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PMID: 10198050 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

Molecular biology of the cell ·Vol. 10 ·No. 4 ·1999-04-00 ·Pages 961-74

Rodal SK, Skretting G, Garred O, Vilhardt F, van Deurs B, Sandvig K

Abstract

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

MeSH Terms
Animals Cell Line Cholesterol/isolation & purification,physiology Clathrin/drug effects,physiology Cyclodextrins/pharmacology Dogs Endocytosis/drug effects,physiology Endosomes/drug effects,physiology,ultrastructure Humans Kidney Kinetics Membrane Lipids/physiology Receptors, Transferrin/genetics,physiology Recombinant Proteins/metabolism Ricin/pharmacokinetics Transfection Transferrin/pharmacokinetics Tumor Cells, Cultured beta-Cyclodextrins
Chemicals
Clathrin Cyclodextrins Membrane Lipids Receptors, Transferrin Recombinant Proteins Transferrin beta-Cyclodextrins methyl-beta-cyclodextrin Ricin Cholesterol
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Rodal S K
Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.
Skretting G
Garred O
Vilhardt F
van Deurs B
Sandvig K
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Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1059-1524
DOI
10.1091/mbc.10.4.961
Published
1999-04-00
Pages
961-74
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC25220
Subset
IM
External Links
PubMed source DOI: 10.1091/mbc.10.4.961 PMC Full Text View journal details
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