Signaling and Development
1. Characterizing Robinow Syndrome-associated DVL1 mutations in Drosophila. Katja MacCharles. Verheyen Lab, SFU
Human development is regulated by intricate, and interconnected, signal transduction networks. Given the complexity, deciphering the effects of mutations that give rise to abnormal development can be challenging. Using Drosophila melanogaster can simplify the puzzle of studying human disorders as flies have little genetic redundancy and are significantly easier, cheaper, and faster to raise than other vertebrate models. I use Drosophila to characterize Dishevelled1 (DVL1) mutations obtained from patients with Robinow Syndrome (RS). RS is a rare genetic disorder associated craniofacial abnormalities and shortened stature. Most of the mutations associated with RS affect components of the non-canonical/Planar Cell Polarity (PCP) pathway of Wnt signaling. Wnt signaling is involved in embryonic development and homeostasis. The two main pathways, canonical and non-canonical/PCP Wnt signaling, require DVL but there is still much to learn about PCP signaling which mediates cytoskeletal rearrangement events and orients cell polarity within the epithelial plane. There are 3 DVL proteins found in vertebrates, and a single ortholog, Dsh, in Drosophila. Each of the DVL1 variants I study have unique frameshift mutations that replace the highly conserved C-terminus with the same novel peptide sequence of no known homology. I use the Gal4-UAS system to express wildtype human DVL1 and three DVL1 variants in Drosophila. My research has shown that these DVL1 patient variants disrupt the stability of Armadillo/β-catenin, ectopically induce PCP/JNK signaling and activate apoptosis. Furthermore, the variants induce several novel phenotypes in wing tissue such as anterior cross vein abnormalities, ectopic bristles, and vein thickening, suggesting novel functions in other conserved signaling pathways. By understanding how conserved signaling pathways are altered by these DVL1 variants, we gain insight into the underlying mechanisms of non-canonical Wnt signaling and more broadly, how development in individuals with RS is altered. This information may guide future therapeutics for RS patients.
2. The PRC2 Complex: Human Phenotypes. William Gibson. Gibson Lab, BC Children's Hospital Research Institute
In children and adults, many rare syndromes that feature both overgrowth and intellectual disability (OGID) are caused by rare functional variants in members of the human Polycomb Repressor Complex. The human EZH2, EED and SUZ12 proteins are the orthologues of Drosophila E(z), esc and Su(z)12 protein, respectively. The PRC2 complex is generally thought of as a transcriptional repressor, since it both reads (via EED) and writes (via EZH2 and SUZ12) methyl groups at lysine residue 27 of histone H3. By laying down H3K27me, H3K27me2 and H3K27me3 marks, PRC2 assists in chromatin compaction and gene repression. Though partial loss-of-function (LoF) variants strongly predispose to OGID, gain-of-function (GoF) variants appear to cause primordial dwarfism (i.e. severe growth retardation). Epigenomewide methylation profiling has revealed reciprocal and reproducible disorder-specific patterns of DNA methylation that are detectable in human peripheral blood mononuclear cells. However, there are a large number of rare coding variants in humans that are currently classified as “Variants of Unknown Significance” (VoUS or VUS). Given the strong evolutionary conservation of these proteins from flies to humans, and the plethora of available strains, Drosophila offers an attractive setting in which to build functional assays for these VoUS. Notably, the ovum is furnished with maternally-derived PRC2 members that are required for early development. This feature of PRC2 complex biology suggests that maternally-encoded PRC2 variants may have transgenerational effects on offspring phenotypes, even when the variants themselves are not transmitted to offspring. The fly also offers a rapid and accessible means of interrogating these variants for transgenerational effects. Well-calibrated, well-validated assays for these effects would be very useful for powering rare variant burden studies that could solve some of the “missing heritability” of complex human phenotypes such as growth, cancer risk and tissue development.
3. Investigating the role of TNF pathway in Drosophila tricellular junction regulation. Zazil Solis Saldivar. Auld Lab, UBC
Establishment and maintenance of permeability barriers are some of the most important functions of epithelial cells. Tricellular junctions (TCJs) are permeability barriers formed at the corners of polarized epithelia where three cells converge. TCJ proteins are uniquely localized at the corners of epithelial cells, yet little is known about the mechanisms that regulate them. Gliotactin is a key protein for TCJ development in Drosophila. When Gliotactin is overexpressed and spreads away from the TCJs, it triggers deleterious phenotypes such as apoptosis, overproliferation and cell migration. Preliminary data showed that the cytokine TNF signaling pathway is involved in the enhancement of these phenotypes. Downregulation of the TNF receptor Grindelwald suppresses the overexpression phenotypes of Gliotactin. Grindelwald mediates the pro-apoptotic functions of Eiger, the unique Drosophila TNF ligand. However, downregulation of Eiger does not rescue the phenotypes mentioned before, raising the question whether Grindelwald may function in an Eiger-independent manner. Genetic and biochemistry approaches in Drosophila wing imaginal discs will be used to address if Gliotactin overexpression phenotypes are Eiger independent. Aberrant TCJ function has been linked to several human genetic diseases, and cancer progression and malignancy. This research could help us understand better how cells undergo apoptosis, and processes in tumor development and progression.
4. Exploring the role of glial Syndecan on Drosophila optic lob development. Duo Cheng. Auld Lab, UBC
To establish a functional brain, neural stem cells (NSC) need to generate an abundant number of neurons and glia under tight temporal and spatial regulations. The Drosophila melanogaster’s optic lobe is a popular model to study neurogenesis as each optic lobe generated by neuroblasts (NB) that differentiate from the columnar neuroepithelium (NE), like that of mammalian neurogenesis during development. Glia provide essential modulation to the NSC niche to ensure appropriate NSC differentiation and homeostasis. Yet the tools by which glia utilize to interpret and control the NSC niche are far from being fully characterized. Here, we explored the role of a transmembrane heparan sulfate proteoglycan (HSPG), Syndecan (Sdc) in regulating NSC niche during development. Using super-resolution microscopy, we revealed Sdc is widely expressed across the brain lobe, though we notice Sdc is particular enriched around the NE. Pan-glial knockdown of Sdc revealed a dramatic decrease in brain lobe volume, accompanied by a reduction of the NB population size within the OPC. Moreover, we saw the lengthening of the ventral nerve cord, indicative of a disruption between glia and the overlying extracellular matrix that coats the entire nervous system. While loss of Sdc in glia is not significantly alter the level of apoptosis, it did lead to a reduction in mitotic events in the OPC. Upon further experiments, we saw pan-glial Sdc knockdown perturbed the NE morphology. Therefore we are investigating the role of Sdc in mediating cross-talk between the glia and the NE. Overall, our results supports a novel aspect of Sdc’s role in regulating the NSC niche though glial regulation of the NE in the developing brain lobe.
