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MATRIX METALLOPROTEINASE REQUIREMENTS for NEUROMUSCULAR SYNAPTOGENESIS by Mary Lynn Dear Dissertation Submitted to the Faculty O

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MATRIX METALLOPROTEINASE REQUIREMENTS for NEUROMUSCULAR SYNAPTOGENESIS by Mary Lynn Dear Dissertation Submitted to the Faculty O MATRIX METALLOPROTEINASE REQUIREMENTS FOR NEUROMUSCULAR SYNAPTOGENESIS By Mary Lynn Dear Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biological Sciences January 31, 2018 Nashville, Tennessee Approved: Todd Graham, Ph.D. Kendal Broadie, Ph.D. Katherine Friedman, Ph.D. Barbara Fingleton, Ph.D. Jared Nordman, Ph.D. Copyright © 2018 by Mary Lynn Dear All Rights Reserved ACKNOWLEDGEMENTS I would like to acknowledge my advisor, Kendal Broadie, and the entire Broadie laboratory, both past and present, for their endless support, engaging conversations, thoughtful suggestions and constant encouragement. I would also like to thank my committee members both past and present for playing such a pivotal role in my graduate career and growth as a scientist. I thank the Department of Biological Sciences for fostering my graduate education. I thank the entire protease community for their continued support, helpful suggestions and collaborative efforts that helped my project move forward. I would like to acknowledge Dr. Andrea Page-McCaw and her entire laboratory for helpful suggestions, being engaged in my studies and providing many tools that were invaluable to my project. I thank my parents, Leisa Justus and Raymond Dear, my brother Jake Dear and my best friend Jenna Kaufman. Words cannot describe how grateful I am for your support and encouragement. Most importantly, I would like to thank my husband, Jeffrey Thomas, for always believing in me, your unwavering support and for helping me endure all the ups and downs that come with graduate school. I am forever grateful and could not have done this without you. iii ACKNOWLEDGEMENT OF FUNDING This work was supported by the National Institutes of Health R01 MH096832 and R01 MH084989 to K.B. PUBLICATIONS Two classes of matrix metalloproteinases reciprocally regulate synaptogenesis. Dear ML, Dani N, Parkinson W, Zhou S, Broadie K. Development. 2016 Jan 1;143(1):75-87. Synaptic roles for phosphomannomutase type 2 in a new Drosophila congenital disorder of glycosylation disease model. Parkinson WM, Dookwah M, Dear ML, Gatto CL, Aoki K, Tiemeyer M, Broadie K. Dis Model Mech. 2016 May 1;9(5):513-27. Neuronal activity drives FMRP- and HSPG-dependent matrix metalloproteinase function required for rapid synaptogenesis. Dear ML, Shilts J, Broadie K. Sci. Signal. 2017 Nov 7;10:eaan3181. iv TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ................................................................................................................... iii ACKNOWLEDGEMENT OF FUNDING ............................................................................................... iv PUBLICATIONS ................................................................................................................................. iv LIST OF FIGURES AND TABLES ....................................................................................................... viii LIST OF SUPPLEMENTARY MATERIAL............................................................................................... x LIST OF ABBREVIATIONS ................................................................................................................. xi Chapter I. Introduction ................................................................................................................................ 1 The Extracellular Matrix ............................................................................................................. 1 Characterizing the Extracellular Matrix ................................................................................... 1 Core Matrisome Components .................................................................................................... 5 Collagens .................................................................................................................................. 5 Proteoglycans .......................................................................................................................... 6 Glycoproteins........................................................................................................................... 8 Matrisome Associated Molecules .............................................................................................. 9 Extracellular Matrix Receptors ................................................................................................ 9 Extracellular Matrix Modifying Enzymes ............................................................................... 12 The Drosophila Neuromuscular Junction as a Genetic Model Synapse ................................... 18 The NMJ System .................................................................................................................... 18 Structural Synaptogenesis ..................................................................................................... 21 Functional Synaptogenesis .................................................................................................... 22 Wnt Trans-Synaptic Signaling ................................................................................................ 25 Heparan Sulfate Proteoglycans in Synaptic Development ....................................................... 32 Secreted HSPGs ..................................................................................................................... 32 Membrane-tethered HSPGs .................................................................................................. 34 HS Biosynthesis and HS-modifying Enzymes ......................................................................... 35 Matrix Metalloproteinases in Neural Development ................................................................ 35 Thesis Outline ........................................................................................................................... 44 v Part One ................................................................................................................................. 44 Part Two................................................................................................................................. 44 References ................................................................................................................................ 45 II. Two Classes of Matrix Metalloproteinases Reciprocally Regulate Synaptogenesis ................ 75 Summary................................................................................................................................... 76 Introduction .............................................................................................................................. 76 Materials and Methods ............................................................................................................ 78 Drosophila Stocks .................................................................................................................. 78 Antibody Production ............................................................................................................. 78 Western Blot Analyses ........................................................................................................... 80 Immunocytochemistry Imaging ............................................................................................. 80 Electrophysiology .................................................................................................................. 81 Electron Microscopy .............................................................................................................. 81 Statistical Measurements ...................................................................................................... 81 Results ...................................................................................................................................... 82 Mmp1 and Mmp2 both regulate synapse morphogenesis ................................................... 82 Mmp1 and Mmp2 both regulate differentiation of synapse function ................................. 85 Mmp1 and Mmp2 both regulate synapse molecular assembly ............................................ 87 Mmp1, Mmp2 and Timp co-dependently localize to the NMJ synaptomatrix ..................... 90 Mmp1 and Mmp2 restrict Wnt trans-synaptic signaling ...................................................... 93 Restoring Wnt co-receptor Dlp levels in Mmp mutants prevents synaptogenic defects ..... 97 Discussion ................................................................................................................................. 99 References .............................................................................................................................. 102 III. Neuronal Activity Drives FMRP- and HSPG-dependent Matrix Metalloproteinase Function Required for Rapid Synaptogenesis ....................................................................................... 109 Summary................................................................................................................................. 110 Introduction ............................................................................................................................ 110 Materials and Methods .......................................................................................................... 112 Drosophila Genetics
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