The molecular mechanisms by which lipid molecules are synthesized, sorted, and transported to various cytoplasmic compartments within animal cells will be studied. A series of fluorescent lipid analogs will be used to correlate the intracellular distribution of lipid metabolites in living cells, as determined by fluorescence microscopy, with traditional lipid biochemical analyses. Where possible, similar studies will be performed using radiolabeled lipids and their precursors. The goals are to: (i) examine the role of vesicular and non- vesicular mechanisms of lipid transport to and from the plasma membrane; (ii) study the metabolism, sorting, and transport of sphingolipids through the ER/Golgi complex; (iii) study the metabolism and transport of a series of fluorescent and radioactive gangliosides in normal and mutant human skin fibroblasts; (iv) examine the sites of synthesis of some sphingolipids and their precursors in subcellular membrane fractions isolated from rat liver; and (v) study the interactions of liposomes with the intracellular membranes of semi-intact (""""""""perforated"""""""") cells. Studies of the fluorescent lipids in living cells will be facilitated by resonance energy transfer microscopy, as recently developed in this laboratory. and by a recently acquired low-light-level digital imaging fluorescence microscope. Some studies will rely on the synthesis of a number of lipid analogs (and their stereoisomers) bearing fluorescent. radioactive, or photoaffinity labels. These will be used to distinguish various transport mechanisms from one another and to help identify the cellular proteins involved in lipid synthesis, sorting, and transport. The studies proposed in this application are basic to understanding membrane assembly and regulation of membrane lipid composition in cells and are fundamental to the development of rational treatments of membrane or cell surface- related disease states.
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