We will examine the distribution and transport of lipids in polarized cells (MDCK, Caco-2, WIF12-1, and hepatocytes). The major goals of this project are (i) to study the movement of lipids along the secretory pathway. Fluorescent ceramide analogs will be used to examine the synthesis, sorting, and transport of newly-synthesized fluorescent sphingolipids in these different cell types. Using the perfused liver system, we will determine whether lipids destined for the apical surface of hepatocytes are first transported to the basolateral surface, as is the case for apically- directed proteins; (ii) to study the effects of cellular sphingolipids and cholesterol on the sorting and transport of lipids along the secretory pathway. Cellular cholesterol and endogenous sphingolipids will be modulated, respectively, by altering growth conditions and by use of an inhibitor of glycosphingolipid synthesis. Since the Golgi apparatus is the major site of sphingolipid biosynthesis and cholesterol also accumulates there, these perturbations may have important effects on lipid and protein (Project 3) sorting and transport through this organelle; (iii) to study processing of lipids along the endocytic pathway. Using two different fluorescent analogs of sphingomyelin, we will study the internalization of lipids from the apical and basolateral membrane surfaces of cells, monitor the intermixing of membrane lipids among populations of endosomes derived from the different plasma membrane domains, and study the process of transcytosis; (iv) to determine the lipid composition of several different intracellular membrane systems including the Golgi apparatus. The lipid composition of Golgi membrane subfractions obtained by immunoisolation (Project 4) will be determined by classical methods. We will also attempt to develop an in situ method for determining lipid composition, using fluorescent lipid analogs and the photoactivatable probe, 5- [125I]iodonaphthyl-1-azide. If successful, this methodology will allow us to determine the lipid composition of endosomes and the plasma membrane; and (v) to study the uptake and metabolism of a fluorescent analog of phosphatidylinositol introduced into the apical or basolateral surfaces of polarized cells. The studies proposed in this application are basic to understanding membrane assembly and regulation of membrane lipid composition in polarized cells, and are fundamental to the future development of rational treatments of membrane or cell surface-related disease states.
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