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Our research examines the establishment and maintenance of the polarized distribution of physiologically important transporting proteins in the apical and/or basolateral plasma membranes of epithelial cells.
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Our research examines the establishment and maintenance of the polarized distribution of physiologically important transporting proteins in the apical and/or basolateral plasma membranes of epithelial cells. The role of the cytoskeleton, GTP-binding proteins and other factors in vesicle transport and membrane recycling processes are being examined. While all renal tubule segments have been studied, effort has been concentrated on the collecting duct (CD) epithelium. Principal cells in the CD respond to vasopressin by increasing the water permeability of their apical plasma membranes via a process involving the exocytotic insertion of a water channel protein, AQP2. This process is being dissected using both in vivo studies and in vitro transfected epithelial cells in which water channel shuttling has been reconstituted by transfection with chimeras of AQP2 cDNA. Cells have also been transfected with chimeras of AQP2 and green fluorescent protein to allow real time analysis of trafficking. Intercalated cells of the CD are responsible for distal acid secretion, and use a similar but distinct vesicle shuttling mechanism to control the number of proton pumps (a vacuolar H+ATPase) in their plasma membrane. The cell biological processes underlying the physiological control and developmental expression of both water permeability and acid secretion have been followed using several convergent techniques. Specific antibodies against water channel proteins (aquaporins) and proton pump subunits are used in high resolution morphological studies, such as immunogold immnocytochemistry and immuno-fluorescence on thin frozen sections. This work is complemented by biochemical assays of protein composition and enzyme activity in isolated purified endosome and membrane preparations, by functional measurements on membrane fluid and electrolyte permeability on the same vesicles using fluorescence quenching assays, by high resolution freeze-fracture techniques, and by molecular biology approaches. While our data is specifically relevant to renal function, the work is also of significance to the biology of epithelial cells in general.
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