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Journal of Biological Chemistry POSTSYNAPTIC EFFECTORS OF NEURON MORPHOLOGY AND FUNCTION: PART I. CHARACTERIZATION OF POSTSYNAPTIC DROSOPHILA SYNDAPIN PART II. CHIMERIC LIGHT-ACTIVATED RECEPTORS FOR THE CONTROL OF 5-HT1A SIGNALING by EUGENE OH Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation advisor: Dr. Stefan Herlitze Department of Neurosciences CASE WESTERN RESERVE UNIVERSITY January 2011 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Eugene Oh candidate for the Doctor of Philosophy degree *. (signed) Dr. Gary Landreth (chair of the committee) Dr. Stefan Herlitze Dr. Jerry Silver Dr. Stephen Maricich (date) November 19th, 2010 *We also certify that written approval has been obtained for any proprietary material contained therein. Copyright © 2011 by Eugene Oh All rights reserved DEDICATION This work is dedicated to my friends and family for their enduring support and encouragement; my children Lucia, Paloma and Elliot who inspire me to be greater person; and most importantly, to my wife Sarah. Nothing would have been possible without her help and unconditional love. iv TABLE OF CONTENTS Title Page ............................................................................................................................ i Signature page ................................................................................................................... ii Dedication ......................................................................................................................... iv Table of Contents ...............................................................................................................v List of Figures ................................................................................................................... ix Acknowledgements .......................................................................................................... xi Abstract ............................................................................................................................ xii CHAPTER 1: INTRODUCTION TO PART I ...............................................................1 The genetic tractability of Drosophila melanogaster ...................................................... 2 The Drosophila neuromuscular junction (NMJ) as a model for synaptic plasticity .............................................................................................................. 6 Regulation of Drosophila NMJ morphology ................................................................... 9 Transsynaptic signaling regulates synaptic growth: ................................... 9 Actin cytoskeleton dependent plasticity ................................................... 11 The function of Drosophila Wsp and linking actin to NMJ growth ........ 15 Endocytic proteins are negative regulators of NMJ growth ..................... 17 Vertebrate Syndapin ....................................................................................................... 20 F-BAR Domain containing proteins: structural bridges between cytoskeleton and cell membranes .................................................................................. 22 Drosophila Syndapin ....................................................................................................... 24 Research aims .................................................................................................................. 26 v CHAPTER 2: DROSOPHILA SYNDAPIN INTERACTS WITH WISKOTT-ALRICH SYNDROME PROTEIN AND REGULATES NEUROMUSCULAR JUNCTION GROWTH POSTSYNAPTICALLY .................28 Introduction ..................................................................................................................... 29 Materials and Methods ................................................................................................... 32 Results .............................................................................................................................. 36 Identification of a Drosophila Syndapin homologue. ............................... 36 Identification P-element inserts near Drosophila Syndapin and characterization of Syndapin hypomorphic alleles ............................ 37 Localization of Syndapin at Drosophila neuromuscular junctions. .......... 39 Loss of Syndapin results in synaptic overgrowth ..................................... 40 Postsynaptic expression of a Synd transgene rescues the overgrowth phenotype .............................................................................. 42 Drosophila Syndapin binds Wsp and the conserved proline487 residue of residue of Synd is required for this interaction ........................ 43 Syndapin and Wsp interact in vivo ........................................................... 44 Synd acts upstream of wsp ........................................................................ 45 Figures .............................................................................................................................. 47 CHAPTER 3: DISCUSSION ..........................................................................................63 Research Conclusions ..................................................................................................... 64 Postsynaptic actin cytoskeleton and the control of synaptic morphology ................. 64 Syndapin is an adaptor protein that recruits Wsp to the postsynaptic membrane .................................................................................................. 66 A putative role for Drosophila Syndapin in synaptic vesicle endocytosis .................. 67 Remaining questions and future directions .................................................................. 68 vi CHAPTER 4: INTRODUCTION TO PART II ............................................................71 The function and importance of the serotonergic system ........................................... 72 The 5-HT1A receptor and its role in disease pathogenesis and treatment ................. 75 Structural determinants of 5-HT receptor function and targeting ............................ 79 G protein-coupled receptors of visual systems as "optogenetic" probes ................... 81 Invertebrate rhodopsin .............................................................................. 83 Vertebrate rhodopsins ............................................................................... 84 Melanopsin ................................................................................................ 90 OptoXRs: chimeric GPCRs for the light-based control of specific GPCR intracellular signaling pathways and behavior .............................. 91 Research goals ................................................................................................................. 94 CHAPTER 5: CONTROL OF 5-HT1A RECEPTOR SIGNALING BY LIGHT-ACTIVATED G PROTEIN-COUPLED RECEPTORS .........................96 Introduction ..................................................................................................................... 97 Materials and Methods ................................................................................................. 100 Results ............................................................................................................................ 108 Cloning of Rh-CT5-HT1A and optimization of the light activation paradigm. ................................................................................ 108 Expression pattern and function of Rh-CT5-HT1A resembles fluorescently tagged 5-HT1A in HEK cells. ............................................ 111 Subcellular targeting of Rh-CT5-HT1A resembles that of 5-HT1A. ........... 114 Rh-CT5-HT1A is functional in cultured hippocampal neurons and competitively inhibits endogenous 5-HT1A receptor. ....................... 116 Rh-CT5-HT1A compensates for the loss of 5-HT1A signaling in cultured hippocampal neurons of 5-HT1A null mice. .............................. 118 Rh-CT5-HT1A functionally substitutes for 5-HT1A signaling in dorsal vii raphe nucleus neurons in brain slices from 5-HT1A null mice. ............... 119 Figures ............................................................................................................................ 121 CHAPTER 6: DISCUSSION ........................................................................................146 Research conclusions .................................................................................................... 147 Critical trafficking domains as molecular tags to direct intracellular targeting ................................................................................................... 148 Endogenous receptor replacement by exogenous receptor expression .................... 149 Remaining questions and future directions ................................................................ 150 REFERENCES ...............................................................................................................156 viii LIST OF FIGURES FIGURES FOR PART I Figure 1 - Amino acid sequence alignment of Drosophila Synd with other known Synd isoforms .................................................................................. 47 Figure 2 - Schematic representation of the gene structure of Drosophila Synd. .. 49 Figure 3 - In wild type embryos and larvae, Synd is distributed in actin-rich, highly curved structures......................................................................
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