Cross-linking of plasmalemmal cholesterol in lymphocytes induces capping, membrane shedding, and endocytosis through coated pits

By use of a nicked and biotinylated perfringolysin O (BC theta), which binds to cholesterol specifically, we studied consequences of cross-linking cholesterol in lymphocytes. When bound with BC theta and then with labeled avidin or streptavidin, capping occurred in most cells within 30 min at 37 degrees C. It was inhibited by cytochalasin D or NaN3, but not by nocodazole. When BC theta-cholesterol was capped, Thy-1 and transferrin receptor, a GPI-anchored protein and a transmembrane protein, respectively, remained evenly distributed. By fluorescence and electron microscopy, a cluster of small vesicles bound with BC theta were observed in the cap. They were then shed in the medium or internalized through coated pits. The result indicates that cross-linking of cholesterol in lymphocytes induces capping, but does not affect distribution of membrane proteins, and that the capped cholesterol molecules are either shed as vesicles or endocytosed. (C) 1999 Academic Press.