ARTICLE |
Correspondence to: Toyoshi Fujimoto, Dept. of Anatomy and Cell Biology, Gunma Univ. School of Medicine, Maebashi 371, Japan.
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Summary |
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-Toxin (perfringolysin O), a cholesterol-binding toxin, was partially proteolyzed and biotinylated (BC
) to eliminate hemolyzing activity and was used as a cytochemical probe. In fixed cells, binding of BC
was intense in the plasma membrane, especially at the base of apical microvilli and in lateral processes. The labeling was abolished by pretreatment with filipin, digitonin, or tomatin. When living cultured cells were treated with BC
and then with either fluorescein-avidin D or colloidal gold-streptavidin, the labeling in fine dots was distributed on the cell surface without local concentration as long as cells were kept on ice. When the temperature was raised to 37C after treatment, the probe formed discrete large patches and became sequestered to caveolae. Binding of BC
alone without the secondary reagents did not cause redistribution even at 37C. Because the plasma membrane maintains integrity even after binding of BC
, the probe can be used not only for cytochemical labeling of fixed cells but for pursuing the behavior of crosslinked cholesterol molecules in living cells. By use of this new probe, the present study revealed that crosslinked cholesterol in the plasma membrane is sequestered to caveolae. (J Histochem Cytochem 45:1197-1205, 1997)
Key Words:
cholesterol, -toxin, caveolae, plasma membrane, histochemistry, electron microscopy, freeze-fracture
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Introduction |
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Cholesterol is a major constituent of the plasma membrane and has been considered to play essential roles in maintaining its integrity (
Relative enrichment of cholesterol in caveolae compared to non-caveolar portions of the plasma membrane has been believed to bring about the above results. Two lines of studies have been cited to support the assumption. First, a morphological study showed that a ring of filipin-sterol complexes is preferentially formed at the orifices of caveolae (
Recently, we showed by immunocytochemistry that antibodies to glycolipids and sphingomyelin bind to the cell surface without local concentration and are sequestered to caveolae when they are crosslinked with secondary antibodies (
Several probes have been used for cytochemical labeling of cholesterol (-toxin of Clostridium perfringens (the derivative is called as BC
hereafter). As described previously for a nicked and methylated
-toxin (
retains specific binding activity to cholesterol but lacks hemolyzing activity (unpublished observations). By using this reagent as a probe, we could analyze the effect of cholesterol crosslinking without damaging the cellular integrity.
A portion of this work was presented previously in preliminary form (
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Materials and Methods |
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Reagents
Recombinant -toxin was prepared from the periplasm of E. coli strain BL21(DE3) harboring pNSP10, a plasmid containing the
-toxin gene (Sekino-Suzuki et al., submitted for publication). The toxin was purified and nicked with subtilisin Carlsberg as reported previously (
-toxin for cholesterol has been reported previously (
Cells
Pam 212, a mouse transformed keratinocyte cell line (
Labeling of Fixed Specimens
Cultured cells were rinsed and incubated in a serum-free medium for at least 2 hr before experiments to minimize the possibility that cholesterol in the bovine serum would affect the results. Mouse erythrocytes were used after extensive washing with PBS. The cells were fixed with 0.1% glutaraldehyde for 10 min. In some experiments, more rigorous fixation (1% glutaraldehyde for 1 hr) was applied, but the result was not changed. For fluorescence microscopy, autofluorescence was quenched with 1 mg/ml sodium borohydride. The samples were treated with 1% bovine serum albumin (fatty acid-free grade; Seikagaku Kogyo, Tokyo, Japan) for 30 min, incubated with 10-30 µg/ml BC for 30 min, and then with either fluorescein-avidin D (20-50 µg/ml) or colloidal gold-streptavidin (diluted to 1:30) for 30 min. For controls, the samples were pretreated with either 300 µM filipin, 160 µM digitonin, or 150 µM tomatin (Sigma Chemical; St Louis, MO) for 30 min, and the BC
solution was also added with 1:10 concentration of the above reagents. All the procedures were done at room temperature.
Treatment of Living Cells
Pam 212 cells were cooled on ice for 15 min, and were treated with 10-30 µg/ml BC and then with either fluorescein-avidin D or colloidal gold-streptavidin, both for 60 min on ice. After rinsing, some samples were immediately fixed with 3% formaldehyde for more than 30 min, and others were incubated for 5-30 min at 37C before fixation.
To exclude the possibility that the final fixation caused redistribution of bound BC, a group of labeled cells was observed without fixation. The result was equivalent to the cells labeled and fixed by the above method. Therefore, the distributional change of BC
observed in the present experiment was not an artifact induced by fixation.
