Defining the 'late' role for UNC-18 in synaptic vesicle exocytosis

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DESCRIPTION (provided by applicant): SM proteins are required for all membrane fusion steps in a cell, yet the mechanism of their action is not known. For example, studies of the SM protein at the synapse - the Unc18 proteins - suggest that these proteins can either promote or inhibit synaptic vesicle fusion. These conflicting results may be due to the multiple binding modes of Unc18. Unc18 can bind syntaxin in a closed form or it can bind to the N-terminus of the open form of syntaxin. Our preliminary data indicate that binding of the C. elegans Unc18 to the closed form of syntaxin is not essential for its function in fusion. The current working model is that Unc18 binding to the N-terminal peptide of syntaxin mediating the role of Unc18 in fusion via SNARE complex interaction. This model predicts, first, that the binding of UNC-18 to syntaxin in C. elegans will be conserved, and that mutations in the binding regions of UNC-18 and syntaxin will disrupt the N-peptide and SNARE interactions. I will assay the specificity of these mutations by using in vivo pull down experiments. Second, I will determine if these mutations disrupt fusion in vivo. Specifically, I will use electron microscopy to characterize vesicle docking at presynaptic terminals; I will measure vesicle priming by quantifying the size of the readily releasable pool; And, I will use in vivo electrophysiology to measure the kinetics of single vesicle fusion events (miniature EPSCs) and calcium evoked events. This analysis will determine the functional significance of Unc18's interaction with the assembled core complex and may elucidate the fusion-promoting role of Unc18 in synaptic vesicle exocytosis.