Extraction of cholesterol with methyl-β-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles

SK Rodal, G Skretting, Ø Garred… - Molecular biology of …, 1999 - Am Soc Cell Biol
SK Rodal, G Skretting, Ø Garred, F Vilhardt, B Van Deurs, K Sandvig
Molecular biology of the cell, 1999Am Soc Cell Biol
The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell
lines by using methyl-β-cyclodextrin (MβCD) to selectively extract cholesterol from the
plasma membrane. MβCD treatment strongly inhibited endocytosis of transferrin and EGF,
whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was
completely reversible. On removal of MβCD it was restored by continued incubation of the
cells even in serum-free medium. The recovery in serum-free medium was inhibited by …
The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-β-cyclodextrin (MβCD) to selectively extract cholesterol from the plasma membrane. MβCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MβCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MβCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MβCD 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 formation of clathrin-coated endocytic vesicles.
Am Soc Cell Biol