Parietal cells are responsible for the secretion of hydrochloric acid in the stomach, using carefully modulated mechanisms to turn secretory activity on and off in coordination with normal dietary intake. To accomplish this the cells must move a mass of membranes containing the acid transport pumps from one cellular compartment to the apical plasma membrane thus remodeling the cell for its functional transport activity. The overall goal of this research is to understand underlying mechanisms of parietal cell activation. Two major projects are developed to discover how the parietal cell is transformed, structurally and functionally, from rest to full secretory activity, and how these events may be applied more generally to processes of membrane trafficking and membrane-cytoskeletal interactions in all secretory cells. The first project examines mechanisms by which a group of proteins known as SNARE proteins target and promote the regulated vectorial translocation of membranes containing the cargo transport proteins. Several parietal cell model systems are used, e.g., gastric glands, cultured parietal cells and purified membrane vesicles, to test whether and how these SNARE proteins participate in regulated trafficking of acid transport pumps. These models provide opportunity for sophisticated cell and molecular approaches to study membrane trafficking and fusion in cells that maintain their full functional secretory phenotype. Fluorescent probes will be incorporated onto a variety of SNARE related proteins, permitting the examination of movement, co-localization, and molecular interaction in live cells, We address the phenomenon of homotypic fusion, the process whereby intracellular vesicles fuse with one another, as well as fuse with the plasma membrane (heterotypic fusion). An in vitro vesicle system will be used to evaluate the influence of specific phospholipids and gastric vesicular proteins reconstituted into a SNARE protein-liposomal fusion system on the kinetics and regulation of the fusion response. The second major project examines the role of the actin cytoskeleton in the apical membrane remodeling underlying acid secretion. The research will ask whether there are essential changes in the dynamic assembly/disassembly of actin microfilaments associated with parietal cell activation, and seek to identify proteins that interact with the cytoskeleton to relay receptor-mediated signals for membrane remodeling and regulated secretion. Ezrin, a phosphoprotein that has been linked to membrane-cytoskeletal interactions and appears to be of special significance in parietal cell activation, will be studied to define how specific phosphorylation sites alter the association of ezrin with actin and accessory proteins. ? ?
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