CANFLY XIII
Montréal, June 21-25, 2015
Program and Abstract book
CANFLY in Québec
The Organizing Committee would like to thank the following for their invaluable
assistance in helping to organize various aspects of CANFLY XIII:
Scott De Vito
Rahul Rote
Stephanie Yee
Heather Collins
The Organizing Committee:
Laura Nilson
Gregory Emery
Gilles Hickson
David Hipfner
Nam-Sung Moon
Frieder Schöck
Front page: Scientific images from the lab of David Hipfner, Nam-Sung Moon and Frieder
Schöck, respectively, photograph by Greg Emery
1 Schedule
Sunday June 21
16:00 - 18:50
Registration
18:50 - 19:00
Introductory Remarks
19:00 - 20:00
Dinner
20:00 - 22:00
Social with bar service
2 Monday June 22
9:00 - 10:25
VIP session for specialized cells (5 talks)
9:00 - 9:05
9:05 - 9:25
Welcome and opening remarks
“Variant ribosomal proteins in germline development”
Paul Lasko, McGill University
9:25 - 9:45 “The transcription factor Hindsight promotes enterocyte
differentiation and is required for intestinal stem cell maintenance”
Bruce Reed, University of Waterloo
9:45 – 10:00 “Traffic Jam regulates actin protrusions of migrating border
cells through PKCδ in the Drosophila ovary”
Felix Gunawan, University of Toronto
10:00 –10:15 “Misshapen regulates Moesin phosphorylation and collective
cell migration in vivo”
Cédric Plutoni, Institute for Research in Immunology and Cancer
10:15–10:30 “PIP5K59B, a conserved phospholipid kinase, directs
dendritogenesis during metamorphosis”
Danielle McEachern, University of British Columbia
10:30 - 11:00
Coffee break
11:00 - 12:25
Transcription and RNA biology (5 talks)
11:00–11:20
“cis-regulatory Integration of spatial and temporal inputs
underlying neuronal terminal differentiation”
Douglas Allan, University of British Columbia
11:20–11:40 “Transvection: making the complex more complicated”
Thomas Merritt, Laurentian University
11:40-11:55 “Examining the role of mRNA Localization in Regulating
Epithelial Cell Polarity”
Ashley Chin, Institute de recherches cliniques de Montréal
11:55-12:10 “Systematic analysis of protein-RNA interactions in Drosophila”
John Laver, University of Toronto
12:10-12:25 “Transcriptional regulation is modulated by Vestigial Family proteins
in the developing wing”
Virginia Pimmett, University of Alberta
13:00 - 14:00
Lunch
3 14:00 - 16:00
14:00-15:00
15:00-16:00
16:00 - 17:05
16:00-16:20
16:20-16:35
16:35-16:50
16:50-17:05
Poster session I
Odd Numbers
Even Numbers
Cell biology (4 talks)
“Polarized endocytic activity directs actomyosin network assembly
during wound repair “
Rodrigo Fernandez-Gonzalez, University of Toronto
“Characterization of Higher-Ordered Septin Structures in
Drosophila cells”
Zlatina Dragieva, Universite de Montreal
“Differential interactions between the core SJ domain proteins, NrxIV and the Na/K ATPase, and the Tricellular J”
Perveen Biln, University of British Columbia
“Interactions between Cell Division and Epithelial Cell Polarity”
Gayaanan Jeyanathan, University of Toronto
17:05 - 17:35
Coffee break
17:35 - 18:40
Plasma membrane signaling (4 talks)
17:35-17:55
17:55-18:10
18:10-18:25
18:25-18:40
“The role of the Moesin complex during neuroblast asymmetric cell
division”
Sarah Hughes, University of Alberta
“A Par-1-Par-3-centrosome cell polarity pathway and its tuning for
isotropic cell adhesion”
Tao Jiang, University of Toronto
“Sac1 selectively regulates trafficking of adhesion molecules in the
developing Drosophila eye”
Lauren Del Bel, University of Toronto
“Regulation of epithelial vesicle trafficking by apical polarity
proteins”
Kenana Al Kakouni, University of Toronto
19:00 - 20:00
Dinner
20:00- 22:00
Social with bar service
4 Tuesday June 23
9:00 - 10:25
Nuclear and Cell division (5 talks)
9:00 - 9:20
“The inner nuclear membrane protein Spegless regulates germline
development”
Helen McNeill, Lunenfeld-Tanenbaum Research Institute
9:20 – 9:40
“Role of Cohesins, Securin and Separase in Drosophila meiosis”
Andrew Swan, University of Windsor
9:40 – 9:55
“Inter-domain allosteric regulation of Polo kinase by Aurora B and
Map205 is required for cytokinesis”
David Kachaner, Institute for Research in Immunology and Cancer
9:55 – 10:10 “Drosophila septins Sep2 and Sep5 are redundant for imaginal cell
proliferation but not oogenesis”
Ryan O'Neill, University of New Brunswick
10:10-10:25 “The role of the protein phosphatase 2A during the exit of mitosis”
Haytham Mehsen, Institute for Research in Immunology and
Cancer
10:25 - 11:00
Coffee break
11:00 - 12:35
Embryogenesis and Morphogenesis (5 talks)
10:00 -11:20 “Post-transcriptional control of gene expression in the early
Drosophila embryo”
Craig Smibert, University of Toronto
11:20-11:35 “Dunk stabilizes the actomyosin network at the leading edge of the
cleavage furrows during Drosophila cellularization”
Bing He, Princeton University
11:35-11:50 “Cell-ECM adhesion coordinates force transmission required for
morphogenesis”
Katharine Goodwin, University of British Columbia
11:50-12:05 “SPARC is required for proper basal lamina assembly in the FB”
Bianca Scuric, University of Toronto
12:05-12:35 “Update on the Rare Diseases: Models & Mechanisms Network”
Howard Lipshitz, University of Toronto
13:00 - 14:00
Lunch
14:00 - 16:00
14:00-15:00
15:00-16:00
Poster session II
Odd Numbers
Even Numbers
Free afternoon/evening
5 Wednesday June 24
9:00 - 10:20
Aging, disease, and behavior (5 talks)
9:00 - 9:20
9:20 – 9:35
9:35 – 9:50
9:50- 10:05
10:05-10:20
“Circular RNA Accumulation in Drosophila is modulated by
Biological Age”
Pedro Miura, University of Nevada
“Commensal L. plantarum significantly reduces lifespan of
Drosophila”
David Fast, University of Alberta
“Role of autophagy proteins beclin 1/Atg6 and Pi3K59F in
Drosophila Parkinson disease models”
Githure M'Angale, Memorial University of Newfoundland
“Non-invasive recording of infra-slow oscillation potentials from the
Drosophila brain”
Shuang Qiu, Queen’s University
“Genetic dissection of aggressive behavior in Drosophila
melanogaster”
Mahmoudreza Ramin, McGill University
10:20 - 10:50
Coffee break
10:50 - 12:10
Physiology and evolution (5 talks)
10:50-11:10
11:10-11:25
11:25-11:40
11:40-11:55
11:55-12:10
12:30 - 13:30
“The sex determination gene transformer controls male-female
differences in body size”
Elizabeth Rideout, University of British Columbia
“Ribosome synthesis in Drosophila muscle is required to maintain
systemic insulin signaling and larval growth”
Abhishek Ghosh, University of Calgary
“The atypical cadherin Dachsous controls Left-Right Asymmetry in
Drosophila”
Nicanor Gonzalez, The Institute of Biology Valrose
“Identification and Functional Characterization of Genes Involved
in Central Nervous System Control of Obesity”
Irene Trinh, University of Toronto
“Drosophila sex comb evolution: how some things change and
some stay the same”
Juan Nicolas Malagon, University of Toronto
Lunch
6 13:45 - 14:45
13:45-14:00
14:00-14:15
14:15-14:30
14:30-14:45
Signaling and development (4 talks)
“Drosophila Smo signals through a novel and conserved
mechanism”
Dominic Maier, Institute de recherches cliniques de Montréal
“Ras activated Dsor1 promotes Wg signalling in Drosophila
development”
Eric Hall, Simon Fraser University
“JAK/STAT signalling determines the cellular response to EGFR
activation in the ovary”
Scott De Vito, McGill University
“Functional understanding of Ral signaling in Drosophila
melanogaster”
Helene Knævelsrud, Institute for Research in Immunology and
Cancer
14:45 - 15:30
Coffee break and business meeting for PIs
15:40 - 16:40
Neurobiology (3 talks)
15:40-16:00
16:00-16:20
16:20-16:40
“Compartments, clocks and circuit assembly in the developing
Drosophila brain “
Teddy Erclik, University of Toronto
“Processing of modality and body position by the Drosophila taste
system”
Michael Gordon, University of British Columbia
“Fruitful investigations using flies to understand the overwhelming
genetic complexity of neurodevelopmental disorders”
Jamie Kramer, Western University
17:15
Buses depart to Montreal Science Centre (banquet dinner)
17:45
Cocktail reception
19:00 - 22:00
Dinner and awards
22:15
Buses depart to New Residence Hall
7 Thursday June 25
Departure
8 Abstracts
Oral presentations
Paul Lasko
Bruce Reed
Felix Gunawan
Cédric Plutoni
Danielle McEachern
p. 13
p. 14
p. 15
p. 16
p. 17
Douglas Allan
Thomas Merritt
Ashley Chin
John Laver
Virginia Pimmett
p. 18
p. 19
p. 20
p. 21
p. 22
Rodrigo Fernandez-Gonzalez
Zlatina Dragieva
Perveen Biln
Gayaanan Jeyanathan
p. 23
p. 24
p. 25
p. 26
Sarah Hughes
Tao Jiang
Lauren Del Bel
Kenana Al Kakouni
p. 27
p. 28
p. 29
p. 30
Helen McNeill
Andrew Swan
David Kachaner
Ryan O'Neill
Haytham Mehsen
p. 31
p. 32
p. 33
p. 34
p. 35
Craig Smibert
Bing He
Katharine Goodwin
Bianca Scuric
Howard Lipshitz
p. 36
p. 37
p. 38
p. 39
p. 40
9 Pedro Miura
David Fast
Githure M'Angale
Shuang Qiu
Mahmoudreza Ramin
p. 41
p. 42
p. 43
p. 44
p. 45
Elizabeth Rideout
Abhishek Ghosh
Nicanor Gonzalez
Irene Trinh
Juan Nicolas Malagon
p. 46
p. 47
p. 48
p. 49
p. 50
Dominic Maier
Eric Hall
Scott De Vito
Helene Knævelsrud
p. 51
p. 52
p. 53
p. 54
Teddy Erclik
Michael Gordon
Jamie Kramer
p. 55
p. 56
p. 57
Poster presentation
1.
Meryl Acker
p.58
2.
Anirban Banerjee
p.59
3.
Matthew N. Baron
p.60
4.
Darius Camp
p.61
5.
Shelag Campbell
p.62
6.
Wen Xi Cao
p.63
7.
Duygu Çevik
p.64
8.
Amel Chaouch
p.65
9.
Tetyana Chorna
p.66
10.
Heather Collins
p.67
11.
Pendleton Cox
p.68
12.
Mehrnoush Dehghani
p.69
13.
Mélanie Diaz
p.70
10 14.
Justin Evans
p.71
15.
Michael J. Fairchild
p.72
16.
Mariana Fregoso Lomas
p.73
17.
Anthony Galenza
p.74
18.
Sarah R. C. Garner,
p.75
19.
Helori Gaudé
p.76
20.
Oxana Gluscencova
p.77
21.
Elizabeth Hoesing
p.78
22.
Silvana Jananji
p.79
23.
Anja Katzemich
p.80
24.
Amel Kechad
p.81
25.
Samantha Koot
p.82
26.
Myreille Larouche
p.83
27.
Kuo-An Liao
p.84
28.
Jiangshu Liu
p.85
29.
Pablo Lopez-Ceballos
p.86
30.
Jing Lu
p.87
31.
Cheng-I Jonathan Ma
p.88
32.
Mary-Rose Bradley-Gill
p.89
33.
Eric M. Merzetti
p.90
34.
Shaza Mokhtar
p.91
35.
Lindsay Petley-Ragan
p.92
36.
Brian Phelps
p.93
37.
Samantha D. Praktiknjo
p.94
38.
Conrad Pridie
p.95
39.
Attey Rostami
p.96
40.
Rahul Rote
p.97
41.
Hunter Shaw
p.98
42.
Arun Shipstone
p.99
43.
Frankie A. Slade
p.100
11 44.
Jennifer D. Slade
p.101
45.
Kristin E Spong
p.102
46.
Jeff Stafford
p.103
47.
David ter Stal
p.104
48.
Junior West
p.105
49.
Nathan Wray
p.106
50.
Chengfeng Xiao
p.107
51.
Norman Yau
p.108
52.
Stephanie Yee
p.109
53.
Carlos Zeledon
p.110
Late abstract:
54.
Caroline Baril
12 Oral presentations
Variant ribosomal proteins in germ line development Jian Kong1, Hong Han1, Julie Bergalet2, Éric Lécuyer2 and Paul Lasko1 1
McGill University 2
Institut de Recherche Clinique, Montréal Genes encoding variant forms of four ribosomal proteins (RpS5b, RpS10a, RpS19b, and RpL22‐like) and variant translation factors have been recovered from high‐throughput screens as highly expressed in primordial germ cells (PGCs) and GSC‐like cells, raising the possibility that GSCs and PGCs use a different translational apparatus than do other cells, and that this may be important in their biology. Supporting this, we have found that a loss‐of‐function mutant in RpS5b is female sterile with defects in oogenesis. RpS5b mutant egg chambers cease development in early vitellogenic stages. Some egg chambers have more than 16 germ line cells and multiple layers of posterior follicle cells are frequently observed. Staining for Orb indicates that anterior‐posterior patterning is disturbed. In contrast, females expressing shRNA targeting the canonical RpS5a in germline are fully fertile, but RNAi‐mediated knockdown of RpS5a in the female germline exacerbates the RpS5b mutant phenotype and causes complete germline lethality, indicating that both isoforms function in germline. High‐level transgenic expression of either RpS5a or RpS5b in germline restored fertility and morphological defects to RpS5b mutant females, indicating some functional redundancy and that differential expression patterns contribute to the specificity of the RpS5b mutant phenotype. To examine whether RpS5a and RpS5b preferentially associate with different populations of mRNAs, we used antisera specific for each isoform to co‐
immunoprecipate mRNAs, and subjected them to high‐throughput sequencing. While a broad spectrum of mRNAs co‐purified with RpS5a, RpS5b‐associated mRNAs were highly enriched for GO terms related to the mitochondrial electron transport chain. Transmission electron microscopy of nurse cell mitochondria showed that RpS5b mutant mitochondria tended to form large clusters and had more heterogeneous morphology than those from controls, and the activity of lysosome was elevated in mutant ovaries. We conclude that in normal development RpS5b‐containing ribosomes play an essential role for the completion of oogenesis, and this is realized by ensuring the proper expression of mitochondrial proteins. 13 The transcription factor Hindsight promotes enterocyte differentiation and is required for intestinal stem cell maintenance Brittany Baechler, Nicole Biro and Bruce Reed University of Waterloo The hindsight (hnt) gene, homologue of mammalian RREB‐1, encodes a C2H2‐type Zinc finger nuclear protein and is required throughout development. Processes known to require hnt function presently include germ band retraction and embryonic tracheal development, retinal morphogenesis in the pupal eye, and development of the follicular epithelium of the adult ovary. In some contexts hnt has been found to be a direct target of the Notch signaling pathway. Given the importance of Notch signalling in the regulation of the adult intestinal stem cells (ISCs), we have investigated the regulation and function of hnt in these multipotent stem cells. We show that hnt is expressed throughout the larval and adult midgut, and we further demonstrate that Hnt is required for ISC establishment and maintenance. To our surprise, we find that the expression of hnt in ISCs does not require Notch signalling, but is dependent on the EGFR/RAS/MAPK signalling pathway. Moreover, we find Hnt overexpression to be a potent effector of ISC loss through enterocyte (EC) differentiation. Last, we show that EC differentiation via Hnt overexpression can suppress ISC tumour formation associated with either the loss of Notch signalling or the activation of the EGFR/RAS/MAPK signalling pathway. 14 Traffic Jam regulates actin protrusions of migrating border cells through PKCδ in the Drosophila ovary Felix Gunawan, Jing Lu, S. Saad Husainie, Dorothea Godt. Department of Cell and Systems Biology, University of Toronto. The border cell cluster (BCC) in the Drosophila ovary is an excellent system to study collective cell migration. The BCC, which contains 6‐8 migratory cells, invades the germline cyst and migrates toward the oocyte using actin‐based cellular protrusions during midoogenesis. Our work identified the transcription factor Traffic Jam (Tj) as an important regulator of BCC migration. Tj appears to be needed at its endogenous level for BCCs to produce proper cellular protrusions. Increased Tj expression largely suppressed major protrusions and caused a high number of thin filopodia‐like projections. Conversely, reduced Tj expression caused broad lamellipodia‐like protrusions with strong F‐actin enrichment to be more numerous than in wild type. Protein Kinase C δ (PKCδ), a serine/threonine kinase, was identified as a putative activation target of Tj in a microarray screen using border cell‐specific RNA.Although both loss and overexpression of PKCδ led to an increased number of cellular protrusions, they differed in their effect on F‐actin organization. BCC protrusions with reduced PKCδ contained fan‐like arrays of unusually prominent actin filament bundles, whereas BCC protrusions with increased PKCδ were poor in actin filaments. Epistatic analysis between Tj and PKCδ indicates that PKCδ is an important downstream factor through which Tj exerts its effects on actin organization. To determine how PKCδ controls F‐actin organization, we studied the distribution of several known actin regulators, and uncovered an effect on the actin‐polymerizing factor Enabled (Ena). Ena is normally enriched in the leading protrusions of BCCs. Protrusions extended by PKCδ‐ or Tj‐depleted BCCs were largely devoid of Ena, whereas Tj or PKCδ overexpression seemed to cause stronger and more punctate cortical Ena localization. We propose a new actin‐regulating pathway in the BCC: Tj directly or indirectly upregulates the expression of PKCδ, which controls the cytoskeletal organization of cellular protrusions, a function that might be mediated through Ena. 15 Misshapen regulates Moesin phosphorylation and collective cell migration in vivo Cédric PLUTONI1, Sarah KEIL1 and Gregory EMERY1 1
IRIC / Montreal University Collective cell migration (CCM) has been characterized as an efficient and highly invasive mode of migration during metastasis progression. We use border cells (BCs) as a model to study CCM in vivo. Indeed, BCs migrate similarly to some metastatic cells: they form small clusters that migrate invasively in a polarized manner. Furthermore, this model allows us to combine fly genetics with cutting‐edge imaging techniques. Recently, we published that the Rab11 acts on BCs cluster to ensure that Rac activity is properly restricted to the leading cell. To regulate this cell‐cell communication event, Rab11 binds and modulates Moesin phosphorylation, an actin cytoskeleton regulator. Accordingly, reducing the level of p‐Moesin also affects cell‐cell communication. Our model suggests that Rab11 controls the sensing of the relative levels of Rac activity, leading to the organization of individual cells in a coherent multicellular motile structure. To investigate how Moesin is phosphorylated in BCs, we performed a screen by knocking down different STE20 kinases, proteins involved in phosphorylation cascades and correlated to tumor aggressiveness. Using p‐Moesin staining as a readout, we found that misshapen (msn) is necessary for Moesin phosphorylation in BCs. While msn was previously involved in BCs migration, our results shed light on a new role of msn in collective cell migration. Indeed, msn knockdown induces a detachment defect and, in accordance with a possible role in cell‐cell communication, uncontrolled protrusions formation. We are currently investigating the role of msn in the restriction of Rac activity to the leading cell of the BC cluster. Misshapen, Moesin and Rac orthologs have all been described as important actors during metastasis. Hence understanding how the msn / Moesin / Rac axis is involved and regulated during collective cell migration in vivo is crucial for our comprehension of tumor progression and metastasis formation. 16 PIP5K59B, a conserved phospholipid kinase, directs dendritogenesis during metamorphosis Danielle R McEachern, Shannon N Wong, Michael D Gordon Department of Zoology, University of British Columbia, Vancouver BC Extensive neuronal remodelling occurs during metamorphosis in Drosophila. As the body plan morphs from larva to fly, neurons also take on their adult morphology by retracting and/or degenerating larval neurites, and extending new dendrites and axons to adult targets. Neuronal morphogenesis has been described in other Drosophila neurons, but none as behaviourally or morphologically tractable as the E49 motor neuron. With unique morphologies during larval and adult stages, and its position within the fly taste system, it is ideally situated to explore novel regulators of neuronal remodelling. Using a behavioural gene screen that capitalizes on the E49 motor neuron’s specific function in feeding behaviour, the phospholipid kinase PIP5K59B was identified as a regulator of E49 motor neuron morphology and function. Further optogenetic and neuron tracing experiments show that PIP5K59B’s role is specific to dendrite and not axon regrowth. PIP5K59B’s phosphorylated substrate, phosphatidylinositol‐(4,5)‐bisphosphate, is one of the most abundant phosphoinositides in the plasma membrane, and has been documented as a regulator of the actin cytoskeleton, ion channels and a variety of signalling pathways. These downstream pathways will be examined further to determine how PIP5K59B directs dendritogenesis. 17 cis‐regulatory Integration of spatial and temporal inputs underlying neuronal terminal differentiation Anthony J.E. Berndt1, Jonathan C.Y. Tang2, Marc S. Ridyard3, Tianshun Lian1, Kathleen Keatings1 and Douglas W. Allan1 1. Department of Cellular and Physiological Sciences, 2401 Life Sciences Centre, 2350 Health Sciences Mall, University of British Columbia, Vancouver, BC, Canada (V6T 1Z3) 2. Current address: Department of Genetics, Room 360, New Research Building, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts, USA (02115) 3. Current address: Gurdon Institute, University of Cambridge,Tennis Court Road, Cambridge, CB2 1QN, UK Terminal differentiation of neurons often requires retrograde signals from the target cells they innervate, which trigger neural subtype‐specific genes upon target contact. Target‐derived BMP signaling and transcription factors including Apterous are required for FMRFa neuropeptide gene expression in Drosophila Tv4 neurons. We modeled the integrative mechanism of these extrinsic and intrinsic inputs at a Tv4‐specific FMRFa enhancer. We show that Tv4‐specific FMRFa expression requires two separable cis‐elements, a BMP‐response element (BMP‐RE) that binds Mad, and a homeodomain response element (HD‐RE) that binds Apterous. We find that concatemers of these two short (~30bp) cis‐elements each independently drive spatial and temporal expression appropriate for Tv4‐specific FMRFa. Thus, specific and robust expression is generated from the synergy of two low‐activity heterotypic cis‐elements that encode the same output from distinct inputs. We further examined the timing mechanism of FMRFa initiation, which models predict would be solely based on target contact. In contrast, we find that the timed downregulation of the nuclear receptor Seven up functions to de‐
repress HD‐RE and BMP‐RE activity immediately prior to target contact. Thus, we reveal that the active suppression of neurotransmitter identity, prior to target contact, is an innate component of the target‐
dependent mechanism for timed gene activation. 18 Transvection: making the complex more complicated Thomas JS Merritt, Teresa Z Rzezniczak and Patrick D. O’Donnell Laurentian University Gene expression determines cellular identity and is, unsurprisingly, complex. In general, gene regulation is primarily cis‐dependent but trans‐regulation can, and does, occur and often substantially modifies gene expression. Transvection, pairing‐dependent inter‐chromosomal gene regulation, is an underappreciated form of epigenetic trans‐regulation that adds complexity to regulation of gene expression. In cases of transvection, the expression of an allele on one chromosome is modified by regulatory sequences on the other through a mechanism that requires close physical proximity of the homologs. The physical requirements of transvection are striking; rearrangements of chromosomal architecture that prevent homolog pairing eliminate transvection effects. Transvection has recently been shown to be widespread in the fly genome and examples of similar pairing‐dependent inter‐chromosomal gene regulation are known in other non‐fly systems, notably some human diseases. My lab is examining the complexity of transvection with a focus on its sensitivity to genetic background and environmental variation. Using a combination of protein activity assays, qPCR, RNAi, and genetic mapping, we find that the effects of transvection are driven by local variation in regulatory regions, vary across genetic backgrounds, and are sensitive to environmental changes, i.e. transvection is itself a complex phenotype. In our model, transvection effects are sensitive to local variation in the effected gene as well as more distant variation that modifies chromosome pairing, the expression of regulatory proteins, or both. Our work suggests that regulation of gene expression is a function of elements on either chromosome acting in both cis‐ and trans‐ and this complexity has implications for how similar trans‐effects may be driving disease states in other systems. 19 Examining the role of mRNA Localization in Regulating Epithelial Cell Polarity Ashley Chin1,2 and Eric Lécuyer1,2,3 1
