activation of Wnt signaling contributes to numerous cancers. According to the current model of canonical Wnt signaling is that in the absence of Wnt, -catenin is retained in

Abstracts- Talks
the cytosol on Axin containing complex, also called destruction complex, where it is being phosphorylated by two kinases of the pathway, CK1 and GSK3 subsequently. These multiple phosphorylations singnal for further polyubiquitination and proteasomal degradation. Binding of Wnt ligands to their receptors initiate cascade of events that result in inhibition of the Axin destruction complex and thus stabilization of -catenin which in turn accumulates in the cytosol, translocates to the nucleus and activates targent genes through interaction with transcription factors. It has been previously found in our lab the presence of several Axin pools within the cell. In my study, I aim to characterize different subcellular Axin complexes, assess their activity in catenin regulation and finally compare and contrast those activities in the present of Wnt. The ants go marching one by one: does dopamine tell them where to? Sofia Ibarraran, McGill Univerisity Division of labour is a key feature of the complex organization of social insects. The worker caste of the ant Pheidole dentata displays temporal polyethism (age related variation in the group tasks in which they engage). Division of labour models hypothesize that differences in response thresholds (the likelihood of reacting to task-associated stimuli) underlie temporal polyethism; and changes in such thresholds could result in the transition between groups of tasks. Biogenic amines have been shown to modulate stimuli-specific response in hymenoptera. In the case of P. dentata an increase in dopamine levels and task repertoire expansion correlate with age. Thus we hypothesize dopamine could be playing a role in temporal polyethism. My goal is to analyze whether there is a causal relationship between changes in the dopaminergic system and task division patterns. Immunohistochemistry and insitu hybridization have been used to characterize the dopaminergic neurons as well as the expression of dopamine receptors; preliminary results show conserved expression patterns with the ant H. saltator and A. mellifera respectively. Colony demography has been shown to have a key role in mediating the rate of behavioural progression in bees, ants, and wasps. Demographic manipulations were used to force behavioural change and analyze the dopaminergic system. We are therefore undertaking the first steps towards verifying the possible role of dopamine as a player in the generation of division of labour patterns in ants. The mapping of ivermectin resistance genes Patrick Janukavicius, Laura Tiseo, Ludmel Urdaneta, Mi Tan, Roger Prichard, Joseph Dent Institute of Parasitology and Department of Biology, McGill Univ. Ivermectin is a drug used to kill parasitic nematodes. However, parasites are evolving resistance to ivermectin. We are studying the mechanisms of resistance in the model organism C. elegans in order to elucidate good targets for future drugs, to which parasites would have more difficulty developing resistance. Typical methods for finding ivermectin resistant C. elegans involve using the mutagen ethyl methane sulfonate. James and Davey (2008) pointed out that parasites develop resistance by being exposed to low concentrations of ivermectin which selects for spontaneous mutations. They propagated C. elegans on low concentrations of ivermectin. After many generations, they created an ivermectin resistant strain, IVR10, which shows ten-fold resistance to ivermectin. James and Davey claim that the increased resistance is largely epigenetic and provide evidence for the increased expression of ABC transporter proteins which are believed to pump ivermectin. To determine if mutations lead to the increased expression of these pumps we performed mapping experiments on the strain IVR10 and found a region of the X-chromosome linked to resistance. We believe that the observed linkage on the X-chromosome is due to a 2 nucleotide deletion in the gene dyf-7. The gene, dyf-7, was first characterized by Starich et al. (1995) for its DYe-Filling defective (dyf) phenotype. When wild-type worms are exposed to dye, the amphid neurons will fill with dye. However, the amphids of dyf mutants do not fill with dye. This phenotype is due to decreased permeability of the dye by the loss of sensory pores in the cuticle of the worm. We believe that increased resistance in dyf mutants is associated with a decrease in the permeability of ivermectin.

