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 freeze-etch electron microscopy of ferritin-conjugates of lectins and antibodies with activity directed against specific glycosphingolipids. Morphological findings will be correlated with biophysical studies of glycosphingolipid organization, using techniques of differential scanning calorimetry, and fluorescence polarization. The organization of glycosphingolipids will be evaluated in multilamellar and unilamellar liposomes consisting of varying mixtures of phospholipids, cholesterol, and glycosphingolipids, with comparison to liposomes containing purified proteins such as red blood cell Band 3 and PAS I glycoproteins and purified sarcoplasmic reticulum Ca++ ATPase. Ferritin conjugated anti-glycosphingolipid markers will be used to evaluate whether the glycosphingolipids form compositional domains and how these are related to the gel-liquid crystalline state of the phospholipid bilayer, and also whether they show any structural relationship to proteins incorporated into the lipid bilayers. The ferritin-conjugated reagents will also be used to evaluate the distribution of glycosphingolipids in the red blood cell membranes of various species and in normal and transformed cells. Glycosphingolipids are an important component of biological membranes, and a study of the organization of these molecules in artificial and natural membranes may help to elucidate their role in such cellular processes as cell-cell recognition and interaction, binding of effector molecules, and oncogenesis.
