The long-term goal of this project is to understand the function of the Golgi complex in terms of its morphology and sorting capacity in different polarized epithelia. Golgi subcompartments from three cell lines which represent models of different epithelial tissues will be investigated. These three types of epithelia (renal tubule, intestinal enterocyte, and hepatocyte) appear to differ in the site at which newly synthesized proteins are sorted to the appropriate plasma membrane domain, and/or the mechanism by which this sorting occurs. Cis and trans Golgi subcompartments will be immunoisolated from these lines using well defined endogenous or transfected marker proteins and antibodies which recognize the cytoplasmic domains of these proteins. The protein and lipid composition of these subcompartments will be determined and compared. The following questions will be asked: 1) Are glycosphingolipids and cholesterol enriched in the trans Golgi from all three cell types? 2) Are there proteins present in the trans Golgi of some of the cell types and not others which might be candidates for the postulated sorter proteins? 3) Are there unique GTP-binding proteins in these Golgi subcompartments? and 4) How does the organization of the cis Golgi compare to the trans Golgi in a given cell type? The morphology of the Golgi complex including its location in the cell during polarization will also be studied. Lastly, the composition of Golgi subcompartments after perturbations including microtubule depolymerization, cholesterol depletion, and inhibition of glycosphingolipid synthesis will be analyzed. The effect of these perturbations on polarized delivery of newly synthesized components in the three cell types will be studied in other projects of this proposal. Malfunction of the sorting machinery in polarized epithelial cells has the potential to cause disease, since the integrity of these cells is required to define and maintain physiological compartments in the organism. Understanding the role of the Golgi complex in directing newly synthesized proteins and lipids to their site of function in polarized cells is instrumental to understanding such disease processes.
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