The Pennsylvania State University Schreyer Honors College
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THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE DEPARTMENT OF BIOLOGY GENETIC ANALYSIS IN DROSOPHILA REVEALS A NOVEL ROLE FOR THE ESCRT COMPONENT VPS24 IN SYNAPTIC TRANSMISSION KERRY CAO SPRING 2013 A thesis submitted in partial fulfillment of the requirements for a baccalaureate degree in Chemistry with honors in Biology Reviewed and approved* by the following: Fumiko Kawasaki Assistant Professor of Biology Thesis Supervisor Bernhard Lüscher Professor of Biology Honors Adviser * Signatures are on file in the Schreyer Honors College. i ABSTRACT Understanding the in vivo molecular mechanisms of synaptic transmission remains a major objective in cellular and molecular neuroscience. Our genetic analysis of synaptic transmission has identified a novel synaptic mechanism involving the Endosomal Sorting Complexes Required for Transport (ESCRT) pathway previously implicated in intracellular membrane trafficking. A forward genetic screen for new mutations affecting synaptic transmission involved chemical mutagenesis with ethyl methanesulfonate (EMS) followed by screening for third chromosome mutations affecting fly behavior. A new mutation was recovered in the vps24 gene, which encodes a conserved protein component of the ESCRT-III complex which functions in targeting proteins to multivesicular bodies (MVBs) for degradation. The new Drosophila vps24 mutant exhibits defects in neurotransmitter release and provides the first evidence implicating the ESCRT pathway in synaptic transmission. Surprisingly, VPS24 was found to be localized in proximity to neurotransmitter release sites at active zones (AZs), suggesting a novel ESCRT-mediated mechanism contributing to neurotransmitter release. Furthermore, our preliminary studies of other ESCRT proteins indicate function and localization of this pathway within the presynaptic terminal. Ongoing studies have involved generation of Drosophila transgenic lines and further in vivo analysis of the VPS24 protein properties and functions in synaptic transmission. Given the strong evolutionary conservation of the ESCRT pathway, it is anticipated that these studies will be of general significance to our understanding of the ESCRT pathway and its importance in neural function. ii TABLE OF CONTENTS List of Figures ................................................................................................................................ iii Acknowledgements ......................................................................................................................... iv Contributions .................................................................................................................................... v Introduction ...................................................................................................................................... 1 Results ............................................................................................................................................ 10 Discussion ...................................................................................................................................... 19 Materials and Methods ................................................................................................................... 23 References ...................................................................................................................................... 26 iii LIST OF FIGURES Figure 1. Multivesicular body biogenesis ........................................................................................ 2 Figure 2. Interactions among the four ESCRT complexes and associated molecules ...................... 3 Figure 3. Sequence of intralumenal vesicle formation ..................................................................... 6 Figure 4. Purse string model of ESCRT-III disassembly ................................................................. 8 Figure 5. Forward genetic screen for new mutations affecting synaptic transmission ................... 10 Figure 6. Behavioral phenotype of 748 mutant .............................................................................. 11 Figure 7. Electrophysiology recordings of 748/Df(3R)Exel6140 flies ........................................... 12 Figure 8. Genetic mapping of 748 mutation ................................................................................... 13 Figure 9. Preservation of presynaptic morphology and organization in vps241 mutant ................. 15 Figure 10. Localization of VPS24 in active zones at neuromuscular synapses .............................. 16 Figure 11. An ultrastructural synaptic phenotype in the vps241 mutant ......................................... 