Dissecting Roles for the Macromolecular Machinery Involved in Neurotransmitter Release

Neurotransmitter release is a tightly regulated process that involves synaptic vesicle docking at presynaptic active zones, priming of the vesicles to a release-ready state, and calcium evoked fusion of the vesicle and plasma membranes. The probability of release is modulated by plastic changes that depend on synaptic activity; these changes shape the properties of neural networks and underlie multiple forms of information processing in the brain. Elucidating the mechanisms of neurotransmitter release and its regulation is thus critical for understanding brain function and establishing fundamental principles of neuronal communication. I have investigated the mechanism by which neurotransmitters send messages between neurons as a specific model system to study the general mechanism of intracellular membrane fusion. In one project, I investigated whether trans-SNARE complexes can be disassembled by NSF-αSNAP. I showed that trans-SNARE complex formation in the presence of NSF-αSNAP requires both Munc18-1 and Munc13-1, and is facilitated by synaptotagmin-1. I proposed a model whereby Munc18-1 and Munc13-1 are critical for mediating vesicle priming as well as precluding de-priming by preventing trans-SNARE complex disassembly. Complexin-1 also impaired de-priming, while synaptotagmin-1 may have assisted in priming and hindered de-priming. Additionally, I used various biophysical approaches including ITC and NMR to shed light into how Complexin has dual roles in fusion. One of my projects investigated the inhibitory role of Complexin and solved a controversy over conflicting ITC data. Another project focused on the Complexin N-terminal and C-terminal domains to try and develop a complete model of how Complexin functions that incorporates all of its known interactions and activating/inhibiting properties. I observed cooperative interactions between Complexin, the SNARE complex, and lipids by forming SNARE complexes anchored on nanodiscs and liposomes. Such cooperative binding of Complexin to membranes and SNAREs may be critical for releasing the inhibition caused by the accessory helix, although the molecular mechanism of action has yet to be determined. Overall, these experiments highlight the importance of interactions between numerous accessory proteins and the trans-SNARE for proper regulation of SNARE complex formation, and therefore fusion.