5. Under Tension: Integrin and Basigin at the Perineurial Glial Membrane. Sophie Roth. Auld Lab, UBC
In Drosophila melanogaster, glial cells play an important role in ensheathing and protecting nerves from damage. To ensure the protection of nerves, the outermost layer of glia known as the perineurial glia (PG), interacts with the extracellular matrix through focal adhesion complexes (FACs), which are comprised of Integrins and intracellular adapter proteins including Talin. In PG FACs, Integrin associates with the transmembrane protein Basigin. Knockdown of Basigin leads to a ruffled phenotype, characterized by areas of constriction across the PG membrane of peripheral nerves. This ruffled phenotype is caused by increased FAC activation since it can be rescued by loss of Integrin or Talin. This leads to the questions: 1. What degree of tension is experienced in the FAC across the PG membrane? 2. Does Basigin play a role in tension regulation in the PG? We hypothesize that the FAC experiences tension to provide stability to the glial membranes. Also, we hypothesize that Basigin negatively regulates Integrin activation in order to modulate tension across the PG membrane. To investigate these questions, a FRET (fluorescence resonance energy transfer) tension sensor will be placed within the protein Talin. The tension sensor will include a donor fluorophore and an acceptor fluorophore, where increased tension leads to a decrease in FRET as the two fluorophores become separated. We are currently testing the degree of tension in the FAC with sensors of varying sensitivity. In the future, we will test the effects on FAC tension in the absence of Basigin. Overall, investigation into tension regulation is an important concept in all animals since nerves are delicate, and therefore too much or too little tension can lead to damage. Since the glia play a main role in protecting the nerves from damage, it is possible that they play a role in modulating tension as well.
6. MAGE- a modulator of Tumour Necrosis Factor Receptor signalling in Drosophila. Tomas Filip. Barker Lab, UBCO
Tumour Necrosis Factor Receptor (TNFR) pathway is a highly conserved and complex signalling decision-maker between cell death and cell survival under physiological conditions and cancer. TNF receptor superfamily in mammals counts to 29 receptors with at least 19 various ligands establishing a significant challenge in deciphering such an essential signalling process. Until recently, mammalian MAGEs have been a large enigmatic family of proteins linked to the incidence of various terminal cancers. Several MAGEs are required for the TNFR signalling, yet the molecular mechanism of their function is unclear. The MAGE protein family includes <50 individual members with high functional redundancy and compensatory kinetics across the protein family. Interestingly, several human MAGEs have been shown to form a complex with E3-ubiquitin ligases, thereby modulating ubiquitination signalling across the cells. Ubiquitination is a major regulatory tool in TNFR signalling. Drosophila has only one MAGE protein and two TNF receptors called Wengen and Grindelwald. TNFR in Drosophila shares a significantly conserved architecture of most of the downstream interactors, signalling mechanisms (including JNK pathway activation), and apoptotic machinery with its mammalian counterparts. Drosophila MAGE is highly structurally similar to most of its mammalian partners, supporting the evolutionary relevance of the MAGEs. In this project, we have established three major aims to investigate: (1) MAGE is required for TNFR signalling in Drosophila as a facilitator of ubiquitination; (2) Identification of the MAGE protein domain required for its action; (3) MAGE modulates a tumour suppressing effect of TNFR in the Drosophila cancer model. The outcomes of this project will lead to a better understanding of the regulatory mechanisms behind the TNFR pathway, which will help to decipher the complexity of TNFR in mammalian systems.
7. Kinetics of blood cell differentiation during hematopoiesis revealed by quantitative long-term live imaging. Kevin Ho. Tanentzapf Lab, UBC
Stem cells typically reside in a specialized physical and biochemical environment that regulates their behaviour. For this reason, stem cells are ideally studied in contexts that maintain this precisely constructed microenvironment while still allowing for live imaging. Here, we develop a long-term multi-organ co-culture system and imaging strategy for hematopoiesis in flies that takes advantage of powerful genetic tools available in this system. Using the culture system, we are able to monitor self-renewal and differentiation of stem cells in real-time with high spatial and temporal resolution under homeostatic condition and upon bacterial infection. We find that the self-renewal division of blood stem cells is linked to cell size and is spatially polarized. Following infection, the stem cell division is altered in their spatial distribution and orientation but the duration of division remains unchanged. Using quantitative imaging to simultaneously track markers for stemness and differentiation in blood stem cells, we identify two types of differentiation that show distinct kinetics during transition of stem cell identity: 1) a linear type differentiation that exhibits a loss of stemness and gain of differentiated state at a constant speed, and 2) a sigmoid type differentiation that exhibits an initial slow phase following by a rapid fast phase during loss of stemness. Following infection, we observe a shift between the two types of differentiation and a change in their spatial distribution and differentiation speed, showing that infection-induced activation of hematopoiesis occurs through modulation of the kinetics of cell differentiation. Overall, these results provide a novel system-level framework for understanding how fly hematopoietic stem cells are regulated in the context of the intact whole organ in real time.
Neuroscience
8. RACK1 knockdown improves motor function and lifespan in an in vivo TDP-43 ALS model. Anaya Saraph. Cashman Lab, UBC
Amyotrophic lateral sclerosis (ALS) is an incurable, adult-onset neurodegenerative disease characterized by the progressive loss of motor neurons. Up to 97% of ALS cases display pathological cytoplasmic depositions of TAR DNA binding protein 43 kDa (TDP-43). This TDP-43 proteinopathy is characterized by the clearance of TDP-43 from the nucleus and the formation of cytoplasmic aggregates, which in turn suppress global protein translation through interactions with receptor activated C kinase 1 (RACK1) protein. Recent work in the Cashman laboratory has shown that RACK1 knockdown (KD) in vitro rescues TDP-43 proteinopathy by reducing cytoplasmic aggregation, promoting nuclear re-entry, and restoring protein translation. Based on these observations, we hypothesized that RACK1 KD would also ameliorate features of TDP-43 pathogenicity in a living organism. We used transgenic Drosophila melanogaster expressing wild type (hTDP-43WT) or mutant (hTDP-43QK) forms of human TDP-43 in motor neurons. hTDP-43QK was co-expressed with RACK1 RNAi (or control RNAi) to achieve cell-specific RACK1 KD. Climbing ability (indicating motor neuron function) and lifespan were used as measures of disease severity. Drosophila expressing hTDP-43 showed significantly reduced climbing ability and lifespan compared to non-transgenic controls; and hTDP-43QK expression conferred greater defects than hTDP-43WT. RACK1 KD in a hTDP-43QK background improved climbing ability and significantly prolonged lifespan. The delayed motor defect and improved lifespan resulting from RACK1 KD confirms the involvement of RACK1 in the mechanism of TDP43 proteinopathy previously seen in vitro. These results support the potential of RACK1 modulation as a therapeutic approach in treating TDP-43-associated ALS.
9. Characterizing appetitive taste memory in Drosophila using a novel optogenic program. Meghan Jelen. Gordon Lab, UBC
Tastes are typically thought to evoke innate appetitive or aversive behaviours, prompting food acceptance or rejection. However, research in Drosophila melanogaster indicates that taste responses can be modified through experience-dependent changes in mushroom body circuits. In this study, we develop a novel taste learning paradigm using closed-loop optogenetics. We find that appetitive and aversive taste memories can be formed by pairing gustatory stimuli with optogenetic activation of sensory or dopaminergic neurons associated with reward or punishment. As with
olfactory memories, distinct dopaminergic subpopulations drive the parallel formation of short- and long-term appetitive memories. Long-term memories are protein synthesis-dependent and have energetic requirements that are satisfied by a variety of caloric food sources or by direct stimulation of MB-MP1 dopaminergic neurons. Our paradigm affords new
opportunities to probe plasticity mechanisms within the taste system and understand the extent to which taste responses are experience dependent.
