LATERAL ORGANIZATION OF EPITHELIAL CELL SURFACES

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Epithelial cells, which comprise 50% of all cells in the body [and account for 90% of all human cancers] have as a defining function the creation and maintenance of physiological compartments of the body. They are functionally and morphologically polarized in order to perform this function. Lipid-anchored proteins, GPI=proteins and glycosphingolipids are concentrated at the apical surfaces of most polarized epithelial cells. We propose to use biophysical techniques to study the formation, and organization of the sorting units for GPI-proteins in the Golgi and plasma membranes and to compare the sorting units in MDCK cells, in which GPI-proteins are delivered directly to the apical surface, and in WIF-B hepatocytes, in which apical proteins take an indirect route to the apical surface. We will also compare the lateral organization of different GPI-proteins in MDCK cells and in liposomes. The following questions will be address: 1) How are GPI-proteins and GSL concentrated and segregated from other proteins in the Golgi? 2) What are the cell & protein specificities of the clustering and polarity of GPI-proteins in cells grown in complete medium and abolish their polarity in cells deprived of LDL? Our techniques to address these questions include fluorescence photo bleaching and recovery, FPR, fluorescence loss in photo bleaching, FLIP, and imaging fluorescence resonance energy transfer, FRET.