The Pennsylvania State University The Graduate School Intercollege Graduate Program in Genetics GENETIC ANALYSIS OF SYNAPTIC TRANSMISSION A Dissertation in Genetics by Janani Iyer 2012 Janani Iyer Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2012 The dissertation of Janani Iyer was reviewed and approved* by the following: Zhi-Chun Lai Professor of Biology, Biochemistry and Molecular Biology Chair of Committee Wendy-Hanna Rose Associate Professor of Biochemistry and Molecular Biology Melissa Rolls Assistant Professor Biochemistry and Molecular Biology Richard Ordway Professor of Molecular Neuroscience and Genetics Dissertation Co-Advisor Fumiko Kawasaki Assistant Professor of Biology Dissertation Co-Advisor Robert Paulson Professor of Veterinary and Biomedical Sciences Genetics Graduate Program Chair *Signatures are on file in the Graduate School. iii ABSTRACT The transmission of electrical impulses at chemical synapses is fundamental to neural function. The elucidation of in vivo molecular mechanisms involved in synaptic transmission has been a major research objective in neuroscience. One important approach to achieve this goal is genetic analysis in Drosophila melanogaster. The synaptic mechanisms of Drosophila are similar to those in vertebrates and, with the advantage of performing in vivo functional analysis of native synapses using genetic, molecular, biochemical, electrophysiological and ultrastructural approaches, Drosophila is a powerful model system. Analysis of temperature sensitive (TS) paralytic mutants of Drosophila has played an important role in elucidating the in vivo molecular mechanisms of synaptic transmission. TS paralytic mutants may allow normal development and function at permissive temperatures and reveal the physiological role of a specific gene product following its acute perturbation at restrictive temperatures. Forward genetic screens for TS paralytic mutants and screens for modifiers of existing TS mutants have revealed novel mechanisms in synaptic transmission. The present work discusses the functional and molecular characterization of two proteins participating in synaptic transmission, DISABLED and COMPLEXIN, in the Drosophila model system. Members of the DISABLED (DAB) family of proteins are known to play a conserved role in endocytic trafficking of cell surface receptors by functioning as monomeric CLATHRIN Associated Sorting Proteins (CLASPs) which recruit cargo proteins into endocytic vesicles. The present study reports analysis of a Drosophila disabled mutant, dabEC1 (enhancer of cac1) (Lisa Posey, Masters Thesis, Penn State University, 2008), which was recovered as an enhancer of the presynaptic TS calcium channel mutant cacTS2. Genetic and functional characterization of dabEC1 has revealed a novel role for DAB proteins in chemical synaptic transmission. dabEC1 exhibits impaired synaptic function including a rapid, activity-dependent reduction in neurotransmitter iv release and disruption of synaptic vesicle endocytosis. In presynaptic boutons, Drosophila DAB (dDAB) and CLATHRIN were highly co-localized within two distinct classes of puncta, including relatively dim puncta which were located at active zones (AZs) which may reflect endocytic mechanisms operating at neurotransmitter release sites. Finally, broader analysis of endocytic proteins including DYNAMIN supported a novel role for CLATHRIN-mediated endocytic mechanisms in rapid clearance of neurotransmitter release sites for subsequent vesicle priming and refilling of the release-ready vesicle pool. The current study of neurotransmitter release mechanisms also provides new insights into the role of COMPLEXIN (CPX) proteins in synaptic transmission. CPX proteins are known to interact with the Soluble NSF Attachment Protein REceptors (SNARE) proteins at the core of the synaptic vesicle fusion machinery and regulate neurotransmitter release. Several studies, including cpx knockout and knockdown studies in mouse, genetic deletion of cpx in Drosophila and in vitro fusion assays, have demonstrated a role for CPX in supporting evoked neurotransmitter release and an inhibitory or clamping role in suppressing spontaneous synaptic vesicle fusion. However, the in vivo mechanisms of CPX function in neurotransmitter release remain controversial. To further investigate these mechanisms, we have conducted a genetic screen to obtain new mutants within the Drosophila cpx gene. Here we report recovery and analysis of a new cpx mutant in Drosophila, cpx1257, revealing spatially defined and separable pools of CPX which make distinct contributions to its activation and clamping functions. The mutation in cpx1257 deletes only the last C-terminal amino acid of CPX within the well conserved CAAX motif for prenylation, a