Abstracts- Talks
DNA Topoisomerase II acts as a mitotic scaffold protein in chromosome assembly in C. elegans Rajesh Ranjan, Jonas F. Dorn, Paul S. Maddox Institute for Research in Immunology and Cancer, University of Montreal, Canada. Assembly of mitotic chromosomes is a dynamic event, occurring rapidly during prophase of each cell cycle. Given the complexity of this process, genetic and biochemical approaches have identified surprisingly few factors involved in condensation, notably histone H1, the condensin complex, DNA topoisomerase II (Topo II) and CENtromere-Protein A (CENP-A). Topo II can cut and reseal DNA and has been hypothesized to act in a structural capacity, but how it acts in mitotic chromosome condensation remains unknown. To define the roles of known and novel proteins in chromosome condensation, we are performing high-resolution live imaging of the C. elegans zygote. Images of fluorescently-tagged core histones are thresholded and segmented to quantify the distribution of chromatin within the prophase nucleus over time. When Topo II is depleted by RNAi, chromosomes collapse prematurely during prophase. When the catalytic activity of Topo II is inhibited with Dexrazoxane, chromosomes do not collapse prematurely; rather, chromatin remains diffuse throughout prophase. These results indicate that independent of its catalytic activity, Topo II acts to scaffold condensation. Its decatenating activity is also important for chromosome resolution during condensation. Interestingly, simultaneous depletion of Topo II and centromeric chromatin partially rescues the Topo II depletion phenotype, but simultaneous depletion of condensin and Topo II does not. This suggests that Topo II with CENPA have opposing contributions to mitotic chromosome assembly. Endogenous Topo II localizes to an axis along metaphase chromosomes distinct from that formed by CENP-A, further indicating that their mechanisms of action are independent. We conclude that metaphase chromosomes are built by the distinct scaffold activities of three proteins: Topo II, CENP-A and Condensin. Importantly, our analysis revealed that depletion of candidate proteins results in quantitatively distinct phenotypes, indicating that they function in discrete events during mitotic chromosome assembly. A Screen for Endocytic Regulators of EGFR signaling in C. elegans Olga Skorobogata and Christian Rocheleau Division of Endocrinology and Metabolism, Department of Medicine, McGill University Epidermal Growth Factor Receptor (EGFR)/Ras/Mitogen Activated Protein Kinase (MAPK) signaling regulates cell proliferation, migration and apoptosis and misactivation can lead to cancer. EGFR endocytosis and trafficking to the lysosome is an important mechanism of signal downregulation. Rab7 regulates EGFR trafficking and degradation, however its effects on signaling are not known. In C. elegans a highly conserved LET-23 EGFR signalling pathway is required for vulval cell fate specification. We found that RAB-7 antagonizes LET-23 EGFR signaling during vulval induction, suggesting a possible role as a tumor suppressor in mammals. The strongest effect of rab-7(ok511) is observed on the LIN-2 CASK/ LIN-7 Veli/ LIN-10 Mint11 complex which is required for basolateral localization of LET-23 in the Vulval Precursor Cells (VPCs). Mutations in lin-2, lin-7, lin-10 or let-23(sy1) result in a strong Vulvaless (Vul) phenotype due to the mislocalization of LET-23 to the apical membrane domain of the VPCs. rab-7(ok511) can strongly suppress the lin-2, lin-7, lin-10 and let-23(sy1) Vul phenotype as well as restore basolateral membrane localization of LET-23 in the VPCs of lin-2(e1309) mutants. To identify additional genes that might regulate LET-23 EGFR trafficking and signaling, we conducted a pilot forward genetic screen for suppressors of the lin-2(e1309) Vul phenotype. We identified two suppressor mutants, vh4 and vh22, that are partial embryonic lethal and display phenotypes suggestive of vesicular trafficking defects. We mapped vh4 and vh22 to distinct regions of chromosome I, devoid of previously identified regulators of LET-23 EGFR signaling, suggesting they are mutations in novel regulators. RNAi and genetic complementation data identified AGEF-1, an Arf-GEF, as a potential candidate for vh4. We have used whole-genome sequencing (WGS) to determine the molecular identity of vh4. WGS approach identified a lesion

