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Mol Biol Cell. Apr 1999; 10(4): 961–974.
PMCID: PMC25220

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

Monty Krieger, Monitoring Editor

Abstract

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.

INTRODUCTION

Endocytosis occurs by clathrin-dependent as well as clathrin-independent mechanisms (van Deurs et al., 1989 blue right-pointing triangle; Sandvig and van Deurs, 1994 blue right-pointing triangle, 1996 blue right-pointing triangle). Clathrin-dependent endocytosis is the best-defined process so far and is responsible for the rapid uptake of, e.g., hormones, growth factors, and transport molecules such as EGF and transferrin. The interaction of the molecules involved in this process has been investigated both in vivo and in vitro, resulting in characterization of a number of important proteins such as clathrin, adaptors, and dynamin (Schmid, 1997 blue right-pointing triangle). Less is known about clathrin-independent endocytosis, but different forms seem to exist; for instance, both dynamin dependent and dynamin-independent mechanisms have been reported (Damke et al., 1994 blue right-pointing triangle, 1995 blue right-pointing triangle; Deirdre et al., 1998 blue right-pointing triangle; Henley et al., 1998 blue right-pointing triangle). Clathrin-independent endocytosis can clearly be different from uptake by invaginated caveolae. Thus, clathrin-independent endocytosis exists both in lymphocytes (Subtil et al., 1994 blue right-pointing triangle) and on the apical side of polarized Madin–Darby canine kidney (MDCK) cells (Eker et al., 1994 blue right-pointing triangle), which do not contain invaginated caveolae (Vogel et al., 1998 blue right-pointing triangle).

Earlier studies have suggested a role for cholesterol in endocytosis (Heiniger et al., 1976 blue right-pointing triangle); however, the type of endocytosis affected by cholesterol depletion was not investigated. Cholesterol present in the membrane of mammalian cells is required for normal cellular function (Bloch, 1991 blue right-pointing triangle) and either is delivered from low-density lipoproteins internalized by receptor-mediated endocytosis via clathrin-coated pits followed by subsequent hydrolysis in lysosomes (Brown and Goldstein, 1980 blue right-pointing triangle) or is synthesized in the endoplasmic reticulum (Reinhart et al., 1987 blue right-pointing triangle). A major fraction of cholesterol is present in the plasma membrane (Lange, 1991 blue right-pointing triangle), and cholesterol is important for various processes at the cell surface. For instance, there seems to be a direct interaction between the oxytocin receptor and cholesterol (Klein et al., 1995 blue right-pointing triangle), and also the acetylcholine receptor was found to have a functional requirement for cholesterol (Criado et al., 1982 blue right-pointing triangle). Furthermore, it has been proposed that annexin II, which is involved in the endocytic pathway (Creutz, 1992 blue right-pointing triangle; Gruenberg and Emans, 1993 blue right-pointing triangle), serves as an interface between membranes containing a high amount of cholesterol and the actin cytoskeleton (Harder et al., 1997 blue right-pointing triangle), and it is clear that cholesterol is essential for the structure and function of invaginated caveolae, including the caveolae-dependent endocytosis that has been reported in some cell types (Rothberg et al., 1990 blue right-pointing triangle; Parton et al., 1994 blue right-pointing triangle; Parton, 1996 blue right-pointing triangle; Schnitzer and Oh, 1994 blue right-pointing triangle; Smaby et al., 1996 blue right-pointing triangle; Chang et al., 1998 blue right-pointing triangle; Hailstones et al., 1998 blue right-pointing triangle).

In the present article, we have studied the role of cholesterol in clathrin-dependent and clathrin-independent endocytosis by using methyl-β-cyclodextrin (MβCD)1 to remove cholesterol from the plasma membrane. It has been shown that both β-cyclodextrin and MβCD remove cholesterol from cultured cells (Ohtani et al., 1989 blue right-pointing triangle; Kilsdonk et al., 1995 blue right-pointing triangle; Klein et al., 1995 blue right-pointing triangle), and we have here used the methylated form because it was found to be more efficient than β-cyclodextrin (Klein et al., 1995 blue right-pointing triangle). Cyclodextrins are cyclic oligomers of glucose that have the capacity to sequester lipophiles in their hydrophobic core (Pitha et al., 1988 blue right-pointing triangle). The water-soluble MβCD is known to form soluble inclusion complexes with cholesterol, thereby enhancing its solubility in aqueous solution (Pitha et al., 1988 blue right-pointing triangle; Irie et al., 1992 blue right-pointing triangle). In this article we demonstrate that cholesterol plays an important role in the invagination of clathrin-coated pits and therefore in clathrin-dependent endocytosis.

