Investigations into the Dynamic Molecular Mechanisms that Govern the Final Stages of Neurotransmitter Release.

Abstract

The release of neurotransmitter from synaptic vesicles is a process that underlies almost all information transfer within the nervous system. This process is exquisitely regulated, both spatially and temporally, such that the proper signals are relayed between neurons to allow for functional integration in higher level organisms. Neurotransmitter release occurs when calcium influx into a presynaptic nerve terminal triggers the fusion of synaptic vesicles with the plasma membrane. SNARE proteins constitute the minimal protein machinery required to catalyze membrane fusion, and formation of SNARE core complexes bridging the vesicle and plasma membranes is thought to generate the energy required to bring these membranes into close proximity and to initiate their fusion. As such, an understanding of SNARE protein function, the dynamic processes that regulate the activity of SNARE complexes, and their relationship to mediating a lipid fusion event, is critical to understanding neurotransmitter release. These topics are the focus of this dissertation, which is comprised of three studies. The first study identified direct interactions between the polybasic juxtamembrane region of the SNARE protein syntaxin1A, and the acidic phospholipids, phosphatidic acid and PI(4,5)P2, and demonstrated that these electrostatic interactions are critical in regulating the energetic requirements for membrane fusion. Notably, the findings of this study bridge the gap between the protein-centered field of SNARE regulation and the lipid-centered field of membrane fusion, by demonstrating that proteins and lipids cooperate to initiate and complete membrane fusion. The second study examined the dynamic regulation of tomosyn, a soluble SNARE protein that may actually inhibit neurotransmitter release through the formation of non-fusogenic SNARE core complexes. Importantly, this study uncovered a novel mechanism by which formation of functional SNARE core complexes can be finely tuned by signaling pathways reflective of secretory demand. The final study of this dissertation focused on development of a novel fluorescence imaging approach, termed sensitized-emission TIRF-FRET. This technique provides the high spatiotemporal resolution required for visualization of changes in molecular interactions in the context of individual secretory vesicles undergoing exocytosis. Importantly, application of this technique is likely to transform our current understanding of the many dynamic molecular processes underlying neurotransmitter release.