Abstracts- Talks
in exon-11 of agef-1 as a missense homozygous mutation CT resulting in Glu1028 to Lys substitution. This finding was further confirmed by Sanger sequencing. Regulation of Rab GTPase-mediated endosomal trafficking by TBC-2 Xiaolin Aileen Wang, Farhad Karbassi, Marc-Andr Sylvain Krittika Bhende, Anna Chavloski and Christian E. Rocheleau Department of Medicine, McGill University and the MUHC Research Institute The Rab5 and Rab7 GTPases are key regulators of endosome to lysosome trafficking, whose activities are positively regulated by Rab Guanine nucletoide Exchange Factors and negatively regulated by GTPase Activationg Proteins (GAP). We are using the nematode Caenorhabditis elegans as a model system to study regulation of the Rab GTPase-mediated endosomal trafficking. We previously demonstrated that TBC-2 functions as a RAB-5 GAP. Loss of tbc-2 activity results formation of enlarged late endosomes in the intestine that require the activities of RAB-5, RAB-7 and components of the HOPS complex and enhanced degradation of Yolk during embryogenesis. TBC-2 colocalizes with RAB-7 on late endosomes, and requires RAB-7 for membrane localization. We hypothesize that RAB-7 recruits TBC-2 to late endosomes to inactivate RAB-5 and possibly RAB-7, to facilitate early to late endosome maturation. We are currently testing if membrane localization is important for TBC-2 function and how TBC-2 is recruited to endosomal membranes. We have yet to detect a significant physical interaction between TBC-2 and RAB-7. Rac1 is an imteresting potential candidate to bridge or stabilize this interaction, as it has been found to physically interact with both Rab7 and Armus (aTBC-2 homolog) in mammals. We found that TBC-2 interacts with all three C. elegans Rac, CED-10, RAC-2, and MIG-2. This is interesting in light of the fact that both TBC-2 and CED-10 are required for efficient phagocytic degreadation of apoptotic corpses. We are currently testing the biological significance of this interaction. To identify additional regulators of endosome to lysosome trafficking, we performed a tbc-2 (tm2241) suppressor screen. We identified vh8, as a mutant that not only suppressed the large late endosome phenotype, but also resulted in a loss of GFP::RAB-7 from endosome membranes, suggesting that vh8 defines a regulator of RAB-7 activity and/or membrane localization. We mapped vh8 to chromosome III, close to the tra-1 gene. We are using RNAi to test candidate genes in this region for similar phenotypes as vh8.

Abstracts- Undergraduate Student Talks Abstracts- Undergraduate Student Talks

Novel Subunits in Drosophila melanogaster as Potential Pesticide Targets Characterization of CG11340, CG7589 and CG6927 Stephanie Bourque, Daniel Feingold and Joseph Dent, Department of Biology, McGill Univerisity Cys-loop ligand-gated ion channels are pentameric neurotransmitter receptors that are currently the target of many successful pesticides. However, the emergence of resistance to these pesticides and issues of off target effects continue to be problems. The focus of my project is to characterize three novel cys-loop ligand-gated ion channel subunits in Drosophila melanogaster CG11340, CG6927 and CG7589 and to determine their potential use as new pesticide targets. Previous work using in-situ hybridization and promoter-GFP constructs has revealed that unlike most channels in this family that are typically expressed in the nervous system, CG7589 and CG11340 are expressed throughout the gut and the Malpighian tubules. Due to the overlapping gene expression of CG11340 and CG7589 we generated a double mutant strain and assessed the impact of these mutations on viability. Since the Malpighian tubules are known to be involved in fluid secretion, we also designed assays to challenge osmoregulation by exposing CG11340 mutants to environments with varying concentrations of salt. Preliminary results indicate that CG11340 mutants are in fact sensitive to salt stress. Short term goals include using the UAS/Gal4 system to selectively express our genes of interest in Malpighian tubule cells. This will enable us to further understand the function of our channels in vivo. Long term goals include the development of a heterologous expression system using the Drosophila cell line, Schneider S2 cells. Subunits will be expressed in different combinations and once stable cell lines are established we will perform high-throughput screens in order to indentify compounds that

target our channels.