10. A molecular mechanism for high salt taste in Drosophila. Sasha McDowell. Gordon Lab, UBC
Dietary salt detection and consumption are crucial to maintaining fluid and ionic homeostasis. To optimize salt intake, animals employ salt-dependent activation of multiple taste pathways. Generally, sodium activates attractive taste cells, but attraction is overridden at high salt concentrations by cation non-selective activation of aversive taste cells. In flies, high salt avoidance is driven by both ‘bitter’ taste neurons and a class of glutamatergic ‘high salt’ neurons expressing pickpocket23 (ppk23). Although the cellular basis of salt taste has been described, many of the molecular mechanisms remain elusive. Here, we show that ionotropic receptor 7c (IR7c) is expressed in glutamatergic high salt neurons, where it functions with co-receptors IR76b and IR25a to detect high salt. Misexpression of IR7c in sweet neurons, which endogenously express IR76b and IR25a, confers responsiveness to non-sodium salts, indicating that IR7c is sufficient to convert a sodium-selective receptor to a cation non-selective receptor. Furthermore, the resultant transformation of taste neuron tuning switches potassium chloride from an aversive to an attractive tastant. This research provides insight into the molecular basis of monovalent and divalent salt taste coding and the full repertoire of IRs needed to form a functional salt receptor.
11. The Neural Basis of Taste Processing in Aedes aegypti mosquitoes. Elsa Cyr. Gordon Lab, UBC
Insects possess gustatory receptor neurons that respond to different taste modalities. Taste processing has been extensively studied in Drosophila melanogaster, which makes it an ideal model to identify molecular mechanisms conserved in other insects. In this study, we explore the neural basis of taste processing in the yellow fever mosquito, Aedes aegypti which utilizes non-volatile chemosensory cues during behaviours such as blood-feeding and egg-laying. We hypothesize that taste processing mechanisms in A. aegypti are broadly conserved from Drosophila, but that mosquito-specific adaptations have evolved to support mosquito-specific behaviours. To test this, we are producing a comprehensive anatomical and functional characterization of gustatory neurons expressing taste receptors in the tarsi of mosquitoes. We use genetic driver lines to label sensory neurons, characterizing their anatomy and neural activity in response to taste stimuli. We expect anatomical and functional maps in mosquitoes to be broadly similar to the Drosophila model, where some receptors label overlapping populations of neurons, while others label more distinct subsets. This work will test our hypothesis that the anatomical and functional patterns of gustatory receptor neurons in mosquitoes are broadly conserved from Drosophila and will provide the functional basis for better understanding of biting behaviour in mosquitoes, ultimately informing the development of better interventions to reduce the burden of mosquito-borne disease.
12. Functional specificity of the sweet taste projection neurons in Drosophila. Jinfang Li. Gordon Lab, UBC
Drosophila taste projection neurons (TPNs), which are key to understanding the flow of taste information from peripheral to higher brain centres, remain significantly understudied. After careful screening, we found several split-GAL4 lines that target different classes of TPNs. Here we examine the tuning properties and connectivity of two different sweet-responsive TPNs—sTPNs and lTPNs. We found that both TPNs respond specifically to sweet taste using calcium imaging. GRASP experiments showed physical proximity between the TPNs and the sweet gustatory receptor neurons (GRNs), and between the TPNs and different subpopulations of the higher-order dopaminergic neurons known to be involved in memory reinforcement. To better understand the function of these novel TPNS, we developed a novel light memory assay, which associates taste values to the flashing of light. Strikingly, the sTPNs and lTPNs are functionally specific to certain types of memory. The sTPNs are specifically required for short term appetitive memory, while the lTPNs are required only for long term appetitive memory. We suggest that parallel sets of taste projection neurons encode sweet information that mediates short- and long-term memory reinforcement.
Metabolism and Physiology
13. Triglyceride Metabolism in Neurons Regulates Sex Differences in Body Fat. Colin Miller. Rideout Lab, UBC
Fat is the primary form of stored energy in animals. Triglycerides (TAGs) are the main form of stored fat. Within cells, TAGs are sequestered into specialized organelles called lipid droplets. The process of storing TAGs is tightly regulated across species. An important but understudied factor in fat metabolism is the sex of the animal. Sex chromosomes establish the sex difference in fat metabolism. Yet the metabolic effectors that contribute to this sex difference in fat metabolism remain unclear. In the Rideout Lab we use Drosophila as a model to identify genes that contribute to sex differences in fat metabolism. Flies are an ideal model to study this problem, as the TAG metabolic pathway is highly conserved in Drosophila and our lab’s research shows sex differences in fat metabolism mirror trends in other animals including humans. Indeed, females flies store more body fat than males, and break down fat more slowly after starvation. The lab’s previous work showed that a TAG lipase brummer (bmm) acts in neurons to regulate the sex difference in fat breakdown. This was the first study to identify a phenotype associated with an intracellular triglyceride lipase in neurons. To build on this finding, I will determine whether neuronal bmm’s function in regulating whole-body fat metabolism is unique to bmm, or if additional genes involved in intracellular TAG metabolism also act in neurons to regulate sex differences in whole-body fat metabolism. The overall objective of my project is to elucidate how TAG metabolism in neurons contributes to sex differences in whole-body fat metabolism. To achieve my objective, I propose two Aims: First, determine whether additional genes associated with TAG metabolism act in neurons to regulate the sex difference in whole-body fat metabolism; secondly, determine whether genes associated with TAG metabolism regulate neuron lipid droplets.
14. Determine the role of Insulin signaling pathway in fat homeostasis in male and female Drosophila. Puja Biswas. Rideout Lab, UBC
Like most animals, female Drosophila stores more triglycerides (TAG) than males. Yet the metabolic pathways that contribute to this sex difference in TAG storage remain unclear. We use fruit flies as a model to identify genes and pathways that contribute to the male-female difference in TAG storage. The conserved insulin/insulin-like growth factor signaling pathway (IIS) is known to coordinate fat metabolism with nutrient intake by acting as lipogenic and anti-lipolytic hormone in flies and many animals. Therefore, we tested whether IIS plays a role in regulating whole-body TAG storage in female flies downstream of food intake by increasing lipogenesis compared with male flies. We found sex differences in several aspects of IIS regulation, and showed that females decreased their whole-body fat storage in the absence of insulin producing cells (IPCs), whereas no change in male fat storage. Females with ablated IPCs had increased lifespan and decreased offspring production which suggested that females required IIS to maintain their fat storage to support their reproduction. As the IIS is nutrient-responsive, particularly sugar, we also tested how changing in dietary sugar levels affected IIS activity and fat storage. we found that females reduced fat levels in low sugar diet compared to regular diet, whereas there was no change in male fat storage. Taken together, our data suggests that sex differences exist in the insulin signaling pathway in adults, as in larvae, and play a role in regulating sex difference in fat metabolism.
