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 (BCθ), which binds to cholesterol specifically, we studied consequences of cross-linking cholesterol in lymphocytes. When bound with BCθ and then with labeled avidin or streptavidin, capping occurred in most cells within 30 min at 37°C. It was inhibited by cytochalasin D or NaN3, but not by nocodazole. When BCθ-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 BCθ 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.