MATERIALS AND METHODS

Materials

MβCD (average degree of substitution: 10.5–14.7 methyl groups per molecule), β-cyclodextrin, α-cyclodextrin, γ-cyclodextrin, water-soluble cholesterol (with ~40 mg of cholesterol per gram solid; balance MβCD), lovastatin, pronase, HEPES, geneticin, lactose, BSA, ricin, and holo-transferrin (iron-saturated) were obtained from Sigma Chemical Co. (St. Louis, MO). [3H]leucine was obtained from the Radiochemical Center (Amersham, Buckinghamshire, UK). Na 125I and 125I-EGF were purchased from DuPont (Brussels, Belgium). Ricin and transferrin were 125I-labeled according to Fraker and Speck (1978) blue right-pointing triangle to a specific activity of 2 × 104–6 × 104 cpm/ng.

Cells

MDCK cells (strain II) transfected with the human transferrin receptor (TfR) (MDCK II hTfR) (from Dr. W. Hunziker, Lausanne, Switzerland) were maintained in DMEM (Flow Laboratories, Irvine, Scotland) supplemented with 5% FCS (Life Technologies, Paisly, Scotland), 100 μg/ml streptomycin, 100 U/ml penicillin, 2 mM l-glutamine (Life Technologies), and 0.25 mg/ml geneticin. HEp-2, A431, and NIH/3T3 cells (American Tissue Type Collection, Rockville, MD) were maintained in the same medium without the addition of geneticin and with different amounts of serum (5% FCS, 10% FCS, and 10% NCS, respectively). The HeLa cell line (tTA-HeLa), stably transfected with the cDNA for dynK44A, was cultured as described previously (Damke et al., 1995 blue right-pointing triangle). For experiments, cell lines were seeded into 24-well plates (Falcon, Franklin Lakes, NJ) at a density of 105 cells/well, or in Falcon 25 cm2 flasks at a density of 1.25 × 106 1 or 2 d before the experiment. The experiments were performed in HEPES-buffered medium.

Measurement of Binding and Endocytosis of 125I-Ricin

Binding of ricin was measured as the amount of cell-associated 125I-ricin after a 30 min incubation at 0° or 37°C with 125I-ricin (40 ng/ml) and four washes with cold HEPES-buffered medium. Endocytosed 125I-labeled ricin was measured after 15 min at 37°C as the amount of toxin that could not be removed after one rapid wash, a 5-min incubation at 37°C, followed by three rapid washes, all with 0.1 M lactose in HEPES-buffered medium at 37°C (Sandvig and Olsnes, 1979 blue right-pointing triangle). The endocytosed ricin was measured in a gamma counter (1261 Multigamma, Wallac, Gaithersburg, MD).

Measurement of Binding and Endocytosis of 125I-Transferrin

Binding of transferrin was measured as the amount of cell-associated 125I-transferrin after a 30 min incubation at 0° or 37°C with 125I-transferrin (100 ng/ml) and four washes with cold HEPES-buffered medium. Endocytosed 125I-labeled transferrin was measured after 5 min at 37°C and calculated as the percentage of total cell-associated (endocytosed and surface-bound) transferrin (Ciechanover et al., 1983 blue right-pointing triangle). Surface-bound transferrin was removed by 1-h incubation with HEPES medium containing 2 mg/ml pronase on ice. After pronase treatment the medium containing the cells was centrifuged for 2 min in an Eppendorf centrifuge (Madison, WI) before the radioactive contents in the cell pellet (endocytosed) and in the supernatant (surface bound) were measured with a gamma counter.

Measurement of Binding and Endocytosis of 125I-EGF

Binding of EGF was measured as the amount of cell-associated 125I-EGF after a 30 min incubation at 0° or 37°C with 125I-EGF (3–4 μCi/ml) and four washes with cold HEPES. Endocytosed 125I-labeled EGF was measured after 10 min at 37°C and calculated as the percentage of total cell-associated EGF. Surface-bound EGF was measured as the amount of EGF that could be released by low pH after three rapid washes with PBS at 0°C. The cells were then incubated at 0°C for 6 min with low pH buffer (0.5 M NaCl and 0.2 M CH3COOH in HEPES, pH 2.5), followed by one rapid wash with the same buffer. Endocytosed EGF was measured as the amount of EGF that could not be removed by this treatment (Sandvig et al., 1987 blue right-pointing triangle).

Measurement of the Potassium Content in Cells

The potassium content in cells was measured after the cells were washed three times with 100 mM MgCl2, air-dried, and dissolved in 0.1 M NaOH as described by Larkin et al. (1983) blue right-pointing triangle. Potassium was then determined by ion selective electrodes (Vitros 250; Johnson-Johnson Clinica Diagnostics, Rochester, NY).