15. Investigating the role of adipose tissue mitochondria in mediating the sex difference in Drosophila body size plasticity. Celena Cherian. Rideout Lab, UBC
Sexual dimorphism has fascinated biologists for centuries and differences in external appearances, behaviour and reproduction have been well documented across many species. However, there is a lack of knowledge surrounding sex differences in developmental and physiological processes. One such physiological aspect is systemic growth. Males and females of many species across the animal kingdom differ in body size and studies using Drosophila have begun to unravel the mechanisms behind this sexual size dimorphism. The nutritional status of an animal has also been shown to play an important role in whole body growth and final body size. When nutrients are abundant, the growth rate is higher and body size is increased. In conditions of starvation, the growth rate is lower and body size is smaller. This ability of an organism to change its body size in response to nutrient availability is called nutrient-dependent body size plasticity. A recent study from our lab has shown that when Drosophila larvae are fed a high-protein diet females are able to significantly increase their body size while males are unable to do so. This sex difference in body size plasticity is mediated by 2 mitochondrial proteins. Mitochondria are classically considered as the powerhouses of the cell and are primarily known for their roles in generating ATP through oxidative phosphorylation. However, they also regulate nutrient-dependent growth through their biosynthetic activities by providing TCA cycle products to fuel the generation of amino acids and lipids. Hence, I aim to understand the role of mitochondria in mediating this sex difference in nutrient-dependent body size plasticity. This research could provide insight into the mechanisms involved in mediating sex differences in the risk of developing mitochondrial-associated diseases and phenotypes affected by mitochondrial function.
16. Investigating the potential mechanism of cyclin dependent kinase 8 in modulating mitochondrial dynamics via lipogenic pathway. Jenny Liao. Verheyen Lab, SFU
Cyclin-dependent kinase 8 (Cdk8) is a well-known serine/threonine protein kinase that functions in the transcriptional regulation. We found a novel function of Cdk8 in regulating mitochondrial morphology, and expression of Cdk8 alleviates the defective phenotypes caused by impaired mitochondria in a pink1 mutant mediated Parkinson’s disease model. We are currently exploring potential mechanisms that can explain these findings. One direction that we are pursuing is the SREBP (sterol regulatory element binding protein) mediated lipogenic pathway. It has been shown that Cdk8 is a negative regulator of SREBP, which is a master transcription factor in fatty acid, cholesterol, glycerolipid, as well as lipid droplet synthesis. Phosphorylation on SREBP by Cdk8 can promote degradation of SREBP, which in turn reduces the lipid anabolic pathway. In addition, since lipid droplets are also a well-known factor associated with neurodegenerative disorders, it is tempting to investigate the potential role of the lipogenic pathway in modulating mitochondrial dynamics. Strikingly, we found mitochondrial morphology shifts from fused and elongated to round-shaped and fragmented when expression of lipogenic pathway is modulated. These findings resembled our previous findings where Cdk8 is knocked down or ectopically expressed, respectively. Furthermore, our preliminary data indicates that suppression of lipogenic pathway can also rescue the climbing and muscle degeneration in the pink1 mutant background, which further suggests that Cdk8 may influence mitochondrial dynamics by negatively regulating the lipogenic pathway.
Ecology and Evolution
17. The invasive spotted wing drosophila and their associated host plant phenology. Warren Wong. Carrillo Lab, UBC
The spotted wing drosophila, Drosophila suzukii, is a major agricultural pest globally. As D. suzukii oviposits in developing fruits, the timing of oviposition in relation to host plant phenology is important to the development of pest management strategies. We conducted a survey in the Fraser Valley of British Columbia to examine the association between host plant phenology and larval development and abundance over the growing season on four host plants - salmonberries, strawberries, raspberries, and Himalayan blackberries.
Immunology (Molecular Virology and Host-Microbe Interactions)
18. When proteins go viral: A viral protein impairs stress granule formation by modulating RanBP2/Nup358. Jibin Sadasivan. Jan Lab, UBC
Viruses have evolved mechanisms to modulate cellular pathways to facilitate infection. One such pathway is stress granule (SG) assembly. SGs are ribonucleoprotein complexes that assemble during translation inhibition following cellular stress. Inhibition of SG assembly has been observed under numerous virus infections across viral families and species suggests an essential conserved fundamental viral strategy. However, the significance of and mechanisms underlying SG modulation during virus infection are not fully understood. The multifunctional 1A protein encoded by the model dicistrovirus, cricket paralysis virus (CrPV) can bind to and degrade Ago-2 in an E3 ubiquitin ligase-dependent manner to block the antiviral RNA interference pathway and the 1A protein also inhibit SG formation. Moreover, the R146 residue of 1A is necessary for SG inhibition and for virus infection in both Drosophila S2 cells and adult flies. In this study, we investigated the molecular mechanism by which CrPV-1A inhibit SGs. We showed that expression of wild-type but not mutant R146A CrPV-1A in S2 cells inhibits SG formation when challenged with potent SG inducers, indicating that 1A-mediated SG inhibition is stress-independent. CrPV-1A’s ability to inhibit SG formation does not require the Ago-2 binding domain but requires the E3 ubiquitin-ligase binding domain. Overexpression and infection studies in both Drosophila and human cells showed that wild-type CrPV-1A but not mutant R146A CrPV-1A localizes to the nuclear periphery which correlates with nuclear enrichment of poly(A)+RNA. Transcriptome analysis demonstrated that the CrPV R146A virus is defective in inducing host transcriptome changes in CrPV-infected cells. Finally, inhibition of SG formation by CrPV-1A requires depletion of NUP358 in an R146-dependent manner. Taking this together, we propose that CrPV utilizes a multiprong strategy whereby the CrPV-1A protein interferes with Nup358 activity to inhibit SG assembly.
19. Distribution of insecticidal activity. Daniel Yanez. Haney Lab, UBC
The genus Pseudomonas includes Gram-negative bacteria the live in a wide range of environments and on diverse hosts. Pseudomonas is among the most complex genera with the largest number of species. Pseudomonas strains exhibit a variety of ecological lifestyles ranging from human and plant pathogens to beneficial plant and animal-associated strains. The Pseudomonas fluorescens group exhibits diverse effects on plant and insect hosts. These range from beneficial effects on plants and infection and killing of insect larvae . How Pseudomonas infect different hosts and overcome each immune system is still largely unknown. However, previous studies on insecticidal activity point to a highly multifactorial nature, with different genes involved in the process. Pseudomonas fluorescens is not the only Pseudomonas clade with insecticidal activity. Insecticidal activity has previously been reported within the P. protegens and P. chlororaphis sub-clades and P. taiwanensis and P. entomophila. Thus, my work aims to explore the distribution and extent of the insecticidal ability throughout the whole Pseudomonas genus by combining bioinformatics and insect oral infection assays. Here, I found a correlation between insecticidal activity and the presence/absence of genes associated with insecticidal activity across some Pseudomonas strains.
