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Vol. 10, Issue 4, 961-974, April 1999

Extraction of Cholesterol with Methyl-beta -Cyclodextrin Perturbs Formation of Clathrin-coated Endocytic Vesicles

Siv Kjersti Rodal,* Grethe Skretting,* Øystein Garred,* Frederik Vilhardt,dagger Bo van Deurs,dagger and Kirsten Sandvig*dagger Dagger

 *Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway; and  dagger Structural Cell Biology Unit, Department of Medical Anatomy, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark

The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-beta -cyclodextrin (Mbeta CD) to selectively extract cholesterol from the plasma membrane. Mbeta CD 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 Mbeta CD 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 Mbeta CD-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 Mbeta 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 formation of clathrin-coated endocytic vesicles.


Dagger    Corresponding author. E-mail address: ksandvig{at}radium.uio.no.


Molecular Biology of the Cell
Vol. 10, 961-974, April 1999
Copyright © 1999 by The American Society for Cell Biology



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