Phenotypic Analysis of a novel EGFR signalling regulator in Drosophila Oogenesis Kevan Lu and Laura Nilson, McGill Univerisity The patterning of the Dorsal-Anterior follicular epithelium of the Drosophila ovary is a powerful model system for the elucidation of epidermal growth factor receptor (EGFR) regulation. Within thissystem, differing levels of EGFR signaling define two dorso-lateral patches that will eventually produce appendages on the surface of the eggshell. While much is known about the regulatory mechanisms that define certain spatial properties of these patches, there is still uncertainty as to how their posterior expansion is determined. We have identified two mutations, called F27 and aldpw+, which exhibit a fusing of these two appendage patches posterior to their endogenous expression pattern. Strangely, while we discovered an early truncation in a gene called midline in F27, we found no coding mutation in aldpw+, which counter-intuitively had a more severe phenotype. To assess whether this phenotypic disparity was due to differences in the genetic background between the two strains, recombination against a wild-type chromosome was performed. Preliminary data suggests that there is little difference in phenotypic severity between the resulting partial recombinants. Given this evidence, we propose that the higher severity of the aldpw+ phenotype is instead due to a regulatory mutation that affects the function of multiple genes.

Abstracts- Posters
EphrinB/EphB Signaling Controls Embryonic Germ Layer Separation by ContactInduced Cell Detachment Nazanin Rohani1, Laura Canty1, Olivia Luu2, Franois Fagotto1, Rudolf Winklbauer2 1 Department of Biology, McGill University, Montreal, Quebec, Canada, 2 Department of Cell and Systems Biology, University of Toronto, Toronto, Canada The primordial organization of the metazoan body is achieved during gastrulation by the establishment of the germ layers. Adhesion differences between ectoderm, mesoderm, and endoderm cells may be sufficient to maintain germ layer integrity and prevent tissue mixing. However, in many organisms, the ectoderm-mesoderm boundary not only keeps these germ layers separated, but the ectoderm also serves as substratum for mesoderm migration, and the boundary must be compatible with repeated cell attachment and detachment. We show that localized detachment resulting from contact-induced signals at the boundary is essential in ectoderm-mesoderm segregation. Cells alternate between adhesion and detachment, and detachment requires ephrinB/EphB signaling. Multiple ephrinB ligands and EphB receptors are expressed on each side of the boundary, and tissue separation depends on forward signaling across the boundary in both directions, involving partially redundant ligands and receptors with subsequent activation of Rac and RhoA. This mechanism differs from a simple differential adhesion process of germ layer formation. Instead, it involves localized responses to signals exchanged at the tissue boundary and an attachment/detachment cycle which allows for cell migration across a cellular substratum. Production of exogenous proteins in the seed coat mucilage secretory cells of Arabidopsis thaliana and Brassica napus Uday K Divi, Asraf A Abdeen and Tamara L Western. Canola (Brassica napus) is a major crop cultivated for oil that is contained in the embryo of the seed. The embryo is surrounded by seed coat or hull which contributes ~16% of total seed mass in Canola. After extraction of oil from the embryo, hull is discarded as waste. Converting the waste hulls to valuable co-products by production of novel bioproducts can enhance the economic value of these crops. The mucilage secretory cells (MSCs) of seed coat present an excellent tool for such manipulation. These cells produce large amounts of pectin and cell wall-related proteins during seed development that are released upon hydration. This would allow easy harvesting of the exogenous protein by wetting of the seed and extracting the water soluble fraction. The current study aims at developing tools for targeting novel proteins to the MSCs of Arabidopsis and B. napus, and determining parameters for maximizing their accumulation. Six Arabidopsis seed coat promoters, including those of two newly identified genes, are being analyzed for their expression in different tissues and various stages during seed development. For all the promoters, a secretable -glucuronidase (GUS plus) reporter gene is used. Different signal sequences were also used to target the proteins to apoplast. Preliminary studies in Arabidopsis using three of the promoters with two different signal sequence combinations showed expression in seed coat for all the combinations. The activity of one of the promoter-signal sequence combinations was confirmed in the seed coat of transgenic B. napus. Further time course and quantitative studies using antibodies and enzymatic assays will identify a suitable promoter-signal sequence combination for exogenous protein production whose application for B. napus will be validated. Rhamnose Synthase genes and Exogenous protein expression in Flax (Linum usitatissimum) Bronwen Forward, Mark Jordan, Tamara L. Western. Biology Department, McGill University, Montreal, Quebec, Canada H3A 1B1 The seeds of linseed flax (Linum usitatissimum L.) are harvested mainly for the oil derived from the embryo. More recently, flax seeds have emerged as a health food product, mainly due to their high levels of lignans, which have been implicated in the reduction of heart disease, prostate, and

