CONTROL OF NEURONAL CIRCUIT ASSEMBLY BY GTPASE REGULATORS Julia E. Sommer Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2011 2011 Julia E. Sommer All rights reserved ABSTRACT Control of Neuronal Circuit Assembly by GTPase Regulators Julia E. Sommer One of the most remarkable features of the central nervous system is the exquisite specificity of its synaptic connections, which is crucial for the functioning of neuronal circuits. Thus, understanding the cellular and molecular mechanisms leading to the precise assembly of neuronal circuits is a major focus of developmental neurobiology. The structural organization and specific connectivity of neuronal circuits arises from a series of morphological transformations: neuronal differentiation, migration, axonal guidance, axonal and dendritic arbor growth and, eventually, synapse formation. Changes in neuronal morphology are driven by cell intrinsic programs and by instructive signals from the environment, which are transduced by transmembrane receptors on the neuronal cell surface. Intracellularly, cytoskeletal rearrangements orchestrate the dynamic modification of neuronal morphology. A central question is how the activation of a neuronal cell surface receptor triggers the intracellular cytoskeletal rearrangements that mediate morphological transformations. A group of proteins linked to the regulation of cytoskeletal dynamics are the small GTPases of the Rho family. Small RhoGTPases are regulated by GTPase exchange factors (GEF) and GTPase activating proteins (GAP), which can switch GTPases into “on or off” states, respectively. It is thought, that GEFs and GAPs function as intracellular mediators between transmembrane receptors and RhoGTPases, to regulate cytoskeletal rearrangements. During my dissertation I identified the GAP α2-chimaerin as an essential downstream effector of the axon guidance receptor EphA4, in the assembly of neuronal locomotor circuits in the mouse. Furthermore, I identified two novel neuronal GAPs, mSYD-1A and mSYD-1B, which interact with components of the presynaptic active zone and which may contribute to presynaptic assembly downstream of synaptic cell surface receptors. TABLE OF CONTENTS CHAPTER 1 INTRODUCTION: THE ASSEMBLY OF NEURONAL CIRCUITS ................................................. 1 PREFACE .................................................................................................................................................... 2 AXON GUIDANCE ....................................................................................................................................... 3 A Historical Perspective of Axon Guidance .............................................................................................. 3 Principles of Axon Guidance .................................................................................................................... 5 The Role of Eph-receptors and Ephrin Ligands in Axon Guidance .......................................................... 9 Steering the Axon Growth Cone ............................................................................................................. 10 SYNAPSE FORMATION ............................................................................................................................ 14 Initiation of Synapse Formation by trans-synaptic signaling .................................................................. 15 Molecular Mechanisms of Presynaptic Assembly downstream of Trans-Synaptic Adhesion Proteins .. 21 SYD-1: A Key Regulator of Presynaptic Assembly in Invertebrates ...................................................... 26 GAPS: COMMON SIGNALING MODULES IN AXON GUIDANCE AND SYNAPSE FORMATION .......... 28 THE DISSERTATION PROJECT: CONTROL OF NEURONAL CIRCUIT ASSEMBLY BY GTPASE REGULATORS .................................... 29 α2-Chimaerin .......................................................................................................................................... 29 Synapse Defective-1 (mSYD-1) ............................................................................................................. 30 FIGURES AND LEGENDS ......................................................................................................................... 31 CHAPTER 2 α2-CHIMAERIN IS AN ESSENTIAL EPHA4 EFFECTOR IN THE ASSEMBLY OF NEURONAL LOCOMOTOR CIRCUITS ............................................................................................................. 38 PREFACE .................................................................................................................................................. 39 INTRODUCTION ........................................................................................................................................ 40 Regulation of Rho GTPase Signaling Downstream of Eph Receptors ................................................... 40 α-Chimaerins are GAPs Involved in Neural Development ..................................................................... 41 RESULTS ................................................................................................................................................... 42 α2-Chimaerin Interacts with Activated Eph-Receptors ........................................................................... 42 i α2-Chimaerin Mutant Mice Exhibit Locomotor Defects .......................................................................... 44 α2-Chimaerin is Required for Ephrin-Induced Growth Cone Collapse ................................................... 45 α2-Chimaerin Mutant Mice Show Aberrant Midline Crossing of Spinal Interneuron Projections ........... 46 Corticospinal Defects in α2-Chimaerin Mutant Mice .............................................................................. 48 α2-Chimaerin Mutants Show Bilateral Hindlimb Movements in Response to Cortical Stimulation ........ 48 Genetic Dissection of the Functional Origin of Hindlimb Synchrony in α2-chimaerin Mutant Mice ........ 49 DISCUSSION AND FUTURE DIRECTIONS .............................................................................................. 50 α2-Chimaerin is an Essential EphA4 Effector in the Assembly of Neuronal Locomotor Circuits ........... 50 Why is there no Redundancy in EphA4 Downstream Signaling in the Neurons Controlling Locomotion? ............................................................................................................................................................... 51 Is the α2-chimaerin Phenotype Selective for Locomotor Circuits? ......................................................... 52 The Creation of an α2-chimaerin Conditional Allele as Tool to Dissect the Function of Neuronal Locomotor Circuits ................................................................................................................................. 53 Conclusion .............................................................................................................................................. 54 FIGURES AND LEGENDS ......................................................................................................................... 56 CHAPTER 3 A POTENTIAL ROLE FOR MSYD-1 IN PRESYNAPTIC ASSEMBLY ........................................ 68 PREFACE .................................................................................................................................................. 69 INTRODUCTION ........................................................................................................................................ 71 Invertebrate SYD-1 in AZ assembly ....................................................................................................... 71 Protein Domain Organization and Function in C. elegans SYD-1 .......................................................... 72 C. elegans SYD-1: a Functional GAP? .................................................................................................. 72 SYD-1: a Presynaptic GAP that Links Trans-Synaptic Adhesion with Cytoskeletal Dynamics? ............ 73 The Actin and Microtubule Cytoskeleton in Presynaptic Assembly ........................................................ 73 RESULTS ................................................................................................................................................... 75 Identification of Potential Vertebrate Orthologues of C. elegans SYD-1 ................................................ 75 Expression Analysis of mouse SYD-1 .................................................................................................... 77 Subcellular Localization of mSYD-1A ..................................................................................................... 80 Functional Analysis of mSYD1A ............................................................................................................. 82 ii mSYD-1A Interaction partners ............................................................................................................... 88 Candidate Screening Approach for mSYD-1A Interaction Partners ....................................................... 88 Yeast Two Hybrid Screen for mSYD-1A Interaction Partners ................................................................ 91 A Functional Interaction between Tankyrase 1 and mSYD-1A and -B ..................................................