Institut de Recherches Cliniques de Montréal (IRCM); 2Division of Experimental Medicine, McGill University; 3Department of Biochemistry, University of Montreal The loss of epithelial polarity is a hallmark of metastatic carcinomas, malignancies of epithelial origin that encompass the vast majority of human cancers. One mechanism involved in establishing epithelial cell polarity is the asymmetric subcellular localization of mRNAs, a process governed by RNA binding proteins (RPBs) that recognize specific Cis‐regulatory elements in mRNAs. A global imaging study in Drosophila embryos identified various mRNAs that are localized to the cell junctions, most of which encode evolutionarily conserved tumour suppressor proteins that dictate epithelial polarity, including Cno, Dlg‐1 and dZO‐1. We hypothesize that i) junctional mRNA targeting and localized translation are essential to the maintenance of epithelial cell polarity and ii) disturbing this process will enhance tumorigenesis. Our first objective is to identify candidate RBPs that are localized to cell junctions via high‐throughput imaging with validated antibodies. Our second objective is to assess the impact of RBP loss‐of‐function on junctional mRNA localization and epithelial cell polarity, in vitro using mammalian and Drosophila epithelial cell lines, and in vivo using transgenic Drosophila ovaries, followed by fluorescence in situ hybridization and immuno‐labeling. We found that 22 out of the 313 RBPs tested exhibit junction‐like localization patterns in our immuno‐labeling studies of two mammalian cell lines, 15 of which have established links to cancers. Moreover, several of these RBPs are targeted to the cell cortex before junctional proteins encoded by localized mRNAs, consistent with the notion that mRNA recruitment precedes protein targeting to cell junctions. Interestingly, RNAi‐mediated knockdown of certain Drosophila RBPs candidates disturbed the distribution patterns of various junctional proteins. To confirm the roles of these RBPs in junctional RNA localization, we are conducting fractionation studies of RBP‐depleted cells, coupled with RT‐qPCR/RNA‐sequencing. Collectively, this project will increase our understanding of cellular organization and epithelial cell polarity, crucial features for unraveling the origins of cancer. 20 Systematic analysis of protein‐RNA interactions in Drosophila John Laver1, Xiao Li1, Hong Na1, Juhyun Jeon1, Fateh Singh1, Timothy Westwood2, Philip Kim1, Sachdev Sidhu1, Quaid Morris1, Craig Smibert1,3, Howard Lipshitz1 1) Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada; 2) Department of Cell and Systems Biology, University of Toronto; 3) Department of Biochemistry, University of Toronto RNA‐binding proteins (RBPs) interact with cis‐elements in their target mRNAs to control their splicing, transport, localization, translation, and stability. The Drosophila genome encodes about 400 proteins with known RNA‐binding domains (RBDs), about three quarters of which are expressed in early embryos, where gene regulation is accomplished exclusively at the post‐transcriptional level. Our long‐term goal is to identify all RBP‐mRNA interactions in early Drosophila embryos. To accomplish this, we are using synthetic antibodies, generated by phage display,to perform RNA co‐immunoprecipitation (RIP) coupled with microarray‐based or next‐generation sequencing analysis (RIP‐Chip or RIP‐seq). After establishing a high‐throughput pipeline for synthetic antibody production, we have generated 326 antibodies against 70 RBPs. 24 out of 25 antibodies tested, representing 13 RBPs, successfully IP their target proteins. We initially focused on three RBPs for genome‐wide identification of associated mRNAs: the double‐
stranded RBP, Staufen (STAU); the PUF protein, Pumilio (PUM); and the TRIM‐NHL protein, Brain Tumor (BRAT). Computational analysis of their targets predicted specific RNA structural motifs for STAU binding, and two distinct single‐stranded motifs, one for PUM binding and one for BRAT binding. Since the latter is the first predicted binding motif for any TRIM‐NHL protein, we confirmed it using an in vitro assay known as RNA compete and showed that it mediates BRAT‐directed repression in S2 cells. Unexpectedly, PUM and BRAT were found to associate with largely distinct sets of mRNAs, whose post‐
transcriptional fates differ. While mRNA targets of both proteins appear to be translationally repressed, the majority of BRAT’s target mRNAs are also degraded during the maternal‐to‐zygotic transition. Indeed, gene expression profiling in brat mutants revealed that BRAT is required for the degradation of hundreds of mRNAs in early embryos. 21 Transcriptional regulation is modulated by Vestigial family proteins in the developing wing Pimmett, V.L.1; Deng, H.2; and Simmonds A.J.1 1
Department of Cell Biology, University of Alberta, Edmonton Alberta CANADA 2
Molecular Biology and Genetics, Johns Hopkins University, Baltimore, Maryland USA Vestigial family proteins are developmentally important transcriptional regulators characterized by the presence of 1+ TONDU domains. The founding family member, Vestigial (vg), is a key fate selector gene in the developing wing. It acts as a transcriptional coactivator along with its DNA binding partner Scalloped (Sd) to direct presumptive imaginal disc tissue towards the wing fate. We have identified several novel mechanisms of identity specification involving Vestigial family proteins. First we have identified specific VG post‐translational modifications that alter both in vitro and in vivo function of the protein. Specific alterations in wing imaginal disc cell fates can be directed by expression of a phosphomimetic VG mutant, indicating this may be a relevant control mechanism for VG activity. Furthermore, we have defined the mechanism behind this post‐translational modification as resulting from the p38‐MAPK pathway. Second, Tondu‐domain‐containing growth inhibitor (Tgi) was recently identified as a second Vestigial family member. TGI function is dependent on SD in the imaginal wing disc, where it acts as a YKI competitor (Koontz et al., 2013). We have discovered that TGI acts antagonistically to Vestigial function in the developing wing disc. From these findings we are able to posit a model of alternative complex formation between VG, SD and TGI that affect transcriptional regulation during development. 22 Polarized endocytic activity directs actomyosin network assembly during wound repair Miranda V. Hunter1, Donghoon M. Lee1, Tony J. C. Harris1, and Rodrigo Fernandez‐Gonzalez1,2,3 1
Department of Cell and Systems Biology, University of Toronto, Toronto, Canada 2
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada 3
Developmental and Stem Cell Biology Program, The Hospital for Sick Children, Toronto, Canada Epithelial tissues have a remarkable ability to rapidly repair wounds, particularly during embryonic development. The mechanisms of embryonic wound repair are conserved and involve the assembly of a supracellular actomyosin cable around the wound. The actomyosin cable contracts, acting as a purse string that promotes wound closure. Actomyosin purse string assembly and contraction are accompanied by the polarized removal of junctional proteins such as E‐cadherin from the interfaces between wounded and adjacent cells. However, the significance and mechanisms of junctional redistribution during wound closure are unclear. We used quantitative imaging to investigate the role of junctional rearrangements during epidermal wound repair in Drosophila embryos. We found that clathrin and dynamin, two important components of the endocytic machinery, accumulated at the wound margin, suggesting a role for endocytosis in wound repair. ARF6, a small GTPase implicated in E‐
cadherin endocytosis, also localized around wounds. When we blocked endocytosis using both pharmacological and genetic approaches, we found that wound closure was significantly delayed. The defect in wound closure was accompanied by both impaired depletion of E‐cadherin from the wound margin, and defective assembly of the actomyosin purse string, suggesting that E‐cadherin endocytosis is necessary for actomyosin purse string assembly and efficient wound closure. Consistent with this, E‐
cadherin overexpression resulted in reduced actin accumulation at the wound margin and slower wound closure. The delay in wound closure was recapitulated when E‐cadherin was stabilized by overexpression of p120‐catenin. Blocking calcium release upon wounding prevented the endocytosis of E‐cadherin from the wound margin. Our results indicate that calcium signaling mediates polarized E‐
cadherin endocytosis, thus enabling actomyosin assembly at the wound margin to promote rapid wound closure. 23 CHARACTERIZATION OF HIGHER‐ORDERED SEPTIN STRUCTURES IN DROSOPHILA CELLS Zlatina Dragieva, Ruella Y, Kechad A, Hickson G, Sainte‐Justine UHC, Montréal ; Dept. Pathology ; Cell Biology, Université de Montréal, Montreal. Introduction: Septins are highly conserved proteins deregulated in cancer. Septins serve as scaffolds and diffusion barriers at the plasma membrane and midbody during cytokinesis. They interact with actin and have been observed to form ordered complexes that can polymerize into higher‐order structures (filaments and rings). The principles of assembly and disassembly of such filaments and rings and their roles are yet to be elucidated. Drosophila offers a simple system, as there are only 5 septin genes: peanut, sep1, sep2, sep4, and sep5 in contrast to 13 in humans. Methods and Results: We find that 20% of Drosophilla S2 cells contain tubular septin structures that label with Peanut immunostaining. These cytoplasmic tubes are present in both mitosis and interphase, suggesting they are cell cycle independent. To investigate their composition and dynamic properties, we have generated cell lines stably expressing Sep2‐GFP, which localizes to septin tubes. Peanut depletion disrupts septin tubes, yielding a uniform GFP signal, whereas Sep1 depletion disperses Sep2‐GFP tubes into clumps. Sep1 and Peanut are thus required for the formation of septin tubes, which is consistent with previously reported Sep1/Sep2/Peanut complexes. FRAP analysis of Sep2‐GFP revealed slow recovery, indicating that tubes are stable. We have explored the potential relationship between septin tubes and the actin cytoskeleton. Although the tubes did not label for F‐actin, treatment with the inhibitor of actin polymerization, Latrunculin A, led to their rapid (30 min) dispersal into rings. Depletion of the actin severing protein Cofilin/twinstar led to a similar phenotype. Thus a dynamic actin cytoskeleton is required for the formation and/or maintenance of higher ordered structures such as rings and tubes. Conclusion and Relevance: Ongoing studies aim to elucidate how septins organize into ordered structures and how actin regulates the process. This will clarify the septin network of interactions and help explain their cellular roles. 24 Chapter 1: Differential interactions between the core SJ domain proteins, Nrx‐IV and the Na/K ATPase, and the Tricellular Junctions Perveen Biln1, Kristen Brown1, Vanessa Auld1,2. 1
Department of Zoology, 2Cell and Developmental Biology, University of British Columbia Permeability barriers form between cells and are essential to prevent both fluid flow and pathogen invasion across tissues including the epidermis, intestine, and brain. These barriers are called tight junctions (TJ) in vertebrates and septate junctions (SJ) in invertebrates. Spanning between the membranes of two adjacent cells are protein strands that adhere to one another to create tight, impermeable junctions. Na/K ATPase is part of the core complex of proteins along with Coracle, Neuroglian, Neurexin IV, and Macroglobulin complement‐related that are required for the formation of SJs. At the sites where the SJs of three or more cells meet, SJ components overlap with proteins that constitute the Tricellular Junctions (TCJs) of which the only known component is Gliotactin (Gli). However, the mechanism by which Na/K ATPase interacts with other barrier components to establish, maintain and regulate these junctions is unknown. We have examined the consequences of loss of Na/K ATPase in the context of several barrier components and developed a model by which Na/K ATPase may be contributing to SJ function. We show that RNAi knockdown of Na/K ATPase in the imaginal wing disc of Drosophila leads to increased immunolabelling of Gli and Disc large and an expansion of their basolateral domain at the TCJ, a relationship unique to the Na+/K+ ATPase. Loss of the canonical core component NrxIV results in a basolateral spread and reduction Gli immunolabeling. These differential effects also extend to interactions with ectopic Gliotactin. The deleterious consequences triggered by spread of Gliotactin away from the TCJ were suppressed by loss of Na/K ATPase and enhanced by loss of NrxIV. These data suggests that Na/K ATPase has a unique role in both the regulation and maintenance of the TCJ. 25 Interactions between Cell Division and Epithelial Cell Polarity Gayaanan Jeyanathan and Ulrich Tepass Cell and Systems Biology, University of Toronto Cell division is essential for proper development of a unicellular zygote into a multicellular organism. An area of interest in the study of cell division is its potential link to epithelial cell polarity. An epithelial cell is polarized into three basic functional domains apical, junctional and basolateral and a breakdown of these domains compromises the function of the epithelium. To what extent cell division challenges epithelial polarity and epithelial tissue integrity is not well‐understood. Recent studies have begun to explore the need for a number of polarity proteins such as Dlg, Lgl, Scribble and Cdc42 for spindle orientation in symmetrically dividing epithelial cells. Interestingly, in our screen to look for novel epithelial polarity determinants, we have identified a gene involved in the mitotic exit machinery. Analysing this mutant we found that dividing cells show a marked reduction in the surface abundance of polarity proteins. Furthermore, we observed that polarity regulators such as Crb and Cdc42 show genetic interactions with the cell cycle regulator Stg/Cdc25. Blocking cell division in mutants rescued polarity defects. Conversely, overexpression of Stg, which increases cell division, worsens polarity defects. We hypothesize that cell division exposes epithelial tissues to significant morphogenetic stress making epithelial tissues that undergo division more sensitive to any polarity defects. We speculate that increased cell division could severely compromise the polarity machinery contributing to epithelial‐
mesenchymal transition and tumour progression. I plan to further characterize the relationship between epithelial integrity and morphogenetic stress induced by cell division and other morphogenetic movements. 26 The role of the Moesin complex during neuroblast asymmetric cell division Namal Abeysundara, Sarah Hughes Department of Medical Genetics, University of Alberta Coordinated self‐renewal and differentiation via asymmetric cell division (ACD) is essential for generating cellular diversity during development. During Drosophila neurogenesis, neuroblasts undergo ACD, self‐renewing the neuroblast and producing a ganglion mother cell (GMC) which further differentiates into the neurons and the glial cells of the adult nervous system. Neuroblast self‐renewal and differentiation is maintained by the appropriate localization of apical and basal polarity proteins, orientation of the mitotic spindle, and segregation of cell fate determinants during mitosis. We find that the actin‐binding protein, Moesin, displays a dynamic localization pattern during neuroblast ACD. During metaphase, the active or phosphorylated form of Moesin (p‐Moesin) is concentrated apically in the neuroblast. As mitosis progresses, the localization of p‐Moesin shifts more basally and at telophase, p‐
Moesin is concentrated at the basal cortex where the GMC buds off from. The loss of Moesin in a neuroblast‐specific manner affects neuroblast cell size and numbers during early larval stages and eventually results in later larval lethality. In addition, the apical and basal polarity proteins, aPKC and Miranda, respectively, do not localize appropriately in neuroblasts undergoing metaphase. Thus, Moesin function appears to be crucial during neuroblast ACD. The loss of the regulatory kinase of Moesin, Slik, reduces p‐Moesin cortical localization and alters neuroblast ACD. Furthermore, knockdown of components of the apical Par complex in the neuroblasts result in the loss of polar p‐Moesin localization during metaphase, suggesting that Moesin may interact with this complex to maintain polarity during ACD. These results reveal a novel role for the actin‐binding protein Moesin in generating cellular diversity through neuroblast ACD. 27 A Par‐1‐Par‐3‐centrosome cell polarity pathway and its tuning for isotropic cell adhesion Tao Jiang1, R. F. Andrew McKinley1, Melanie A. McGill1, Stephane Angers2,3 and Tony J. C. Harris1 1
Department of Cell ; Systems Biology 2
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy 3
Department of Biochemistry University of Toronto, Toronto, Ontario, Canada Epithelial plasma membranes contain apical and basolateral domains separated by cell‐cell junctions. Despite close molecular links with the polarity networks that generate single polarized domains, cell‐cell junctions are organized as multiple clusters with an isotropic distribution around each cell circumference. How junctions avoid the influence of polarity networks to maintain their isotropy has been unclear. In Drosophila, aPKC plays a central role in preventing the hyper‐polarization of junctions as epithelia develop from cellularization to gastrulation. We show that aPKC does so by inhibiting a positive feedback loop between the junctional‐organizer Bazooka (Baz)/Par‐3 and centrosomes. Without aPKC, Baz and centrosomes lose their isotropic distributions and recruit each other to single plasma membrane domains. Surprisingly, our loss‐ and gain‐of‐function analyses show that the Baz‐centrosome positive feedback loop is driven by Par‐1, a basolateral kinase known to phosphorylate Baz and inhibit its basolateral localization. The ability of Par‐1 to both locally promote and globally inhibit Baz complex assembly suggests a simple circuit for ‘Turing‐type polarization’. More specifically, we find that the phosphorylation of Baz by Par‐1 is needed for Baz to join the positive feedback loop. aPKC seems to restrain this mechanism at gastrulation by expelling Par‐1 from the apical domain and allowing Baz and junctions to distribute isotropically around the apical circumference of epithelial cells. 28 Sac1 selectively regulates trafficking of adhesion molecules in the developing Drosophila eye Lauren Del Bel (1), Ronit Wilk (2), Jason Burgess (1, 2), Gordon Polevoy (2), Ho‐Chun Wei (2), Julie A. Brill (1, 2) 1) Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada 2) Program in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada Phosphatidylinositol phosphates (PIPs) are membrane lipids that regulate many cellular events. They can act as signaling molecules, organelle identifiers and recruiters of effector proteins. Phosphatidylinositol (PI) 4‐phosphate (PI4P) is the most abundant cellular PIP and a critical regulator of intracellular trafficking. In Drosophila melanogaster, three PI 4‐kinases are responsible for generating PI4P, while a single phosphatase, Sac1, maintains PI4P levels. Sac1 is essential in the mouse and fly; thus, little is known about its roles during animal development. By generating Drosophila mutants, we discovered that proper regulation of PI4P by Sac1 is critical for eye development. Flies bearing a temperature‐
sensitive (ts) allele of sac1 exhibit an eye pigmentation defect, as well as a rough eye phenotype due to defects in pigment cell morphogenesis.We found that during early stages of pupal eye development, sac1ts mutants display abnormal distribution of the Neph1 homolog Roughest (Rst), a transmembrane protein required for ommatidial patterning and pigment cell specification. As a result, sac1ts mutant pupal eyes exhibit irregular, multilayered interommatidial cells (IOCs, pigment cell precursors), as well as disorganized bristles. Interestingly, insac1tsmutants the Rst paralog Kirre is normally distributed, suggesting that Rst regulation and trafficking alone is sensitive to alterations in PI4P. Furthermore, at later stages of pupal eye development, we discovered that sac1ts IOCs exhibit ER stress and accumulate Rst and Kirre intracellularly due to defects in the endo‐lysosomal pathway. Our work demonstrates that proper regulation of PI4P by Sac1 is essential for normal distribution of adhesion molecules and ER homeostasis during key stages of Drosophila eye development. 29 Regulation of epithelial vesicle trafficking by apical polarity proteins Kenana Al Kakouni1 and Kathryn Harris2 1
University of Toronto 2
Massachusetts Institute of Technology Epithelial cells form sheets of tightly adhering cells that act as barriers regulating transport to and from the lumen. They are characterized by their highly polarized and distinct apical and basolateral membranes. Polarity proteins associated with the apical membrane include atypical protein kinase C (aPKC), Cdc42, and the transmembrane protein Crumbs. The vesicle trafficking machinery regulates epithelial polarity by facilitating the transport of proteins and lipids to their respective apical and basolateral domains. Howerver, the mechanism by which polarity proteins interact with the trafficking machinery remains unclear. Our previous work showed that aPKC acts as an effector of the GTPase Cdc42, which is required for epithelial integrity by modulating the endocytosis and/or recycling of apical components, including Crumbs. This finding led us to hypothesize that aPKC has phosphorylation targets in the vesicle trafficking machinery. Bioinformatic analysis suggests that aPKC may have several phosphorylation targets in the trafficking machinery, including the retromer component Vps26. The retromer protein complex is involved in retrieving endocytosed proteins from endosomes and transporting them back to either the Golgi or plasma membrane. Our data suggests that aPKC, when activated by Cdc42, phosphorylates Vps26, and increases the activity of the retromer complex. We found that vps26 and vps35 maternal/zygotic mutants show loss of epithelial integrity and defects that resemble those observed with loss of crumbs or other apical polarity regulators. In addition, wild‐type, phosphomimic and non‐phosphorylatable forms of Vps26 form a complex with aPKC in vivo. Interestingly, only expression of phosphomimic Vps26 suppresses the Cdc42 loss‐of‐function phenotype. Together, these results suggest a model in which the retromer is phosphorylated by aPKC to maintain and regulate epithelial polarity. 30 The inner nuclear membrane protein Spegless regulates germline development Robyn Rosenfeld1, Lala Fabian2, Julie Brill2, Brian Ciruna2, Yonit Tsatskis1, Rod Bremner1, Anne‐Claude Gingras1, Laurence Pelletier1, Eric Schirmer3, Chikin Kuok1, Helen McNeill1 1
LTRI and University of Toronto 2
Sickkids and University pf Toronto 3
University of Edinburgh We have identified Spegless ( Sperm and Eggless) as a highly conserved nuclear membrane protein that is essential for both male and female fertility. Spegless encodes a protein with 5 transmembrane domains and a nucleoplasmic domain, that is almost entirely composed of a Domain of Unknown Function, Duf2215. Duf2215 is highly conserved, and is found in plants and animals, but not in yeast. Using high‐resolution OMX microscopy we have shown that Spegless is a nuclear envelope protein and it is restricted to the Inner nuclear membrane in S2 cells. We used ends‐out recombination to generate a null allele of Spegless. Loss of Spegless results in misregulation of chromatin organization, and weakening of the nuclear envelope. BioID analysis reveals that mammalian Speg homologs bind to LEM domain proteins, suggesting that Spegless is important for stabilizing LEM domain‐ nuclear envelope interactions Testes development is defective in Spegless mutants, and stem cells are lost from both the germline and somatic compartments. Thus Speg is needed to maintain stem cells. Strikingly, Speg null germline cells fail to reconstitute a nuclear envelope. These data suggest that Speg ( and metazoan DUF2215 proteins) are essential to maintain normal chromatin structure, to stabilize the nuclear envelope and to reconstitute the nuclear envelope after mitosis. 31 Role of Cohesins, Securin and Separase in Drosophila meiosis Zhihao Guo*, Osama Batiha*, Mohammed Bourouh*, Eric Fifield* and Andrew Swan* * University of Windsor, Windsor, ON Chromosome segregation in mitosis depends on the breaking of ring‐like structures, Cohesins, that link sister chromatids. Cohesin loss in anaphase depends on the ubiquitin‐mediated destruction of Securin, releasing bound Separase. Separase cleaves Rad21, the α‐kleisin component of the cohesin complex leading to dissolution of cohesin complexes and sister chromatid segregation. In meiosis, cohesin release occurs in two steps: first in anaphase I, arm cohesion is released to permit homologue segregation; and then in anaphase II centromeric cohesion is released, allowing sister chromatids to segregate. While core components of the cohesin complex are used in both mitosis and meiosis, in diverse organisms ranging from yeast to mammals, Rad21 is substituted for a meiosis‐specific α‐kleisin, rec8. Drosophila conspicuously lacks a rec‐8 homologue, raising the possibility that meiotic cohesion and its release are regulated differently in Drosophila. Here we investigate the role of Cohesin as well as Separase and Securin in Drosophila female meiosis. Using FISH against centromeric and arm regions of a single chromosome we find that stabilization of Securin or loss of Separase results in a delay in arm release in anaphase I and a complete failure of centromeric cohesion release in anaphase II, resulting in the production of 2 rather than 4 meiotic products. We find that the core Cohesin component SMC3 and the mitotic α‐kleisin, Rad21 are required for homologue pairing in meiosis, consistent with roles in maintaining arm cohesion. On the other hand, neither appears necessary for centromeric cohesion. Interestingly, both SMC3 and Rad21 are required immediately following meiosis to maintain centromeric cohesion on the arrested meiotic products within the polar body. Our results suggest that in Drosophila meiosis, Separase mediates the two‐step release of cohesion first by targeting Rad21 and then by targeting an unknown component of a novel cohesion complex. 32 Inter‐domain allosteric regulation of Polo kinase by Aurora B and Map205 is required for cytokinesis David Kachaner1, Xavier Pinson1, Khaled Ben El Kadhi1, Karine Normandin1, Lama Talje1, Hugo Lavoie1, Guillaume Lépine1, 2, Sébastien Carréno1, 4, Benjamin H. Kwok1, 5, Gilles R. Hickson3, 4 and Vincent Archambault1, 2 1