1. Characterizing Robinow Syndrome-associated DVL1 mutations in Drosophila. Katja MacCharles. Verheyen Lab, SFU
Human development is regulated by intricate, and interconnected, signal transduction networks. Given the complexity, deciphering the effects of mutations that give rise to abnormal development can be challenging. Using Drosophila melanogaster can simplify the puzzle of studying human disorders as flies have little genetic redundancy and are significantly easier, cheaper, and faster to raise than other vertebrate models. I use Drosophila to characterize Dishevelled1 (DVL1) mutations obtained from patients with Robinow Syndrome (RS). RS is a rare genetic disorder associated craniofacial abnormalities and shortened stature. Most of the mutations associated with RS affect components of the non-canonical/Planar Cell Polarity (PCP) pathway of Wnt signaling. Wnt signaling is involved in embryonic development and homeostasis. The two main pathways, canonical and non-canonical/PCP Wnt signaling, require DVL but there is still much to learn about PCP signaling which mediates cytoskeletal rearrangement events and orients cell polarity within the epithelial plane. There are 3 DVL proteins found in vertebrates, and a single ortholog, Dsh, in Drosophila. Each of the DVL1 variants I study have unique frameshift mutations that replace the highly conserved C-terminus with the same novel peptide sequence of no known homology. I use the Gal4-UAS system to express wildtype human DVL1 and three DVL1 variants in Drosophila. My research has shown that these DVL1 patient variants disrupt the stability of Armadillo/β-catenin, ectopically induce PCP/JNK signaling and activate apoptosis. Furthermore, the variants induce several novel phenotypes in wing tissue such as anterior cross vein abnormalities, ectopic bristles, and vein thickening, suggesting novel functions in other conserved signaling pathways. By understanding how conserved signaling pathways are altered by these DVL1 variants, we gain insight into the underlying mechanisms of non-canonical Wnt signaling and more broadly, how development in individuals with RS is altered. This information may guide future therapeutics for RS patients.
2. The PRC2 Complex: Human Phenotypes. William Gibson. Gibson Lab, BC Children's Hospital Research Institute
In children and adults, many rare syndromes that feature both overgrowth and intellectual disability (OGID) are caused by rare functional variants in members of the human Polycomb Repressor Complex. The human EZH2, EED and SUZ12 proteins are the orthologues of Drosophila E(z), esc and Su(z)12 protein, respectively. The PRC2 complex is generally thought of as a transcriptional repressor, since it both reads (via EED) and writes (via EZH2 and SUZ12) methyl groups at lysine residue 27 of histone H3. By laying down H3K27me, H3K27me2 and H3K27me3 marks, PRC2 assists in chromatin compaction and gene repression. Though partial loss-of-function (LoF) variants strongly predispose to OGID, gain-of-function (GoF) variants appear to cause primordial dwarfism (i.e. severe growth retardation). Epigenomewide methylation profiling has revealed reciprocal and reproducible disorder-specific patterns of DNA methylation that are detectable in human peripheral blood mononuclear cells. However, there are a large number of rare coding variants in humans that are currently classified as “Variants of Unknown Significance” (VoUS or VUS). Given the strong evolutionary conservation of these proteins from flies to humans, and the plethora of available strains, Drosophila offers an attractive setting in which to build functional assays for these VoUS. Notably, the ovum is furnished with maternally-derived PRC2 members that are required for early development. This feature of PRC2 complex biology suggests that maternally-encoded PRC2 variants may have transgenerational effects on offspring phenotypes, even when the variants themselves are not transmitted to offspring. The fly also offers a rapid and accessible means of interrogating these variants for transgenerational effects. Well-calibrated, well-validated assays for these effects would be very useful for powering rare variant burden studies that could solve some of the “missing heritability” of complex human phenotypes such as growth, cancer risk and tissue development.
3. Investigating the role of TNF pathway in Drosophila tricellular junction regulation. Zazil Solis Saldivar. Auld Lab, UBC
Establishment and maintenance of permeability barriers are some of the most important functions of epithelial cells. Tricellular junctions (TCJs) are permeability barriers formed at the corners of polarized epithelia where three cells converge. TCJ proteins are uniquely localized at the corners of epithelial cells, yet little is known about the mechanisms that regulate them. Gliotactin is a key protein for TCJ development in Drosophila. When Gliotactin is overexpressed and spreads away from the TCJs, it triggers deleterious phenotypes such as apoptosis, overproliferation and cell migration. Preliminary data showed that the cytokine TNF signaling pathway is involved in the enhancement of these phenotypes. Downregulation of the TNF receptor Grindelwald suppresses the overexpression phenotypes of Gliotactin. Grindelwald mediates the pro-apoptotic functions of Eiger, the unique Drosophila TNF ligand. However, downregulation of Eiger does not rescue the phenotypes mentioned before, raising the question whether Grindelwald may function in an Eiger-independent manner. Genetic and biochemistry approaches in Drosophila wing imaginal discs will be used to address if Gliotactin overexpression phenotypes are Eiger independent. Aberrant TCJ function has been linked to several human genetic diseases, and cancer progression and malignancy. This research could help us understand better how cells undergo apoptosis, and processes in tumor development and progression.
4. Exploring the role of glial Syndecan on Drosophila optic lob development. Duo Cheng. Auld Lab, UBC
To establish a functional brain, neural stem cells (NSC) need to generate an abundant number of neurons and glia under tight temporal and spatial regulations. The Drosophila melanogaster’s optic lobe is a popular model to study neurogenesis as each optic lobe generated by neuroblasts (NB) that differentiate from the columnar neuroepithelium (NE), like that of mammalian neurogenesis during development. Glia provide essential modulation to the NSC niche to ensure appropriate NSC differentiation and homeostasis. Yet the tools by which glia utilize to interpret and control the NSC niche are far from being fully characterized. Here, we explored the role of a transmembrane heparan sulfate proteoglycan (HSPG), Syndecan (Sdc) in regulating NSC niche during development. Using super-resolution microscopy, we revealed Sdc is widely expressed across the brain lobe, though we notice Sdc is particular enriched around the NE. Pan-glial knockdown of Sdc revealed a dramatic decrease in brain lobe volume, accompanied by a reduction of the NB population size within the OPC. Moreover, we saw the lengthening of the ventral nerve cord, indicative of a disruption between glia and the overlying extracellular matrix that coats the entire nervous system. While loss of Sdc in glia is not significantly alter the level of apoptosis, it did lead to a reduction in mitotic events in the OPC. Upon further experiments, we saw pan-glial Sdc knockdown perturbed the NE morphology. Therefore we are investigating the role of Sdc in mediating cross-talk between the glia and the NE. Overall, our results supports a novel aspect of Sdc’s role in regulating the NSC niche though glial regulation of the NE in the developing brain lobe.