Measurement of Protein Synthesis

Protein synthesis was measured by incubating the cells for 10 min in a HEPES-buffered medium with 1 μCi/ml [3H]leucine. The medium was then removed, and 5% trichloroacetic acid was added. This solution was removed 10 min later, and the cells were washed twice with the same solution to remove free radioactive leucine. The precipitated protein was then dissolved in 0.1 M KOH, and the radioactivity associated with the cells was measured in a β-counter (MINAXI, TRI-CARB 4000 SERIES, United Technologies, Packard, Meriden, CT).

Electron Microscopy

HEp-2 cells for EM were preincubated with and without MβCD (10 or 15 mM) for 15 or 30 min at 37°C, washed with HEPES-buffered medium, and fixed with 2% formaldehyde and 0.1% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2. The cells were then washed, scraped off the flasks, pelleted, and post-fixed with OsO4, contrasted en bloc with 1% uranyl acetate, dehydrated in a graded series of ethanols, and embedded in Epon. Sections were further contrasted with lead citrate and uranyl acetate and examined in a Philips CM 100 electron microscope (Philips, Eindhoven, the Netherlands). In some experiments, control cells and cells treated with MβCD were fixed, washed with PBS, and incubated with anti-human TfR antibody B3/25 (Boehringer Mannheim, Mannheim, Germany), 2 μg/ml PBS, for 1 h at room temperature. Then the cells remaining in monolayer in the culture flasks were washed with PBS and incubated for 2 h at room temperature with goat anti-mouse IgG coupled to 10 nm gold (Amersham), washed, scraped off, and processed for EM. Quantification of the immunogold labeling for TfRs was performed as described by Hansen et al. (1992) blue right-pointing triangle.

Effect of MβCD on Cellular Cholesterol and Invaginated Caveolae

HEp-2 cells were preincubated in HEPES-buffered DMEM with 0.2% BSA for 10 min and then incubated for 15 min at 37°C in HEPES-buffered DMEM with 1 μCi (1α,1β(n)-[3H])-cholesterol (toluene solution; Amersham) to label the cell surface (Lange, 1991 blue right-pointing triangle; Debry et al., 1997 blue right-pointing triangle). To equilibrate with the cholesterol of intracellular compartments, cells were incubated for 20 h in DMEM containing 2.5% delipidated calf serum (Sigma C-1696) and 1 μCi [3H]cholesterol (Debry et al., 1997 blue right-pointing triangle; Keller and Simons, 1998 blue right-pointing triangle). In both instances, cells were subsequently washed thoroughly and incubated at 37°C with agitation in 1 ml DMEM without MβCD, or with 10 mM MβCD for 15, 30, and 60 min. After washing, the cells were lysed in 250 μl 2% NP-40, 0.2% SDS in distilled water, and the lysates were then centrifuged to remove insoluble material before aliquots were mixed with EcoLite+ scintillation mixture, 1:10, and counted in a scintillation counter (Beckman, Fullerton, CA).

In parallel experiments (i.e., same cell passage, same day, and same MβCD solution), HEp-2 cells were washed and incubated with 10 mM MβCD in DMEM for 0, 15, 30, and 60 min at 37°C, washed again, fixed, and further processed for electron microscopy as described above. The amount of characteristically invaginated caveolae on the plasma membrane (Vogel et al., 1998 blue right-pointing triangle) was subsequently quantitated.

RESULTS

Effect of MβCD on Endocytosis of Transferrin, EGF, and Ricin

To study whether incubation with MβCD would affect endocytosis of transferrin and EGF, cells were preincubated for 15 min with MβCD at 37°C, and then 125I-transferrin or 125I-EGF was added. Endocytosis of transferrin was strongly reduced with increasing concentrations of MβCD (50% reduction at 10 mM MβCD) (Figure (Figure1).1). As shown in Figure Figure2,2, 10 mM MβCD also gives a strong reduction of 125I-EGF endocytosis (~40%). In agreement with the result that MβCD inhibits transferrin endocytosis, pretreatment of cells with 10 mM MβCD for 15 min at 37°C increased the cell surface binding of 125I-transferrin twofold (Figure (Figure3).3). To further investigate the effect of MβCD on clathrin-dependent endocytosis, uptake of 125I-transferrin was measured in different cell lines preincubated with 10 mM MβCD for 15 min at 37°C. As shown in Figure Figure4,4, this concentration of MβCD inhibited endocytosis of transferrin in all cell lines by ~50%.

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. ...
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 ...
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 ...
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. ...

