This program project aims to understand and define some of the pathways, mechanisms and signals involved in the sorting, traffic and localization of membrane lipids and proteins to their functional surface domains in polarized epithelial cells. Six highly interrelated projects served by 4 core units will address and compare the problems of molecular targeting and localization in a variety of polarized epithelial cells [MDCK (kidney tubule epithelium), Caco-2 and HT-29 (intestinal epithelium) and WIF12-1 (hepatocyte)] in vitro. These projects address: 1) The lateral organization of membrane proteins and the function of this organization in the sorting and targeting of newly synthesized proteins. 2) The amino acid sequences involved in determining the distribution of an apical (dipeptidylpeptidase-IV) and a basolateral (Na,K-ATPase) protein and of a protein (LEP-100) cycling between lysosomes and the plasma membrane via endosomes. The intracellular transport of these proteins in differential epithelial cells will also be compared. (3) Molecule and vesicle traffic in different epithelial cell types, and the perturbation of this traffic resulting from changes in lipid metabolism or in microtubule architecture. This project will include work on a newly-derived hepatocyte cell line WIF12-1. 4) The isolation and characterization of cis- and trans- Golgi complex membranes as a function of cell type and extent of polarity. 5) The distribution and transport of lipids. Special emphasis will be placed on the movement of fluorescent sphingolipid analogs along secretory pathways and the effects which alterations in cellular sphingolipids and cholesterol may have on lipid sorting and transport. 6) The organization of microtubules, the routes of biosynthetic and endocytic vesicle transport and the mechanoenzymes required for this transport. These six projects will be supported by cores for cell culture, electron microscopy and other imaging, fluorescence measurements of molecular behavior, and administration. The structure, central support and resources proposed here will result in a highly collaborative and interactive approach to the problems of the establishment and maintenance of polarity of epithelial cells.
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