Institut de recherche en immunologie et en cancérologie, 2Department of Biochemistry and Molecular Medicine, 3Centre Hospitalier Universitaire Sainte‐Justine, 4Department of Pathology and Cell Biology, 5
Department of Medicine, Université de Montréal, Montréal, Québec, Canada Drosophila Polo and its human ortholog Polo‐like kinase 1 fulfill essential roles during cell division. Members of the Polo‐like kinase (Plk) family contain an N‐terminal kinase domain (KD) and a C‐terminal Polo‐Box Domain (PBD) which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood. Here we show that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD‐
bound microtubule‐associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that inter‐domain allosteric mechanisms can play important roles in controlling the cellular functions of Plks. 33 Drosophila septins Sep2 and Sep5 are redundant for imaginal cell proliferation but not oogenesis Ryan O’Neill, Denise Clark University of New Brunswick Septins are proteins with many functions, including cytoskeleton and membrane organization, protein scaffolding, and cytokinesis. Septins assemble into rod‐like complexes which can further assemble into higher‐order structures. The septin family is divided into subgroups, where subgroup members appear to occupy the same position and are interchangeable within septin complexes. There are five septin genes in Drosophila melanogaster: Sep1, Sep2, Pnut, Sep4, and Sep5. Sep5 is the most recent of the Drosophila septins and arose via retrotransposition of Sep2 in the ancestor of the Sophophora. As members of the same subgroup, Sep2 and Sep5 are predicted to be interchangeable within septin complexes. Multiple sequence alignment of Sep2 and Sep5 shows that ~14% of amino acid sites are different between paralogs, yet are conserved between orthologs, suggesting functional divergence. We investigated the functional divergence and redundancy of Sep2 and Sep5. Sep5 mutants are viable and fertile. Sep2 mutant females are semisterile, with defects in egg chamber formation. Sep2 cDNA or Sep2‐GFP transgenes rescue the Sep2 mutant egg chamber phenotype, whereas Sep5 cDNA transgenes do not rescue, showing that Sep2 and Sep5 are not functionally identical. Sep2 Sep5 double mutants are early pupal lethal and the larvae lack imaginal discs, similar to mutants for Pnut and other genes required for cell proliferation. Therefore, Sep2 and Sep5 have a redundant function in imaginal cell proliferation. We characterized the localization of Sep2‐GFP and Sep5‐GFP in oogenesis. Sep2‐GFP and Sep5‐GFP have similar localization, including localization to the germline ring canals, suggesting that Sep2 and Sep5 have a conserved mechanism for localization and that some other aspect of their function has diverged. 34 The role of the protein phosphatase 2A during the exit of mitosis. Mehsen Haytham1, Boudreau, V.1,2,3, Wang P.1, 2, Archambault V.1, 2 1: Institute for Research in Immunology and Cancer. Université de Montréal 2: Department of Biochemistry and Molecular Medicine. Université de Montréal 3: Department of Biology, University of North Carolina at Chapel Hill Introduction: Mitosis is coordinated by kinases and phosphatases. The cyclin dependent kinase 1 (CDK1), upon activation by cyclin B binding, triggers mitosis by phosphorylating multiple substrates. Several of these proteins are dephosphosphorylated at mitotic exit. The protein phosphatase 2A carrying the B55 regulatory subunit (PP2A‐B55) plays a major role in this process. However, the identity of the important substrates of PP2A‐B55 is still elusive. Methods and Results: We conducted a genetic screen to identify genetic interactors of tws (B55) in Drosophila melanogaster. We found lamin, which encodes an important structural protein of the nuclear envelope. Most eggs laid by mothers heterozygous for hypomorphic mutations in both lamin and tws failed to hatch, while single mutant controls showed no defects. Immunostaining in embryos revealed developmental failures at the syncytial stage. Defects observed include frequent free centrosomes, fragmented nuclei, and abnormal nuclear positioning. In fly cells in culture, simultaneous depletion of lamin and Tws by RNAi induced synthetic lethality accompanied by fragmented nuclei. Conclusion and Significance: Our results suggest that PP2A‐Tws and lamin are required for centrosome attachment to nuclei, maintaining nuclear envelope integrity and nuclear migration during early embryonic development. Our results fit a model in which PP2A‐Tws dephosphorylates CDK sites on lamin known to be important for lamina disassembly in mitosis. This study may reveal the roles of PP2A‐
B55 in the reformation of the nuclear envelope at mitotic exit. Other genetic interactions identified will serve as starting point to dissect other aspects of PP2A‐Tws function in the cell cycle. 35 Post‐transcriptional control of gene expression in the early Drosophila embryo Craig Smibert1,2, John Laver2, Alexander Marsolais1, Jason Dumelie1,Xiao Li2, Emiley Chen2, Mariana Kekis2, Hong Na2, Juhyun Jeon2, Fateh Singh2, Timothy Westwood3, Philip Kim2, Sachdev Sidhu2, Quaid Morris2, Timothy Hughes2, Howard Lipshitz2. 1) Department of Biochemistry, University of Toronto; 2)Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada; 3) Department of Cell and Systems Biology, University of Toronto Early development of the Drosophila embryo is directed by RNA‐binding proteins (RBPs) that control the translation, stability and sub‐cellular localization of maternally‐loaded mRNAs. My lab employs genome‐wide approaches to gain a panoramic view of the post‐transcriptional regulatory landscape of the embryo. Microarrays allow us to define stable and unstable mRNAs, and polysome gradients coupled to microarrays allow us to define mRNA translational status. Finally, the mRNA targets of an RBP can be defined by immunoprecipitating that RBP and identifying co‐purifying mRNAs using microarrays, in an approach known as RIP (ribonucleoprotein immunoprecipitation)‐Chip. Application of these approaches to the RBP Smaug indicates that Smaug binds to thousands of mRNAs in the early embryo to repress their translation and induce their degradation. Analysis of Smaug target mRNAs suggests a role for Smaug in regulating the proteasome, the chaperonin TCP1 ring complex, lipid droplets and metabolism. To characterize all RBP/mRNA interactions that take place in the early embryo we are generating synthetic antibodies against Drosophila RBPs. To date we have generated such antibodies against ~70 RBPs and used them to identify the target mRNAs of Pumilio, Brain tumor (BRAT) and Staufen via RIP‐
Chip. Computational analysis of bound mRNAs identified the RNA motifs that these proteins interact with. The regulatory status of Pumilio and BRAT‐bound mRNAs suggests that both proteins induce the degradation of their target mRNAs and we confirmed this by comparing the transcriptomes of wild‐type, brat and pumilio mutant embryos. Our data also suggests that Pumilio activity is temporally regulated by microRNAs expressed from the zygotic genome. In addition, smaug mRNA is a target of both Pumilio and BRAT‐mediated decay and we have evidence that they prevent inappropriate expression of Smaug which would otherwise repress the expression of potential Smaug target transcripts. 36 Dunk stabilizes the actomyosin network at the leading edge of the cleavage furrows during Drosophila cellularization Bing He1, Adam Martin2, Eric Wieschaus1,3 1. Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. 2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA 3. HHMI, Princeton University, Princeton, NJ 08544, USA. Actomyosin contractility underlies force generation in morphogenetic processes ranging from cytokinesis of a single cell to tissue‐scale epithelial extension or invagination. In Drosophila, the cleavage of the syncytial blastoderm is initiated by a dynamic network of actin‐myosin at the base of membrane furrows that invaginate from the surface of the embryo to partition syncytial nuclei into individual cells. We show that recruitment of myosin to the invagination front is biphasic in wild type embryos. During phase 1, a tension‐driven flow brings cortical myosin to the base of the newly invaginated furrows and establishes an interconnected network across the entire invagination front. In phase 2, additional myosin is directly recruited from the cytoplasm to this network. The two phases are distinguished in their requirement for the novel blastoderm‐specific gene dunk, which we show to be transcriptionally activated immediately before cellularization. Mutation in dunk causes instability and loss of myosin from the cortex during phase 1, leading to rupture of the actomyosin network and a misdirected flow of myosin toward network vertices. Myosin is replenished to the network in phase 2 by slam‐dependent, dunk‐independent direct recruitment, but the initial myosin loss disrupts the hexagonal packing of the ingressing furrows and results in uncoordinated furrow invagination. The expression of dunk is restricted at the blastoderm stage, and its protein product co‐localizes with actin and myosin at the invagination front. Computer simulation demonstrates that the biased myosin distribution towards vertices seen in mutants is a mechanically feasible consequence of altering the balance between myosin recruitment and dissociation in networks under tension. We propose that Dunk‐dependent stabilization of cortical myosin establishes a tension balance at the invagination front, which controls hexagonal symmetry of the ingressing furrows. 37 Cell‐ECM adhesion coordinates force transmission required for morphogenesis Katharine E. Goodwin1, Stephanie J. Ellis1, Emily Lostchuck1, Teresa Zulueta‐Coarasa2, James J. Feng3,4, Rodrigo Fernandez‐Gonzalez2,5,6, Guy Tanentzapf1,*. 1. Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada, V6T 1Z3 2. Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada M5S 3G9 3. Department of Mathematics, University of British Columbia, Vancouver, Canada, V6T 1Z2 4. Department of Chemical and Biological Engineering, University of British Columbia, Vancouver, Canada, V6T 1Z3 5. Department of Cell and Systems Biology, University of Toronto, Toronto, Canada, M5S 3G5 6. Developmental and Stem Cell Biology Program, The Hospital for Sick Children, Toronto, Canada M5S 1X8 Tissue morphogenesis requires force‐generating mechanisms to drive the organization of cells into complex structures. Changes in cell and tissue morphology are achieved by coordinated action of the cytoskeleton and cell adhesion to neighbouring cells and to the extra‐cellular matrix (ECM). Integrin‐
mediated cell‐ECM adhesion is known to transduce traction forces in spreading and migrating cells in vitro. The work we will present illustrates that cell‐ECM adhesion also transduces forces in vivo in the context of animal morphogenesis. To study the mechanisms by which cell‐ECM adhesion regulates tissue mechanics, we use the model system of Dorsal Closure (DC), an integrin‐dependent morphogenetic process in the Drosophila embryo. We show that failure to regulate Cell‐ECM adhesion results in abnormal levels of tension in the amnioserosa (AS), an extraembryonic epithelium required for DC. Using mutations to either increase or decrease Cell‐ECM adhesion, we show that DC is defective and that AS cell dynamics are altered in both cases. Quantitative image analysis and in‐vivo laser ablation experiments reveal a relationship between cell deformation and the level of tension in the AS. We propose a mechanism in which cell‐ECM adhesion regulates cell deformation, which in turn influences the magnitude and distribution of tension required to complete DC. Our results suggest that Cell‐ECM adhesion controls the transmission of forces across developing tissues to in order to promote specific outcomes. 38 SPARC is required for proper basal lamina assembly in the FB Bianca Scuric, Maurice Ringuette, Alexa Chorian, Sepher Jamali University of Toronto SPARC (secreted protein, acidic, and rich in cysteine), is a collagen and Ca2+‐binding glycoprotein that is expressed and secreted by a variety of cell types and is associated with several cellular processes including tissue remodelling, cell turnover, and tissue repair. SPARC is a transient component of Drosophila extracellular matrix (Bornstein 1995).Proper expression by haemocytes and fat body cells, is required for basal lamina assembly and homeostasis. Our lab has recently shown that SPARC null mutants are larva lethal. Loss of function leads to a cell‐autonomous rounding of polygonal fat body cells and an aberrant accumulation of fibrous extracellular matrix components, enriched in type IV Collagen. We are carrying out structure‐ function analysis of SPARC, to determine which amino acid residues and domains are required to maintain basal lamina homeostasis in the fat body. 39 Update on the Rare Diseases: Models & Mechanisms Network Howard Lipshitz University of Toronto http://rare‐diseases‐catalyst‐network.ca/ 40 Circular RNA Accumulation in Drosophila is Modulated by Biological Age Kevin So, Lucas Bishop, Hannah Jordan and Pedro Miura University of Nevada, Reno, Department of Biology Circular RNAs (circRNAs) are a newly appreciated class of noncoding RNAs. They are widely expressed in diverse species, from C. elegans to humans, yet their functions remain poorly understood. Previous work suggests that some circRNAs can act as microRNA sponges in the brain. In the fruit fly, Drosophila melanogaster, most circRNAs are generated from back‐splicing events of exons from known protein coding genes. CircRNAs accumulate in the central nervous system of adult Drosophila during normal aging. Aging can be altered by environmental factors in flies, including temperature, caloric intake, and oxidative stress. We hypothesized that such alterations in lifespan would alter circRNA expression during aging. Flies were subjected to various environmental conditions and circRNA accumulation was monitored by quantitative RT‐PCR analysis of dissected heads. Interestingly, conditions that increase longevity of flies were found to slow down the accumulation of circRNAs. Thus, circRNA accumulation in the central nervous system is influenced by biological age. Ongoing studies are testing whether the accumulation of circRNAs during aging in the brain are detrimental. 41 Commensal L. plantarum significantly reduces lifespan of Drosophila David Fast, Aashna Duggal, Dr. Edan Foley University of Alberta Background: Intestinal microbes play a critical role in the normal physiological and immunological function of the gastrointestinal tract. Studies have shown that disruptions to the gastrointestinal microflora greatly impact host viability and longevity. Pathobionts are symbiotic members of the microflora that can cause disease in response to uncharacterized factors. Recently, we established that the commensal bacterial species, Lactobacillus plantarum, significantly shortens the lifespan of adult Drosophila. We showed that inoculation of germ‐free flies with our commensal strain of L. plantarum caused intestinal dysplasia and greatly shortened the lifespan of the host. This observation offers an opportunity to study the impact that a single bacterial species has on host health. Results: From qPCR of fly guts, I found that L. plantarum does not elicit an immune response in the gut. However, the microbe is associated with elevated levels of insulin signaling and has a possible influence on epidermal growth factor signaling. The interruption of Ras signaling in intestinal progenitor cells reverts the shortened lifespan of flies associated with L. plantarum. Conclusion: This research demonstrates the impact that a single commensal bacterial species has on a host. Drosophila associated with L. plantarum have a significantly shortened lifespan that is possibly associated with insulin and epidermal growth signaling. Studying the pathobiont, L. plantarum’s, interactions with Drosophila will allow for greater understanding of how even seemingly beneficial microbes can negatively impact their host. 42 Role of autophagy proteins beclin 1/Atg6 and Pi3K59F in Drosophila Parkinson disease models Githure M’Angale1, Brian E. Staveley1 1
Department of Biology, Memorial University of Newfoundland, St. John’s, Newfoundland ; Labrador, Canada, A1B 3X9 Parkinson disease is the second most common human neurodegenerative disease: the result of degeneration and subsequent loss of the dopamine producing neurons. Autophagy is one of the key cellular mechanisms implicated in the pathology of Parkinson disease. Both beclin/Atg6 and Pi3K59F are proteins involved in the initiation of the autophagosome, an extremely important component in autophagic cell death. Drosophila melanogaster possesses a single homologue of each of these protein‐
encoding genes. RNA interference was used to knock down the production of these proteins in the dopaminergic neurons. This led to reduced survival and highly compromised locomotor abilities. We attempted to rescue/enhance the decreased survival and loss in climbing ability by co‐expressing Buffy, the single Drosophila pro‐survival Bcl‐2 homologue, with beclin 1/Atg6 and Pi3K59F. In both cases, Buffy co‐
expression rescued the loss in locomotor ability for both Beclin‐1 and Pi3K59F. While co‐expression of Buffy did not alter the survival of beclin/Atg6‐inhibited flies, expression of Buffy significantly improved survival of Pi3K59F flies. In the α‐synuclein‐induced Drosophila model of PD, we altered the expression of beclin/Atg6 and Pi3K59F in the dopamine producing neurons and analyzed longevity and climbing ability of these flies. When these two genes were reduced in the dopamine producing neurons, they significantly increased survival and slightly improved climbing over time in the α‐synuclein‐induced model of PD. 43 Non‐invasive recording of infra‐slow oscillation potentials from the Drosophila brain Shuang Qiu, Chengfeng Xiao and R Meldrum Robertson Department of Biology, Queen’s University, Kingston, Ontario K7L 3N6 Canada Synchronized neural activity is associated with important functions such as sensory processing and arousal. Here we demonstrate that coherent neural activity from the fly brain can be monitored non‐
invasively by recording from the hemolymph in the head. Periodic potential oscillations at 0.03‐0.1Hz, in the frequency range of infra‐slow oscillations (ISOs), were observed in the hemolymph and the brain in wild‐type flies. ISOs were globally synchronized events rather than locally distributed spontaneous activities. ISOs recorded from the hemolymph and directly from the brain displayed exactly the same peak frequencies with zero phase delay. Thus, the ISOs recorded from the hemolymph were faithful replicas of brain activities. Importantly, hemolymph recording of ISOs was non‐invasive, whereas brain recording induced spontaneous direct current potential shifts, indications of coma, with a chance of 7.5% in young (5‐10 days) and 30% in aged (40‐45 days) flies. The ISOs displayed several features related to different physiological states. The peak frequencies of ISOs in the night (0.038 mn; 0.011 Hz) were reduced compared to those in the daytime (0.063 mn; 0.007 Hz). The variance of peak frequencies increased with age (p>0.05, F test). A 10s anoxic exposure induced the loss of ISOs without the occurrence of anoxic coma, indicating that the ISOs were sensitive to oxygen deprivation. We also demonstrate that ISOs were lost in the mutant strain w1118. A serial genetic analysis indicates that the white gene was dissociated from the generation of ISOs. Rather, the genetic background on second and third chromosomes in wild‐type was strongly associated with the appearance of ISOs. In summary, we describe a noninvasive electrophysiological method to evaluate neural activity from the intact fly brain. Using this method we have characterized brain ISOs and revealed a strong association between genetic background, excluding the white gene, and ISO occurrence in wild‐type flies. 44 Genetic dissection of aggressive behavior in Drosophila melanogaster Mahmoudreza Ramin, Claudiu Domocos, David Slawaska‐Eng and Yong Rao Centre for Research in Neuroscience, Department of Neurology and Neurosurgery, McGill University Health Centre, Montreal, Quebec, Canada Aggression is an innate behavior that helps animals survive. It occurs when animals compete for limited resources, such as food, mating partners and habitats. Accumulated evidence supports that the control of aggressiveness involves both genetic and epigenetic factors. Recent studies have begun to reveal molecular and cellular mechanisms that modulate the level of aggressiveness in experimental model systems such as Drosophila and mice. We utilize Drosophila as a model to understand the mechanisms underlying the control of aggressive behavior. To investigate if visual perception is required for social suppression of fly aggression, we manipulated visual circuit activity and examined its effects on the behaviors of grouped flies. Our results showed that the blockade of visual circuit activity does not affect social suppression of aggression. We also found that temporal blockade of vision significantly increased the level of aggressiveness of flies without social experience. Moreover, to gain insights into molecular networks that control fly aggression, we performed a systematic genetic screen to identify genomic regions that are involved in the suppression of fly aggression. This suggested us some potential candidates for further studies. 45 The sex determination gene transformer controls male‐female differences in body size Elizabeth J. Rideout1 and Savraj S. Grewal2 1. Dept. Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC V6T 1Z3 2. Dept. Biochemistry and Molecular Biology , lark H. Smith Brain Tumour Center, Southern Alberta Cancer Research Institute, University of Calgary, Calgary, AB T2N 4N1 Almost all animals show sex differences in body size. For example, in Drosophila, females are larger than males. Although Drosophila is widely used as a model to study growth, the mechanisms underlying this male‐female difference in size remain unknown. Here, we describe a novel role for the sex determination gene transformer (tra) in promoting female body growth. Normally, Tra is expressed exclusively in females. We find that loss of Tra in female larvae decreases body size, while ectopic expression of Tra in males increases body size. Although we find that Tra exerts autonomous effects on cell size, we also discovered that Tra expression in the fat body and in neurons augments female body size in a non cell‐autonomous manner. These effects of Tra do not require its only known target genes doublesex and fruitless. Instead, Tra expression in the fat body promotes growth by stimulating the secretion of insulin‐like peptides from neurosecretory cells in the brain. Our data suggest a model of sex‐specific growth in which body size is regulated by a previously unrecognized branch of the sex determination pathway, and identify Tra as a novel link between sex and the conserved insulin signaling pathway. 46 Ribosome synthesis in Drosophila muscle is required to maintain systemic insulin signaling and larval growth. Abhishek Ghosh, Elizabeth J. Rideout, Savraj S. Grewal Department of Biochemistry and Molecular Biology, Clark H. Smith Brain Tumour Centre, Southern Alberta Cancer Research Centre, 3330 Hospital Drive NW, HRIC 2A33A, Calgary, AB T2N 4N1. The conserved Target‐of‐Rapamycin (TOR) kinase signaling pathway links nutrient availability to cell, tissue and body growth in animals. One important growth‐regulatory target of TOR signaling is ribosome biogenesis. Studies in yeast and mammalian cell culture have described how TOR controls ribosomal RNA (rRNA) synthesis via the RNA Polymerase I transcription factor Transcription Initiation Factor ‐ IA (TIF‐IA). However, the role of TOR‐dependent ribosome synthesis to tissue and body growth in animals is less clear. Here we show in Drosophila larvae that ribosome synthesis in muscle is required non‐
autonomously to maintain normal body growth and development. We find that amino acid starvation and TOR inhibition lead to reduced TIF‐IA levels and decreased rRNA synthesis in larval muscle. When we mimic this decrease in muscle ribosome synthesis using RNAi‐mediated knockdown of TIF‐IA, we observe delayed larval development and reduced body growth. This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin‐like peptides (dILPs) from the brain and increased expression of Imp‐L2 ‐ a secreted factor that binds and inhibits dILP activity ‐ from muscle. Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF‐IA function. These data suggest that muscle ribosome synthesis functions as a nutrient‐dependent checkpoint for overall body growth: Upon nutrient rich conditions, TOR is required to maintain levels of TIF‐IA and ribosome synthesis to keep high levels of systemic insulin, but upon starvation, reduced muscle ribosome synthesis triggers an endocrine response that limits systemic insulin signaling to restrict growth and maintain homeostasis. 47 The atypical cadherin Dachsous controls Left‐Right Asymmetry in Drosophila González‐Morales N.1,2,3, Géminard C.1,2,3, Lebreton G.1,2,3, Coutelis J.B.1,2,3, Cerezo D.1,2,3 and Noselli S.1,2,3,4 1