To test whether MβCD would affect all endocytic activity, we studied the uptake of a molecule that does not only enter by clathrin-dependent endocytosis. Experiments performed with increasing concentrations of MβCD showed that the endocytosis of the general membrane marker ricin (Sandvig and Olsnes, 1979 blue right-pointing triangle; Sandvig and van Deurs, 1996 blue right-pointing triangle) was less affected by increasing concentrations of MβCD than that of transferrin in MDCK II cells (Figure (Figure1).1). In addition, there was no effect on the binding of 125I-ricin (Figure (Figure3).3). Endocytosis of 125I-ricin was also measured in different cell lines preincubated with 10 mM MβCD for 15 min at 37°C. As shown in Figure Figure4,4, MβCD inhibited endocytosis of ricin in the various cell types by ~20%. Because MβCD strongly decreased the number of invaginated caveolae (see below), the data suggest that there is another clathrin-independent form of endocytosis that is resistant to removal of cholesterol.

The data described above suggest that removal of cholesterol has a much stronger effect on clathrin-dependent than on clathrin-independent endocytosis. To further investigate this question we studied the effect of MβCD on transferrin and ricin endocytosis in HeLa K44A cells. These cells express mutant dynamin on removal of tetracycline, and clathrin-dependent endocytosis will then become inhibited (Damke et al., 1994 blue right-pointing triangle). HeLa K44A cells grown with and without tetracycline were incubated with and without MβCD for 15 min at 37°C, and then 125I-transferrin or 125I-ricin was added. As shown in Figure Figure5A,5A, ricin endocytosis was essentially unaffected by MβCD in the absence of tetracycline (mutant dynamin is expressed); however, MβCD decreased the endocytosis of ricin by ~20% in the presence of tetracycline (both clathrin-dependent and clathrin-independent endocytosis are functioning) (Figure (Figure5A).5A). As shown in Figure Figure5B,5B, endocytosis of transferrin is inhibited by MβCD to more than half in the presence of tetracycline, whereas transferrin endocytosis is blocked (>80%), as expected, when mutant dynamin is expressed and inhibits the clathrin-dependent pathway.

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 ...

The Effect of MβCD on Endocytosis Is Related to Removal of Cholesterol

In agreement with previous studies (Neufeld et al., 1996 blue right-pointing triangle; Keller and Simons, 1998 blue right-pointing triangle) we found that MβCD efficiently extracts cholesterol from HEp-2 cells (Figure (Figure6A).6A). Caveolae with the characteristic deeply invaginated shape were frequently observed in control HEp-2 cells. As an alternative approach to evaluating the effect of MβCD on plasma membrane cholesterol, we used the observation that typically invaginated caveolae (Figure (Figure6B)6B) can only be maintained in the presence of cholesterol (Rothberg et al., 1990 blue right-pointing triangle; Chang et al., 1998 blue right-pointing triangle; Hailstones et al., 1998 blue right-pointing triangle). Figure Figure6C6C shows that MβCD extraction of cholesterol has a pronounced effect on the number of invaginated caveolae.

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 [ ...

Although previous studies have shown that MβCD specifically removes cholesterol from the plasma membrane (Ohtani et al., 1989 blue right-pointing triangle; Kilsdonk et al., 1995 blue right-pointing triangle; Klein et al., 1995 blue right-pointing triangle; Neufeld et al., 1996 blue right-pointing triangle; Keller and Simons, 1998 blue right-pointing triangle), we investigated whether the decreased transferrin endocytosis in the presence of MβCD was due to loss of cholesterol from the plasma membrane. Two other cyclodextrins, α-cyclodextrin and γ-cyclodextrin, do not bind cholesterol in the same way as MβCD (Ohtani et al., 1989 blue right-pointing triangle), and we therefore tested whether they would affect transferrin endocytosis. In contrast, β-cyclodextrin, like MβCD, will extract cholesterol from the plasma membrane (Ohtani et al., 1989 blue right-pointing triangle; Klein et al., 1995 blue right-pointing triangle). HEp-2 cells were preincubated with 10 mM α-, β-, methyl-β-, or γ-cyclodextrins for 15 min at 37°C, before the addition of 125I-transferrin. Five minutes later, the amount of endocytosed transferrin was measured. As shown in Figure Figure7,7, both MβCD and β-cyclodextrin inhibited endocytosis of transferrin (>50%), whereas neither α- nor γ-cyclodextrin had any significant effect. Furthermore, when transferrin endocytosis was measured after preincubation of the cells with a MβCD-complex that was already saturated with cholesterol before it was added to the cells, there was no reduction in the endocytosis of transferrin (our unpublished results).

