ARHGAP4 IS A SPATIALLY REGULATED RHOGAP THAT INHIBITS NIH/3T3 CELL MIGRATION AND DENTATE GRANULE CELL AXON OUTGROWTH By DANIEL LEE VOGT Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Neuroscience CASE WESTERN RESERVE UNIVERSITY August, 2007 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Daniel Lee Vogt ______________________________________________________ candidate for the Ph.D. degree *. (signed) (chair of the committee)________________________________ Stefan Herlitze ________________________________________________Alfred Malouf Robert Miller ________________________________________________ ________________________________________________Thomas Egelhoff ________________________________________________Susann Brady-Kalnay ________________________________________________ (date) _______________________6-21-2007 *We also certify that written approval has been obtained for any proprietary material contained therein. ii Copyright © 2007 by Daniel Lee Vogt All rights reserved iii Table of contents Page # Title page i Table of contents iv List of figures vii Abstract 1 Chapter one: General introduction 2 Hippocampal axon pathways and development 3 Guidance cues in hippocampal axon outgrowth 6 Slit/Robo 7 Semaphorins, plexins and neuropilins 8 Ephrins and ephs 11 Other guidance cues in the hippocampus 13 GTPases: structure and function of ras superfamily members 15 Ras GTPases 17 Ran GTPases 18 Arf GTPases 18 Rab GTPases 19 Rho GTPases 20 GTPase regulatory proteins and the role of GAPs 23 RasGAP family functions, localizations and binding partners 24 RanGAP family 25 ArfGAP family 26 iv RabGAP family 26 RhoGAP family 27 GAP protein mutations and developmental consequences 29 Regulation of GAP proteins 29 Phosphorylation 30 Protein-protein interactions 31 Other types of regulation 32 Actin and microtubule dynamics in growth cone and fibroblast motility 33 General actin dynamics 33 General microtubule dynamics 36 Coordinated signaling regulated by Rho-family GTPases 40 Cell migration and functional parallels to growth cone guidance 41 Previous work on ARHGAP4 45 Conserved domains of ARHGAP4 48 FCH domain, extended FCH domain and ARNEY domain 48 RhoGAP domain 51 SH3 domain 52 Proline rich (PxxP) domains 52 Research goals 53 Figures 55 Chapter two: ARHGAP4 is a spatially regulated GTPase activating protein (GAP) expressed in NIH/3T3 fibroblasts and dentate granule neurons 71 Summary 72 v Introduction 72 Materials and methods 74 Results 82 Discussion 89 Figures 97 Chapter three: ARHGAP4 is an inhibitor of NIH/3T3 cell motility and Dentate granule cell axon outgrowth 123 Summary 124 Introduction 124 Materials and methods 126 Results 130 Discussion 133 Figures 139 Chapter four: Mechanistic insights into the function of ARHGAP4 155 Summary 156 Introduction 156 Materials and methods 158 Results 161 Discussion 165 Figures 169 Chapter five: General Discussion 179 Chapter six: Bibliography 195 vi List of figures Figure Page # 1.1 Hippocampal anatomy and pathways 55 1.2 Common guidance cues and receptors found in the hippocampus utilize various GTPases to effect growth cone guidance 57 1.3 The GTPase cycle and role of GAPs, GEFs and GDIs 59 1.4 Rho-family GAP proteins and their domain assortments 61 1.5 Actin and microtubule cytoskeletons in migrating cells and growth cones 63 1.6 ARHGAP4’s amino-terminus contains FCH, extended FCH and ARNEY domains 65 1.7 ARHGAP4 GAP domain alignment 67 1.8 ARHGAP4 SH3 domain alignment 69 2.1 ARHGAP4 constructs used in transfections and bacterial protein expression 97 2.2 In vitro GAP assay of WT and R562A ARHGAP4 GAP domains 99 2.3 Endogenous ARHGAP4 localizes to the leading edge of NIH/3T3 cells 101 2.4 Amino acids 1-71 are necessary and sufficient to target ARHGAP4 to the tips of NIH/3T3 cell cytoplasmic extensions 103 2.5 ARHGAP4 is enriched in the mossy fiber axons of the hippocampus 105 2.6 Endogenous ARHGAP4 is enriched in mossy fiber growth cones 107 2.7 ARHGAP4 expressed proteins localize to growth cones via amino acids 1-71 109 2.8 Amino acids 1-71 are necessary and sufficient to target ARHGAP4 to growth cones 111 vii 2.9 Amino acids 1-71 can associate indirectly but do not bind microtubules in an in vitro microtubule cosedimentation assay 113 2.10 Leading edge distribution of microtubules and F-actin in the presence of nocodazole or cytochalasin-D 115 2.11 Full length ARHGAP4 (1-965) leading edge distribution in the presence of nocodazole or cytochalasin-D 117 2.12 1-770 leading edge distribution in the presence of nocodazole or cytochalasin-D 119 2.13 72-965 leading edge distribution in the presence of nocodazole or cytochalasin-D 121 3.1 Wound assay model system and quantification of cell migration in individually transfected