Abstracts- Posters
breast cancers. In addition, the seed coat of flax secretes a thick, pectinaceous mucilage that can be used as soluble fibre, emulsifier, and a substitute for animal products in food. The mucilage secretory cells (MSCs) of Arabidopsis thaliana have been well characterized and many genes involved in the synthesis and secretion of mucilage have been determined. This process remains poorly understood in flax, and this study of MSC differentiation in flax has been initiated alongside an investigation of potential RHAMNOSE SYNTHASE (RHM) genes to stage the development in flax MSCs and to gain a better understanding of the genes involved in pectin synthesis. In addition to studying the mechanism of pectin secretion and synthesis, we are also interested in harnessing seed coat specific promoters in the expression of exogenous proteins as a means of producing value-added oilseed crops. Thus far, candidate promoters for protein expression in the MSCs include GLABRA2 (GL2), a promoter driving expression in the seed epidermis in Arabidopsis, PINORESINOL LARICIRESINOL REDUCTASE (PLR), an endogenous promoter responsible for the accumulation of a flax lignan, and a novel group of potential RHM genes found recently in flax. Preliminary work using a secreteable -glucuronidase is being utilized to optimize expression of exogenous proteins which are targeted for secretion to the apoplastic space in the seed coat. Thus far, the Gl2 promoter appears to be a promising candidate, as preliminary GUS data shows that it targets protein secretion in the mucilage, and is excluded from the embryo as the seed matures. This could be useful in producing easily isolated coproducts which would be removed before the embryos are crushed for oil extraction. As the other candidate promoters are studied in more detail, this study proposes to determine the most appropriate system for the expression of exogenous proteins in flax. Uncovering the roles of RhoA and Anillin during morphogenesis in C.elegans. Nellie Fotopoulos, Concordia University Cytokinesis is regarded as the final stage in the process of cell division. In order for the cell to properly divide itself into two daughter cells, it must undergo cytoskeletal rearrangements involving actin and myosin. It is understood that active Rho initiates the cytokinetic pathway to lead to the formation of a contractile ring. This ring will allow the ingression of the furrow and cause the cell to change shape. Morphogenesis, also known as elongation, is an analogous process that permits cells to adopt the required shape for the proper development of all multicellular organisms. We are investigating the mechanisms involved in the elongation pathway among C.elegans nematodes. This crucial process is what transforms the ovoid embryo into a characteristic worm shape. The cells initially appear box shaped and change to more of a cylindrical shape in order to accommodate their obligated roles. In addition, anillin has been shown to play a role in cytokinesis by acting as a scaffolding protein that aids in the proper formation of the contractile ring. Biochemical evidence supports that anillin is conserved from our model organism to humans. However, we suspect that anillin has non-mitotic roles and may function in morphogenesis since it is observed in many differentiated cells. Biomechanics of Stems and Hypocotyls in Arabidopsis thaliana (L.) Heynh. N. Hristozov1*, T. R. Faisal2, D. Pasini2, and T. L. Western1.1Department of Biology, McGill University, Montreal, Quebec, Canada H3A 1B1 (e-mail: nicolay.hristozov@mail.mcgill.ca); 2Department of Mechanical Engineering, McGill University, Montreal, Quebec, Canada H3A 2K6. While biomechanical factors significantly influence growth, morphogenesis and mechanical stress response in plants, the mechanical properties of plant structures remain largely unexplored. Arabidopsis thaliana (L.) Heynh, being amenable to subtle structural modification and well-controlled experimentation, will be used as a model system to elucidate these properties in stems. Stems are understood to be complex hierarchical systems displaying four to seven integrated levels of structural organization, each of which contributes to the mechanical properties of the whole organ. Firstly, to better understand the mechanical significance of these structural features, mechanical tests will be performed on segments of primary inflorescence stem from a set of candidate mutant lines. Ten mutants displaying tissue-level structural defects will be tested, as well as seven mutants displaying altered cell wall chemistry. Samples will be