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 ...

To further investigate the dependence of endocytosis on cholesterol, we studied whether the inhibition of transferrin endocytosis by MβCD was reversible. First we tried to compensate for the loss of plasma membrane cholesterol by adding cholesterol to the cells. The cells were preincubated with MβCD for 15 min at 37°C, and then HEPES-buffered medium with or without 5% fetal calf serum or 400 μg/ml water-soluble cholesterol was added before endocytosis of 125I-transferrin was measured. As shown in Figure Figure8A,8A, there was a time-dependent recovery of the transferrin endocytosis. The uptake of transferrin was still inhibited by ~20% after a 1-h incubation even when serum or cholesterol was added; however, after 3-h incubation, the inhibition by MβCD was totally reversed. The finding that transferrin endocytosis recovered even in the absence of added serum or cholesterol suggested that the recovery could be due to cholesterol synthesis in the cells. This hypothesis was strengthened by the finding that lovastatin, an inhibitor of cholesterol synthesis, did inhibit the recovery of transferrin endocytosis unless the water soluble form of cholesterol was added (Figure (Figure8B).8B). Unless the cells were pretreated with MβCD, lovastatin only decreased transferrin endocytosis by ~10%, suggesting that there is only a small decrease in the level of plasma membrane cholesterol during 3 h in the absence of cholesterol synthesis. Thus, our results clearly indicate that the effect of MβCD observed is due to extraction of cholesterol.

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 ...

It was recently reported that removal of cholesterol with MβCD did not induce release of lactate dehydrogenase, suggesting that there are no large changes in the permeability of the plasma membrane (Keller and Simons, 1998 blue right-pointing triangle). This is in agreement with our finding that up to 10 mM MβCD did not reduce protein synthesis in our cells to any significant extent (Figure (Figure9A).9A). Also, as shown in Figure Figure9B,9B, 10 mM MβCD did not cause any significant membrane leakage of potassium. Furthermore, in MDCK II cells there was no increase in the cell-associated amount of 45Ca2+ even when the isotope was added to cells in a Ca2+-free buffer (our unpublished results). In HEp-2 cells there was a small increase (20%) under the same conditions, but there was no significant change when the isotope was added to a buffer containing normal amounts of CaCl2 (2 mM) (our unpublished results). Thus, in agreement with the data published by Keller and Simons (1998) blue right-pointing triangle, our data indicate that MβCD strongly decreases clathrin-dependent endocytosis without causing any large permeability changes.

Figure 9
Effect of MβCD on protein synthesis (A) and on the intracellular potassium content (B). (A) HEp-2 (○), NIH/3T3 ([down-pointing small open triangle]), MDCK II (●), and A431 ([filled triangle]) cells were washed briefly in HEPES-buffered medium and incubated with ...

Effect of MβCD on Clathrin-coated Pits and TfR Distribution

The inhibition of endocytosis of transferrin (and EGF) by MβCD could reflect that removal of cholesterol prevented clathrin-coated pit formation, i.e., assembly of clathrin coats on the cytoplasmic face of the membrane; however, clathrin-coated pits were readily observed by electron microscopy in MβCD-treated cells, although they clearly appeared more flattened than in control cells. To investigate how MβCD affected the morphological appearance of clathrin-coated pits quantitatively, we classified the pits as follows: shallow pits, which are flattened or only slightly invaginated (Figure (Figure10,10, A and B); invaginated pits, that is, when the pits are clearly invaginated but the necks connecting them with the exterior are still open wide (slightly wider or narrower than the diameter of the pit itself) (Figure (Figure10C);10C); and pits that are almost or completely pinched off, where the neck is very narrow (Figure (Figure10D),10D), or absent in the particular plane of sectioning (which could very well mean that the pit is surface-connected in another plane of sectioning; see Petersen and van Deurs [1983]) (Figure (Figure10E).10E). These three categories of clathrin-coated pits are shown schematically in Figure Figure10F10F and will be referred to below as type 1, 2, or 3. When control HEp-2 cells and cells treated with 10 mM MβCD for 15 min at 37°C were compared with respect to the relative frequency of the three types of clathrin-coated pits (Figure (Figure10G),10G), the effect of MβCD was striking: type 2 and in particular type 3 dominated in control cells, whereas MβCD caused a clear predominance of type 1 pits.

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 ...