University of Nice Sophia Antipolis, institut de Biologie Valrose, iBV, 06108, Nice, France 2
CNRS, institut de Biologie Valrose, iBV, UMR 7277, 06108 Nice, France 3
INSERM, institut de Biologie Valrose, iBV, U1091, 06108 Nice, France 4
Corresponding author. Email: noselli@unice.fr Left‐Right (LR) asymmetry is essential for organ development and function in metazoans. Yet, how initial LR cue is relayed to tissues still remains unclear. Here, we uncover a mechanism by which the Drosophila LR determinant Myosin ID (MyoID) transfers LR information to neighboring cells through the planar cell polarity (PCP) atypical cadherin Dachsous (Ds). Molecular interaction between MyoID and Ds in a specific LR organizer controls dextral cell polarity of adjoining hindgut progenitors and is required for organ looping in adults. Loss of Ds blocks hindgut tissue polarization and looping, indicating that Ds is a crucial factor for both LR cue transmission and asymmetric morphogenesis downstream of MyoID. We further show that the Ds/Ft and Frizzled PCP pathways are required for the spreading of LR asymmetry throughout the hindgut progenitor tissue. These results identify a direct functional coupling between the LR determinant MyoID and PCP, essential for non‐autonomous propagation of early LR asymmetry. 48 Identification and Functional Characterization of Genes Involved in Central Nervous System Control of Obesity Irene Trinh1,2, Oxana Gluscencova2, Gabrielle L. Boulianne1,2 1
Department of Molecular Genetics, University of Toronto, Toronto, Canada, 2 Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Canada The increasing prevalence of obesity as well as its association with many chronic diseases have turned obesity into a major health concern worldwide. Obesity has many underlying environmental and genetic factors that both contribute to disturb the homeostatic mechanisms that maintain a balance between energy intake and energy expenditure which is controlled by the central nervous system. The goal of my project is to help further our understanding of these CNS mechanisms by using the powerful tools available in Drosophila melanogaster to identify neuronal genes involved in energy homeostasis. An RNAi screen of 1748 genes using the neuronal fru‐Gal4 driver produced 25 hits that resulted in obese or lean phenotypes defined as increases or decreases in triacylglycerol (TAG) levels respectively. One of the hits identified was Diacylglycerol kinase (Dgk) that converts diacylglycerol (DAG) to phosphatidic acid (PA). Knockdown of Dgk using fru‐Gal4 results in increased TAG, glycogen and circulating trehalose levels. These effects of Dgk may be mediated by changes in DAG and/or PA levels. I am currently working towards elucidating the mechanisms through which Dgk affects energy homeostasis. 49 Drosophila sex comb evolution: how some things change and some stay the same. Juan Nicolas Malagon and Ellen Larsen Department of Cell and System Biology, University of Toronto, Toronto, ON, Canada M5S 3G5 In evolution, trait characteristics are thought to be determined by two opposing forces, buffering mechanisms that reduce the phenotypic variation caused by genetic or environmental perturbations and selection of variation for a more adapted phenotype. The mechanisms underlying phenotypic stability and phenotypic modification in evolution are rarely understood at the cellular level. Here we present evidence that the straight shape of sex combs during evolution is maintained by controlling comb length and that evolutionary changes of comb position on the leg can be explained by modulating tissue area in the epithelium surrounding the comb. By analyzing genetic perturbations, time lapse movies, and computer simulations, we suggest that the morphological stasis of comb shape and the morphological variation of comb position in evolution can be understood in terms of simple mechanical models of cell and tissue behavior. These studies highlight the potential for developmental processes to modulate simple physical forces in producing adaptive phenotypes. 50 Drosophila Smo signals through a novel and conserved mechanism. Dominic Maier1,2, Anastasia Adriyanta1,3 and David R. Hipfner1,2,4,* 1. Institut de recherches cliniques de Montréal (IRCM), Montreal, QC, Canada 2. Department of Anatomy ; Cell Biology, McGill University, Montreal, QC, Canada 3. Department of Physiology, McGill University, Montreal, QC, Canada 4. Department of Medicine, Université de Montréal, Montreal, QC, Canada. The Hh signaling pathway plays crucial roles in controlling tissue growth and patterning during embryonic development and adult tissue homeostasis in most bilaterian metazoans. The general architecture of the pathway is well conserved, with intracellular Hh signaling initiated by Smoothened (Smo), a distant member of the G protein‐coupled receptor (GPCR) protein family. Smo orthologs of all three bilatarian lineages share an evolutionarily conserved core sequence but show substantial differences at the C‐terminal end of their cytoplasmic tails. In Drosophila the non‐conserved portion harbors domains crucial for Smo activation and downstream signaling, including PKA phosphorylation sites and binding sites for the downstream effectors Costal2 (Cos2) and Fused (Fu). How mammalian Smo orthologs activate downstream signaling without these domains is still poorly understood. In recent years G protein‐coupled receptor kinases (GRKs) have emerged as new, positive components of the Hh pathway that phosphorylate and activate Smo in several organisms. We mapped sites in the cytoplasmic tail of Smo that are phosphorylated by Gprk2, a Drosophila GRK. The functionally crucial sites we identified overlap with GRK2 phosphorylation sites in the murine Smo protein and fall into a highly conserved region found in all bilaterian Smo orthologs. We demonstrate that Gprk2 phosphorylation triggers the recruitment of Cos2 to a novel binding site in Drosophila Smo. This interaction is most likely mediated by a short stretch of amino acids adjacent to critical Gprk2 phosphorylation sites that are predicted to form an α‐helix. Disruption of this helix has the same effect as abolishing Gprk2 phosphorylation, causing a reduction in Smo activity and impairing Cos2 recruitment. We propose that Drosophila Smo signals through a novel signaling mechanism which does not rely on fly‐specific Smo domains or downstream effectors (i.e. Fu) and is thus likely employed by all bilaterian Smo proteins. 51 Ras activated Dsor1 promotes Wg signalling in Drosophila development Eric Hall, Esther Verheyen Simon Fraser University Wnt/Wingless (Wg) and Ras‐MAPK signaling pathways both play fundamental roles in growth, cell‐fate determination, and when dysregulated, can lead to tumorigenesis. Several conflicting modes of interaction between Ras‐MAPK and Wnt signaling have been identified in specific cellular contexts, causing synergistic or antagonistic effects on target genes. Examination of Wg pathway activity in developing Drosophila imaginal discs revealed genetic evidence of a novel interaction in which the Drosophila dual specificity kinase MEK, Downstream of Raf1 (Dsor1), is required for Wg signal transduction. Knockdown of Dsor1 resulted in loss of Wg target gene expression, as well as reductions in stable Armadillo (Arm; Drosophila β‐Catenin).In the absence of Wg, a complex of proteins targets Arm for destruction. Wg induces the recruitment and inactivation of this complex allowing the stabilization of newly synthesized Arm, which then enables it to promote transcriptional activation of target genes. Dsor1 reduction failed to recruit and inhibit the destruction complex proteins to the Wg receptors. We identified a close physical interaction between Dsor1 and Arm at the cell membrane, and found that catalytically inactive Dsor1 caused reductions in active Arm. These results suggest that Dsor1 normally counteracts the Arm destruction complex through its membrane recruitment. As part of the MAPK signaling cascade, Dsor1 requires activation via upstream signaling components. We find that Ras‐Dsor1 activity is independent of upstream activation by EGFR, rather it appears to be activated by the insulin‐
like growth factor receptor to promote Wg signaling. Together our results suggest novel crosstalk between Insulin‐like growth factors and Wg signaling in the developing Drosophila via Dsor1 recruitment of the destruction complex to the membrane. 52 JAK/STAT signalling determines the cellular response to EGFR activation in the ovary Scott De Vito1, Mariana Fregoso Lomas1, Jean François Boisclair Lachance2, Josée Houde1, Laura Nilson1. 1) Deparment of Biology, McGill University, Montreal, QC, Canada 2) Ben May Department for Cancer Research, University of Chicago, Chicago, Il. During oogenesis, the follicle cells that overly the oocyte are exposed to EGFR activation by a graded ligand, Gurken(Grk), secreted from the oocyte. The follicle cell response to Grk/EGFR activation depends on the developmental stage when this signal is received. During mid‐oogenesis, dorsal anterior follicle cells are exposed to Grk/EGFR activation. These cells respond by expressing the transcription factor Mirror and take on a dorsal anterior fate. However, posterior follicle cells are also exposed to Grk/EGFR activation during early‐oogenesis. These cells respond by expressing the transcription factors H15 and Midline(Mid) and take on a posterior fate. Our current model is that the specific fate response to Grk/EGFR activation depends on the mutual repression of Mirror and Mid, constituting a molecular switch that allows EGFR signaling to toggle between two different outcomes. These two mutually exclusive cellular responses from a single signal suggest a requirement for additional input from other signalling pathways. Since posterior but not anterior cells experience JAK/STAT signalling, we asked whether induction of H15 and Mid expression also requires this additional signalling input. Using mosaic analysis, we show that the expression of H15 and Mid requires the JAK/STAT signalling members Hopscotch and Stat92E. These results show that input from JAK/STAT signalling dictates the cellular response to Grk/EGFR signalling at the posterior, leading to the expression of H15 and Mid. However, it has been shown that JAK/STAT signalling is capable of repressing Mirror. This may suggest that repression of Mirror by JAK/STAT signalling at the posterior may lead to the de‐repression of H15 and Mid. The absence of JAK/STAT signalling in the anterior allows for the induction of Mirror expression by dorsal anterior Grk/EGFR signalling. Expression of Mirror in the dorsal anterior thereby prevents the expression of H15 and Mid allowing for the development of dorsal anterior fates. 53 Functional understanding of Ral signaling in Drosophila melanogaster Helene Knævelsrud1 and Marc Therrien1,2 1
Institute for Research in Immunology and Cancer, Laboratory of Intracellular Signaling, Université de Montréal, PO box 6128, Station Centre‐Ville, Montreal, Quebec H3C 3J7, Canada 2
Département de pathologie et de biologie cellulaire, Université de Montréal The small GTPase Ral regulates important membrane trafficking events, including exocytosis, endocytosis and autophagy. In mammalian cells Ral is activated downstream of active Ras, which directly interacts with RalGEFs. Over the recent years it has become clear that Ral plays an important role in signal transduction leading to cancer formation and metastasis, both in Ras‐dependent and independent manners. Yet, little is known about the Ral signaling network. To understand more about Ral signaling in development and carcinogenesis, we have characterized the effect of expressing constitutively active RalG20V and dominant negative RalS25N in various fly tissues, including wings, notum and eyes. Furthermore, we have analyzed the dependence of these phenotypes on known and predicted regulators and effectors of Drosophila Ral. Upon RalG20V expression we observed misorientation of bristles in the notum, wing vein defects and misaligned ommatidia. Conversely, expression of RalS25N led to loss of notum bristles, small and malformed wings and mild eye roughening. We further characterized the RalS25N‐induced balding of the notum and found it to be suppressed by coexpression of the RalGEF Rgl or wild‐type Ral, by introducing mutations in the exocyst component Sec5 ‐ a known Ral effector ‐ or by reducing JNK signaling a known downstream pathway negatively regulated by Ral signaling. In conclusion, we present fly models for Ral signaling amenable to genetic screening that will allow us to identify new components of the Ral signaling network. 54 Compartments, clocks and circuit assembly in the developing Drosophila brain Ted Erclik1, Xin Li2, Claire Bertet3, Zhenqing Chen2 and Claude Desplan3 1
University of Toronto, Mississauga 2
University of Illinois 3 New York University In the Drosophila optic lobes, 800 retinotopically organized columns act in the medulla as functional units for the processing of both color and motion information from each of the 800 unit eyes (ommatidia). These columns are contributed to by over 70 neuronal cell types, which comprise uni‐
columnar neurons, with a stoichiometry of one per column, and multi‐columnar neurons that are fewer in number and extend arborizations to multiple columns. Here, we show that combinatorial inputs from spatial and temporal axes generate neuronal diversity in the medulla: (1) in the temporal axis, neuroblasts sequentially switch between at least five distinct fates to produce neurons of different identities; (2) in the spatial axis, the neuroepithelium that generates medulla neuroblasts is sub‐divided into at least six compartments by the spatially restricted expression of transcription factors and signaling molecules. The sequential progression of neuroblasts is identical in each compartment and produces distinct types of neurons in each temporal window. However, although uni‐columnar neurons are refractory to spatial inputs and remain at their site of birth, multi‐columnar neurons are generated by neuroblasts in a region‐specific manner. For instance, the youngest neuroblasts that express Homothorax (Hth) generate uni‐columnar Mi1 neurons throughout the larval crescent. In the central compartment that expresses Vsx1, they additionally produce non‐columnar Pm3 neurons while, in the Rx compartments, they produce Pm1 neurons ventrally and Pm2 neurons dorsally. These multi‐
columnar neurons, generated in restricted compartments, subsequently migrate to take up their final position in the adult medulla, or send long projections to innervate the whole retinotopic field. This combination of regional and temporal neuronal specification represents a powerful mechanism for the generation of the proper stoichiometry of neuronal types and the establishment of the retinotopic map. 55 Processing of modality and body position by the Drosophila taste system. Michael D Gordon1, Bonnie Chu1, Emily E LeDue1, Vincent Chui1, Kevin Mann1, Yu‐Chieh Chen2, Aera Y Jung1, Anupama Dahanukar2,3 1
Department of Zoology, Cell and Developmental Biology, University of British Columbia, V6T 1Z3 2
Interdepartmental Neuroscience Program, University of California, Riverside, CA 92521 3
Department of Entomology, University of California, Riverside, CA 92521 The sense of taste is critical in determining the nutritional suitability of foods. Flies detect tastes using specialized chemosensory bristles located in their legs, labellum, and a set of pharyngeal sense organs. Within each of these bristles generally reside four gustatory receptor neurons, each tuned to a discrete taste modality. Stimulation of each taste neuron type leads to a specific behavioural outcome. For example, activation of sweet neurons promotes feeding, while activation of bitter neurons inhibits feeding and promotes avoidance. Here, I will present evidence for a synaptic gain control mechanism underlying the integration of sweet and bitter tastes. This mechanism relies on feedback inhibition by the inhibitory neurotransmitter GABA onto sweet taste neuron terminals, following detection of both sweet and bitter tastes. I will also present work describing the function of a previously uncharacterized class of pharyngeal sweet taste neurons. These neurons respond to an array of sweet compounds and function to sustain ingestion of appetitive food sources. Interestingly, these neurons account for the majority of sugar consumption previously attributed to taste‐independent post‐ingestive mechanisms, raising questions about the true impact of nutrient signaling on feeding decisions. 56 Fruitful investigations using flies to understand the overwhelming genetic complexity of neurodevelopmental disorders Maria Knip1, Tara Edwards1, and Jamie M. Kramer1,2 1
Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry 2
Department of Biology, Faculty of Science Western University, London, Ontario Intellectual Disability (ID) is a neurodevelopmental disorder defined by limitations in intellectual functioning and adaptive behaviour. A large proportion of ID has a genetic cause. There are currently over 650 genes that are known to cause ID when mutated. Recent genomic studies have demonstrated that up to 60% of ID cases are cause by dominant mutations that are acquired de novo. Of the 650 known ID genes, about 130 are affected by dominant mutations. Our bioinformatic analysis has revealed that almost half of the dominant ID genes are present in a single protein‐protein interaction network that predominantly consists of genes involved in chromatin modification and transcription. Strikingly, some chromatin regulating complexes contain a very high proportion of known ID genes. In particular, the SWI/SNF chromatin remodelling complex is the most enriched protein complex in all ID. This suggests that disruption of epigenetic mechanisms or chromatin regulation is one of the most important mechanisms underlying ID. This has implications in the clinic since a single therapy targeting the commonly disrupted mechanism might be applicable to a diverse array of genetic defects. However, our current understanding of the function of these protein networks in the brain is highly limited. In my lab, we take a systems biology approach to understanding the role of epigenetic regulatory complexes in brain development and function using the highly efficient model organism, Drosophila melanogaster. Results will be presented from various projects investigating the function of epigenetic regulators and ID gene orthologs in Drosophila neurobiology. 57 Poster presentation
1. Reversible Immobilization of Intact Larvae for Live Imaging Meryl Acker1,Duygu Çevic1, Valentin Demidov2, Alex Vitkin2 and Roger Jacobs1 1
Biology, McMaster University 2
Medical Biophysics, University of Toronto Microscopy of fluorescently‐tagged proteins enable scientists to observe developmental events at the cellular level in intact living embryos. However, its use in larvae is limited by the peristaltic waves of contraction in the body wall during locomotion. Immobilizing larvae with adhesives or sealants stresses the animal and does not eliminate motility. Fictive peristalsis of immobilized larvae transiently arrests heart contraction. In the interest in monitoring cardiac physiology in intact larvae, we have explored the use of different anesthetic approaches that are simple, robust and reproducible. Chloroform (CHCl3), also known as trichloromethane, is a fast acting, non‐flammable anesthetic. We have found that calibrated doses of airborne chloroform released in a closed chamber can immobilize Drosophila melanogaster larvae. Optimal chloroform exposure time was determined by balancing repeated short exposure periods to obtain maximum immobilization time and maximum eclosion success of the exposed larvae. Our protocol enables immobilization of five larvae at a time for thirty minutes without causing long‐term damage, such as neurological or cardiac defects. We will also show how anaesthetised larvae can be readily handled and imaged in a microfluidics based chamber, eliminating the need for adhesives. Using chloroform along with an inexpensive set up, researchers can eliminate movement artefacts and acquire higher quality images compared to standard imaging techniques. 58 2. Mapping patterns of phosphorylation of proteins important in Drosophila melanogster development Anirban Banerjee, Dan Bath, Anthony Percival‐Smith The University of Western Ontario, London, ON, Canada During formation of the anterior‐posterior axis, HOX proteins determine the identity of body segments in Drosophila. A HOX‐derived protein, Fushi tarazu (FTZ), a HOX co‐factor, Extradenticle (EXD) and the HOX proteins are transcription factors that regulate target gene expression during development. Besides a highly conserved DNA‐binding homeodomain, HOX proteins also contain other conserved motifs called Short Linear Motifs (SLiMs) whose sequences when altered have an effect on overall HOX activity. SLiMs are proposed to be sites of phosphorylation and this is thought to regulate the activity of HOX proteins. Previous studies have used 2‐D gels, in vitro kinase assays, yeast two‐hybrid screens and phosphorylation prediction programs to show or suggest that FTZ, EXD, UBX, SCR and ANTP are phosphoproteins. But, the above assays have a limitation in not directly mapping the site of phosphorylation. Mass spectrometry (MS) is the best technique for mapping the exact sites of phosphorylation in vivo. A MS phosphoproteome analysis of homogenized Drosophila embryos showed that UBX is phosphorylated at T170, S174 and S177 and SCR is phosphorylated at S216 (Zhai et al., 2008). S216 lies in the SCR SLiM DISPK which is the first data point supporting the hypothesis that SLiMs are sites of phosphorylation. But, the above MS analysis involved the whole proteome of the embryo which most likely only mapped a small fraction of phosphorylation. A specific and targeted analysis of FTZ protein extracted from the developing Drosophila embryo was performed by Dan Bath in our laboratory. Tandem mass spectrometry (MS/MS) mapped six putative sites of phosphorylation: S107 or T108, T115, T280 and Y399 or H402 in FTZ. Phosphorylation of T115 (which maps to the FTZ‐F1 site) abolishes FTZ activity suggesting that phosphorylation of the FTZ‐F1 binding site may negatively regulate the FTZ and FTZ‐F1 interaction (Bath, 2010). I want to extend this analysis to EXD and the HOX proteins too. The primary aim of my work is to develop a comprehensive, unbiased catalogue of the phosphorylation sites for FTZ, EXD and HOX proteins extracted from developing embryos. I will then test the hypothesis that HOX SLiMs are sites of phosphorylation (Sivanantharajah ; Percival‐Smith, 2015) by analyzing whether SLiMs are phosphorylated and whether SLiMs are preferential sites of HOX phosphorylation. All the fly lines to express these 10 proteins have been established. I have also performed a MS/MS analysis which confirmed a previously predicted site of phosphorylation (S165) in the maternal‐effect protein, Bicoid (BCD) which along with 6 other putative sites is thought to be involved in the Torso‐induced strengthening of BCD morphoghenic acitivity (Janody et al., 2000). I also found S343 is a site of phosphorylation in BCD. Finally, with the data on truly phosphorylated residues in HOX, I will genetically modify important sites of phosphorylation and investigate their role during development. Phosphorylated S/T/Y residues in important motifs will be genetically modified to alanine (abolish phosphorylation) and to aspartic acid (mimic phosphorylation). For instance, the UBX homeodomain and the conserved YPWM motif are required for effective interaction with EXD protein (Johnson et al., 1995). If the Y residue in a HOX YPWM motif is found to be phosphorylated, it would show an additional mechanism for the regulation of the HOX‐EXD interaction. Moreover, unpublished data from our laboratory had shown that the expression of the constitutively active RASv12 molecule results in an exd phenocopy which indicates that EXD may be negatively regulated by a MAP kinase. This means that phosphorylated EXD is inactive which can be tested by my experiments. These are some types of assays that will be performed after mapping the phosphorylation sites. 59 3. Modelling peroxisome biogenesis disorders in Drosophila melanogaster Matthew N. Baron, Di Cara F, Simmonds AJ, and Rachubinski R Deptartment of Cell Biology, Univeristy of Alberta, Edmonton, Alberta Peroxisomes are small ubiquitous organelles responsible for the oxidation of very‐long chain fatty acids (VLCFAs) and the synthesis of plasmalogens and bile acids, which are crucial for pathways involved during early childhood development. Mutations in the peroxisome biogenesis factors, or PEX genes, results in a class of diseases called Peroxisome Biogenesis Disorders (PBDs). PBDs are rare genetic disorders with a conservative birth incidence estimate of 1/50 000. Albeit rare, these diseases exact a large toll on the health care system and families of those afflicted due to the severe nature of the symptoms. The symptoms include craniofacial abnormalities, hearing and vision loss, skeletal dysmorphia, and cognitive and developmental impairments. To date there is no cure for PBDs and the treatments that are available are limited. The molecular mechanism of peroxisome biogenesis is strongly conserved across species. This conservation has fostered multiple pioneering studies using yeast that have advanced our understanding of peroxisome biogenesis; however, these studies are limited in their scope because of the unicellularity of yeast. Flies represent one of the simplest animals with organ systems, including a brain capable of learning and memory similar to the brains of much more complex and expensive laboratory animals, like mice. We have been developing an effective and relatively inexpensive animal model in the genetically tractable organism, Drosophila melanogaster, to study the PBDs and to investigate the requirements for peroxisomes in tissue development and homeostasis. This model will allow future development of unique ways to probe tissue‐specific defects associated with the PBDs given the genetic techniques available in flies that we can use to inform further experimentation in mammalian models. 60 4. Systematic analysis of protein turnover and its role for long‐term muscle maintenance Darius Camp1 and Guy Tanentzapf1 1) Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada Animal muscles must maintain their function while bearing substantial mechanical loads and undergoing numerous contraction and extension cycles. How muscles withstand persistent mechanical strain is not well understood. Previous studies have shown that ongoing synthesis of key structural muscle components are required to maintain muscle integrity and function. We therefore hypothesize that cytoskeletal proteins undergo renewal via protein turnover and that this is required to maintain muscle function. To visualize long‐term turnover of muscle proteins requires the ability to label proteins in vivo as well as the ability to turn this label on and off. We have therefore developed a novel system that allows us to conditionally control the expression of endogenously GFP tagged sarcomeric proteins in the adult flight muscles of Drosophila melanogaster. Specifically, we controlled the expression of GFP‐
