The aims of the research outlined in this proposal are to freeze fracture and deep etch techniques to examine the molecular structure and disposition of a variety of different intrinsic and cytoskeletal proteins in both intact and isolated preparations. 1) the disposition and possible interactions of myocardial membrane pumps and ion channels. will be studied using freeze fracture, deep etch and high resolution shadowing of immunolabeled intact tissue, cultured myocytes and isolated membranes in order to clarify the mechanism of excitation contraction coupling. 2) Site specific immunolabeling of purified intermediate filament proteins will be studied using quick frozen, deep etched preparations in order to clarify the structure of purified polypeptides and the mechanisms of assembly of neurofilaments. 3) The role of myosin light chain phosphorylation in the regulation of smooth muscle contractility will be examined by using freeze fracture and immunolabeling of chemically demembranated, 'skinned' smooth muscle cells and isolated contractile filaments. 4) Freeze fracture will also be used to correlate changes in membrane capacitance with secretory vesicle release in cultured bovine chromaffin cells. 5) Freeze fracture of developing heart tissue and cultured myocytes will be used to correlate changes in the molecular structure of myocardial membranes with electrophysiological measurements. Freeze fractures and deep etch techniques are now being, rediscovered and recognized as powerful tools in providing extremely high resolution structural information that is impossible to obtain using conventional thin section electron microscopy. The wide ranging projects outlined in this proposal will provide unique insights about the molecular structure and mechanisms of several extremely important biological processes.
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