The polArity of epithelial cells is key to the function of adult epithelial organs, such as kidney, liver, intestine and exocrine glands. The hallmark of epithelial cell polarity is the asymmetric distribution of structural features and functional markers between the apical and the basolateral domains of the plasma membrane. The molecular basis of this asymmetric distribution is currently under intensive investigation. This laboratory has recently shown that a novel group of integral plasma membrane proteins anchored to the bilayer via glycosylphosphatidylinositol (GPI) is targeted preferentially to the apical surface of the polarized dog kidney epithelial cell line MDCK. This proposal aims to study the possible role of GPI as an apical targeting signal in epithelial cells. The surface localization of GPI-anchored proteins in epithelial cells of diverse tissue origins will be analyzed by a biotin polarity assay and immunocytochemistry. Antibodies against GPI anchors will be raised and used to localize them in frozen sections of epithelial monolayers. The intracellular sorting and surface expression of exogenous GPI-anchored proteins, (e.g., Thy-1, Decay Accelerating Factor -DAF-, etc.) will be studied after transfection of their c-DNAs into MDCK cells). GPI-deficient mutants of these proteins either naturally occurring or constructed in vitro by deletion of a C- terminal signal for GPI-addition will be transferred and tested for polarity of secretion. GPI will be added, by recombinant DNA procedures, to the C-terminal end of a variety of secretory and truncated plasma membrane proteins and the polarized expression of these fusion proteins studied after transfection into MDCK cells. The hypothesis that lateral clustering of glycolipids, including GPI, contribute to the sorting of apical glycoproteins will be analysed by biophysical techniques (fluorescence recovery after photobleaching -FRAP-, and fluorescence energy transfer -FET) and video enhanced microscopy in the apical surface of intact monolayers and after incorporation of glycolipids and GPI-anchored proteins into large unilamellar liposomes. It is expected that these studies will contribute fundamental information to understand the process of epithelial cell differentiation.
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