tagged proteins at the mRNA or protein level using either RNAi or nanobody‐based approaches that destroy the tagged protein but do not affect muscle function or organization. Using this approach confirmed for the first time that the sarcomeric cytoskeleton undergoes turnover throughout the life of the organism. Furthermore, this approach allow us to determine the reasons why turnover is functionally relevant. We are in particular interested in the effects of age and exercise on muscle protein turnover. We therefore plan to study muscle protein turnover in old and young flies and in mutant backgrounds that speed or delay aging. To understand the effects of exercise we will use behavioral assays and mutations that specifically modulate the exercise level of the fly without affecting muscle development. Together these studies will provide detailed insight into how muscles, as well as other cell and tissues types, maintain their structure and function throughout life. 61 5. Role of Myt1 kinase during pre‐meiotic G2 phase arrest in Drosophila spermatocytes Shelag Campbell, Ramya Varadarajan, Joseph Ayeni, Zhigang Jin, Ellen Homola, Shelagh D Campbell *Biological Sciences, University of Alberta Germline cells often undergo developmentally programmed pre‐meiotic G2 phase arrest before initiating MI. In organisms such as X. laevis and C. elegans, immature oocytes arrest by a checkpoint mechanism involving inhibitory phosphorylation of the Cdk1 by the Myt1 kinase. In Drosophila, spermatocytes (not oocytes) undergo a prolonged G2 phase arrest and require Myt1 activity for male fertility. Using myt1 mutants generated in our laboratory we have now further characterized this role of Myt1. Surprisingly, loss of Myt1 activity did not markedly affect the timing of MI cell division. Instead, we discovered that Myt1 is required for maintaining an organelle checkpoint during pre‐meiotic G2 phase arrest. These findings provide new insights into cell cycle checkpoint mechanisms that spatially and temporally coordinate meiotic progression during Drosophila spermatogenesis. 62 6. Temporal and spatial control of the Smaug RNA binding protein in the early embryo Wen Xi Cao1, Alexander Marsolais2, Matthew Cheng2, Najeeb Siddiqui1, Craig Smibert1,2, Howard Lipshitz1 1) Molecular Genetics, University of Toronto, Toronto, Ontario, Canada 2) Biochemistry, University of Toronto, Toronto, Ontario, Canada Maternally loaded mRNAs control early embryo development in many animals. RNA binding proteins and small RNAs regulate the translation, stability and subcellular localization of these maternal mRNAs. The highly conserved RNA binding protein, Smaug (SMG), is synthesized upon egg activation, and is required for translational repression and degradation of a large subset of the maternal transcripts during the maternal‐to‐zygotic transition (MZT). Both SMG protein and smg mRNA are cleared at the end of the MZT although SMG is transported into, and persists in, the pole plasm and pole cells. We have shown that protein instability is directed by the C‐terminal portion of SMG whereas RNA instability is directed by elements in smg’s 3’ UTR. When either the C‐terminus of the protein or the 3’UTR elements are deleted, SMG protein persists in the soma beyond blastoderm cellularization. We are mapping both the protein and RNA instability elements; identifying the mechanisms and factors that confer instability; and investigating the biological functions of SMG clearance from the soma. In addition we are investigating the mechanisms and functions of SMG accumulation in the pole plasm. 63 7. Hemocyte‐pericardial cell interaction during the growth of the dorsal vessel Duygu Çevik, Megan Collie, Roger Jacobs Biology, McMaster University Drosophila melanogaster has a tubular heart called the dorsal vessel, which is composed of contractile cardiomyocytes and hemolymph filtering pericardial cells. During larval development the dorsal vessel (heart) grows in size, and the luminal space inside the heart expands, however it has not been clear which cells that surround the heart tube are responsible for laying the extracellular matrix (ECM) during this expansion. Hemocytes (white blood cells), pericardial cells and cells of the fat body are candidate cell types that may secrete ECM for assembly during the growth of the heart lumen. Studies of fluorescently tagged hemocytes in intact larvae reveal that hemocytes aggregate around pericardial cells of the dorsal vessel in 3rd instars. Confocal studies of dissected larval hearts indicate that hemocytes aggregate within infoldings of basement membrane associated with pericardial cells. With cell tracking and gene knock‐down techniques we are exploring whether hemocytes participate in assembly of the heart ECM at this location. Hemocyte‐pericardial cell association could indicate that hemocytes assemble ECM that is produced by pericardial cells or the pericardial cells could be a previously unidentified hemocyte stem cell niche. 64 8. Drosophila melanogaster as an animal model organism to study mutations of Histone H3.3 involved in glioblastoma Amel Chaouch1, Nada Jabado2, Paul Lasko1 Department of Biology, McGill University, Montreal, Quebec, Canada 1. Department of Biology, McGill University, Montreal, Quebec, Canada. 2. Departments of Human Genetics and Experimental Medicine, McGill University, Montreal,Quebec, Canada Drosophila melanogaster has proven to be a powerful model system to investigate the molecular bases of many human diseases. Recently, mutations in the H3F3A gene encoding the histone variant H3.3 were identified in whole exome sequencing of pediatric glioblastomas. These mutations create substitutions at either of two amino acids within the N‐terminal tail of the H3.3 located at critical positions implicated in major regulatory post‐translational modifications. Drosophila melanogaster, like human, has two genes encoding the same H3.3 protein, and these proteins are identical in flies and human. We have generated flies expressing these two mutations, H3.3 K27M and H3.3 G34R, using the GAL4‐UAS system for a targeted gene expression. Ubiquitous expression of either transgene causes complete lethality at the larval and pupal stage for the H3.3 K27M and the G34R mutations respectively. The expression of either transgene in the brain using the pan neuronal driver Elav‐GAL4 and the glial driver Repo‐GAL4 did not show any noticeable phenotype. However, expression of the H3.3 K27M mutation in muscles was lethal at the pupal stage, but no phenotype was observed with the H3.3 G34R mutation. H3F3A mutations in pediatric glioblastomas were associated with mutations in the ATRX‐
DAXX remodeling complex and TP53 gene. We are now investigating the over‐expression of the two mutated histone H3.3 K27M and G34R in association with the loss of expression of XNP and Dmp53 (the Drosophila homologs of ATRX and p53) and determine whether this can lead to the development of tumors. 65 9. REDUNDANT FUNCTIONS OF PI4KII AND PI4KIIIβ (Fwd) IN DROSOPHILA CELLS Tetyana Chorna1, Jason Burgess2, Julie Tan2, Karen Oh3, David R. Hipfner3 and Julie A. Brill1 1 Cell Biology Program, Hospital for Sick Children, Toronto, Ontario 2
Department of Molecular Genetics, University of Toronto, Ontario 3 IRCM, Montreal, Quebec Phosphatidylinositol 4‐kinases (PI4Ks) synthesize phosphatidylinositol 4‐phosphate (PI4P), which binds and recruits factors required for post‐Golgi trafficking. Drosophila melanogaster has three PI4Ks, single type II enzyme (PI4KII), PI4KIIIα and PI4KIIIβ (Fwd). Previously, our lab showed that PI4KII and fwd are non‐essential, as the null mutants are viable. Fwd localizes to the Golgi, where it is required for spermatocyte cytokinesis, but is dispensible for glue granule formation in salivary glands. PI4KII localizes to the Golgi and endosomes and is required during glue granule biogenesis, but is dispensible for spermatocyte cytokinesis. In contrast, PI4KIIIα is essential and generates a pool of PI4P at the plasma membrane that is needed for cortical integrity during oogenesis. Because Fwd and PI4KII are non‐
essential, we hypothesized that these enzymes might have overlapping functions. Thus, the main objective of this study was to investigate potential redundancy of Fwd and PI4KII in Drosophila. Consistent with our hypothesis, GFP‐Fwd and endogenous or mCherry‐tagged PI4KII localized near each other at the Golgi in salivary cells and spermatocytes. Double knockdown of fwd and PI4KII by RNAi in Drosophila S2 cells displaced a fluorescent PI4P marker (YFP‐PH‐FAPP) from the Golgi into the cytoplasm, indicating that the two PI4Ks are redundant for synthesizing Golgi PI4P. fwd PI4KII double mutants die shortly after pupariation. In double mutants and double mutant clones, localization of PI4P effector, Rotini, was cytosolic in salivary cells. In addition, double mutants showed severe Golgi phenotypes in egg chambers. Moreover, nurse cell membranes, which were not affected in fwd and PI4KII single mutant ovaries, appeared thinner and discontinuous in fwd PI4KII double mutant germ line clones, revealing overlapping roles of these enzymes in plasma membrane stability. Our results indicate that Fwd and PI4KII are redundant for synthesizing an essential pool of PI4P needed for Golgi and PM integrity in Drosophila. 66 10. dCAF1 is required for the cell death sensitivity of rbf1 mutant cells. Heather Collins, Nam‐Sung Moon McGill University, Biology Department, 1205 Avenue Docteur Penfield. Montreal, QC. H3A 1B1 The loss of the tumor suppressor Retinoblastoma (Rb) leads to cell proliferation and differentiation defects but also causes cell death in specific contexts. The Rb homologue in flies, rbf1, also produces these phenotypes when absent and offers a simpler system for studying the role of Rb in cell death. Components of the histone chaperone complex dCAF1 are upregulated in rbf1 mutants and are required for the stripe of cell death endogenous to rbf1 mutant eye imaginal discs. Here we show that this interaction extends to other developmental contexts, demonstrating that the role of dCAF1 in rbf1 mutant cell death is likely more general. We investigate the role of the pro‐apoptotic gene hid which was previously identified as a key determinant of cell death in rbf1 mutant cells. We are currently using chromatin immunoprecipitation to understand the mechanism by which hid expression may be regulated by dCAF1 to drive cell death susceptibility. 67 11. Examining Relationships Between Nitric Oxide, Iron and Ecdysone Biosynthesis Pendleton Cox and Kirst King‐Jones. Dept. of Biological Sciences, University of Alberta, Edmonton. Pulses of ecdysone, a steroid hormone, represent the principal driver of insect development. A key interest in our lab is how these ecdysone pulses are regulated. We recently found that a short pulse of nitric oxide (NO) in the larval prothoracic gland (PG), the principal source of larval ecdysone, precedes the major hormone pulse that triggers metamorphosis. Expressing NOS RNAi (NOSi) in the larval PG causes third instar larvae to arrest development. In addition, NOSi PGs are overgrown and exhibit a red‐
brownish color. Under UV light, NOSi PGs autofluoresce in a bright red, and this autofluorescence largely originates from mitochondria. We show here that this phenotype is caused by a buildup of heme precursors, suggesting that loss‐of NOS‐function impairs heme biosynthesis. Heme is required for the production of ecdysone, and by extension iron, a key component of heme, is also needed in large amounts. We thus hypothesize that NO acts as a cellular signal to ramp up iron availability and heme production prior to a major increase in ecdysone production. Previous work has established that NO can directly modulate the activity of the iron regulatory protein (IRP), and we are currently examining whether NO‐dependent IRP activation is required for an ecdysone peak to occur. We are also testing whether the requirement for NO can be bypassed, by activating IRP by other means, either by reducing dietary iron levels, or by providing active IRPs ectopically. Preliminary data showed that a reduction in 50% available iron rescued NOSi larvae to adulthood. We are now using holidic diets with defined iron concentrations to validate these findings. Finally, we are testing whether NO blocks ferritin mRNA translation in order to increase cellular iron availability. These results will establish for the first time whether NO regulates iron homeostasis in vivo to promote steroid hormone synthesis. 68 12. From an in vivo structure‐function analysis of Vasa to exploring protein‐protein interactions Mehrnoush Dehghani, Paul Lasko McGill University, Biology Department, Montreal, QC, Canada, H3G 0B1 The DEAD‐box RNA helicase Vasa (Vas) has been implicated in germ cell development in many species. In Drosophila, females homozygous for a hypomorphic allele, vas1, produce embryos which do not form germ cells and are not viable due to the defects in abdominal segmentation. In addition, vas‐null females, which rarely complete oogenesis, exhibit defects in mitotic progression of germ line stem cells, transposon silencing and Gurken translation. Multi‐functional proteins, such as Vas, often use different motifs in their sequence to interact with the other components in various pathways. Through a systematic mutagenesis approach we generated several egfp‐vas alleles with point mutations or deletions in different regions of Vas and studied their function in vivo. These analyses indicated that the divergent N‐ and C‐terminal sequences yet contain conserved motifs which play crucial roles in germ cell development, embryonic patterning and piRNA biogenesis. We have selected a subset of these vas alleles to be further characterized by different genetic and molecular approaches. In particular we are using a yeast two‐hybrid screen to identify proteins directly interacting with Vas and to compare them between wild type and the mutant alleles. Furthermore, the CRISPR‐Cas9 system has enabled us to generate genomic alleles of vas, which facilitate genetic interaction experiments, and mimic the expression pattern from the endogenous wild type gene. 69 13. Cyclin B involvement in the mitotic end process – Cytokinesis and Abscission Mélanie Diaz1, Charles Bruneau2 and Gilles Hickson1 1 CHU Sainte‐Justine Centre de recherche, Centre de cancérologie 2
Dept. molecular biology, Université de Montréal, Montréal, Québec, Canada A disturbance of the cell cycle can cause cancer. During cytokinesis, a contractile ring containing actin and myosin filaments, contracts and forms a midbody ring that drives cell separation via abscission. A Rho GTPase‐dependent protein network activates cytokinesis while cyclins‐Cdks inhibit cytokinesis. In Drosophila there are three mitotic cyclins: A/B/B3, which are successively degraded at the end of mitosis to permit cytokinesis initiation. Cyclin B appears to act as a central brake to cytokinesis since failure to degrade it at the metaphase‐anaphase transition derails cytokinesis (Parry ; O’Farrell, Curr Biol 2001). CycB also appears to control later events of abscission through poorly understood mechanisms involving the ESCRT‐III machinery and Aurora B kinase (Mathieu et al., 2013; Thoresen et al., 2014). Our aim is to determine how cyclin B acts during cytokinesis and abscission. We first generated Drosophila S2 cell lines expressing non‐degradable cyclin B (CycB‐stable) or wild‐type (CycB) coupled to GFP, and depleted endogenous CycB by RNAi to test the recruitment of each variant. Live‐cell spinning‐disc confocal microscopy indicated that CycB‐stable delays and slows the contraction of the contractile ring. Moreover, this CycB‐stable localized transiently at the early midbody, whereas wt CycB did not but rather appeared much later at the midbody ring immediately prior to abscission. Preliminary FRAP analysis indicates a dynamic localization of CycB at the late midbody. Consistent with previous studies, our results show an intervention of cycB during contractile ring closure and abscission. It is likely that the CycB interacts with different proteins in the two processes. This work will shed light on how cytokinesis and abscission are regulated in time and space by CyclinB. 70 14. AMP‐activated protein kinase protects against anoxia in Drosophila melanogaster Justin Evans, Chengfeng Xiao, Shuang Qiu, R. Meldrum Robertson Department of Biology, Queen’s University, Kingston, ON, Canada During anoxia, proper energy maintenance is essential in order to avoid neuronal damage. To reduce damage Drosophila melanogaster enter a neuroprotective coma which results in the arrest of neural and muscular activity. We examined the protective role of AMP‐activated protein kinase (AMPK), an evolutionarily conserved indicator of cellular energy change, during short anoxic stress. Post‐anoxia locomotion was used as an indicator of behavioural recovery. We found that,compared to controls, time to recovery was significantly reduced in AMPK activated treatments: percent reduction for starvation (32.7%), metformin (27.3%), AICAR (17.0%), and pan‐glial upregulation of the α‐subunit of AMPK (26.8%). In order to evaluate the neural mechanisms that speeded recovery of post‐anoxia locomotion, we measured direct current (DC) potential shifts, an indicator of ionic disturbance, in the brain during anoxia. The DC potential shifts, similar to mammalian anoxic events, are characterized by a modest positive shift preceding neural failure followed by a large negative shift coincident with neuronal depolarization and coma. We found that during anoxia AMPK activation was associated with a smaller area of ionic disturbance (7.5 mn; 1.5 mV x min) and a longer decay time (0.51 mn; 0.07) compared to controls (25.0 mn; 4.2 mV x min)( 0.10 mn; 0.05 min). This suggests that AMPK activation may enhance recovery by supplying energy to delay neural failure. These results are consistent with models that suggest AMPK enhances neuronal energy metabolism and supports the conclusion that the AMPK cascade is neuroprotective in Drosophila. 71 15. Stem cell niche homeostasis is dependent on a somatic permeability barrier around the differentiating germline Michael J. Fairchild, Lulu Yang, Guy Tanentzapf University of British Columbia ‐ Department of Cellular ; Physiological Sciences Interactions between the soma and germline are essential for gametogenesis. In the Drosophila testis germline stem cells (GSC) are arrayed around a cluster somatic cells termed the hub that acts as a stem cell niche. The hub maintains GSCs using a combination of adhesion proteins and secreted signalling molecules. The size of the hub determines the number the stem cells available to undergo spermatogenesis; however relatively little is known about how the size of the stem cell niche is regulated over the life of the organism. As germ cells leave the vicinity of the niche they are encapsulated by two somatic cyst cells and begin to differentiate. Using a permeability assay we find that the germline is isolated from the surrounding environment by the encapsulating somatic cells. This soma‐germline permeability barrier is dependent on both proper encapsulation and the formation of septate junctions by the somatic cells. Disruption of either results in the germline failing to differentiate due in part to an increase in the signaling range of stem cell maintenance factors that emanate from the stem cell niche. We additionally find that when the permeability barrier is disrupted the number of hub cells increases over time leading to a larger stem cell niche that maintains more GSCs. We demonstrate that the origin of new hub cells is from conversion of somatic cyst cells into hub cells. Furthermore we provide evidence that growth of the hub is due to disruption of the differentiating germ cells outside of the niche, indicating a potential feedback loop that links the outcome of stem cell function to stem cell niche homeostasis. Taken together our results reveal an ongoing feedback mechanism between germline differentiation and niche size. This mechanism may ensure a proper balance between stem cell differentiation and maintenance during an organism’s lifetime. 72 16. Fine‐tuning EGFR output through regulation of the Mid/Mirr molecular switch Mariana Fregoso Lomas1, Scott De Vito1, Jean François Boisclair Lachance2, Josée Houde1, Laura Nilson1. 1) Department of Biology, McGill University, Montreal, QC, Canada. 2) Ben May Department for Cancer Research, University of Chicago, Chicago, Il. We are generally interested in studying how tissues are patterned during development, and more specifically how the same signal can induce different cell fates within the same tissue. We focus on the determination of cell fate in the Drosophila ovarian follicular epithelium in response to localized activation of the epithermal growth factor receptor (EGFR). Interestingly EGFR activation generates different follicle cell fates in the anterior and posterior regions of this tissue, and these fates are associated with distinct transcriptional outcomes. We recently identified an important EGFR‐induced determinant of the posterior fate: the T‐box transcription factor Midline (Mid). At the anterior, EGFR activity does not induce Mid but instead induces the transcription factor Mirror (Mirr), a determinant of dorsal anterior fates. Therefore anterior and posterior regions represent two contexts with fundamentally different EGFR outputs, but why anterior and posterior cells respond differently to EGFR activity is not known. Here we identify two extrinsic inputs, TGF‐b and JAK/STAT pathway activation, that occur at different locations in the epithelium (anterior and posterior respectively) and influence the EGFR signaling outcome. We also show that activation of these pathways is integrated with EGFR activity at the level of their common targets, Mid and Mirr. A crucial part of our data shows that these two transcription factors can mutually repress each other and explain, in principle, how these two EGFR targets are mutually excluded within the tissue. Our preliminary data show that JAK/STAT signaling promotes posterior Mid expression through repression of Mirr, while TGF‐b pathway activation promotes anterior Mirr expression through repression of Mid. We propose that this reciprocal repression between Mid and Mirr acts as a molecular switch, and that which of these two EGFR targets is ultimately expressed depends on input from the JAK/STAT and TGF‐b signaling pathways. 73 17. Living the sweet life: the many benefits of glucose for Drosophila. Anthony Galenza, Jaclyn Hutchinson, Edan Foley University of Alberta Background. Host‐microbiome interactions are remarkably complex and have a profound impact on the wellbeing of the host. Disruptions to these interactions lead to chronic inflammation, autoimmune disease, and even cancer. In mammals, high microbe diversity, limited genetic tools, and long lifespans make this system difficult to study. The organization of the fly gut is remarkably similar to its mammalian counterparts and serves as a useful model for these interactions. In this project, I used a recently described chemically defined medium to investigate the effects that different dietary modifications have on the fly and its microbiome. Results. By deep‐sequencing microbial DNA isolated from fly guts, I found that different diets had a drastic impact on the composition of the intestinal microbiome. Longevity studies of flies on these different diets brought glucose to my attention. When raised on a medium with an increased level of glucose, flies lived longer, were more active, and even were more resistant to infection. Conclusions. This project demonstrates how important diet is to the hosts overall health. By taking a diet that contains all required nutrients and increasing the glucose, I saw numerous benefits to the flies. The diet of the host is essential to the interplay between the host and its microbiome. 74 18. Male‐specific programmed cell death underlies female‐specific generation of a motoneuron population in Drosophila Garner, SRC, Castellanos, MC, Allan DW Dept. Cellular and Physiological Sciences, 2350 Health Sciences Mall, Life Sciences Institute, University of British Columbia, Vancouver, Canada, V6T 1Z3 Differences between male and female behaviour is intimately associated with gender‐specific neuronal circuitry at a genetic and cellular level. In Drosophila melanogaster, the neuronal circuitry underlying courtship behaviour in males has served as a powerful model for identifying sexually dimorphic neuronal populations, but generation and function of the female‐specific neuronal populations has largely been underrepresented in the literature. A primary mechanism for constructing a male‐specific neuronal population is the programmed cell death (PCD) of specific neuroblast or neuronal populations in females, which is triggered by the female‐specific isoform of doublesex. In order to study the genetic and cellular mechanisms that construct a female‐specific circuitry, we look to a dimorphic set of motoneurons in females that are critical for egg‐laying, the post‐embryonic Ilp7 motoneurons. These neurons were identified in the posterior abdominal nerve cord and were found to be necessary for oviduct innervation and egg‐laying, a female‐specific behaviour. We present data that the Ilp7‐