5. Under Tension: Integrin and Basigin at the Perineurial Glial Membrane. Sophie Roth. Auld Lab, UBC
In Drosophila melanogaster, glial cells play an important role in ensheathing and protecting nerves from damage. To ensure the protection of nerves, the outermost layer of glia known as the perineurial glia (PG), interacts with the extracellular matrix through focal adhesion complexes (FACs), which are comprised of Integrins and intracellular adapter proteins including Talin. In PG FACs, Integrin associates with the transmembrane protein Basigin. Knockdown of Basigin leads to a ruffled phenotype, characterized by areas of constriction across the PG membrane of peripheral nerves. This ruffled phenotype is caused by increased FAC activation since it can be rescued by loss of Integrin or Talin. This leads to the questions: 1. What degree of tension is experienced in the FAC across the PG membrane? 2. Does Basigin play a role in tension regulation in the PG? We hypothesize that the FAC experiences tension to provide stability to the glial membranes. Also, we hypothesize that Basigin negatively regulates Integrin activation in order to modulate tension across the PG membrane. To investigate these questions, a FRET (fluorescence resonance energy transfer) tension sensor will be placed within the protein Talin. The tension sensor will include a donor fluorophore and an acceptor fluorophore, where increased tension leads to a decrease in FRET as the two fluorophores become separated. We are currently testing the degree of tension in the FAC with sensors of varying sensitivity. In the future, we will test the effects on FAC tension in the absence of Basigin. Overall, investigation into tension regulation is an important concept in all animals since nerves are delicate, and therefore too much or too little tension can lead to damage. Since the glia play a main role in protecting the nerves from damage, it is possible that they play a role in modulating tension as well.
6. MAGE- a modulator of Tumour Necrosis Factor Receptor signalling in Drosophila. Tomas Filip. Barker Lab, UBCO
Tumour Necrosis Factor Receptor (TNFR) pathway is a highly conserved and complex signalling decision-maker between cell death and cell survival under physiological conditions and cancer. TNF receptor superfamily in mammals counts to 29 receptors with at least 19 various ligands establishing a significant challenge in deciphering such an essential signalling process. Until recently, mammalian MAGEs have been a large enigmatic family of proteins linked to the incidence of various terminal cancers. Several MAGEs are required for the TNFR signalling, yet the molecular mechanism of their function is unclear. The MAGE protein family includes <50 individual members with high functional redundancy and compensatory kinetics across the protein family. Interestingly, several human MAGEs have been shown to form a complex with E3-ubiquitin ligases, thereby modulating ubiquitination signalling across the cells. Ubiquitination is a major regulatory tool in TNFR signalling. Drosophila has only one MAGE protein and two TNF receptors called Wengen and Grindelwald. TNFR in Drosophila shares a significantly conserved architecture of most of the downstream interactors, signalling mechanisms (including JNK pathway activation), and apoptotic machinery with its mammalian counterparts. Drosophila MAGE is highly structurally similar to most of its mammalian partners, supporting the evolutionary relevance of the MAGEs. In this project, we have established three major aims to investigate: (1) MAGE is required for TNFR signalling in Drosophila as a facilitator of ubiquitination; (2) Identification of the MAGE protein domain required for its action; (3) MAGE modulates a tumour suppressing effect of TNFR in the Drosophila cancer model. The outcomes of this project will lead to a better understanding of the regulatory mechanisms behind the TNFR pathway, which will help to decipher the complexity of TNFR in mammalian systems.
7. Kinetics of blood cell differentiation during hematopoiesis revealed by quantitative long-term live imaging. Kevin Ho. Tanentzapf Lab, UBC
Stem cells typically reside in a specialized physical and biochemical environment that regulates their behaviour. For this reason, stem cells are ideally studied in contexts that maintain this precisely constructed microenvironment while still allowing for live imaging. Here, we develop a long-term multi-organ co-culture system and imaging strategy for hematopoiesis in flies that takes advantage of powerful genetic tools available in this system. Using the culture system, we are able to monitor self-renewal and differentiation of stem cells in real-time with high spatial and temporal resolution under homeostatic condition and upon bacterial infection. We find that the self-renewal division of blood stem cells is linked to cell size and is spatially polarized. Following infection, the stem cell division is altered in their spatial distribution and orientation but the duration of division remains unchanged. Using quantitative imaging to simultaneously track markers for stemness and differentiation in blood stem cells, we identify two types of differentiation that show distinct kinetics during transition of stem cell identity: 1) a linear type differentiation that exhibits a loss of stemness and gain of differentiated state at a constant speed, and 2) a sigmoid type differentiation that exhibits an initial slow phase following by a rapid fast phase during loss of stemness. Following infection, we observe a shift between the two types of differentiation and a change in their spatial distribution and differentiation speed, showing that infection-induced activation of hematopoiesis occurs through modulation of the kinetics of cell differentiation. Overall, these results provide a novel system-level framework for understanding how fly hematopoietic stem cells are regulated in the context of the intact whole organ in real time.
Neuroscience
8. RACK1 knockdown improves motor function and lifespan in an in vivo TDP-43 ALS model. Anaya Saraph. Cashman Lab, UBC
Amyotrophic lateral sclerosis (ALS) is an incurable, adult-onset neurodegenerative disease characterized by the progressive loss of motor neurons. Up to 97% of ALS cases display pathological cytoplasmic depositions of TAR DNA binding protein 43 kDa (TDP-43). This TDP-43 proteinopathy is characterized by the clearance of TDP-43 from the nucleus and the formation of cytoplasmic aggregates, which in turn suppress global protein translation through interactions with receptor activated C kinase 1 (RACK1) protein. Recent work in the Cashman laboratory has shown that RACK1 knockdown (KD) in vitro rescues TDP-43 proteinopathy by reducing cytoplasmic aggregation, promoting nuclear re-entry, and restoring protein translation. Based on these observations, we hypothesized that RACK1 KD would also ameliorate features of TDP-43 pathogenicity in a living organism. We used transgenic Drosophila melanogaster expressing wild type (hTDP-43WT) or mutant (hTDP-43QK) forms of human TDP-43 in motor neurons. hTDP-43QK was co-expressed with RACK1 RNAi (or control RNAi) to achieve cell-specific RACK1 KD. Climbing ability (indicating motor neuron function) and lifespan were used as measures of disease severity. Drosophila expressing hTDP-43 showed significantly reduced climbing ability and lifespan compared to non-transgenic controls; and hTDP-43QK expression conferred greater defects than hTDP-43WT. RACK1 KD in a hTDP-43QK background improved climbing ability and significantly prolonged lifespan. The delayed motor defect and improved lifespan resulting from RACK1 KD confirms the involvement of RACK1 in the mechanism of TDP43 proteinopathy previously seen in vitro. These results support the potential of RACK1 modulation as a therapeutic approach in treating TDP-43-associated ALS.
9. Characterizing appetitive taste memory in Drosophila using a novel optogenic program. Meghan Jelen. Gordon Lab, UBC
Tastes are typically thought to evoke innate appetitive or aversive behaviours, prompting food acceptance or rejection. However, research in Drosophila melanogaster indicates that taste responses can be modified through experience-dependent changes in mushroom body circuits. In this study, we develop a novel taste learning paradigm using closed-loop optogenetics. We find that appetitive and aversive taste memories can be formed by pairing gustatory stimuli with optogenetic activation of sensory or dopaminergic neurons associated with reward or punishment. As with
olfactory memories, distinct dopaminergic subpopulations drive the parallel formation of short- and long-term appetitive memories. Long-term memories are protein synthesis-dependent and have energetic requirements that are satisfied by a variety of caloric food sources or by direct stimulation of MB-MP1 dopaminergic neurons. Our paradigm affords new
opportunities to probe plasticity mechanisms within the taste system and understand the extent to which taste responses are experience dependent.