post-translational lipid modification implicated in membrane targeting of CPX and other cytosolic proteins. Our immunocytochemical studies have revealed that CPX is highly enriched at active zone (AZ) regions of the presynaptic plasma membrane containing neurotransmitter release sites and is also detected in presynaptic plasma membrane compartments outside of the AZ, including synaptic vesicles. Biochemical studies v confirmed membrane association of CPX and robust interactions of CPX with all the three SNARE proteins. In contrast, at cpx1257 mutant synapses, AZ localization of CPX persists but there is selective loss of broader presynaptic membrane localization of CPX and, surprisingly, the bulk of the CPX-SNARE protein interactions are also abolished in this mutant. Functional analysis of cpx1257 began with electrophysiological studies at adult neuromuscular synapses in a previously reported cpx null mutant as a basis for comparison. These experiments demonstrated severe reduction in the activation of evoked neurotransmitter release, as indicated by a marked reduction in the amplitude of the Excitatory PostSynaptic Current (EPSC). This phenotype is accompanied by a marked increase in the frequency of spontaneous fusion events, reflecting a loss of the CPX clamping function. Further, the null mutant exhibited an altered EPSC waveform, observed as a slowing of the EPSC rise and decay times with respect to wild-type. Interestingly, this is a presynaptic phenotype and thus indicates a role for CPX in determining the kinetics of neurotransmitter release. In contrast to the cpx null, the cpx1257 mutant exhibited a wild-type EPSC amplitude and waveform, demonstrating that cpx1257 retains the CPX activation function in evoked neurotransmitter release. However, cpx1257 exhibited a selective loss of clamping function as indicated by a marked increase in the frequency of spontaneous neurotransmitter release. Together with the preceding immunocytochemical and biochemical analysis, these findings indicate that spatially distinct and separable interactions of CPX with presynaptic membranes and SNARE proteins appear to mediate separable activation and clamping functions of CPX in neurotransmitter release. We have used Drosophila melanogaster as a model system to investigate the in vivo mechanisms of synaptic transmission. Our analysis of DISABLED and COMPLEXIN function in neurotransmitter release advances our understanding of synaptic function and illustrates the power of forward genetic analysis in defining the molecular basis of physiological processes. vi TABLE OF CONTENTS LIST OF FIGURES ................................................................................................................. viii LIST OF TABLES ................................................................................................................... xi ACKNOWLEDGEMENTS ..................................................................................................... xii Chapter 1 Introduction ............................................................................................................ 1 1.1 Chemical Synaptic Transmission ............................................................................... 1 1.1.1 Synapses and Synaptic Transmission ............................................................. 1 1.1.2 Synaptic Vesicle Membrane Trafficking Cycle.............................................. 6 1.1.3 Synaptic Vesicle Pools ................................................................................... 9 1.1.4 Drosophila melanogaster: A genetic model system for the study of synaptic transmission ....................................................................................... 12 1.2 Molecular Mechanisms of Synaptic Vesicle Exocytosis ........................................... 14 1.2.1 Membrane fusion ............................................................................................ 14 1.2.2 The Soluble N-ethylmaleimide Sensitive Factor (NSF) Attachment proteins Receptors (SNAREs) .......................................................................... 16 1.2.3 NSF and Soluble NSF Attachment Protein (SNAP) ....................................... 22 1.2.4 Presynaptic voltage gated calcium channel .................................................... 23 1.2.5 Regulators of Synaptic Vesicle Exocytosis .................................................... 24 1.2.5.1 Synaptotagmin ................................................................................... 24 1.2.5.2 Complexin .......................................................................................... 25 1.3 Synaptic Vesicle Endocytosis ...................................................................................