The objective of the proposed research is to study the organization of glycosphingolipids in liposomal systems and in plasma membranes of mammalian cells. Morphological techniques for visualization of glycosphingolipids will be utilized, including localization by thin-section and freeze-etch electron microscopy of specific markers for glycosphingolipids, such as lectins, toxins, and antibodies conjugated to ferritin or adsorbed to colloidal gold. Morphological findings will be correlated with biophysical studies of glycosphingolipid organization, using techniques of differential scanning calorimetry, fluorescence polarization, and bilayer molecular transfer. The organization of glycosphingolipids will be evaluated in multilamellar and unilamellar liposomes consisting of mixtures of phospholipids, cholesterol, and proteins into which pure glycosphingolipids are incorporated. Electron microscopic techniques will be used to evaluate whether glycosphingolipids form compositional domains and to study the relationship of such domains to the gel-liquid crystalline state of the phospholipid bilayer and to cholesterol and proteins incorporated into the lipid bilayer. The distribution of glycosphingolipids in biological membranes, including plasma membranes of MDCK epithelial tissue culture cells and oncogenically transformed cells, will be determined, and the process of receptor-mediated endocytosis of glycosphingolipids in tissue culture cells will be investigated. Glycosphingolipids are an important component of biological membranes, and a study of the organization of these molecules in bilayer systems and natural membranes may help to elucidate their role in such cellular processes as cell-cell recognition, binding of effector molecules, and oncogenesis.
