CELLULAR ROLE OF THE DROSOPHILA EFR3, ROLLING BLACKOUT (RBO) IN SYNAPTIC TRANSMISSION By Niranjana Vijayakrishnan Dissertation Submitted to the Faculty of the Graduate School at Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY In Neuroscience May, 2010 Nashville, Tennessee Approved by : Professor Roger J.Colbran Professor Kendal S.Broadie Professor Randy D.Blakely Professor Todd R.Graham To Sri Akka, my parents, Raji and Dr.K.G.Vijayakrishnan and my husband Ramadass Prabhakar, with my love and gratitude. ii ACKNOWLEDGEMENTS I am grateful to my thesis advisor Dr. Kendal Broadie for letting me join his lab and work on this project. I am thankful for his support and input during the course of this study. I must acknowledge Elvin A. Woodruff III, the lab EM technician for his vital contribution to this project. I am deeply grateful to Drs. Jeffrey Rohrbough and Heinrich Matthies who taught me much about this project in my initial years in the lab. Jeff has a keen eye for detail and very patiently and painstakingly taught me several experimental techniques I used during the course of this study. Heiner initiated me into this project, I am extremely thankful to him for his mentorship, his infectious enthusiasm for science, and for his critical comments on my data. I also acknowledge Drs. Ralf Mohrmann, Fu-de Huang, Scott Phillips, Cheryl Gatto and Charles Tessier for useful discussion on data quantification and experimental design. I am grateful to Dr. Elaine Sanders- Bush for accepting me into the Neuroscience program and funding me during my first two years in the program. I thank the National Institutes of Health (NIH) for funding support during the course of this study. I am thankful to my dissertation committee members for their keen involvement in this project and for their critical comments on my dissertation. I thank Vandana Grover, for our daily ritual coffee break and discussions of life in graduate school and Joshua Decker for proofreading this dissertation. iii TABLE OF CONTENTS Page DEDICATION……………………………………………………………………….......ii ACKNOWLEDGEMENTS………………………………………………………….....iii LIST OF TABLES………………………………………………………………………vi LIST OF FIGURES…………………………………………………………………….vii LIST OF ABBREVIATIONS……………………………………………………….......ix CHAPTER I.INSIGHTS INTO THE SYNAPTIC VESICLE CYCLE: DROSOPHILA TEMPERATURE-SENSITIVE PARALYTIC MUTATIONS .................................... 1 Introduction to the synaptic vesicle cycle ............................................................ 1 Synaptic vesicle exocytosis ................................................................................ 2 The SNARE hypothesis .................................................................................. 4 Syntaxin 1A .................................................................................................... 8 Lipids in exocytosis ....................................................................................... 11 Endocytosis ...................................................................................................... 12 Clathrin-mediated endocytosis ......................................................................... 13 Accessory factors in clathrin-mediated endocytosis ......................................... 15 AP-2 ............................................................................................................. 15 AP180(CALM) .............................................................................................. 17 Amphiphysin ................................................................................................. 18 Dynamin ....................................................................................................... 20 Endophilin and synaptojanin ......................................................................... 27 Bulk endocytosis ............................................................................................... 30 Drosophila temperature-sensitive paralytic mutants ......................................... 31 Rolling Blackout (RBO): acute requirement in neurotransmission .................... 33 Drosophila phototransduction ........................................................................... 38 Visual signal transduction ............................................................................. 40 Genetic approaches for studying Drosophila vision ...................................... 43 Phototransduction-related phenotypes in rbots ............................................. 45 II.THE DROSOPHILA EFR3 ROLLING BLACKOUT IS REQUIRED FOR BULK ENDOCYTOSIS IN NON-NEURONAL CELLS AND NEURONAL SYNAPSES . 48 Introduction ................................................................................................... 48 Results ............................................................................................................. 51 RBO is required for endocytosis at the neuromuscular synapse .................. 51 RBO is required for endocytosis in central brain synapses .......................... 56 RBO is required for endocytosis in non-neuronal cells ................................. 61 iv RBO is required for bulk endocytic formation of endosomes at the synapse 69 Discussion .................................................................................................... 73 Materials and methods ..................................................................................... 80 Drosophila stocks ......................................................................................... 80 Immunohistochemistry .................................................................................. 81 FM dye imaging assays ................................................................................ 81 Garland cell tracer endocytosis assays ........................................................ 82 Electron microscopy ..................................................................................... 83 Tannic acid impregnation and horse radish peroxidase uptake methods ..... 83 FM1-43 photoconversion .............................................................................. 84 III. ROLLING BLACKOUT POSSESSES LIPASE DOMAIN-DEPENDENT AND INDEPENDENT ENDOCYTIC FUNCTIONS DOWNSTREAM OF DYNAMIN ... 86 Introduction ................................................................................................... 86 Materials and methods ..................................................................................... 90 Drosophila stocks ........................................................................................ 90 Molecular techniques .................................................................................... 90 Behavioral assays ........................................................................................ 91 Immunihistochemistry ................................................................................... 92 FM 1-43 dye loading assays ......................................................................... 92 Garland cell tracer assays ............................................................................ 93 Results ............................................................................................................. 93 Putative lipase-dead RBO does not rescue rbo null embryonic lethality ....... 93 Putative lipase-dead RBO is unable to rescue garland cell endocytosis defects ................................................................................................................ 98 Putatitve lipase-dead RBO rescues endocytic defects at the NMJ ............. 100 shibirets1 is epistatic to rbots in synaptic vesicle endocytosis ...................... 103 shibirets1 is epistatic to rbots in non-neuronal cell endocytosis .................... 104 Discussion .................................................................................................. 108 IV. CONCLUSIONS AND FUTURE DIRECTIONS ........................................... 115 REFERENCES ................................................................................................. 126 v LIST OF TABLES Table Page 1. Drosophila TS paralytic mutants ..................................................................... 34 vi LIST OF FIGURES Figure Page 1. The synaptic vesicle cycle ................................................................................ 3 2. SNARE assembly and disassembly during exocytosis ..................................... 7 3. Domain structure of syntaxin 1A ....................................................................... 9 4. Steps involved in clathrin-mediated endocytosis ............................................ 16 5. Domain structure of dynamin ......................................................................... 22 6. Domain structure of proteins involved in clathrin-mediated endocytosis ...... ..28 6. Domain structure of proteins involved in clathrin-mediated endocytosis …….29 7. Localization of RBO at the Drosophila 3rd instar larval NMJ ........................... 37 8. Phototransduction cascade in Drosophila ...................................................... 42 9. PI turnover pathway ........................................................................................ 44 10. Initial model for RBO function based on synergism with syx3-69.................... 46 11. RBO facilitates FM1-43 endocytosis at the NMJ (a-c) .................................. 52 11. RBO facilitates FM1-43 endocytosis at the NMJ