PI's Abstract): The long-term goal of this research plan is to understand the molecular mechanism of exocytosis. Using electron microscopy, atomic force microscopy and immunoisolation procedures, a """"""""new structure"""""""" associated with the neuronal fusion machinery has been identified and its participation in membrane fusion is suggested. The immediate research goal is to characterize and determine the involvement of this structure in the fusion of synaptic vesicles with the presynaptic membrane. Impairment of the exocytotic process in cells is involved in a number of disease states. Understanding this vital cellular process will eventually lead to effective diagnosis and treatment of disease resulting from secretory defects. Although SNARE proteins have been implicated as the minimal fusion machinery, their participation leading to the fusion of lipid bilayers is in the order of hours, as opposed to minutes or milliseconds for membrane fusion occurring in cells. Therefore, the involvement of additional proteins in membrane fusion is suggested. The studies by the Investigators demonstrate coiling and supercoiling of the """"""""new structure"""""""" with SNAREs in neuronal tissue. Increased coiling of SNAREs was observed in neurons compared to the pancreas, a slow secretory cell. The Investigators hypothesize that the extent of coiling and supercoiling of SNAREs with the """"""""new structure"""""""" may dictate the potency and efficacy of membrane fusion in cells. They seek to test this hypothesis and to gain further understanding of the native membrane fusion machinery in neurons.
The specific aims are the following: 1) to determine the role of coiling and supercoiling in membrane fusion, 2) to determine the nature of coiling of SNAREs, 3) to determine the role of the """"""""new structure"""""""" in membrane fusion and 4) to determine the biochemical composition of the """"""""new structure."""""""" Electron microscopy and atomic force microscopy examination of the extent of coiling of these structures in stimulated and resting neurons will determine the role of coiling in membrane fusion in cells. Atomic force microscopy will be used to determine the nature of binding interactions and the coiling and supercoiling of native and recombinant SNAREs, in their hydrated states in buffer. Atomic force microscopy studies on coiling of the """"""""new structure"""""""" with recombinant full-length and truncated SNARE proteins will help to define their interaction with each other. The function of the """"""""new structure"""""""" in membrane fusion will be examined using the in vitro fusion assay of reconstituted lipid bilayer vesicles. The biochemical composition of the """"""""new structure"""""""" will be determined by microsequencing electrophoretically resolved proteins immunoisolated from brain tissue, using antibodies to the SNARE proteins.
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