10. A molecular mechanism for high salt taste in Drosophila. Sasha McDowell. Gordon Lab, UBC
Dietary salt detection and consumption are crucial to maintaining fluid and ionic homeostasis. To optimize salt intake, animals employ salt-dependent activation of multiple taste pathways. Generally, sodium activates attractive taste cells, but attraction is overridden at high salt concentrations by cation non-selective activation of aversive taste cells. In flies, high salt avoidance is driven by both ‘bitter’ taste neurons and a class of glutamatergic ‘high salt’ neurons expressing pickpocket23 (ppk23). Although the cellular basis of salt taste has been described, many of the molecular mechanisms remain elusive. Here, we show that ionotropic receptor 7c (IR7c) is expressed in glutamatergic high salt neurons, where it functions with co-receptors IR76b and IR25a to detect high salt. Misexpression of IR7c in sweet neurons, which endogenously express IR76b and IR25a, confers responsiveness to non-sodium salts, indicating that IR7c is sufficient to convert a sodium-selective receptor to a cation non-selective receptor. Furthermore, the resultant transformation of taste neuron tuning switches potassium chloride from an aversive to an attractive tastant. This research provides insight into the molecular basis of monovalent and divalent salt taste coding and the full repertoire of IRs needed to form a functional salt receptor.
11. The Neural Basis of Taste Processing in Aedes aegypti mosquitoes. Elsa Cyr. Gordon Lab, UBC
Insects possess gustatory receptor neurons that respond to different taste modalities. Taste processing has been extensively studied in Drosophila melanogaster, which makes it an ideal model to identify molecular mechanisms conserved in other insects. In this study, we explore the neural basis of taste processing in the yellow fever mosquito, Aedes aegypti which utilizes non-volatile chemosensory cues during behaviours such as blood-feeding and egg-laying. We hypothesize that taste processing mechanisms in A. aegypti are broadly conserved from Drosophila, but that mosquito-specific adaptations have evolved to support mosquito-specific behaviours. To test this, we are producing a comprehensive anatomical and functional characterization of gustatory neurons expressing taste receptors in the tarsi of mosquitoes. We use genetic driver lines to label sensory neurons, characterizing their anatomy and neural activity in response to taste stimuli. We expect anatomical and functional maps in mosquitoes to be broadly similar to the Drosophila model, where some receptors label overlapping populations of neurons, while others label more distinct subsets. This work will test our hypothesis that the anatomical and functional patterns of gustatory receptor neurons in mosquitoes are broadly conserved from Drosophila and will provide the functional basis for better understanding of biting behaviour in mosquitoes, ultimately informing the development of better interventions to reduce the burden of mosquito-borne disease.
12. Functional specificity of the sweet taste projection neurons in Drosophila. Jinfang Li. Gordon Lab, UBC
Drosophila taste projection neurons (TPNs), which are key to understanding the flow of taste information from peripheral to higher brain centres, remain significantly understudied. After careful screening, we found several split-GAL4 lines that target different classes of TPNs. Here we examine the tuning properties and connectivity of two different sweet-responsive TPNs—sTPNs and lTPNs. We found that both TPNs respond specifically to sweet taste using calcium imaging. GRASP experiments showed physical proximity between the TPNs and the sweet gustatory receptor neurons (GRNs), and between the TPNs and different subpopulations of the higher-order dopaminergic neurons known to be involved in memory reinforcement. To better understand the function of these novel TPNS, we developed a novel light memory assay, which associates taste values to the flashing of light. Strikingly, the sTPNs and lTPNs are functionally specific to certain types of memory. The sTPNs are specifically required for short term appetitive memory, while the lTPNs are required only for long term appetitive memory. We suggest that parallel sets of taste projection neurons encode sweet information that mediates short- and long-term memory reinforcement.
Metabolism and Physiology
13. Triglyceride Metabolism in Neurons Regulates Sex Differences in Body Fat. Colin Miller. Rideout Lab, UBC
Fat is the primary form of stored energy in animals. Triglycerides (TAGs) are the main form of stored fat. Within cells, TAGs are sequestered into specialized organelles called lipid droplets. The process of storing TAGs is tightly regulated across species. An important but understudied factor in fat metabolism is the sex of the animal. Sex chromosomes establish the sex difference in fat metabolism. Yet the metabolic effectors that contribute to this sex difference in fat metabolism remain unclear. In the Rideout Lab we use Drosophila as a model to identify genes that contribute to sex differences in fat metabolism. Flies are an ideal model to study this problem, as the TAG metabolic pathway is highly conserved in Drosophila and our lab’s research shows sex differences in fat metabolism mirror trends in other animals including humans. Indeed, females flies store more body fat than males, and break down fat more slowly after starvation. The lab’s previous work showed that a TAG lipase brummer (bmm) acts in neurons to regulate the sex difference in fat breakdown. This was the first study to identify a phenotype associated with an intracellular triglyceride lipase in neurons. To build on this finding, I will determine whether neuronal bmm’s function in regulating whole-body fat metabolism is unique to bmm, or if additional genes involved in intracellular TAG metabolism also act in neurons to regulate sex differences in whole-body fat metabolism. The overall objective of my project is to elucidate how TAG metabolism in neurons contributes to sex differences in whole-body fat metabolism. To achieve my objective, I propose two Aims: First, determine whether additional genes associated with TAG metabolism act in neurons to regulate the sex difference in whole-body fat metabolism; secondly, determine whether genes associated with TAG metabolism regulate neuron lipid droplets.
14. Determine the role of Insulin signaling pathway in fat homeostasis in male and female Drosophila. Puja Biswas. Rideout Lab, UBC
Like most animals, female Drosophila stores more triglycerides (TAG) than males. Yet the metabolic pathways that contribute to this sex difference in TAG storage remain unclear. We use fruit flies as a model to identify genes and pathways that contribute to the male-female difference in TAG storage. The conserved insulin/insulin-like growth factor signaling pathway (IIS) is known to coordinate fat metabolism with nutrient intake by acting as lipogenic and anti-lipolytic hormone in flies and many animals. Therefore, we tested whether IIS plays a role in regulating whole-body TAG storage in female flies downstream of food intake by increasing lipogenesis compared with male flies. We found sex differences in several aspects of IIS regulation, and showed that females decreased their whole-body fat storage in the absence of insulin producing cells (IPCs), whereas no change in male fat storage. Females with ablated IPCs had increased lifespan and decreased offspring production which suggested that females required IIS to maintain their fat storage to support their reproduction. As the IIS is nutrient-responsive, particularly sugar, we also tested how changing in dietary sugar levels affected IIS activity and fat storage. we found that females reduced fat levels in low sugar diet compared to regular diet, whereas there was no change in male fat storage. Taken together, our data suggests that sex differences exist in the insulin signaling pathway in adults, as in larvae, and play a role in regulating sex difference in fat metabolism.
