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The Discovery and Regulation of Modes of Exocytosis Through the Lens of Computer Vision

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The Discovery and Regulation of Modes of Exocytosis Through the Lens of Computer Vision THE DISCOVERY AND REGULATION OF MODES OF EXOCYTOSIS THROUGH THE LENS OF COMPUTER VISION Fabio Urbina A thesis submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the department of Cell Biology and Physiology in the School of Medicine. Chapel Hill 2020 Approved by: Stephanie Gupton Patrick Brennwald Doug Cyr Keith Burridge Shawn Gomez © 2020 Fabio Urbina ALL RIGHTS RESERVED ii ABSTRACT Fabio Urbina: Discovery and Regulation of the Modes of Exocytosis through the lens of computer vision (Under the direction of Stephanie Gupton) The formation of the nervous system involves establishing complex networks of synaptic connections between proper partners, which requires the rapid expansion of the plasma membrane surface area as neurons grow. Critical to the expansion of the plasma membrane is exocytic vesicle fusion, a regulated mechanism driven by soluble N-ethylmaleimide-sensitive factor attachment proteins receptors (SNAREs). Multiple modes of exocytosis have been proposed, with full-vesicle fusion (FVF) and kiss-and-run (KNR) being the best described. The basis of SNARE-mediated fusion, the opening of a fusion pore, and its contribution to plasma membrane expansion remains enigmatic, as vesicle fusion is spatially small and temporally fast. We exploited TIRF microscopy to image VAMP-pHluorin mediated exocytosis in murine embryonic cortical neurons and developed computer-vision software and statistical tools to perform unbiased, efficient identification of exocytic events and uncover spatiotemporal aspects of exocytosis during neuron development. We further developed novel classification algorithms to describe. and classify individual exocytic events. Vesicle fusion behavior differed between vesicle types, cell types, developmental stage, and extracellular environment. Spatial statistics uncovered distinct spatiotemporal regulation of exocytosis in the soma and neurites of neurons that was modulated by developmental stage, the guidance cue netrin-1, and the E3 ubiquitin ligase TRIM9. Four distinguishable fusion modes were uncovered in developing neurons: two FVF-like modes that insert membrane material (FVFi and FVFd) and two KNR-like modes that do not (KNRi iii and KNRd). Experiment-based mathematical calculations and experiments indicated that FVF and FVFd VAMP2-mediated vesicle fusion supplied sufficient material for the plasma membrane expansion that occurred early in neuronal morphogenesis. The mode of exocytosis is dependent on the E3-ubiquitin ligase TRIM67. Neurons lacking Trim67 have increased KNRi and KNRd as well as smaller surface area, presumably due to lowered rates of FVFi and FVFd. Our data suggest this is accomplished in part by limiting incorporation of the Qb/Qc SNARE SNAP47 into SNARE complexes, and thus, SNAP47 involvement in exocytosis. SNAP47 levels are elevated in neurons lacking TRIM67, and overexpression of SNAP47 in neurons expressing wildtype levels of TRIM67 have increased KNRi and KNRd and decreased FVFi. Thus, we establish a regulatory mechanism of TRIM67, distinct from TRIM9. iv To my mother and father, who have supported me and pushed me to always strive to be a better person. To my love Erin Pedone, who is my bedrock and my anchor. To all of my brothers, especially Armando, who have been a constant source of familiarity and fun and who remind me to never take life so seriously. To my best friend Katie Stember, who has put up with my inability to put work down and all of my flaws throughout, yet still pushes me to be more than I am. To Bast and Orion, the best and worst roommates I could ever have. To Billy and all the joy he has brought me. v ACKNOWLEDGMENTS I would like to first and foremost thank my mentor, Dr. Stephanie Gupton. Dr.Gupton has been one of the greatest mentors I could have asked for, and put in so much effort to train me to become the scientist that I am today. I will always be grateful for her mentorship and guidance. I would like to also thank all of the members of the Gupton lab, past and present, who have been a source of sanity and ideas. I would like to thank the members of my thesis committee, Patrick Brennwald, Doug Cyr, Shawn Gomez, and Keith Burridge for their input and invaluable advice given to me over the years. I especially want to thank Patrick Brennwald for his thought and ideas and encouragement and all he’s done for not just me, but graduate students at large. vi TABLE OF CONTENTS LIST OF FIGURES ................................................................................................................................... xii LIST OF ABBREVIATIONS .................................................................................................................... xiv CHAPTER 1: SNARE-MEDIATED EXOCYTOSIS IN NEURONAL DEVELOPMENT1 .................. 1 1 The Remarkable Mammalian Neuron .......................................................................... 1 1.1 Development of Neurons .......................................................................................... 1 2 SNAREs and Exocytosis ............................................................................................. 5 2.1 Discovery of the Fusion Machinery ......................................................................... 5 2.2 SNAREs in Complex ................................................................................................. 5 3 SNARE Roles in Different Stages of Development ...................................................... 8 3.1 SNARE Expression Changes Throughout Development .................................... 8 3.2 The Mature Neuron ................................................................................................. 12 3.3 Sites of Membrane Growth .................................................................................... 14 3.4 Evidence for the Growth Cone .............................................................................. 15 3.5 Evidence for the Soma ........................................................................................... 15 3.4 A Point of Tension ................................................................................................... 18 4 Modes of Fusion .........................................................................................................19 4.1 Beyond Two Modes of Fusion ............................................................................... 23 5 The Fusion Pore: Kinetics, Dynamics, and Models of Formation ................................25 5.1 The Fusion Pore: Expansion and Contraction .................................................... 25 5.2 Regulation of the Fusion Pore ............................................................................... 26 vii CONCLUSION ..............................................................................................................30 CHAPTER 1 FIGURES .................................................................................................32 REFERENCES ..............................................................................................................37 CHAPTER 2: SPATIOTEMPORAL ORGANIZATION OF EXOCYTOSIS EMERGES DURING NEURONAL SHAPE CHANGE ........................................................................ 48 1 Introduction.................................................................................................................48 2 Results .......................................................................................................................50 2.1 Automated identification and analysis of exocytosis .......................................... 50 2.2 Confirmation of exocytic detection ........................................................................ 51 2.3 Confirmation of detection of VAMP7-mediated exocytosis. .............................. 53 2.4 Spatiotemporal changes in exocytosis occur during development .................. 54 2.5 A mathematical approximation of membrane expansion .................................. 56 2.6 Netrin-1 modulates the spatiotemporal distribution of exocytic events in neurites .......................................................................................... 58 2.7 TRIM9 is required for netrin-dependent exocytic changes in neurites ............ 59 2.8 Different domains of TRIM9 modulate specific parameters of exocytosis in neurites ............................................................................................... 60 2.9 Exocytosis in melanoma cells is distinctly organized ......................................... 61 3 Discussion ..................................................................................................................62 3.1 Dynamic spatial regulation of exocytosis in developing neurons ..................... 63 3.2 Calculating membrane growth in developing neurons ....................................... 64 3.3 Exocytosis in the soma and neurites are distinctly regulated ........................... 64 3.4 Polarized exocytosis in non-neuronal migrating cells ........................................ 65 4 Materials and Methods ...............................................................................................66 4.1 Animal Work ............................................................................................................. 66 viii 4.2 Media, culture, and transfection of primary neurons and immortalized cell lines ...........................................................................
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