These experiments are designed to better understand the role that structural components such as intracellular tight junctions (TJ) and cytoskeletal elements play in the dynamic expression of permeability, barrier and transport characteristics of intestinal epithelia in health and in disease. To accomplish this goal, a variety of electron microscopic, fluorescence localization and electrophysiological experiments will be conducted in parallel. First, the hierarchical order by which various Na+-cotransported nutrients physiologically regulate TJ will be defined and the mechanisms of such regulation explored. Second, cultured epithelial models will be developed which allow exploration of the role of both Na+-glucose cotransport and myosin light chain kinase- regulated cytoskeletal tension on TJ structure and function. Thirdly, the contribution of the TJ specific protein Z)-1 to long term TJ regulation elicited by gammaIFN and the mechanisms by which elevated intracellular Ca++ regulate TJ will be defined. Fourth, we will define, in detail, the integrated microanatomy of the perijunctional cytoskeleton in the baseline state and in the above states of TJ regulation. Fifth, we will utilize a cell culture model of epithelial wound healing to explore cytoskeletal contributions to restitution and de novo intercellular formation of junctions. Sixth, we will follow up recent observations that the cytoskeleton also modulates transcellular movement of C1-. Lastly, we will develop fractionation procedures for our cultured epithelial model (T84) which will permit future extensions of the above work to the subcellular level. These experiments designed to correlate epithelial cell biology with dynamic epithelial physiological responses should yield insights into intestinal epithelial function in health and disease.
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