Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formationof clathrin-coated endocytic vesicles

The importance of cholesterol for endocytosis has been investigated in HEp- 2 and other cell lines by using methyl-beta-cyclodextrin (M beta CD) to sel ectively extract cholesterol from the plasma membrane. M beta CD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was c ompletely reversible. On removal of M beta CD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-fr ee medium was inhibited by addition of lovastatin, which prevents cholester ol synthesis, but endocytosis recovered when a water-soluble form of choles terol was added together with lovastatin. Electron microscopical studies of M beta CD-treated HEp-2 cells revealed that typical invaginated caveolae w ere no longer present. Moreover, the invagination of clathrin-coated pits w as strongly inhibited, resulting in accumulation of shallow coated pits. Qu antitative immunogold labeling showed that transferrin receptors were conce ntrated in coated pits to the same degree (approximately sevenfold) after M beta CD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the fo rmation of clathrin-coated endocytic vesicles.