Synaptic vesicle exocytosis is a special case of membrane fusion. Despite extensive research in neurons and other cells, controversies remain concerning the basic mechanism of vesicle fusion. Two models have been proposed to describe the process of how vesicles fuse with a target membrane. In one model, cognate SNARE proteins on apposed membranes form a trans complex that is necessary and sufficient to catalyze the fusion of the two membranes. In a second model, fusion is initiated by protein-protein interactions between integral membrane V0 sectors of the vacuolar-type ATPase in apposed membranes. The goal of this proposal is to test the hypothesis that V0 proteins are involved in synaptic vesicle fusion in the nematode C. elegans. To test this we will characterize the impact vacuolar ATPase mutations have on the fusion of synaptic vesicles. We will use with electrophysiological imaging techniques to assay vesicle fusion at the neuromuscular junction. Since the V0 sector is involved with acidifying synaptic vesicles when associated with the V1 ATPase sector, we will also examine whether the genetic mutants in the V0 solely affect vesicle acidification. This approach will resolve a major controversy in synaptic vesicle exocytosis; specifically whether the V-ATPase participates solely function in neurotransmitter loading or whether it has in additional role in priming and/or fusion. First, using a vesicle-targeted pH-sensitive GFP, we will measure the effects V-ATPase mutations have on vesicle acidification. Second, we will measure spontaneous postsynaptic miniature currents to determine whether V0 mutations decrease the frequency of vesicle fusion. Third, fusion rates in V-ATPase mutants will be measured more directly by measuring the exocytosis of fluorescent dye tracer FM4-64 from the membranes of stained vesicles.