Having shown that MβCD does not affect endocytosis in general or does prevent assembly of clathrin-coated pits at the cell surface but interferes with the ability of flattened pits to invaginate, it was important to determine whether TfRs could still accumulate in the pits of MβCD-treated cells. We therefore used an immunogold-labeling protocol to detect TfRs on the surface of HEp-2 cells (Hansen et al., 1992 blue right-pointing triangle). As shown in Figure Figure11A,11A, TfR gold labeling was distinct in the shallow, clathrin-coated pits mostly found in the MβCD-treated cells. Indeed, gold labeling was also seen on the cell surface outside clathrin-coated pits and on microvilli (Figure (Figure11,11, B and C). In control HEp-2 cells, 8.7% of the TfRs as detected by immunogold labeling (using 2 μg/ml of the B3/25 anti-TfR antibody on prefixed cells) are localized to coated pits that occupy 1.1–1.4% of the total surface area of these cells (Hansen et al., 1992 blue right-pointing triangle). Accordingly, TfRs are concentrated approximately sevenfold in the coated pits (Table (Table1).1). After incubation with 10 mM MβCD, we found that 11.5% of the TfR gold labeling was localized to coated pits (Table (Table1);1); however, because the surface area now occupied by coated pits had increased by 25% (presumably reflecting the fact that efficient invagination and pinching off are perturbed), the amount of golds in coated pits still corresponds to a sevenfold TfR concentration (Table (Table1).1). It should also be noted that we found a 60% increase in the total TfR cell-surface labeling in MβCD-treated cells as compared with control cells (Table (Table1),1), in agreement with the biochemical data reported above. Thus MβCD treatment does not interfere with the ability of TfRs to become concentrated efficiently in coated pits. Taken together, our findings therefore indicate that rather than blocking all types of endocytosis or preventing TfRs from entering clathrin-coated pits, MβCD perturbs invagination of the coated pits.

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 ...
Table 1
Concentration efficiency of TfR in clathrin-coated pits in HEp-2 cells as determined by immunogold labelinga

DISCUSSION

In the present study we have investigated the role of cholesterol in endocytosis by using MβCD, which specifically removes cholesterol from the plasma membrane (Ohtani et al., 1989 blue right-pointing triangle; Kilsdonk et al., 1995 blue right-pointing triangle; Klein et al., 1995 blue right-pointing triangle; Neufeld et al., 1996 blue right-pointing triangle). Our results show that endocytosis of transferrin and EGF was strongly reduced by addition of MβCD. This reduction could be due 1) to a general inhibition of all endocytic activity, 2) to a perturbation of the ability of clathrin-coated membrane areas to form, invaginate, or pinch off to form free, coated vesicles, or 3) to an exclusion of the receptors from clathrin-coated pits. The first possibility can be excluded because the general membrane marker ricin is still internalized. Furthermore, MβCD clearly reduced ricin endocytosis to a much larger extent when both clathrin-dependent and clathrin-independent endocytosis were operating than when only clathrin-independent endocytosis was functioning. The results indicate that MβCD strongly reduces clathrin-dependent endocytosis, whereas there is a clathrin-independent endocytic pathway that is less affected. Because invagination of caveolae also is affected by MβCD, this clathrin-independent endocytosis seems to be caveolae-independent. It has previously been found that a downregulation of clathrin-dependent endocytosis can be associated with an upregulation of clathrin-independent endocytosis (Damke et al., 1995 blue right-pointing triangle), and there is a possibility that this might occur also on treatment with MβCD. Second, EM revealed that coated pits were indeed formed in MβCD-treated cells, but a marked flattening of these structures had taken place. Third, the quantitative immunogold data clearly show that the TfRs are not prevented from being concentrated in (the shallow) coated pits after MβCD treatment. A concentration of TfRs in clathrin-coated pits of approximately sevenfold both in MβCD-treated cells and in control cells is in agreement with previous data (Hansen et al., 1992 blue right-pointing triangle).

TfRs undergo endocytosis and recycling, and the number of TfRs on the surface is consequently dependent on both the amount of receptors which is internalized, and the amount that is recycled. The finding that binding of transferrin to the cell surface was increased nearly twofold by MβCD indicates that MβCD inhibits endocytosis without reducing the recycling of the TfRs to the same extent. There seems to be a somewhat stronger effect of MβCD on the morphology of the clathrin-coated pits than on the uptake of transferrin. This might be due to the formation of some invaginated coated pits that rapidly pinch off. Our results strongly suggest that removal of cholesterol perturbs clathrin-coated pit invagination and, therefore, coated vesicle formation.