Abstracts- Posters
tested in tension, torsion and three-point bending. Additionally, wild type stem segments will be tested from different stages and portions of the stem, representing different degrees of tissue development. Secondly, to better understand the role of cell wall structure and anisotropy in plant cell growth, tests will be performed on etiolated hypocotyls from the aforementioned set of cell wall mutants. Samples will be tested in tension and subjected to creep testing, which simulates turgordriven elongation. Thirdly, the role of cellulose microfibril angle (MFA) in cell wall anisotropy will be examined using confocal microscopy and pontamine fast scarlet dye. This angle will be measured across the cell wall layers in hypocotyls before and after creep testing. Mutants with altered cell wall anisotropy may display differences in MFA, and may also display difference in MFA reorientation during growth. The data collected from these experiments will serve to develop and validate a multiscale mechanics model of plant stems that integrates the entire hierarchy of structural organization. The integrated model will be used in the development of novel biomimetic materials and structures. Statics and Dynamics of DNA in a Nanopit Lattice Alex Klotz, Department of Physics, McGill Univerisity Single polymers have been proposed as a tool for self-assembly in nanotechnology. Here, entropic trapping is used to affect the behaviour of polymers at the single molecule level. In these experiments, DNA is confined to a nanofluidic slit embedded with a lattice of entropic traps and the behaviour of single polymers in this system is observed. Polymers self-assemble into nanostructures with discrete configurational states. It is shown that such a system can be used to entropically trap single polymers into stable nanostructures. Observations of the diffusivity of a molecule with respect to the confinement geometry show interesting non-monotonic behaviour. An analysis of maternally contributed mRNAs in early Drosophila embryogenesis and germ cell specification. Michelle Kowanda, McGill Univerisity The targeting of developmental signals to subcellular regions in oogenesis and embryogenesis is imperative to the establishment of Drosophila melanogaster morphology. Germline specification by the localization of particular mRNAs at the posterior of the embryo ensures the generation of primordial germ cells, also known as pole cells, that later combine with somatic cells to create the gonads. A number of mRNAs are localized to the posterior of the embryo, some of whose function in pole cell development have not been determined. Stephanie Yee and I have been investigating the localization of 43 D. melanogaster posterior mRNAs in two other Drosophila species, to utilize the conservation of localization as a potential indicator of their requirement in germline development. Additionally, a mechanism of active transport has been found to localize a subset of germ plasm mRNAs to the pole cells. Active transport along astral microtubules creates RNA islands in stage 3 embryos, ensuring that specific mRNAs are incorporated into the primordial germ cells. Interestingly, many well-characterized mRNAs that perform crucial roles in pole cell development are recruited to RNA islands. I have choosen several mRNA transcripts for further studies from the list of conserved RNA island localizing mRNAs. This research will identify novel Drosophila primordial germ cell specification genes by investigating poorly characterized posterior mRNAs in early embryogenesis. Acetylcholine-Gated Chloride Channels (ACCs) may be involved in C.elegans development and behaviour, suggesting a central role for fast inhibitory cholinergic neurotransmission in C.elegans. Claudia M. Wever, Patrick Janukavicius, Jin-Kyung Chang, Danielle Farrington, Julian Gitelman, Igor Putrenko and Joseph A. Dent , McGill University, Department of Biology, Montreal QC