cells 139 3.2 siRNA mediated knockdown of ARHGAP4 increases cell migration in NIH/3T3 cells 141 3.3 ARHGAP4 inhibits NIH/3T3 cell migration via its GAP domain 143 3.4 ARHGAP4 inhibits axon outgrowth from dentate explant cultures through its FCH, GAP and SH3 domains 145 3.5 Dentate explant axon outgrowth is inhibited by ARHGAP4 in an FCH, GAP and SH3 domain manner 147 3.6 Dentate explant astrocyte outgrowth is not altered by ARHGAP4 149 3.7 ARHGAP4 inhibits axon outgrowth in dissociated granule cells in a GAP dependent manner 151 3.8 ARHGAP4 significantly inhibits axon outgrowth in dissociated granule cells viii in a GAP dependent manner 153 4.1 ARHGAP4 amino-terminal protein design and expression 169 4.2 Amino acids 1-289 are the minimal unit required to inhibit NIH/3T3 cell migration 171 4.3 Amino acids 1-71 and 1-289 of ARHGAP4 result in different actin phenotypes in migrating NIH/3T3 cells 173 4.4 Full length ARHGAP4 overexpression decreases levels of GTP-bound RhoA in NIH/3T3 cells 175 4.5 Trends in axon outgrowth and branching are altered in DRGs expressing the R562A mutant on laminin and aggrecan 177 5.1 Model of ARHGAP4 activity 193 ix ARHGAP4 is a spatially regulated RhoGAP that inhibits NIH/3T3 cell migration and dentate granule cell axon outgrowth Abstract By DANIEL LEE VOGT Cell migration and axonal growth cone guidance are tightly regulated events that share many similarities. While some factors are not always shared between migrating cells and growth cones, the signaling events that are required for cell migration and growth guidance each utilize GTPases to regulate the actin and microtubule cytoskeletons. GTPases are expressed ubiquitously but signal in discrete subcellular locales to control directed cell migration and growth cone guidance. This strict control of GTPase signaling is controlled by GAPs, GEFs and GDIs, which can assemble signaling complexes and localize to unique regions due to an array of conserved functional domains. The Rho-family GAP ARHGAP4 contains FCH and SH3 domains, as well as other conserved domains of unknown function. The FCH domain has been attributed to binding actin, microtubules and lipids, but there is no consensus on its function. Here we show that ARHGAP4 localizes to the leading edges of NIH/3T3 fibroblasts and to growth cones of dentate granule neurons, via its FCH domain. Overall, ARHGAP4 inhibits NIH/3T3 cell migration and dentate granule cell axon outgrowth in a GAP dependent manner, and this inhibition is temporally and spatially regulated by ARHGAP4’s conserved functional domains. 1 Chapter 1 General Introduction 2 Unraveling the intricacies of neural networks and their functions during development and in the adult has been an ongoing task since the infancy of developmental biology. Deciphering how neurons migrate, send axons and dendrites to their proper targets and communicate with the vast array of non-neuronal cells is still not well understood. Although many signaling events have been characterized and candidate proteins identified, the detailed mechanisms whereby all the players in neuronal development and maturation are brought together is still far from complete. The mechanisms that regulate axon outgrowth and guidance require tight regulation of cytoskeletal elements, and these mechanisms are shared among many migratory cells as well. Conserved families of proteins that include guidance and growth cues with their respective receptors, actin and microtubule regulatory proteins, GTPases and their regulators the GEFs, GAPs and GDIs, are all involved in guiding an axon or migrating cell to its determined location. These signaling cascades require spatial and temporal control to utilize the potential of the GTPases, and regulatory proteins must have functional domains that allow for spatial control and assembly with signaling components. The RhoGAP family of proteins have diverse tissue expression, subcellular location and signaling partners, and achieve this through a myriad of functional domains. ARHGAP4 is a RhoGAP that inhibits axon outgrowth and cell motility, and whose actions are temporally and spatially regulated by its conserved functional domains. Hippocampal axon pathways and development One of the classic areas studied for axon outgrowth is the hippocampus. With its stereotyped pattern of axonal fibers, adult neurogenesis and axon outgrowth, and rich 3 history of electrophysiological mapping of circuits, the hippocampus has been an excellent model to study axon outgrowth and regeneration. The hippocampus arises from the invaginating dorsal midline of the telencephalon, and its initial formation is dependent on the patterning of the dorsal telencephalon (Theil et al., 1999). The mature hippocampus