It has previously been reported that the membrane in coated pits is not disrupted by the sterol-binding drugs filipin or digitonin, although the membrane adjacent to these specialized domains readily binds these agents (Montesano et al., 1979 blue right-pointing triangle). On the other hand, endosomal membrane becomes susceptible to filipin disruption as soon as the clathrin coat is released (McGookey et al., 1983 blue right-pointing triangle), suggesting that the clathrin coat directly or indirectly binds cholesterol and prevents access to filipin. In agreement with those results, nystatin was reported not to affect transferrin endocytosis (Deckert et al., 1996 blue right-pointing triangle), and our own unpublished data showed that filipin has no effect on transferrin endocytosis in HEp-2 cells. Also, it is not clear that complexes between cholesterol and sterol-binding drugs would affect endocytosis in the same way as removal of cholesterol by MβCD.

Sterol-binding drugs such as filipin, nystatin, and digitonin have been found to inhibit endocytosis from invaginated caveolae and caveolae-like domains (Schnitzer et al., 1994 blue right-pointing triangle; Deckert et al., 1996 blue right-pointing triangle; Orlandi and Fishman, 1998 blue right-pointing triangle). Invaginated caveolae disappear in cells that are depleted of cholesterol or exposed to sterol-binding agents (Rothberg et al., 1990 blue right-pointing triangle, 1992 blue right-pointing triangle; Schnitzer et al., 1994 blue right-pointing triangle). In agreement with those data, our results clearly show that MβCD leads to disappearance of invaginated caveolae in HEp-2 cells. That MβCD treatment leads to loss of invaginated caveolae is in agreement with the recent results of Hailstones et al. (1998) blue right-pointing triangle showing that different cholesterol depletion treatments, including β-trimethyl cyclodextrin treatment, remove morphologically recognizable invaginated caveolae. Interestingly, they found that invaginated caveolae only form when the cholesterol level is >50% of control values. Also, Orlandi and Fishman (1998) blue right-pointing triangle found that the activity of cholera toxin was completely inhibited by β-cyclodextrin, and they concluded that productive entry of cholera toxin occurs from caveolae. Furthermore, they reported that diphtheria toxin retained 80% of its toxic activity in the presence of β-cyclodextrin, and they therefore assumed that entry from clathrin-coated pits was essentially normal. This apparently contradicts our results; however, no direct measurement on uptake from clathrin-coated pits after β-cyclodextrin treatment was performed by Orlandi and Fishman (1998) blue right-pointing triangle, and the apparent discrepancy can be explained if removal of cholesterol both reduces uptake from coated pits and at the same time facilitates membrane translocation of the toxin. In fact, filipin, which does not affect uptake from clathrin-coated pits, increased the toxicity of diphtheria toxin (Orlandi and Fishman, 1998 blue right-pointing triangle).

Heiniger et al. (1976) blue right-pointing triangle reported that L cells, in which hydroxymethylglutaryl-CoA reductase activity and cholesterol synthesis were inhibited by addition of 25-hydroxycholesterol, had a reduced rate of endocytosis of the fluid-phase marker HRP. The uptake of HRP was restored by the addition of cholesterol (Heiniger et al., 1976 blue right-pointing triangle). HRP is endocytosed, however, by both clathrin-dependent and clathrin-independent endocytosis (Oliver, 1982 blue right-pointing triangle), and the fact that cholesterol depletions of L cells seem to have a stronger effect on total endocytosis in these cells (50%) than the effect we observed on HRP endocytosis in the cell types we have tested could be due to a larger proportion of uptake by clathrin-dependent endocytosis in L cells. Interestingly, Chang et al. (1992) blue right-pointing triangle reported that removal of cholesterol drastically increased the number of low-density lipoprotein receptors in MA104 cells. The authors reported a slightly reduced internalization index, but because the calculation of such an index only takes into account the amount of surface-bound ligand versus internalized ligand, in this case at the end of a long incubation, the data might actually be in agreement with a reduced rate of endocytosis of the receptor from clathrin-coated pits. Two other cyclodextrins (Ohtani et al., 1989 blue right-pointing triangle)—α-cyclodextrin, which has been reported to remove phospholipids from the plasma membrane, and γ-cyclodextrin, which is also not specific for cholesterol—had much less effect on transferrin endocytosis than MβCD. This supports the notion that it is the removal of cholesterol per se that is essential for the inhibitory effect on clathrin-dependent endocytosis in the MβCD-treated cells. On removal of MβCD, the transferrin endocytosis was fully restored by continued incubation of the cells, even in serum-free medium. The recovery was inhibited by addition of lovastatin, suggesting that the cholesterol level in the plasma membrane was restored by newly synthesized cholesterol. This idea is supported by the finding that endocytosis did recover when a water-soluble form of cholesterol was added together with lovastatin. When lovastatin is used to inhibit cholesterol synthesis, fatty acid modification of intracellular proteins also is inhibited (Alberts et al., 1980 blue right-pointing triangle; Brown and Goldstein, 1980 blue right-pointing triangle), and it is therefore essential for the conclusion, that cholesterol is required for clathrin-dependent endocytosis, that addition of cholesterol can counteract the effect of treatment with lovastatin.