motoneurons initially arise in both males and females, but subsequently undergo programmed cell death in males to give rise to the female‐specific subset. We will describe our exploration of the cellular and genetic regulatory mechanisms underlying male‐specific death. Our data provide the first evidence of a pro‐apoptotic role for the male‐specific splice isoform of the sex‐determination factor, fruitless, yet our allelic analysis of the fruitless locus points to novel genetic activities that are necessary for pro‐
apoptotic function. 75 19. PP2A dephosphorylates Yurt to control epithelial cell polarity Helori Gaudé and Patrick Laprise Département de Biologie Moléculaire, Biochimie Médicale et Pathologie and Centre de Recherche sur le Cancer, Université Laval, and 2 Axe Oncologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Québec, Québec G1R 3S3, Canada. Epithelial cell polarity is characterized by the asymmetric distribution of many cellular constituents, thereby defining apical, lateral and basal membrane domains. The polarized architecture of epithelial cells sustains epithelial tissue physiology and morphogenesis. During fly embryogenesis, Yrt is dynamically excluded from the apical domain by aPKC‐dependent phosphorylation in maturing epithelial cells. This allows the Crb and aPKC‐containing apical machinery to assemble and expand the apical domain. During terminal differentiation of epithelial tissues, Yrt is no longer phosphorylated and is recruited apically by Crb. This defines a negative feedback loop preventing over‐elongation of the apical membrane. The precise temporal control of Yrt phosphorylation is thus crucial for the establishment and maintenance of epithelial cell polarity. Hence, identification of the phosphatase dephosphorylating Yrt and opposing aPKC signaling is an outstanding puzzle to be solved to delineate the molecular mechanisms regulating epithelial cell polarity. The heterotrimeric serine‐threonine phosphatase PP2A is an interesting candidate. Indeed, PP2A opposes aPKC and Par‐1 signaling to control the polarity of neuroblasts and photoreceptor cells. However, the role of PP2A in sustaining apical‐basal polarity in simple epithelia remains poorly defined. We show that endogenous Yrt and the structural subunit of PP2A (PP2A‐A) form a complex in fly embryos. Moreover, chemical inhibition of PP2A with cantharidin or mutation of the PP2A‐A gene increases Yrt phosphorylation in mature epithelia (stages 15‐17). This demonstrates that PP2A is required for Yrt dephosphorylation in vivo and suggests that Yrt is a substrate of this phosphatase. Our data also establish that mutation of PP2A‐A enhances the yrt hypomorphic phenotype, resulting in the formation of convoluted cuticle extensions. This indicates excessive apical membrane growth, as observed in yrt null embryos. Together, our results suggest that PP2A plays a role in epithelial cell polarity regulation by dephosphorylating and activating Yrt. 76 20. Deciphering the Role of Phospholipase A2 in Infantile Neuroaxonal Dystrophy and Related Neurodegenerative Disorders Oxana Gluscencova1, Konstantin Iliadi1 and Gabrielle Boulianne1,2 1
the Hospital for Sick Children, Toronto 2
Department of Molecular Genetics, University of Toronto, Ontario, Canada Infantile neuroaxonal dystrophy (INAD) is a fatal neurodegenerative disorder that typically begins within the first few years of life and leads to progressive impairment of movement and cognition. The pathological hallmark of the disease is the presence of large axonal spheroids in the brain, spinal cord and peripheral nerves. These spheroids accumulate material normally found in axons and nerve terminals. Several years ago, it was shown that ~80% of patients with INAD have mutations in the phospholipase gene, PLA2G6. Interestingly, mutations in PLA2G6 have also been shown to be causative in two other related neurodegenerative diseases. Atypical neuroaxonal dystrophy (aNAD) gives rise to similar phenotypes as those observed in INAD but develops during early childhood to late teens while Dystonia‐parkinsonism develops between 20‐30 years of age. PLA2G6 encodes a group VIA calcium‐
independent phospholipase A2‐beta, which catalyzes the hydrolysis of glycerophospholipids to produce fatty acids and lysophospholipids. Although mutations in PLA2G6 are expected to have a significant negative impact on the synthesis of lipid mediators and membrane homeostasis, the precise mechanisms underlying disease pathogenesis are unknown. Here, we are using Drosophila to gain insight into the normal and pathological functions of PLA2G6 using a series of genetic, molecular and biochemical tools. We present selected results of the functional characterization of Drosophila Calcium‐
Independent Phospholipase A2. 77 21. Regulators or Non Muscle Myosin II Regulate Wg Targets Elizabeth Hoesing, Eric Hall, Esther Verheyen Simon Fraser University Wnt, or Wingless (Wg) in Drosophila, is a morphogen conserved in all metazoans that can signal through either canonical or non‐canonical pathways. Canonical Wg signaling affects a wide range of processes including cell fate determination, primary axis formation, stem cell renewal and organogenesis. It is tightly regulated by a series of phosphorylation events so kinases and phosphatases play important roles in its activity. To explore this regulation, the Verheyen lab conducted a comprehensive in vivo RNAi screen of the Drosophila kinome and phosphatome. The screen identified several non‐muscle myosin II (MyoII)‐associated proteins as potential modulators of Wg target genes. One such protein is the myosin phosphatase targeting subunit (MYPT)‐75D. MYPT‐75D binds specifically to the catalytic subunit of a Protein Phosphatase 1 isoform PP1β. Through MYPT‐75D, PP1β can then dephosphorylate MyoII resulting in MyoII inactivation. This occurs mainly in the periphery of the cells affecting the actomyosin network in these regions. Immunohistochemistry following MYPT75D over‐expression and RNAi knockdown showed perturbation of Wg targets, while total Wg protein levels appear unaffected. Knockdown of the myosin II regulatory light chain, spaghetti‐squash (sqh) also show similar disruptions to Wg targets. Though PP1 β was previously shown to affect Wnt signaling, no mechanism has yet been described for how this may occur. Given the role of MyoII and the observation that both Myo II and its regulatory proteins can influence Wg targets, we aim to elucidate when and how actomyosin itself may be involved in the modulation of Wg signal transduction. 78 22. Mapping of Anillin Binding sites on F‐actin Silvana Jananji2, Robert Van Sciver1, Guillaume Laflamme4, Louis‐Philippe Picard2, Abe Albaghjati2, Gilles R. X. Hickson2,3,5, Benjamin H. Kwok4,5 and Vitold E. Galkin1,5 1. Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA 23507, USA 2. Sainte‐Justine Hospital Research Center, 3175 Chemin de la Côte Ste‐Catherine, Montreal, QC H3T 1C5, Canada. 3. Département de Pathologie et Biologie Cellulaire, Université de Montréal, P.O. Box 6128, Station Centre‐Ville, Montréal, QC H3C 3J7, Canada. 4. Institute for Research in Immunology and Cancer (IRIC), Département de médecine, Université de Montréal, P.O. Box 6128, Station Centre‐Ville, Montréal, QC H3C 3J7, Canada. Cytokinesis requires an assembly of the actomyosin contractile ring (CR) which promotes daughter cell segregation. Assembly of the ring is tightly controlled in space and time. Anilin is a scaffolding protein that is involved in organizing contractile ring components including filamentous actin, myosin and septins. Anillin has been shown to bundle actin filaments, but molecular details of its interaction with F‐
actin remains unknown. We used electron microscopy and image analysis to rigorously evaluate the interaction of anillin actin bundling domain (BD) with F‐actin. We show that anillin BD harbors three actin binding sites. These sites bind to multiple places on the surface of the actin filament in a mutually exclusive fashion. We show that polymorphic binding of anillin BD to F‐actin allows anillin to form different types of bundles out of F‐actin. Importantly, we demonstrate that the bundling activity of the anillin fragments is essential for their localization in the cortex and cleavage furrow of the dividing cells. Our structural data suggests that anillin can function as an actin remodeling factor in the contractile ring upon cytokinesis. 79 23. Possible functions of obscurin in non‐muscle tissue Anja Katzemich and Frieder Schoeck McGill University Obscurin is a large, multi‐domain protein, originally identified in vertebrate striated muscles and part of the titin family. Members of this family are made up of tandem immunoglobulin (Ig) and fibronectin‐like (Fn3) domains and can have one or two kinase domains near the C‐terminus. Other signaling domains, such as Rho guanine nucleotide exchange factors (Rho‐GEFs), can also be present. In the muscle sarcomere, obscurin predominantly localizes to the M‐line and plays essential roles in myofibrillogenesis, cytoskeletal organization, and Ca2+‐homeostasis. Obscurin has a similar modular structure in invertebrates and vertebrates, but the number of modules and the position of signaling domains vary. Drosophila obscurin has SH3 and Rho‐GEF signaling domains at the N‐terminus and two kinase domains at the C‐terminus, as well as 21 Ig‐domains. With the generation of specific antibodies directed against epitopes in the N‐ and C‐terminus as well as the middle of the protein, we have undertaken a systematic approach to characterize the expression profile of obscurin in muscle and non‐
muscle tissue of Drosophila. So far, five large isoforms have been detected in Drosophila muscles with a size of ~475 kDa. Isoforms below 250 kDa were not detected in adult or larval muscles. Smaller isoforms, ranging from 250 kDa to 48 kDa, were predominantly expressed in non‐muscle tissue, such as the salivary glands, as well as in the early and late stages of the embryo. Different isoforms are present in different subcellular locations, including epithelial membranes and nuclei, suggesting additional roles for obscurin in nuclear architecture and epithelial organization. Furthermore, obscurin co‐localizes with non‐muscle myosin in blastoderm embryos. Co‐immunoprecipitation demonstrated that both proteins are associated. Currently, mutagenesis approaches including the CRISPR/Cas9 system are being undertaken, in order to further analyze the function of obscurin in different tissues and cellular processes. 80 24. The role of Anillin in coordinating closure of the contractile ring with maturation of the midbody during cytokinesis Amel Kechad, Silvana Jananji and Gilles Hickson CHU Sainte Justine . Université de Montréal Cytokinesis is the fundamental conserved process by which a cell physically separates into two. It occurs in distinct steps. First, signalling between the mitotic spindle (MS) and the cortex provides cues to position the cleavage furrow. Then, the cortex undergoes complex remodelling events and an acto‐
myosin contractile ring (CR) is assembled to constrict the cell at its equator. After the CR constricts, it matures into a more stable structure, the midbody ring (MR), while the spindle midzone matures to form the midbody (MB), which together with the MR comprises the intercellular bridge that link sister cells until abscission. Maturation of microtubule structures (MS, MB) and cortical structures (CR, MR) appear to occur together, through poorly understood mechanisms. Anillin is a scaffold protein essential for MR formation. We hypothesise that Anillin also coordinates the maturation of the MB. Anillin binds many other cortical and MB proteins, including RacGAP50C/Tum via its conserved C‐terminus. Preliminary results show that Anillin is required to maintain the RacGAP50C at the MB of Drosophila S2 cells. Furthermore, analysis of RacGAP50C truncation mutants revealed that the Anillin binding region of RacGAP50C is required for CR formation and that this function depends on Anillin. Ongoing studies will test the role of the interaction between Anillin and RacGAP50C and determine whether it is involved in coordinating CR closure with maturation of the microtubule‐based MB. This will further our understanding of the complex and robust molecular circuitry of cytokinesis. 81 25. Investigating HOX protein requirement for tarsus determination in Drosophila melanogaster Samantha Koot, Anthony Percival‐Smith University of Western Ontario Understanding how evolutionarily conserved processes work in the model organism, Drosophila melanogaster fruit fly, can help to explain how these processes work in other eukaryotes, including humans. Many aspects of early development are regulated by Hox genes, which are arranged in conserved clusters and involved in controlling pattern formation that results in segmental identity. In Drosophila, Sex‐combs reduced (Scr), Antennapedia (Antp), and Ultrabithorax (Ubx) are required for the identity of thoracic segments T1, T2, and T3, respectively. It is yet unclear which HOX activity, SCR or ANTP or both, are required for leg tarsus determination and the development of all legs of Drosophila and whether UBX has a redundant role in leg development. A heat shock‐inducible nanobody, a very small form of an antibody, was developed to specifically recognize and degrade the green fluorescent protein (GFP). The anti‐GFP nanobody degrader is used to degrade GFP‐tagged SCR, ANTP, and UBX proteins during larval development in all cells. These were created using CRISPR (clustered regularly interspaced short palindromic repeats) which causes targeted double‐stranded breaks, and homologous recombination (HR). With expression of the nanobody and degradation of HOX::GFP fusion proteins, I will be able to determine through phenotypic analysis in Drosophila whether ANTP is required for leg development, or whether SCR is required instead. In addition, I can also determine whether ANTP, UBX and SCR are all required or whether no HOX protein is required for tarsus determination in Drosophila and whether this requirement is conserved. 82 26. Cell cycle control by the Greatwall‐PP2A axis Myreille Larouche, Peng Wang, Maxime Cormier & Vincent Archambault Institut de recherche en immunologie et en cancérologie, Département de biochimie et biologie moléculaire, Université de Montréal The cell division cycle is regulated by reversible phosphorylation. Cyclin B‐Cdk1 triggers the events of mitotic entry including chromosome condensation, spindle assembly and nuclear envelope breakdown, by the phosphorylation of multiple substrates. At mitotic exit, several of these proteins are dephosphorylated. The Protein phosphatase 2A in complex with its regulatory subunit B55/Tws (PP2A‐
Tws) plays a major role in this process. To facilitate the accumulation of phosphorylated Cdk1 substrates, PP2A‐Tws is inhibited at mitotic entry by a system recently discovered in Drosophila by us and others. The Greatwall (Gwl) kinase, following its activation by Cdk1, phosphorylates Endos which then acts as a potent and specific competitive inhibitor of PP2A‐Tws. How this pathway is regulated in time and space is not completely understood. While PP2A‐Tws is concentrated in the cytoplasm, Gwl is enriched in the nucleus in interphase. We found that, following its activation by Cyclin B‐Cdk1 at the G2/M transition, Gwl suddenly relocalizes to the cytoplasm just a few minutes before nuclear envelope breakdown. This spatial regulation of Gwl is essential for its function, and we wish to understand why. In addition, we are dissecting the molecular mechanisms mediating the changes in Gwl localization at mitotic entry and mitotic exit. Our work deciphers the Gwl‐Endos‐PP2A module, which was an important missing link in our understanding of the basic cell cycle machinery common to many eukaryotes including humans. This knowledge serves as a basis to better understand aberrant cell division in cancer and envision new therapeutic avenues. 83 27. Alp/Enigma family proteins cooperate in Z‐disc formation and myofibril assembly Kuo‐An Liao and Frieder Schöck Department of Biology, McGill University, 1205 Dr. Penfield Avenue, Montreal, Quebec H3A 1B1 Zasp (Z‐band alternatively spliced PDZ‐motif protein) is a member of the Alp/Enigma family, which comprises Enigma, ENH, ZASP, Alp, CLP36, RIL, and Mystique in vertebrates. In Drosophila, Zasp52 colocalizes with integrins to myotendinous junctions and with alpha‐actinin to Z‐discs, and is required for muscle attachment and Z‐disc assembly and maintenance. There is additionally a closely related PDZ‐
only domain protein in Drosophila, called Zasp66, which also localizes to Z‐discs. Here we show that Zasp52 and Zasp66 precisely colocalize at the Z‐disc and that depletion of Zasp66 by RNAi causes mild defects in Z‐disc structure. Additionally, knockdown of Zasp66 with one copy of Zasp52 removed causes higher pupal lethality than knockdown of Zasp66 alone, demonstrating a strong genetic interaction. Finally, the Zasp52 Zasp66 double mutant shows higher embryonic lethality than the Zasp52 mutant alone, suggesting that these two proteins act partially redundantly in Z‐disc assembly. To assess the mechanism of this redundancy, we have started to analyze proteins interacting with Zasp52 and Zasp66 by affinity purification and mass spectrometry. In particular, we demonstrate that both Zasp52 and Zasp66 biochemically interact with alpha‐actinin, which tethers thin filaments to the Z‐disc. This interaction appears to be mediated by the PDZ domain, which is present in both Zasp52 and Zasp66. In this report, we also show that Zasp52 directly binds to Zasp52 and Zasp66 through the Zasp motif. To test the dimerization of Zasp52, we performed a chemical cross‐linking assay and blue native gel electrophoresis. Both assays demonstrate Zasp52 forms dimers under reducing (physiological) conditions. Given Zasp52 null mutant larvae show a more severe defect in myofibril assembly compared to alpha‐
actinin null mutant larvae, we have reevaluated the role of Zasp52 in myofibril assembly, particularly actin thin filament assembly, and will discuss our results. 84 28. Stepping stone: a cytohesin adaptor for membrane cytoskeleton restraint in the syncytial Drosophila embryo Jiangshu Liu, Donghoon M. Lee, Caoguo Yu, Tony J.C. Harris Department of Cell and Systems Biology, University of Toronto Stephane Angers Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy Department of Biochemistry, University of Toronto Cytohesin Arf‐GEFs are conserved plasma membrane regulators. The sole Drosophilia cytohesin, Steppke, restrains Rho1‐dependent membrane cytoskeleton activity at the base of plasma membrane furrows of the syncytial embryo. By mass spectrometry, we identified a single major Steppke‐interacting protein from syncytial embryos, which we named Stepping stone (Sstn). By sequence, Sstn seems to be a divergent homologue of the mammalian cytohesin adaptor FRMD4A. Our experiments supported this relationship. Specifically, heterophilic coiled‐coil interactions linked Sstn and Steppke in vivo and in vitro, whereas a separate C‐terminal region was required for Sstn localization to furrows. Sstn mu­tant and RNAi embryos displayed abnormal, Rho1‐dependent membrane cytoskeleton ex­pansion from the base of pseudocleavage and cellularization furrows, closely mimicking Step­pke loss‐of‐function embryos. Elevating Sstn furrow levels had no effect on the steppke phenotype, but elevating Steppke furrow levels reversed the sstn phenotype, suggesting that Steppke acts downstream of Sstn and that additional mechanisms can recruit Steppke to furrows. Finally, the coiled‐coil domain of Steppke was required for Sstn binding and in addi­tion homodimerization, and its removal disrupted Steppke furrow localization and activity in vivo. Overall we propose that Sstn acts as a cytohesin adaptor that promotes Steppke activ­ity for localized membrane cytoskeleton restraint in the syncytial Drosophila embryo. 85 29. Elucidating the mechanism by which mechanical force stabilizes Cell‐ECM adhesion during development. Pablo Lopez‐Ceballos1, Katrin Hakonardottir1, Alejandra Herrera‐Reyes2, Daniel Coombs2 and Guy Tanentzapf1. 1 Department of Cellular and Physiological Scicences, University of British Columbia. 2 Department of Mathematics,University of British Columbia. Cells in multicellular organisms are arranged in complex‐three dimensional shapes. To achieve such complexity, cells must form adhesive contacts with the extracellular matrix (ECM). During early embryonic development Cell‐ECM adhesions are typically dynamic and transient but later on, as tissue architecture is consolidated, they become stable and long lasting. We are interested in the mechanisms that regulate this developmental transition. The principal mediators of Cell‐ECM adhesion are the integrin family of transmembrane adhesion receptors. Integrin‐based adhesions undergo assembly and disassembly, or turnover; this provides an important means of regulating the duration and strength of Cell‐ECM attachment. We are particularly interested in how the turnover of Cell‐ECM adhesion is controlled by mechanical force experienced by cells. To investigate the turnover of the integrin adhesion complex in vivo we perform FRAP on the myotendionous junctions (MTJs) in live embryos and use conditional mutants to alter the force acting on the MTJs. Using this approach in combination with mathematical modeling we elucidate the kinetics of adhesion complex turnover. Our analysis has uncovered a role for mechanical force in regulating integrin turnover and in stabilizing cell adhesions over the course of development. We hypothesized that conformational changes in Cell‐ECM adhesion proteins transmit mechanosensory information downstream of integrin to regulate adhesion‐complex turnover. Our work provides mechanistic insight into how the transition from dynamic to stable Cell‐