15. Investigating the role of adipose tissue mitochondria in mediating the sex difference in Drosophila body size plasticity. Celena Cherian. Rideout Lab, UBC
Sexual dimorphism has fascinated biologists for centuries and differences in external appearances, behaviour and reproduction have been well documented across many species. However, there is a lack of knowledge surrounding sex differences in developmental and physiological processes. One such physiological aspect is systemic growth. Males and females of many species across the animal kingdom differ in body size and studies using Drosophila have begun to unravel the mechanisms behind this sexual size dimorphism. The nutritional status of an animal has also been shown to play an important role in whole body growth and final body size. When nutrients are abundant, the growth rate is higher and body size is increased. In conditions of starvation, the growth rate is lower and body size is smaller. This ability of an organism to change its body size in response to nutrient availability is called nutrient-dependent body size plasticity. A recent study from our lab has shown that when Drosophila larvae are fed a high-protein diet females are able to significantly increase their body size while males are unable to do so. This sex difference in body size plasticity is mediated by 2 mitochondrial proteins. Mitochondria are classically considered as the powerhouses of the cell and are primarily known for their roles in generating ATP through oxidative phosphorylation. However, they also regulate nutrient-dependent growth through their biosynthetic activities by providing TCA cycle products to fuel the generation of amino acids and lipids. Hence, I aim to understand the role of mitochondria in mediating this sex difference in nutrient-dependent body size plasticity. This research could provide insight into the mechanisms involved in mediating sex differences in the risk of developing mitochondrial-associated diseases and phenotypes affected by mitochondrial function.
16. Investigating the potential mechanism of cyclin dependent kinase 8 in modulating mitochondrial dynamics via lipogenic pathway. Jenny Liao. Verheyen Lab, SFU
Cyclin-dependent kinase 8 (Cdk8) is a well-known serine/threonine protein kinase that functions in the transcriptional regulation. We found a novel function of Cdk8 in regulating mitochondrial morphology, and expression of Cdk8 alleviates the defective phenotypes caused by impaired mitochondria in a pink1 mutant mediated Parkinson’s disease model. We are currently exploring potential mechanisms that can explain these findings. One direction that we are pursuing is the SREBP (sterol regulatory element binding protein) mediated lipogenic pathway. It has been shown that Cdk8 is a negative regulator of SREBP, which is a master transcription factor in fatty acid, cholesterol, glycerolipid, as well as lipid droplet synthesis. Phosphorylation on SREBP by Cdk8 can promote degradation of SREBP, which in turn reduces the lipid anabolic pathway. In addition, since lipid droplets are also a well-known factor associated with neurodegenerative disorders, it is tempting to investigate the potential role of the lipogenic pathway in modulating mitochondrial dynamics. Strikingly, we found mitochondrial morphology shifts from fused and elongated to round-shaped and fragmented when expression of lipogenic pathway is modulated. These findings resembled our previous findings where Cdk8 is knocked down or ectopically expressed, respectively. Furthermore, our preliminary data indicates that suppression of lipogenic pathway can also rescue the climbing and muscle degeneration in the pink1 mutant background, which further suggests that Cdk8 may influence mitochondrial dynamics by negatively regulating the lipogenic pathway.
Ecology and Evolution
17. The invasive spotted wing drosophila and their associated host plant phenology. Warren Wong. Carrillo Lab, UBC
The spotted wing drosophila, Drosophila suzukii, is a major agricultural pest globally. As D. suzukii oviposits in developing fruits, the timing of oviposition in relation to host plant phenology is important to the development of pest management strategies. We conducted a survey in the Fraser Valley of British Columbia to examine the association between host plant phenology and larval development and abundance over the growing season on four host plants - salmonberries, strawberries, raspberries, and Himalayan blackberries.
Immunology (Molecular Virology and Host-Microbe Interactions)
18. When proteins go viral: A viral protein impairs stress granule formation by modulating RanBP2/Nup358. Jibin Sadasivan. Jan Lab, UBC
Viruses have evolved mechanisms to modulate cellular pathways to facilitate infection. One such pathway is stress granule (SG) assembly. SGs are ribonucleoprotein complexes that assemble during translation inhibition following cellular stress. Inhibition of SG assembly has been observed under numerous virus infections across viral families and species suggests an essential conserved fundamental viral strategy. However, the significance of and mechanisms underlying SG modulation during virus infection are not fully understood. The multifunctional 1A protein encoded by the model dicistrovirus, cricket paralysis virus (CrPV) can bind to and degrade Ago-2 in an E3 ubiquitin ligase-dependent manner to block the antiviral RNA interference pathway and the 1A protein also inhibit SG formation. Moreover, the R146 residue of 1A is necessary for SG inhibition and for virus infection in both Drosophila S2 cells and adult flies. In this study, we investigated the molecular mechanism by which CrPV-1A inhibit SGs. We showed that expression of wild-type but not mutant R146A CrPV-1A in S2 cells inhibits SG formation when challenged with potent SG inducers, indicating that 1A-mediated SG inhibition is stress-independent. CrPV-1A’s ability to inhibit SG formation does not require the Ago-2 binding domain but requires the E3 ubiquitin-ligase binding domain. Overexpression and infection studies in both Drosophila and human cells showed that wild-type CrPV-1A but not mutant R146A CrPV-1A localizes to the nuclear periphery which correlates with nuclear enrichment of poly(A)+RNA. Transcriptome analysis demonstrated that the CrPV R146A virus is defective in inducing host transcriptome changes in CrPV-infected cells. Finally, inhibition of SG formation by CrPV-1A requires depletion of NUP358 in an R146-dependent manner. Taking this together, we propose that CrPV utilizes a multiprong strategy whereby the CrPV-1A protein interferes with Nup358 activity to inhibit SG assembly.
19. Distribution of insecticidal activity. Daniel Yanez. Haney Lab, UBC
The genus Pseudomonas includes Gram-negative bacteria the live in a wide range of environments and on diverse hosts. Pseudomonas is among the most complex genera with the largest number of species. Pseudomonas strains exhibit a variety of ecological lifestyles ranging from human and plant pathogens to beneficial plant and animal-associated strains. The Pseudomonas fluorescens group exhibits diverse effects on plant and insect hosts. These range from beneficial effects on plants and infection and killing of insect larvae . How Pseudomonas infect different hosts and overcome each immune system is still largely unknown. However, previous studies on insecticidal activity point to a highly multifactorial nature, with different genes involved in the process. Pseudomonas fluorescens is not the only Pseudomonas clade with insecticidal activity. Insecticidal activity has previously been reported within the P. protegens and P. chlororaphis sub-clades and P. taiwanensis and P. entomophila. Thus, my work aims to explore the distribution and extent of the insecticidal ability throughout the whole Pseudomonas genus by combining bioinformatics and insect oral infection assays. Here, I found a correlation between insecticidal activity and the presence/absence of genes associated with insecticidal activity across some Pseudomonas strains.