Abstracts- Posters
Acetylcholine is an abundant neurotransmitter in C.elegans that is involved in many of the organisms behaviours including feeding, locomotion and egg-laying. Over a third of the neural cells in C.elegans release acetylcholine and acetylcholine is the only essential neurotransmitter in C.elegans. We previously cloned and characterized members of the acetylcholine-gated chloride channel (ACC) family in C.elegans (Putrenko et al., 2005) and the discovery of these channels was the first evidence that C.elegans employs acetylcholine as a fast inhibitory neurotransmitter. We have shown that ACC-1, ACC-2, ACC-3 and K10D6.1 subunits form homomeric acetylcholinesensitive channels in Xenopus Laevis oocytes and that ACC-1 and ACC-3 subunits interact in oocytes to form a heteromeric channel. Expression data from promoter-GFP fusion constructs revealed that ACC-1 and F47A4.1 are co-expressed in M3 neurons. Supporting these results, when co-expressed in oocytes, the ACC-1 and F47A4.1 subunits form a functional heteromeric chloride channel that is less sensitive to acetylcholine than the ACC-1 homomer. Interestingly, the membrane currents generated by the ACC-1/F47A4.1 heteromer are over five times larger in magnitude than those generated by the ACC-1 homomer. In addition to characterizing the electrophysiological properties of the ACC channels, we have obtained strains with deletions in the various ACC genes in order to begin to understand their functions in vivo. Worms with deletions in ACC-2 have delayed developmental timing, taking longer to reach gravidity compared to N2 worms. F47A4.1 promoter-GFP fusions are expressed in HSN neurons, which are a central component of the egg-laying circuit. We therefore examined egg-laying behaviour in the F47A4.1 and ACC-1 deletion strains. Both strains lay fewer eggs than N2. The decrease in the number of eggs laid appears to be the result of an ovulation defect as opposed to a deficit in the egg-laying circuit itself. Rescue experiments as well as experiments to find and characterize new phenotypes associated with deletions in the ACC genes are ongoing. We are also currently generating double and triple mutant strains to uncover any phenotypes that are only apparent when multiple members of the ACC family are affected. (Putrenko et al., 2005, J. Biol. Chem. 280, 6392-6398) This work is supported by NSERC and Chemtura Co. A Global Analysis of mRNA Localization in Drosophila Germ Line Specification Stephanie Yee and Paul Lasko, McGill Univerisity During early Drosophila embryogenesis, the molecular asymmetry created by the localization of maternal transcripts to the pole cells of the embryo is essential for specification of germ cells. According to two ongoing large-scale in situ hybridization (ISH) screens performed on Drosophila melanogaster embryos, a small fraction of maternally contributed mRNAs localize to the pole cells (Lecuyer et al., 2007; Tomancak et al., 2007). The combined results from the screens yield a list of 320 mRNAs that localize to blastoderm-stage pole cells with an overlap of only 58 mRNAs. Beginning with the list of 58 mRNAs procured from the two screens, fluorescent ISH was used to determine which of those also localize to the pole cells of blastoderm-stage embryos in two other species, D. simulans and D. virilis. The proportion of mRNAs that localize to the pole cells in three Drosophila species may serve as a measure of the functional significance of mRNA localization to germ cell specification. Preliminary results indicate that the pole cell localization of most mRNAs is conserved across Drosophila species. Since cis-acting elements involved in translational control or localization are typically found in the untranslated regions, the corresponding sequences are currently being analyzed to identify conserved localization elements.

Abstracts- Undergraduate Student Posters

In vivo analysis of the mitotic function of the Drosophila melanogaster chromokinesin Nod Kelsey Thibault, McGill Univerisity The Drosophila melanogaster nonmotile chromokinesin, Nod, has been shown to have critical functions in the proper segregation of non-exchange chromosomes in female meiosis. Previous RNAi-based studies have suggested that Nod may also function in mitotic cell division. This work further investigates the potential mitotic role of Nod. Here, we saw that knocking down Nod protein levels using RNAi in Schneider 2 (S2) cells produced mutant mitotic spindle phenotypes similar to those seen previously in screens1,2. Immunofluorescence studies in early stage nod mutant D. melanogaster embryos using an anti-tubulin antibody allowed visualization of mitotic spindles in the embryos and will allow for future comparisons with the S2 cell spindles. The mutant mitotic spindle phenotypes observed after RNAi knockdown were identified as chromosome misalignments, monopolar spindles, and tripolar spindles. These results provide specific evidence of a crucial role for this chromokinesin in mitosis. Finally, a nod antigen was designed for future injection into rabbits and production of an anti-Nod antibody. The antibody will be used for Nod localization studies in S2 cell and embryonic mitotic spindles. Future research will investigate if Nod functions in neural development, a hypothesis suggested by online database mRNA expression profiles.
1. Goshima, G., and R.D. Vale. 2003. The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line. The Journal of Cell Biology 162(6): 1003-1016. 2. Goshima, G., Wollman, R., Goodwin, S.S., Zhang, N, Scholey, J.M., Vale, R.D., and N. Stuurman. 2007. Genes required for mitotic spindle assembly in Drosophila S2 cells. Science 316(5823): 417-421.