Fusion and fission of membrane-bounded cells and organelles are fundamental events governing the compartmentalization of biological space. It is proposed to use video microscopy and an ultra-sensitive, electrical assay of the cell surface to study, in living cells, the mechanism of membrane fusion during exocytosis of single secretory vesicles. In particular we will explore a novel hypothesis: that the first step in membrane fusion is the formation of an ion channel (the fusion pore) that connects the inside of the vesicle to the cell exterior much like a gap junction would connect two adjacent cells, and that subsequent event in exocytosis are a consequence of electrolyte fluxes through the fusion pore. Patch clamp studies of single secretory vesicles will reveal the type of ion channels present in the vesicle membrane. We will also explore """"""""fusion pores"""""""" possibly formed by virus fusion proteins stably expressed in a fibroblast-derived cell line, in order to test whether there are similarities between fusion events induced by viruses and fusion events during exocytosis. Lastly, we plan to explore the role of cytosolic (Ca++) and other cytoplasmic messengers in the control of pinocytosis and membrane turnover.
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