ECM adhesion is achieved at the end of embryogenesis and suggests that mechanical force can act to stabilize and maintain tissue architecture over the lifetime of the organism. 86 30. PKCδ regulates actin protrusions of migrating border cells in the Drosophila ovary Jing Lu, Felix Gunawan and Dorothea Godt University of Toronto The border cell cluster (BCC) in the Drosophila ovary allows us to study the molecular mechanisms that regulate cell migration in vivo. During mid‐oogenesis, the BCC, which contains approximately six migratory rosette cells, delaminates from the follicular epithelium and migrates toward the oocyte with the help of actin‐rich cellular protrusions. Our lab previously showed that the large Maf transcription factor Traffic jam (Tj) contributes to the regulation of BCC migration. A microarray screen for putative Tj targets in BCCs suggests that expression of PKCδ, a member of the protein kinase C family, is sensitive to changes in Tj expression level. Our work suggests that PKCδ is involved in regulating the organization of the actin cytoskeleton in the cellular protrusions of rosette cells. To determine the expression pattern of PKCδ in Drosophila ovaries, I generated antibodies that target PKCδ, and examined immuno‐stained BCCs with increased and reduced expression of PKCδ, respectively. BCCs that overexpressed PKCδ showed an enrichment of the immuno‐signal in the cortical region, as well as in some cellular protrusions. To address whether the effects of Pkcd depletion or overexpression on rosette cell behavior are cell‐autonomous or not, we currently conduct a clonal analysis. A quantitative analysis of the compactness, actin organization and protrusive activity of mutant rosette cells and their location within a mosaic BCC will give a better understanding of the function of PKCδ in BCC migration. 87 31. Roles of PI4P in glue granule biogenesis. Cheng‐I Jonathan Ma1,2, Changhua C. Chen1,3, Jason A. Burgess4, Julie A. Brill1,2,4 1. Program in Cell Biology, The Hospital for Sick Children, Toronto, ON,M5G 1L7 2. Institute of Medical Science, University of Toronto, Toronto, ON,M5S 1A8 3. Department of Biochemistry, University of Toronto, Toronto, ON,M5S 1A8 4. Department of Molecular Genetics, University of Toronto, Toronto, ON, M5S 1A8 Phosphatidylinositol 4‐kinases (PI4Ks) are responsible for production of the lipid phosphatidylinositol 4‐
phosphate (PI4P), a crucial resident of Golgi membranes that regulates membrane trafficking events such as secretion. We have discovered that PI4KII but not fwd function is required for normal development of the Drosophila larval salivary gland. In PI4KII mutants, mucin‐containing glue granules were considerably smaller than in wild type. Furthermore, some of the glues were mistrafficked to enlarged late endosome and lysosomes. The glue granule associated SNARE SNAP24 was also found on enlarged vesicles in PI4KII mutants. To further examine the role of PI4P and endocytic machinery in glue granule biogenesis, I employed a reverse genetic screen to identify potential genetic interactions using publicly available transgenic RNAi lines. A selection of candidate genes including Rab small GTPases, SNAREs, and various endocytic proteins were screened. 9 Rab GTPases resulted in either small or improper glue granule production after RNAi knock down. I am currently investigating the localization of these Rab GTPases in the larval salivary gland and I am overexpressing the dominant negative mutant proteins to see whether I could recapitulate the RNAi phenotype. I have also identified a number of endocytic and Golgi SNAREs that are important for proper glue granule formation. Interestingly, PI4KII was found on enlarged vesicles when Syntaxin‐7 and ‐16 were knocked down, which further suggests co‐
regulation between PI4KII and these SNAREs. Moreover, another positive hit SNAP29 localized to glue granules when overexpressed in wild type and I am currently investigating the localization of SNAP29 in our PI4KII mutant. In conclusion, I have identified a number of potential regulators of secretory granule biogenesis and I plan to further examine these hits to determine whether they are PI4KII dependent and whether they are directly involved in the biogenesis of glue granules. 88 32. Identification of de2f1 regulatory elements required for imaginal disc development Mary‐Rose Bradley‐Gill, Minhee Kim, Christine Yergeau, Nam‐Sung Moon McGill University E2F family transcription factors are evolutionarily conserved regulators of the cell cycle that are required for development in most species. E2F proteins can be divided into two groups based on their ability to either activate or repress transcription. While most research on E2Fs has focused on regulating E2F activity during the cell cycle, little is known about how its expression is controlled during development. In Drosophila, there is only one activator E2F, dE2F1, which provides all the pro‐proliferative activity of E2F. Interestingly, the de2f1 gene can be transcribed from multiple promoters resulting in six alternate transcripts. We focused on the de2f1 promoter region named RA. The levels of transcripts originated from this region increase at the larval stage when the fly primarily increases its body mass. Analysis of GFP reporters covering different segments of the RA region revealed tissue specific enhancer activities. Genomic deletion of the regulatory elements that drive expression of de2f1 in imaginal discs severely decreased the size of the discs demonstrating the functional importance of this region. Overall, our study identifies the de2f1 transcript and regulatory element that are required for the proliferation of imaginal discs and demonstrates that transcription of de2f1 is highly regulated during development. 89 33. Power loss: the PGC‐1α homologue spargel as a model of Parkinson Disease in Drosophila melanogaster Eric M. Merzetti, Brian E. Staveley Memorial University of Newfoundland Parkinson Disease (PD) is a severe and progressive neurodegenerative disorder resulting in symptoms of bradykinesia, rigidity, resting tremor, postural instability and eventual decline of cognitive functions including memory. PD symptoms are caused by a lack of dopamine, a neurotransmitter, present in the striatum of the brain. This decrease is a by‐product of impaired function or premature death in dopamine producing neurons. Current understanding of PD provides therapeutic options to combat symptoms of disease but does not address the underlying cause of these symptoms. Dopaminergic neurons have high energetic requirements and improper mitochondria function has been linked to a number of disease phenotypes. Mitochondrial repair is a complex process partially regulated by the genes Pink1, Parkin, and the peroxisome proliferation activated co‐receptor gamma (PGC) family of proteins. Mammalian PGC‐1α has been characterised as a modulator of mitochondrial biogenesis. In mammals, the PGC family contains three genes with partial functional redundancy, making it difficult to study the effects of gene alteration in cells. In Drosophila the only PGC gene family member present is the PGC‐1α homologue spargel (srl) which codes for a 1,088 amino acid protein with 68% homology in the COOH‐terminal RNA‐binding motif, shares an arginine‐serine rich domain, an acidic NH2 terminal domain and a leucine‐rich domain with the mammalian PGC‐1α. We have investigated the effect of altered srl activity in dopaminergic neurons. Surprisingly, tissue specific expression of srl‐RNAi leads to a significant increase in mean lifespan while tissue specific expression of srl‐EY in dopaminergic neurons causes a decrease in mean lifespan and locomotor ability, indicating a new model of PD. This model may be useful in identifying the relationship of other genes and their role in mitochondrial biogenesis and degeneration. Funded by a School of Graduate Studies Fellowship to EMM and an NSERC Discovery Grant to BES. 90 34. Investigating the Pathological Mechanism of Spinal and Bulbar Muscular Atrophy via Androgen Receptor Humanized Fly Model Shaza Mokhtar1, 2, Miltiadis Paliouras1, 2, 3, Mark Trifiro1, 2, 3 1) Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec 2) Department of Human Genetics, McGill University, Montreal, Quebec 3) Department of Medicine, McGill University, Montreal, Quebec Introduction: The function of androgen receptor (AR) in disease pathology is defined by its mutational status and androgen hormone dependent activity. A number of loss‐of‐function mutations that are linked to the disorder known as Androgen Insensitivity Syndrome, while gain‐of‐function mutations are commonly found in prostate cancer. There is another class of mutations in AR, an expansion of a polymorphic tricnucleotide CAG repeat, coding for a polyglutaminde tract in exon 1 of the the AR gene. Normally, the CAG tract 20 repeats, whereas in the disease known as spinal and bulbar muscular atrophy (SBMA) the repeat is expanded to greater than 37 repeats. SBMA is an X‐linked recessive neurodegenerative, trinucleotide repeat disorder. The toxic AR gain‐of‐
function that is characteristic of SBMA results in a slow progressive muscle weakness, dysarthria ; dysphagia, reduced fertility, gynecomastia. Aims of the Study: We will study the role of polyQ‐expanded‐AR (polyQ‐AR) in RNA splicing by using AR‐
humanized Drosophila‐GAL4 genetic model that allows us to exogenously express the polyQ‐AR in selective fly tissue/organs. Results: A proteomics‐coupled‐systems biology approach was used to chatracterize proteome of wild‐
type‐AR vs. polyQ‐AR complexes, and have identified a number of androgen‐dependent protein interactors involved in RNA splicing. PolyQ‐AR flies have been crossed with RNAi and homologous fly genes encoding polyQ‐AR candidate protein interactors, to identify the enhancers or suppressors of polyQ‐AR phenotype. Conclusion: AR functionality has been extended beyond its classical role as a transcription factor, to include RNA splicing, suggesting that the complexity of the AR‐interacting protein involve in different pathways that impact on AR biological outputs. Studying the role of AR in RNA binding and splicing is a novel discovery. Furthermore, using a Drosophila as a genetic interaction screen, will help us to understand the mechanism that is involved in SBMA disease. 91 35. Basigin as an integrin co‐receptor regulating glial‐ECM adhesion Lindsay Petley‐Ragan1, Mriga Das1, Catharine Rankin2 and Vanessa Auld1 1. Department of Zoology, University of British Columbia 2. Department of Psychology, University of British Columbia Glial cells ensheathe neurons to insulate the transmission of electrical signals and protect neurons against pathogens and physical stress. A basement membrane containing extracellular matrix (ECM) proteins such as perlecan, collagen and laminin covers the glia and encapsulates the entire Drosophila nervous system. The basement membrane is essential for the structure and function of the nervous system, however the specific roles of each ECM component and their respective receptors are still being investigated. We have investigated the role of basigin as a regulator of integrin binding to the ECM in the Drosophila nervous system. Integrin is a well‐established receptor of ECM components and basigin has been demonstrated to genetically interact with integrin during dorsal closure. Basigin is a transmembrane protein with two extracellular Ig domains and a small intracellular domain. Our PLA data suggests that basigin and integrin bind specifically in the perineurial glia of the Drosophila peripheral nervous system (PNS). Knockdown of basigin using RNAi in glia has demonstrated that basigin is a critical regulator of the structure and function of the larval nervous system. Analysis using fluorescent and electron microscopy has demonstrated that knockdown of basigin results in glial folds or ruffles in the PNS likely due to a collapsed actin cytoskeleton. We have determined that proteins associated with the integrin adhesion complex are located in the glial ruffles and we have investigated whether basigin knockdown affects the ability of integrin to bind ECM components such as collagen and perlecan. Additionally, we have expressed mutant basigin constructs in glia and determined which mutations affect the interaction between basigin and integrin and overall morphology of the nervous system. Our data explores a novel role for basigin in mediating glial‐ECM binding and highlights the importance of regulation of specific ECM‐receptor binding for the structure and function of underlying tissues. 92 36. Nuclear receptor DHR51 regulates ecdysone and heme biosynthesis in Drosophila melanogaster Brian Phelps and Kirst King‐Jones Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada Steroid hormones are signaling molecules that regulate developmental transitions such as puberty in humans and metamorphosis in insects. While the actions of steroid hormones are well understood, less is known about the regulatory pathways that control steroid hormone production. Recently, our lab began investigating the role of heme in the regulation of insect steroid hormone (ecdysone) production, since heme acts as a prosthetic group in most steroid hormone‐producing enzymes. Steroid hormone‐
producing glands may anticipate the need for heme in order to synthesize adequate amounts of steroidogenic enzymes. In addition, when cellular heme levels are too low, cells must be able to respond and upregulate heme biosynthesis, suggesting the existence of a heme‐sensing mechanism. We show here that DHR51 (Drosophila hormone receptor 51), a nuclear receptor, is a likely candidate for such a heme sensor in the steroid hormone‐producing gland. Specifically, RNA‐Seq analysis of steroid hormone‐
producing glands showed that loss‐of‐DHR51 resulted in a similar transcription response when compared to disrupting a heme enzyme via RNAi, indicating that interfering with DHR51 function impairs heme biosynthesis. We show that the induction of the rate‐limiting enzyme of heme biosynthesis, ALAS, is dependent on DHR51. The RNA‐Seq data also showed that key genes associated with steroid hormone production were downregulated when DHR51 function was impaired. Feeding larvae ecdysone in their diet rescued the developmental defects associated with loss‐of‐DHR51, suggesting that DHR51 was necessary for steroid hormone production. Taken together, DHR51 may coordinately regulate steroid hormone and heme biosynthesis, allowing steroidogenic cells to adapt their heme requirements prior to the formation of a steroid hormone pulse. 93 37. Characterization of heterotrimeric G Protein‐dependent signaling in the Hedgehog pathway Samantha D. Praktiknjo1,2, Dominic Maier1,2, Karen Oh1, David R. Hipfner1,2,3 1
Institut de recherches cliniques de Montréal (IRCM), Montreal, Quebec, Canada 2
Department of Anatomy ; Cell Biology, McGill University, Montreal, Quebec, Canada 3
Department of Medicine, Université de Montréal, Montreal, Quebec, Canada The Hedgehog signaling pathway has a central role in embryonic development and is required for stem cell maintenance and tissue homeostasis. Misregulation of Hedgehog signaling has therefore been related to a variety of diseases and cancers. In flies, Hedgehog signaling is activated by multisite phosphorylation of the G protein‐coupled receptor (GPCR)‐like seven‐pass transmembrane protein Smoothened in its cytoplasmic C‐terminus by the cAMP‐dependent Protein kinase A and Casein kinase I. Once activated, Costal‐2 and Fused associate with Smoothened at specific sites and regulate downstream signaling controlling target gene expression. We have recently shown that phosphorylation at a highly conserved core region of Smoothened by Gprk2, one of the two GPCR kinases in flies, is essential for high‐threshold target gene expression and for the recruitment of Costal‐2. To assess to what extent Smoothened might actually function as a GPCR, we investigated the ability of Smoothened to couple to heterotrimeric G Proteins to control cAMP production (via Gαs or Gαi). We found that upon activation in response to Hedgehog, Smoothened significantly increased cellular levels of cAMP in a Gαs‐
dependent manner. These changes occurred gradually over a span of a few hours, matching the time course of Smoothened phosphorylation and activation. Similar results were obtained by using a C‐
terminally truncated form of Drosophila Smoothened consisting only of the highly conserved core region. Interestingly, a Gprk2 non‐phosphorylatable form of Smoothened had the opposite effect, causing cAMP levels to drop below baseline in a Gαi‐dependent manner. This suggests that Smoothened can couple to either Gαs or Gαi, with the choice of effector determined by Gprk2 phosphorylation. Our results suggest that G protein‐dependent signaling is an important and common feature of the Hedgehog pathway. As this mechanism appears to be highly conserved, further characterization could provide significant insights into the fundamental mechanisms of this signaling pathway. 94 38. Spatio‐temporal characterization of peroxin expression patterns during D. melanogaster development Conrad Pridie, Andrew Simmonds and Richard Rachubinski University of Alberta Peroxisomes are almost ubiquitous eukaryotic organelles involved in the β‐oxidation of fatty acids and detoxification of reactive oxygen species. In humans, a spectrum of diseases called Peroxisome Biogenesis Disorders (PBDs) arise when genes encoding Peroxins, the proteins responsible for peroxisome biogenesis and function, acquire hypomorphic or amorphic mutations. PBD symptoms manifest at birth and appear tissue‐specific, thus they have a developmental origin. Disease symptoms include ataxia, craniofacial malformation, dwarfism, renal cysts and death within a year of birth. Studying the developmental effects of peroxin (pex) mutation requires a model organism. Drosophila melanogaster (Dmel) is an excellent candidate for pex study because it has tissues analogous to those affected by PBDs, it is a proven disease model, it is an excellent intermediate between the yeast and mammal models, and little is known presently about the genetic regulation of Dmel peroxisome biogenesis and function. My project will define peroxisome requirements during Dmel development in order to advance knowledge of the Dmel peroxin genes and to further the model’s utility in modeling PBDs to pursue therapeutic interventions. My hypothesis is that tissue‐specific differences in Drosophila peroxisome requirements reflect differential peroxin regulation. I am presently examining endogenous embryonic and larval pex expression via fluorescent in situ hybridization (FISH) and quantitative reverse transcriptase PCR (qRT‐PCR). I will also observe endogenous embryonic and larval peroxisome content and assay protein/mRNA co‐localization by immunofluorescence (IF). Preliminary FISH results suggest the expression of several Dmel pex genes is regulated in a tissue‐specific manner. Once I have generated a spatio‐temporal profile of endogenous Dmel pex expression and peroxisome content, I will alter peroxin expression via RNAi and targeted mutagenesis, i.e. CRISPR/Cas9, and observe developmental effects. 95 39. Investigating the role of glycogen accumulation in brain aging and neurodegeneration Attey Rostami and Gabrielle L. Boulianne Program in Developmental ; Stem Cell Biology, Sick Kids Hospital and Department of Molecular Genetics, University of Toronto Biological aging is a complex process. Degeneration of cells of a living organism can lead to aging of the whole organism. I am interested in mechanisms that underlie physiological degeneration and more specifically in mechanisms that regulate brain aging and neurodegeneration. Recently, studies have shown that increases in glycogen levels may be associated with brain aging and neurodegeneration. Specifically, it has been shown that both flies and mice accumulate glycogen in their brain as they age. More importantly, inhibiting glycogen synthase (GS), which prevents glycogen accumulation, increases the lifespan of flies and mice. Increased glycogen has also been linked to neurodegenerative diseases such as Lafora Disease (LD) and adult polyglucosan body disease, to name a few. Specifically, glycogen granules have been found in the brain of patients with LD and studies have shown that glycogen accumulation may be responsible for neurodegeneration. It has also suggested that glycogen synthesis is associated with the formation of several age dependent protein‐based aggregates, including alpha‐
synuclein, which is involved in Parkinson disease. Whether, glycogen accumulation occurs in all neurodegenerative diseases and contributes to the pathogenesis of disease, is not known. Thus, my main objective is to determine whether accumulation of glycogen within the brain leads to premature aging and neurodegeneration and whether neurodegenerative phenotypes can be ameliorated by targeting GS and thereby, reducing glycogen levels. 96 40. Regulation Of Actomyosin Network By Homophilic Cell Adhesion Molecule Echinoid During Epithelial Morphogenesis Rahul Rote, Arsida Nocka, Laura Nilson Department of Biology, McGill University, Montreal, Quebec, Canada Cell shape changes during epithelial morphogenesis are driven by differential distribution of cell adhesion molecules, cytoskeletal components and polarity factors. Echinoid (Ed) is a homophilic cell adhesion molecule required in normal epithelial morphogenesis during embryonic dorsal closure and ovarian follicular epithelium development. Both of these processes are characterized by loss of Ed from a defined group of cells, which leads to the formation of a supracellular actomyosin cable and smooth contour at the interface of Ed‐positive (Ed) and Ed‐negative (no‐Ed) cells. Ed is also lost from the Ed‐
positive cells at this interface, due to the absence of a homophilic binding partner in the adjacent cells, resulting in a planar polarized distribution of Ed in these cells. We are testing two alternate hypotheses to explain this phenotype. The first is that planar polarized distribution of Ed guides the formation of actomyosin cable at the interface devoid of Ed. The second hypothesis is that Ed simply functions as a negative regulator of the actomyosin network and absence of Ed at an interface leads to actomyosin enrichment at that particular interface. Quantification of actin using fluorescently‐labeled phalloidin, and myosin using fluorescently labeled heavy or light chain of Myosin II, revealed that significant enrichment of actin and myosin occurs at interfaces between Ed/no‐Ed cells as well as no‐Ed/no‐Ed cells. Cells lacking Ed are also apically constricted, indicating higher actomyosin contractility. These observations seem to support the negative regulator hypothesis. However, Ed/no‐Ed interfaces exclusively display a smooth border, loss of the polarity protein Par3/Bazooka, enrichment of the actomyosin contractility regulator Rho‐Kinase, and in some cases, disruption of adherens junctions. These observations seem to support the planar polarization hypothesis. Thus, the regulation of actomyosin network by Ed possibly encompasses a variety of interactions between downstream effector molecules involved in cell polarity and cytoskeletal dynamics. 97 41. The Ig Transmembrane Protein Borderless Is Required for Synaptic Development and Function in the Drosophila Visual System Hunter Shaw, Scott Cameron, Wen‐Tzu Chang, and Yong Rao Centre for Research in Neuroscience, Department of Biology, Department of Neurology and Neurosurgery, McGill University, 1650 Cedar Avenue, Montreal, Quebec H3G 1A4, Canada Development of the Drosophila melanogaster visual system is an excellent model to understand the molecular and cellular mechanisms controlling neural network formation during embryonic development. The Drosophila visual system is comprised of the compound eye and optic lobe. The compound eye consists of ~800 ommatidia or single eye units, each containing eight photoreceptor cells (R cells). R1‐R6 axons synapse in the superficial lamina and detect motion, while the R7 and R8 axons target the deeper medulla layer and detect ultra violet and blue/green light respectively. Once R‐cell axons reach their target in the brain, they must recruit the proper pre‐synaptic machinery in order to establish fully functional synapses. Recent studies in our lab identified a novel Ig transmembrane protein, Borderless (Bdl), as a critical molecule in visual system development and function. Bdl mediates homophilic cell‐cell adhesion in vitro, and down‐regulation of Bdl is required for R7 axonal tiling and layer‐specific target selection in vivo. In this study, we show that Bdl is also required for proper assembly of pre‐synaptic machinery in R8 axons. Behavioral assays reveal that bdl mutants display impaired blue and green light detection, suggesting a defect in R8 photoreceptor functionality and connectivity. At the anatomical level, we discover that loss of bdl causes the mislocalization of synaptic vesicles in the R8 axon. Genetic mosaic analysis and cell‐type‐specific rescue experiments indicate that Bdl is required in both R8 axons and their target region. We propose that homophilic adhesion between Bdl on the R8 axon and its target region mediates the proper recruitment of presynaptic machinery to R8 synapses in the medulla, thereby ensuring R8 functionality and blue/green light detection in the fruit fly visual system. 98 42. Analyzing the role of the mechanosensory domain of α ‐Catenin through live imaging of early Drosophila embryos Ritu Sarpal, Ridhdhi Desai, Arun Shipstone and Ulrich Tepass University of Toronto Cell‐cell contacts known as adherens junctions (AJs) keep epithelial cells tightly bound within a sheet‐like layer, yet allow for dynamic cellular rearrangements during morphogenetic developmental processes. A physical link between F‐actin and the AJs through α‐catenin is not only essential for adhesion and epithelial integrity but also allows actomyosin contraction to elicit cell shape changes during development. Tension due to actomyosin contraction causes the central region of α‐catenin to stretch into an ‘open’ conformation, revealing cryptic binding sites for proteins that bind to actin, thereby allowing α‐catenin to act as a mechanosensor. We aim to use Drosophila as an in vivo model to investigate the role of the mechanosensory domain of α‐Catenin in connecting the AJ to the actomyosin network. By manipulating this domain through deletion of subdomains and through mutations predicted to stabilize the ‘open’ or ’closed’ conformation, we aim to tease apart how mechanosensing by α‐
