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. 1999 Apr;10(4):961-74.
doi: 10.1091/mbc.10.4.961.

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

S K Rodal  1 , G Skretting, O Garred, F Vilhardt, B van Deurs, K Sandvig
Affiliations
Free PMC article

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

S K Rodal et al. Mol Biol Cell. 1999 Apr.
Free PMC article

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.

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Figures

Figure 1
Figure 1
Effect of MβCD on transferrin (●) and ricin (○) endocytosis. MDCK II cells were washed briefly in HEPES-buffered medium before incubation with and without the indicated concentrations of MβCD for 15 min at 37°C. 125I-labeled transferrin or 125I-labeled ricin was then added to the cells, and the amounts of endocytosed transferrin or ricin were measured as described in MATERIALS AND METHODS. The error bars show deviations between duplicates.
Figure 2
Figure 2
Effect of MβCD on endocytosis of EGF. HEp-2 cells were washed briefly in HEPES-buffered medium and incubated with and without MβCD (10 mM) for 15 min at 37°C. 125I-labeled EGF was then added to the cells, and the amounts of endocytosed EGF were measured as described in MATERIALS AND METHODS. The error bars show deviations between duplicates.
Figure 3
Figure 3
Effect of MβCD on binding of transferrin (●) and ricin (○). MDCK II cells were incubated with and without the indicated concentrations of MβCD in HEPES-buffered medium at 37°C for 15 min. Then the cells were cooled down to 0°C, 125I-labeled transferrin or 125I-labeled ricin was added, and binding was measured after 30 min at 0°C as described in MATERIALS AND METHODS. The error bars show deviations between duplicates.
Figure 4
Figure 4
Effect of MβCD on transferrin and ricin endocytosis in different cell lines. HEp-2, NIH/3T3, MDCK II, and A431 cells were washed briefly in HEPES-buffered medium and incubated with and without MβCD (10 mM) for 15 min at 37°C. 125I-labeled transferrin or 125I-labeled ricin was then added to the cells, and the amounts of endocytosed transferrin or ricin were measured as described in MATERIALS AND METHODS. The error bars show deviations between duplicates.
Figure 5
Figure 5
Effect of MβCD on endocytosis of ricin (A) and transferrin (B) in HeLa cells stably transfected with the cDNA for mutant dynamin (dynK44A). The cells were washed briefly in HEPES-buffered medium and incubated with and without MβCD (10 mM) for 15 min at 37°C. 125I-labeled transferrin or 125I-labeled ricin was then added to the cells, and the amounts of endocytosed transferrin or ricin were measured as described in MATERIALS AND METHODS. The error bars show deviations between duplicates.
Figure 6
Figure 6
Effect of MβCD on cellular cholesterol and invaginated caveolae. A shows the percentage of [3H]cholesterol remaining in HEp-2 cells after extraction with MβCD as indicated. Black bars represent cells incubated with [3H]cholesterol for 20 h, whereas open bars are cells incubated for 15 min. Data from three independent experiments with samples performed in quadruplicate. Mean ± SD; n = 3. B shows characteristic invaginated caveolae (arrowheads) as observed in HEp-2 cells (without incubation with MβCD). Bar, 100 nm. In C, the frequency of such caveolae has been quantified after cholesterol extraction with MβCD as indicated; pooled data from two of the three experiments shown in A.
Figure 7
Figure 7
Effect of α-, β-, methyl-β-, and γ-cyclodextrins on transferrin endocytosis. HEp-2 cells were washed briefly in HEPES-buffered medium and incubated with and without α-, β-, methyl-β-, or γ-cyclodextrins (10 mM) for 15 min at 37°C. 125I-labeled transferrin was then added to the cells, and the amounts of endocytosed transferrin were measured as described in MATERIALS AND METHODS. The error bars show deviations between duplicates.
Figure 8
Figure 8
Reversal of MβCD-induced inhibition of transferrin endocytosis in the absence (A) or presence (B) of lovastatin. In both cases MDCK II cells were washed briefly in HEPES-buffered medium and incubated with and without MβCD (10 mM) for 15 min at 37°C. The cells were then incubated in HEPES-buffered medium with and without 5% FCS or water-soluble cholesterol (400 μg/ml) in the presence or absence of lovastatin (5 μg/ml) from 15 min to 3 h (A) or for 3 h (B) before 125I-labeled transferrin was added. The amounts of endocytosed transferrin were measured as described in MATERIALS AND METHODS. The error bars show deviations between duplicates.
Figure 9
Figure 9
Effect of MβCD on protein synthesis (A) and on the intracellular potassium content (B). (A) HEp-2 (○), NIH/3T3 (▿), MDCK II (●), and A431 (▾) cells were washed briefly in HEPES-buffered medium and incubated with and without MβCD (10 mM) for 15 min at 37°C. [3H]leucine was then added to the cells, and after 15 min further incubation the amounts of incorporated [3H]leucine were measured as described in MATERIALS AND METHODS. (B) HEp-2 (●), MDCK II (○), and A431 (▾) cells were washed briefly in HEPES-buffered medium and incubated with and without MβCD (10 mM) for 15 min at 37°C. The cells were then washed with MgCl2, and the amounts of intracellular potassium were measured as described in MATERIALS AND METHODS.
Figure 10
Figure 10
Examples of clathrin-coated pits at various degrees of invagination (A–D); E shows an apparently free, clathrin-coated vesicular profile that, however, may be surface-connected in another plane of sectioning. The clathrin coat is indicated by arrowheads in A and B. The small arrows in C and D indicate the necks connecting the pits to the exterior. Bar, 100 nm. (F) For quantification, the coated pits and apparently free vesicles (A–E) were subdivided into three types. (G) Coated pits in control HEp-2 cells and cells treated with 10 mM MβCD for 15 min (approximately 200 coated pits in each experiment) were scored at random and classified as 1, 2, or 3 as defined in F. The relative frequency of the different types of coated pits is shown. Bar, 100 nm.
Figure 11
Figure 11
Effect of MβCD on clathrin-coated pits and TfR distribution. (A) Examples of shallow coated pits characteristic of MβCD-treated cells and of immunogold labeling for TfRs in the coated pits. Arrowheads mark the extension of the pits. Asterisk marks for comparison a clearly noncoated vesicular profile. (B and C), Examples of TfR immunogold labeling of portions of the cell surface without coated pits and of microvilli (Mv). HEp-2 cells were incubated for 15 or 30 min at 37°C with 10 mM MβCD, fixed, incubated with B3/25 anti-TfR antibody and then with goat anti-mouse antibody conjugated to 10 nm gold, and processed for EM. Bar, 100 nm.
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