PROJECT SUMMARY The proposed research applies biophysical methods toward elucidating the underlying mechanism of Ca2+ triggered exocytosis of neurotransmitters during signal transmission at chemical synapses. Neurotransmitters (e.g., glutamate, GABA, catecholamine) within unstimulated neurons are sequestered within secretory vesicles docked at the plasma membrane of the presynaptic terminal. Upon the arrival of an action potential at the axon terminal, voltage-dependent calcium channels open and the resulting influx of calcium triggers a biochemical cascade that causes the neurotransmitter- containing vesicles to fuse to the plasma membrane, releasing their contents into the synaptic cleft. This process is mediated by SNARE proteins expressed on the plasma membrane and their counterparts on the vesicle's surface. Trans pairing of the neuronal v-SNARE (VAMP2) and t-SNAREs (syntaxin and SNAP-25) has been shown to form the essential molecular machinery necessary to induce vesicle fusion at the presynaptic terminal. The fusogenic function of the SNAREs is regulated by associated accessory molecules, including synaptotagmin and complexin. Although there have been considerable advances toward identifying the individual components of the fusion machinery in recent years, there is still numerous gaps in our understanding of SNARE-mediated membrane fusion and how the process is regulated. The central hypothesis of our research is that the interaction of the SNARE proteins generates a mechanical force that destabilizes the apposing membranes and thus lowers the energy requirement for membrane fusion. Calcium bound synaptotagmin promotes fusion by further lowering this energy requirement, whereas complexin inhibits fusion by preventing the SNARE complex from completely annealing. To test this hypothesis, the proposed research will employ state-of-the-art atomic force microscopy techniques to measure the force generated by the interactions of the cognate SNAREs. Results will determine if it is sufficient to bring the membranes to close proximity in order to initiate the SNARE-facilitated membrane fusion process. Moreover, we will determine by direct force measurements how the SNAREs, along with complexin and synaptotagmin, alter the energetics of the membrane fusion process, hence revealing the mechanism of SNARE- mediated membrane fusion.