The objective of the proposed research is to investigate the molecular organization of glycosphingolipids in reconstituted bilayer systems and in biological membranes. Morphological techniques will be utilized that involve labeling the glycosphingolipid molecules with reagents that bind specifically to their carbohydrate moieties and that can be visualized by freeze-etch and thin-section electron microscopy. Double-label techniques will be used to examine mixtures of glycosphingolipids inserted into phospholipid bilayers to determine whether each glycosphingolipid forms a separate domain or whether there is mixing of glycosphingolipids in domains. The effect of addition of cholesterol on the organization of glycosphingolipids in phospholipid bilayers will be investigated. Scanning tunneling and atomic force microscopy will be utilized in an attempt to directly visualize glycosphingolipids and proteins incorporated into lipid bilayers, without the need for secondary labels. Morphological findings will be correlated with biophysical studies of glycosphingolipid organization using the technique of resonance energy transfer. The MDCK polarized epithelial cell system will be used to study mechanisms by which glycosphingolipids may influence the segregation of proteins to the apical or basolateral surfaces of cells. Double-label ultrastructural techniques will be used to search for co-localization of glycosphingolipids and proteins in domains in the trans-Golgi network and in intracellular transport vesicles targeted to the apical plasma membrane of MDCK cells. The spatial relationship of glycosphingolipids to specific apical and basolateral membrane-associated proteins inserted into phospholipid bilayers will be studied. Glycosphingolipids are an important component of biological membranes, and a study of the organization of these molecules in bilayer systems and cellular membranes may help to elucidate their role in such cellular processes as cell-cell recognition, binding of effector molecules, and sorting of proteins to specific domains on the surface of cells.