Catenin contributes to morphogenesis and the maintenance of the epithelium. Live imaging analysis of mesoderm invagination and germband extension inDrosophilaembryos expressing fluorescent probes for actomyosin network components, α‐Catenin binding partners and the AJ protein DE‐cadherin will be used. Visualizing the localization of α‐Catenin mechanosensory domain binding partners may also be useful as a readout for tension at the AJ. 99 43. Hunting in the Park: Investigating the relationship between Huntington Disease and Parkinson Disease genes in D. melanogaster Frankie A. Slade and Brian E. Staveley Memorial University of Newfoundland Huntington Disease (HD) and Parkinson Disease (PD) are progressive neurodegenerative disorders with severe implications to human health including impaired cognition, motor deficiencies, dementia and premature death. Treatments provide temporary relief of symptoms, yet there is no cure for HD or PD. The onset of HD results from premature destruction of spiny GABAergic neurons in the striatum and cortex of the brain. PD is a common neurodegenerative disease, characterized by the loss of dopaminergic neurons in the brain and the formation of Lewy bodies. Genetically, HD is inherited in an autosomal dominant fashion while PD can be inherited either autosomal‐dominantly or autosomal‐
recessively, with different genes appearing to play a role in inheritance. In healthy individuals, the Huntingtin protein (Htt) interacts with a number of cellular proteins, including Huntingtin interacting protein 1 (Hip1). In HD patients an increased number of CAG residues in the Htt gene results in an expanded number of glutamine residues (polyQ) in the protein. This expansion results in the formation of aggregates, and affects the binding ability of Htt. Downstream effects include apoptosis and improper regulation of neurogenesis. Hip1R, an orthologue of Hip1 interacts with huntingtin and is involved in intrinsic cell‐death pathways. Hip1R has been identified in a genome‐wide association study as a candidate gene for PD, but its role in disease pathogenesis has yet to be identified. In Drosophila melanogaster Hip1 is the only known homologue of both mammalian Hip1 and Hip1R. We investigate the effects of altered Hip1 expression upon D. melanogaster lifespan and motor ability in both HD and PD models. Characterizing the interactions between HD and PD in D. melanogaster may lead to new therapeutic targets and novel treatment options. Funded by a School of Graduate Studies Fellowship to FAS and an NSERC Discovery Grant to BES. 100 44. Avoiding the die in diet: Starvation resistance in Drosophila as a model to understand eating disorders Jennifer D. Slade and Brian E. Staveley Memorial University of Newfoundland Disordered eating may lead to diagnosable medical conditions such as anorexia and bulimia, to extreme weight loss or to obesity. While no animal model is able to mimic the entirety of any complex human disease or behaviour, Drosophila melanogaster can serve as an uncomplicated model to study the biological basis of abnormal eating patterns. The conserved insulin receptor pathway and its endpoint effector the foxo transcription factor are pivotal for survival during nutritional stress. The loss of foxo function results in a defective survival response to amino‐acid starvation. Two modifiers of foxo activity, the Akt1 kinase and the Sir2 deacetylase, may adjust the activity of foxo to enhance survival. Novel Akt1 mutant lines exhibit a moderate decrease in lifespan and growth when aged upon standard media, yet they show a significant increase in survival on amino‐acid deprived media. Replacement of Akt1 activity is sufficient to suppress these phenotypes. Combination of the novel Akt1 hypomorphs and the null foxo mutant reveal an epistatic relationship. Biometric analysis and longevity evaluation of these double mutants indicate a phenotype similar to the original foxo mutant signifying its necessity in the Akt1 phenotype. Unlike the Akt1 mutants, Sir2 mutant heterozygotes do not have altered growth when raised upon standard conditions. However, the Sir2 heterozygotes exhibit a greatly extended lifespan when reared on both a standard diet and when starved of amino‐acids. These results indicate that the subtle manipulation of foxo, by either Akt1 or Sir2, can enhance survival during adverse nutrient conditions to model the survival of individuals undergoing nutrient deprivation. Ultimately, we believe that a Drosophila model of disordered eating could generate new avenues of potential therapies for related human conditions. Funded by an NSERC PGSD and a School of Graduate Studies Fellowship to JDS and an NSERC Discovery Grant to BES. 101 45. Drosophila melanogaster as a model to investigate mechanisms underlying stress‐
induced neural failure Kristin E Spong and R. Meldrum Robertson Department of Biology, Queens University, Kingston, ON, Canada Disruptions in ionic homeostasis can trigger massive neuronal depolarization which leads to an arrest of electrical activity. During severe metabolic stress, such as anoxia, neural function is not restored until the stress is removed. In less severe conditions the ionic disturbance and associated depression in electrical activity is observed to propagate through neural tissue and is thus termed spreading depression (SD). Elucidating control mechanisms involved in such events is of great importance due to their association with human pathologies such as stroke, migraine, and traumatic brain injury. We have developed an experimental protocol where repetitive SD can be triggered in the brain of Drosophila melanogaster by disrupting the extracellular potassium concentration ([K+]o) either directly or by inhibiting the Na+/K+‐ATPase with ouabain. Using a pressure injection system, small volumes (~2‐5nL) of either KCl or ouabain were administered into the fly head and SD was monitored by measuring the [K+]o in the brain using K+‐sensitive microelectrodes or by recording direct current (DC) potential. We demonstrate that ouabain induces SD in a concentration‐dependent manner and show that these events propagated throughout the fly brain at a rate similar to what is observed in mammalian cortex. Lastly, to investigate the role that the protein kinase G (PKG) pathway plays during SD we are using the foraging mutants (rover and sitter) which are naturally occurring populations differing in their levels of PKG. Preliminary results suggest that flies with greater levels of PKG (rover) are more susceptible to the ionic disturbance compared to flies with lower levels (sitter) suggesting that the PKG pathway may be important in modulating these events. This work was supported by NSERC. 102 46. Jhe is a conserved regulator of hunger‐induced behavior Jeff Stafford1, Alex Keene2, Carl Lowenberger3 and Michael Gordon1 1. University of British Columbia 2. University of Nevada, Reno 3. Simon Fraser University Although feeding behavior is a matter of life and death for animals, the genetic factors that control it remain poorly understood. Here we describe an approach to finding novel regulators of hunger‐induced behavior through comparison of transcriptomic changes in the fruit fly Drosophila melanogaster and the yellow fever mosquito Aedes aegypti. Quantification of tissue sugar levels allowed us to sequence head mRNA for each insect at equivalent levels of starvation. Using data gleaned from the protein orthology database OrthoDB, we looked for gene pairs in which both A. aegypti and D. melanogaster orthologs were siginificantly regulated. This identified Juvenile Hormone Esterase (Jhe) as a possible modulator of hunger‐induced behavior. Confirming this, pan‐neuronal knockdown of Jhe resulted in increased food consumption and caused enhancement of starvation‐induced sleep suppression in Drosophila. This phenotype is not a developmental or metabolic defect, and was reproduced by feeding adult Drosophila methoprene, a synthetic Juvenile Hormone analog. Our analysis suggests that Jhe (and Juvenile Hormone by extension) is a novel and biologically relevant regulator of hunger‐induced behavior. 103 47. The phosphorylation state of Crb plays a regulatory role in its abundance and function at the apical domain during embryogenesis David ter Stal1, Milena Pellikka1, Patrick Laprise2, Yang Hong3 and Ulrich Tepass1 1. University of Toronto 2. University of Laval 3. University of Pittsburg Crumbs (Crb) is a key regulator of epithelial polarity with functions in apical basal polarity, regulation of apical membrane size, and several aspects of epithelial morphogenesis. Crb acts as an apical determinant and the apical membrane is very sensitive to the concentration of Crb in the plasma membrane. Crb is a large transmembrane protein with a short cytoplasmic tail that has a PDZ binding motif (PDB) that interacts with Stardust, which is critical for Crb function, and FERM domain‐binding site (FDB) which interacts with several FERM proteins that both positively and negatively modulate Crb activity. We show that interactions through the FDB of Crb are important for Crb function as well as activity. The Crb FDB contains four highly conserved Threonine and Serine residues, which have been shown to be putative phosphorylation targets of aPKC. To test the role of phosphorylation of the Crb FDB during embryogenesis, we have generated a number of non‐phosphorylatable and phosphomimetic forms of the Crb FDB. We show that the membrane accumulation of Crb, as well as Crb activity are regulated by both interaction through and phosphorylation of the Crb FDB. Crb, through its FDB, interacts with a number of different FERM domain containing proteins. Of particular interest to us are Yurt, a negative regulator of Crb function, and the actin binding protein, Moesin. We tested whether phosphorylation of Crb‐FDB is involved in coordinating the interactions between the Crb‐FDB and FERM domain proteins. We have analyzed the interactions between our Crb phosphomutants in vitro and have found that phosphorylation of the Crb FDB plays a role in coordinating the interaction between Crb and these and other FERM domain proteins. Our results suggest that the phosphorylation state of the Crb FDB can play a regulatory role in the dynamics of apical domain regulation. 104 48. Steppke uses its Arf‐GEF activity to align cells during epithelial morphogenesis Junior West and Tony Harris Department of Cell and Systems Biology, University of Toronto Epithelia undergo dynamic morphogenetic movements to form the animal body plan. It is well understood that regulation of cell adhesion and the cytoskeleton are important for these processes, however, the local intracellular signalling events for this regulation are not clear. During Drosophila germband retraction, epithelial cells undergo a wave of morphogenesis that transforms a cobblestone array of ectodermal cells into a tiled rectangular array needed for dorsal closure and subsequent morphogenesis. We have identified the Arf‐GEF Steppke as a regulator of this cell re‐alignment. In steppke zygotic mutant embryos, the cells fail to form a widespread rectangular array and instead often form multicellular rosettes. These defects persist from germband retraction into dorsal closure and ultimately the mutant embryo dies with failed head involution. These defects can be rescued by expressing a wild type Steppke construct, however, a Steppke construct lacking its GEF activity cannot rescue either phenotype, indicating that downstream Arf signalling is critical for the cell re‐alignment. Steppke colocalizes with the cortical actomyosin networks at adherens junctions of epithelial cells. Thus, we hypothesize that Steppke induces local Arf‐induced endocytosis affecting either the cytoskeleton, adherens junctions, or both for proper epithelial morphogenesis. In this way Steppke may inhibit the formation of multicellular rosettes to allow morphogenesis of a tiled rectangular array for dorsal closure and subsequent development. 105 49. Hipk promotes tumorigenesis through JAK/STAT signaling in Drosophila Nathan Wray, Jessica Blaquiere and Esther Verheyen Simon Fraser University Homeodomain‐interacting protein kinases (Hipk) are evolutionarily conserved serine‐threonine kinases involved in regulating signaling pathways through their ability to phosphorylate target proteins and affect their activity. Previously, Hipk has been shown to modulate signaling pathways such as Hippo, Wingless, and Notch, and as such, it is a strong regulator of tissue growth. Furthermore, in some contexts we find that Hipk can promote cell migration and epithelial‐to‐mesenchymal transition. Because of these roles, we propose that Hipk activity must be tightly controlled, as mis‐expression can lead to severe consequences such as tumours. However, the context specific regulation of Hipk remains unclear. This problem led us to investigate two distinct questions: What are the Hipk interacting genes necessary for normal development and what effect do these genes have on Hipk‐mediated growth and metastasis? To tackle these questions, we used the model organism Drosophila melanogaster to perform an in vivo kinase RNAi screen to identify modifiers of a Hipk over‐expression phenotype. From this screen, we found multiple potential repressors of Hipk activity, one of which was Hopscotch (Hop) kinase, a core component of the JAK/STAT cascade. Similar to Hipk, the JAK/STAT pathway can promote tissue growth. A gain‐of‐function allele of hop causes constitutive activation of the pathway and induces tumors in Drosophila. Interestingly we observed that hipk can induce tumors of similar appearance, thus we investigated a possible interaction between these two proteins. We used clonal analyses to modulate levels of hipk and then assayed the expression of the JAK/STAT activity reporter 10xStat92E‐GFP. Through these results and genetic interactions studies we found that Hipk is a positive regulator of the JAK/STAT pathway. Ongoing genetic experiments are attempting to determine where hipk is acting on the JAK/STAT cascade. We will discuss the implications of these results, with emphasis on Hipk being a positive regulator of tumorigenesis. 106 50. Behavioral representation of spatial restriction in Drosophila adult Chengfeng Xiao and R Meldrum Robertson Department of Biology, Queen’s University, Kingston, ON K7L 3N6 Drosophila adults display an unwillingness to enter confined spaces. The characteristics of locomotion with spatial restriction in Drosophila are unknown. Here we described features of locomotor activity with spatial restriction. Intense and persistent locomotion was observed in small circular arenas (1.27cm diameter), whereas locomotion was greatly reduced in large circular arenas (3.81cm diameter). The increased locomotion induced by spatial restriction was seen in males but not females, indicating sexual dimorphism of locomotion with spatial restriction. In large arenas, male flies increased locomotion in arenas pre‐used by male but not female individuals. In small arenas, male flies displayed no additional increase of locomotion in pre‐used arenas compared with that in fresh arenas. Thus male flies were highly sensitive to spatial restriction. In arenas pre‐used by a pair of siblings, wildtype Canton‐S males had the same locomotion in fresh and pre‐used arenas despite the arena size. However, mutant w1118 increased locomotion in pre‐used large arenas and reduced the locomotion in pre‐used small arenas compared with fresh counterparts. Therefore the sensitivity to spatial restriction was strain‐specific. During locomotion with spatial restriction, CS male flies traveled slower and steadier than w1118 flies. Additionally, CS flies showed higher boundary preference than w1118 flies. Genetic analysis revealed that the white gene was dissociated with boundary preference in CS flies. We also demonstrated that expression of mCD8‐GFP proteins in neuronal cytoplasmic membrane in central nervous system impaired the locomotion with spatial restriction, suggesting that integrity of neuronal membrane is critical for sustained locomotion. In summary, Drosophila adults displayed a rich repertoire of behavioral consequences representing spatial restriction. We showed that the white gene was dissociated with boundary preference in circular arenas, and that integrity of neuronal membrane was critical for sustained locomotion with spatial restriction. 107 51. The Atypical Cadherin Fat Directly Regulates Mitochondrial Function and Metabolic State Anson Sing1,2,5, Yonit Tsatskis1,5, Lacramioara Fabian3, Ian Hester1, Robyn Rosenfeld1,2, Mauro Serricchio4, Norman Yau1,2, Mailis Bietenhader4, Riya Shanbhag4, Andrea Jurisicova1, Julie A. Brill2,3, G. Angus McQuibban4, and Helen McNeill1,2 1
Lunenfeld‐Tanenbaum Research Institute, Mt. Sinai Hospital, Toronto, ON, M5G 1X5, Canada 2
Department of Molecular Genetics, University of Toronto, Toronto, ON, M5S 1A8, Canada 3
Cell Biology Program, The Hospital for Sick Children, Toronto, ON, M5G 0A4, Canada 4
Department of Biochemistry, University of Toronto, Toronto, ON, M5S 1A8, Canada 5
Co‐first author Proper development of multicellular organisms requires a coordination of conserved developmental pathways involved in proper tissue growth and patterning. Genetic analyses in Drosophila melanogaster have identified Fat (Ft), a ~560kDa atypical cadherin that functions to regulate tissue growth and planar cell polarity (PCP) tissue organization. Mutations of Ft cadherins in Drosophila have been implicated in polarity defects, tissue overgrowth, and spindle orientation defects. Mitochondria are critical in cell growth and proliferation. They generate ATP by oxidative phosphorylation where the proton gradient generated by Complexes I through V of the electron transport chain (ETC) is used by Complex V to make ATP. Using yeast‐two hybrid analyses, we have identified three mitochondrial interactors of Drosophila Ft whose knockdown by RNAi lead to PCP defects similar to those observed in ft‐/‐ mutants: the Complex I component Ndufv2, the Complex V component CG1746, and mitochondrial processing peptidase (MPP). Structure‐function analyses of the intracellular domain (ICD) of Ft identified two predicted mitochondrial targeting sequences (MTS), and a highly conserved region that is necessary for Ndufv2 binding. We showed that the ICD of Ft is cleaved to release a labile 68kDa cytosolic fragment that is imported into mitochondria (Ftmito). Using biochemical techniques, we also show that Ftmito is required for Complex I and V stability and regulates Complex I activity. Based on theses results, I will be conducting CRISPR mutagenesis and genetic analyses to ablate the functions of the two MTS sites and other conserved motifs within the ICD to investigate the effects of Ft on mitochondrial function. 108 52. Identification and characterization of neuronal mRNAs regulated by d4E‐BP Stephanie Yee1, Nahum Sonenberg1, Christos Gkogkas2, and Paul Lasko1 1
McGill University, Montreal, Quebec, Canada 2
University of Edinburgh, Edinburgh, Scotland, UK A significant proportion of the cellular abundance of proteins is established at the level of protein synthesis or messenger RNA (mRNA) translation; highlighting the importance of translational control. Translation initiation is the phase that is most highly regulated and rate‐limiting. The 5’ cap structure of the mRNA is recognized by eukaryotic initiation factor 4E (eIF4E). 4E binding protein (4E‐BP), a downstream target of Target of Rapamycin (TOR), can bind to eIF4E to impair the assembly of the preinitiation complex, thereby inhibiting cap‐dependent translation. TOR/eIF4E/4E‐BP are components of an essential signaling pathway that regulates protein synthesis by responding to environmental and intracellular cues. Moreover, this pathway regulates the translation of specific mRNAs, termed eIF4E‐
sensitive, rather than impacting translation globally. Recent studies in 4E‐BP2 knockout and eIF4E overexpression mouse models demonstrated that increased eIF4E‐dependent translation of specific transcripts was linked to the development of autism spectrum disorders (ASD)‐like phenotypes. These findings provide an impetus to identify and characterize specific neuronal mRNAs regulated through 4E‐
BP to gain insight into the mechanisms of cap‐dependent translation downstream of TOR. To systematically identify neuronal transcripts regulated by d4E‐BP, we have prepared ribosome profiling sequencing libraries from d4E‐BPnull flies. Preliminary results suggest that transcripts whose translation is upregulated in d4E‐BPnull flies encode for factors involved in translation. Target mRNAs identified by ribosome profiling will be validated with immunoblotting, polysome profiling and translating ribosome affinity purification. The architecture of the 5’UTR of these transcripts will be further examined to identify cis‐regulatory elements that confer sensitivity to eIF4E levels. 109 53. IMPLICATION OF THE ARFGAPS: DRONGO AND ARFGAP1 IN COLLECTIVE MIGRATION OF BORDER CELLS Carlos Zeledon, Xiaojuan Sun and Gregory Emery Institut de recherche en immunologie et en cancérologie (IRIC),Université de Montréal Introduction: Cell migration is implicated in various important biological processes, notably it is central for cancer and formation of metastases. Recently, our lab has showed that endocytosis regulates cell guidance and cell‐cell coordination during collective cell migration. Our hypothesis is that other events of vesicular trafficking might be implicated in collective cell migration. Mainly, we propose that small GTPases Arfs, important for the formation of vesicles and sorting of cargo in these vesicles, and their regulators might regulate trafficking and localization of determinants of collective cell migration. Methods and Results: As a model, we use the egg chambers of Drosophila melanogaster. Indeed, during oogenesis a group of cells, named border cells, migrate collectively towards the oocyte. By doing an RNAi screen specifically in borders cells, we found migration defects when we deplete the Arfs, three ArfGAPs and two ArfGEFs. However, we found that depletion of ARFs affect various determinant of border cell migration. Whereas, depletion of the ArfGAPs: Drongo and ArfGAP1 affect specifically active RTKs localisation. Surprisingly, live‐imaging shown that we have distinct migration defects when we deplete these two ArfGaps. Indeed, Drongo depletion leads to a detachment defect whereas when ArfGAP1 is depleted, cells have no detachment defect but lose directionality during migration. These differential phenotypes are possibly due to their different localisation in border cells or the fact that they might act on different Arfs. Conclusion and Relevance: Loss‐of‐function of Arf proteins induce pleiotropic effects, so that it is difficult to determine their exact role during collective cell migration. However their regulators have more specific effects, possibly through the regulation of precise vesicular transport events in the cell. 110 54. Characterization of Nup98‐HOXA9 leukemogenic activity in Drosophila Caroline Baril, Gwenaëlle Gavory, Helene Knaevelsrud and Marc Therrien IRIC‐Université de Montreal HOX transcription factors play crucial roles during hematopoiesis and also behave as oncogenes in hematological malignancies such as acute myeloid leukemia. Despite intensive work in mammals, the molecular mechanisms through which HOX operates during leukemogenesis remain largely unresolved. To better understand HOX‐dependent leukemia, we generated transgenic Drosophila lines that specifically express the human Nup98‐HOXA9 (NA9) translocation in larval hemocytes of the hematopoietic organ called the lymph gland (LG) as well as in circulating hemocytes. NA9 fuses the N‐
terminal part of the nuclear pore component Nup98 to the C‐terminal DNA binding domain of HOXA9 and serves as a prototypical translocation to study HOX‐mediated leukemia in mammals. Interestingly, NA9 expression in Drosophila stimulated cell proliferation and led to hyperplasia of the LG as well as up to a 10‐fold increase in the number of circulating hemocytes. In this system, expression of NA9 is restricted to the differentiated cell compartment, called the cortical zone (CZ). While differentiation per se did not appear to be compromised, NA9 promoted the expansion of the CZ at the expense of the adjacent medullary zone (MZ) composed of progenitor cells. NA9 also induced in a non‐cell autonomous manner the enlargement of a third compartment called the posterior signaling center, which serves as the hematopoietic niche. The NA9‐dependent phenotypes were suppressed by mutagenesis of critical residues required for NA9 binding to DNA or to PBX cofactor. In addition, co‐overexpression of HTH (fly orthologue of Meis also acting as a HOXA9 cofactor) considerably enhanced NA9 phenotypes. Finally, we found that increased PDGF\VEGF (PVR) receptor signaling suppressed NA9 phenotypes in the LG, which suggests a functional interplay between PVR and NA9 activities. Together, these results recapitulate the salient phenotypes observed in NA9 murine models and therefore make this fly model a promising system for genetically dissecting NA9 function. 111 Mc Gill’s New Residence Hall
Centre des Sciences (banquet)
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