17 Figure 12. Localization of VPS28 at neuromuscular synapses ...................................................... 18 Figure 13. The synaptic vesicle cycle ............................................................................................. 21 iv ACKNOWLEDGEMENTS First and foremost, I would like to thank Dr. Fumiko Kawasaki and Dr. Richard Ordway for their boundless knowledge, guidance, patience, and constant support throughout my time in this research lab. I am fortunate to have two established faculty as open and accessible resources. I would also like to thank Dr. Bernhard Lüscher for his generosity, patience, and flexibility during this process. I am grateful for Dr. Janani Iyer, Rie Danjo, and Yunzhen Zheng for making me feel like a welcomed member of the lab and for providing insight into the theory behind the methods of the lab. I am thankful for Sean Stevens for being one of many mentors, being someone to talk to during my early days in the lab, and showing me the protocols of the lab. I am grateful for and appreciate Chris Wahlmark and Giselle Ahnert for partnering with me during our molecular biology and flywork experiments, which made learning new techniques less stressful and also more informative. I would like to thank Alex Strauss, Julie Oliver, and Ximena Chica for being the role models I strove to emulate. I am thankful for Rolfy Perez and the other undergraduate members of the lab for being my friends during this journey as well as providing a unique and pleasant dynamic in the lab. I would like to thank Dr. Noreen Reist for providing the anti-SYT antibody, Dr. Helmut Kramer for providing the anti-VPS28 antibody, and the Developmental Studies Hybridoma Bank for supplying the anti-BRP antibody. I would also like to thank the Bloomington Drosophila Stock Center for supplying the deficiency and P element stocks used in this thesis as well as the Drosophila Genomics Resource Center (Bloomington, IN) for providing us with the vps24 cDNA needed to conduct our transgenic experiments. v CONTRIBUTIONS Sean Stevens introduced me to the majority of the flywork done in this thesis. He, along with Giselle Ahnert and Christ Wahlmark, assisted with the execution of the forward genetic screen and behavior test. Giselle Ahnert and Chris Wahlmark were responsible for the mutagenesis of Iso 3 male flies used in the genetic screen. Giselle Ahnert and Chris Wahlmark were also collaborators for the molecular biology experiments conducted to generate the UAS-EGFP-vps24 construct used to generate the transgenic flies mentioned in this thesis. Chris Wahlmark assisted with the larval dissections used for immunocytochemical analysis and the rescue of the vps241 mutant. Dr. Fumiko Kawasaki conducted the electrophysiological experiments and performed confocal microscopy on the larval and adult immunocytochemical preparations. 1 INTRODUCTION Eukaryotic endosomes coordinate the movement of proteins between the plasma membrane, the trans-Golgi network, and the lysosome/vacuole [1]. The endosomal system conducts this intracellular trafficking by internalizing membrane proteins and sorting these to multivesicular bodies (MVBs), which consist of membrane enclosing intralumenal vesicles (ILVs) formed by the invagination of the outer membrane into the lumen [2]. Ubiquitination serves as the major entry signal for proteins into the MVB pathway [3,4]. A set of approximately 20 proteins, the majority of which are vacuolar protein sorting (Vps) gene products, conserved from yeast to mammals is thought to regulate and drive formation of ILVs (Figure 1a). These include components of four ESCRT complexes (endosomal sorting complexes required for transport-0, I, II, and III), the AAA ATPase VPS4, and several associated proteins. Mutations in genes encoding ESCRT subunits cause enlarged, “Class E,” endosomal compartments, and result in sorting defects [5,6]. Recently, non-MVB functions for several ESCRT components have been identified [7]. Because the required membrane curvature is opposite to that of clathrin-mediated endocytosis and other coat- mediated vesicle formation, it is likely that novel mechanisms are involved. Importantly, the role of the ESCRT machinery extends to topologically similar cellular processes including autophagy, cytokinesis, and viral budding (Figure 1b-d). For example, ESCRT-III has been implicated in the autophagy pathway and in the clearance of protein aggregates that characterize neurodegenerative disorders [8-10]. Mammalian ESCRT-III also has a role in retroviral budding, as do ESCRT-0, -I, and the ESCRT- associated protein AIP/ALIX [11,12]. ESCRT-III components have also been shown to concentrate at the mid-body during cell abscission in cytokinesis [13,14], a process that is topologically similar to retroviral budding and ILV formation. Interest