It was reported recently that extraction of cholesterol with MβCD did not affect the amounts of lactate dehydrogenase in baby hamster kidney cells (Keller and Simons, 1998 blue right-pointing triangle). In agreement with those findings, our results show that treatment with 10 mM MβCD had no effect on the intracellular potassium content nor on the protein synthesis, indicating that the plasma membrane permeability was not strongly affected. Furthermore, the lack of change in transepithelial resistance in MβCD-treated MDCK I cells (our unpublished results) and MDCK II cells (Keller and Simons, 1998 blue right-pointing triangle), together with our finding that binding of ricin and Shiga-toxin to the cell surface was unaffected by MβCD (our unpublished results), indicates that there are no large changes in the bilayer structure in cells treated with MβCD compared with the control cells. All of these data indicate that the flattening of the clathrin-coated pits is directly correlated to the removal of cholesterol.

It has been suggested that annexin II, which seems to be involved in both the regulated exocytic pathway (Creutz, 1992 blue right-pointing triangle) and the endocytic pathway (Gruenberg and Emans, 1993 blue right-pointing triangle), interacts directly with cholesterol-rich domains of the bilayer, and perhaps with cholesterol itself (Harder et al., 1997 blue right-pointing triangle). Such a direct protein–cholesterol interaction was recently revealed for the membrane protein VIP21-caveolin (Murata et al., 1995 blue right-pointing triangle). The cholesterol-binding drug filipin induces dissociation of annexin II and actin-binding proteins from endosomes (Harder et al., 1997 blue right-pointing triangle). In contrast, there is no evidence that cholesterol is important for binding of clathrin. As shown here, the clathrin coat stays at the plasma membrane after treatment of cells with MβCD. It is only the curvature that is affected. Actin was recently reported to be involved in clathrin-dependent endocytosis, because endocytosis was inhibited by perturbation of the actin cytoskeleton (Parton et al., 1994 blue right-pointing triangle; Schnitzer and Oh, 1994 blue right-pointing triangle; Deckert et al., 1996 blue right-pointing triangle). Nevertheless, actin does not seem to be required for the formation of the coated-pit invagination (Lamaze et al., 1997 blue right-pointing triangle). Thus, the effect of MβCD described here is probably not due to an effect on the actin cytoskeleton; however, cholesterol could be important for the function of a protein involved in inducing curvature of clathrin-coated pits, such as affecting the activity of GTP-binding proteins that regulate coated-pit function (Schmid, 1997 blue right-pointing triangle). Also, removal of cholesterol from the membrane might affect association of other molecules with the clathrin-coated pits, it might affect the interaction of membrane-associated proteins with each other, or it might somehow directly affect the ability of the other lipids in the clathrin-coated membrane domains to form an invaginated structure. As mentioned in the INTRODUCTION, cholesterol is important for the structure and function of membrane proteins (Muller and Shinitzky, 1979 blue right-pointing triangle; Criado et al., 1982 blue right-pointing triangle; Bloch, 1991 blue right-pointing triangle; Kilsdonk et al., 1995 blue right-pointing triangle), and furthermore, a change in the cholesterol content will affect the membrane viscosity, the exposure of membrane proteins to the surroundings, and therefore also the interaction between membrane proteins and cytosolic proteins (Shinitzky and Inbar, 1974 blue right-pointing triangle; Shinitzky and Rivnay, 1977 blue right-pointing triangle). Such changes might be responsible for the inhibition of formation of clathrin-coated pits observed here.

In conclusion, our results clearly indicate that the concentration of cholesterol in the plasma membrane of mammalian cells is essential for clathrin-dependent endocytosis, and it will be important for the understanding of this pathway to investigate why this is the case.

ACKNOWLEDGMENTS

We are grateful to Anne-Grethe Myrann, Ulla Hjortenberg, Mette Ohlsen, Kirsten Pedersen, and Keld Ottosen for their excellent technical assistance. This work was supported by the Norwegian Research Council for Science and the Humanities, The Norwegian Cancer Society, The Danish Cancer Society, The Danish Medical Research Council, the Novo-Nordisk Foundation, Blix legacy, Torsteds legacy, the Jahre foundation, the Nordic Cancer Union, a NATO Collaborative Research Grant (CRG 900517), a Human Frontier Science Program grant (RG404/96 M), and Jeanette and Søren Bothners legacy.

Abbreviations used:

MβCD
methyl-β-cyclodextrin
TfR
transferrin receptor

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