SNARE complex formation in living cells

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Cells are able to transmit information to each other via the highly regulated process of exocytosis. Exocytosis allows cells to release hormones, neurotransmitters, and other chemicals into the extracellular space by transporting a vesicle to the cell surface that fuses with the plasma membrane and releases its contents. SNARE proteins are essential for the regulation of membrane fusion and do so by forming a complex between proteins on the vesicle and proteins on the plasma membrane that brings the two membranes in close proximity. The proposed work is designed to determine the molecular mechanism of SNARE complex formation in living INS-1 cells by: (1) characterizing the dependence of SNARE pre-complexes on syntaxin and synaptotagmin I concentration, (2) identifying which pre-complexes facilitate exocytosis. The proposed experiments offer a method of directly measuring SNARE complexes and exocytosis by using fluorescently labeled SNAREs that will be illuminated using total internal reflection microscopy. Ultimately, these results could reveal that control of secretion partially lies in the relative amounts of SNAREs present in a cell.