In an experiment, living Pam 212 cells were labeled with BC on ice as above, and their apical plasma membranes were isolated on Alcian blue-coated coverslips (
Microscopic Observation
For fluorescence microscopy, samples were observed and photographed with a Zeiss Axiophot microscope. For thin-section electron microscopy, the colloidal gold-labeled samples were further fixed with 2% glutaraldehyde, postosmicated with 1% osmium tetroxide, stained en bloc with 0.5% aqueous uranyl acetate, dehydrated, and embedded in Spurr's resin. For freeze-fracture electron microscopy, glutaraldehyde-fixed samples were soaked in 25% glycerin, inverted on a mixture of polyvinyl alcohol and glycerin (
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Results |
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Labeling of Fixed Cells with BC
When fixed cells were incubated with BC, and then with fluorescein-avidin D, labeling occurred on the entire cell surface. Figure 1A shows the labeling of 3Y1 cells, a rat fibroblast cell line. Previous results have shown that a nicked
-toxin binds specifically to membrane cholesterol in living cells (
treatment did not cause labeling (Figure 1B). Second, the specimens were pretreated with reagents specific for cholesterol and then incubated with BC
; filipin, digitonin, and tomatin were applied at the concentration used previously for cytochemical labeling (
. These results showed that the labeling with BC
is specific for cholesterol.
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In Pam 212 cells, in addition to general labeling, intense fluorescence was observed as speckles on the apical surface; lateral processes also showed strong labeling (Figure 2). Next, fixed Pam 212 cells were treated with BC and then with colloidal gold-streptavidin and were observed by label-fracture electron microscopy (
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Treatment of Living Cells with BC
Antibodies to GPI-anchored proteins, glycolipids, and sphingmyelin bound to the entire surface of Pam cells, but became concentrated to caveolae when further treated with secondary antibodies and incubated at 37C ( and then with fluorescein-avidin D. When the cells were treated with BC
on ice and fixed immediately, the labeling was seen as fine dots distributed on the cell surface without local concentration (Figure 4A). In contrast, when the temperature was raised to 37C after treatment, the labeling became concentrated at the cell periphery; the concentration was obvious at 10 min and increased further at 30 min (Figure 4B and Figure 4C).
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Because caveolae are accumulated in the vicinity of intercellular contact in Pam 212 cells ( labeling on ice (Figure 4A) are not likely to match caveolae. To confirm this, the apical plasma membranes of the cells treated with BC
alone on ice were isolated on adherent coverslips and immunolabeled for caveolin. Caveolin was localized as dots, more concentrated in some areas compared to other areas of the isolated membrane (Figure 5B). In the same membranes, binding of BC
, which was visualized by fluorescein-avidin D after fixation, appeared in speckles, and their distribution did not coincide with caveolin (Figure 5A).
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Next, to observe the fine distribution of crosslinked BC, the specimen incubated for 30 min at 37C was subjected to label-fracture electron microscopy. Compared to the labeling in fixed cells (Figure 3B), gold particles were observed more concentrated in the caveola-rich area (Figure 6A and Figure 6B). They were seen on the bumps themselves and on the flat membrane area close to the bumps. The same specimen was processed for thin-section electron microscopy. Most gold particles were seen in caveolae (Figure 6C).
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Discussion |
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BC as a Cytochemical Probe for Cholesterol
Many probes have been used to visualize the two- dimensional distribution of 3ß-OH sterols (mainly cholesterol) in biomembranes. They are classified into three categories: polyene antibiotics (
In the present study, we utilized a nicked and biotinylated bacterial toxin, BC, which retains specific cholesterol-binding activity but lacks hemolyzing effect (unpublished observations). When used as a probe to living cells, BC
appeared less damaging to the membrane than filipin or digitonin. A dye exclusion test showed that more than 95% of lymphocytes excluded trypan blue after incubation with 30 µg/ml BC
for 30 min at 37C; even when they were further treated with avidin D and incubated for 30 min at 37C, more than 80% of the cells excluded the dye (Fujimoto et al., unpublished observations). Because BC
is a protein of 53 kD, it cannot penetrate the plasma membrane and its binding is limited to the cell surface. Therefore, BC
may not be useful as a general cytochemical probe, but this property is an advantage when applied to living cells. Another difference between BC
and filipin or digitonin is that localization of BC
itself can be visualized by streptavidin conjugated to colloidal gold. Because the detection does not require membrane deformation, the rigidity of the membrane should not affect the labeling.
On the other hand, although the binding of BC to cholesterol was shown to be specific, several problems remain to be solved. First, stoichiometry of the binding has not been determined. Therefore, whether the binding of BC
alone crosslinks multiple cholesterol molecules is not known. Second, it has been shown that nicked
-toxin binds to cholesterol with two different affinities (
may detect some populations of cholesterol more efficiently than others (
, its binding to cholesterol is very probably affected by steric hindrance. Fourth, it is not clear whether BC
can recognize cholesterol bound to membrane proteins. This factor is important in interpreting the results concerning caveolae because caveolin is a cholesterol-binding protein (
with an equal probability.
In the present study, when cells were first fixed and then labeled, intense labeling around the base of microvilli was seen. On the other hand, such concentration was not observed in cells treated with the same combination of the reagents on ice and then fixed. Fixation may affect the state of plasmalemmal cholesterol molecules. For example, reactivity to cholesterol oxidase dramatically increased after fixation (
Caveolar Sequestration of Crosslinked Cholesterol in Living Cells
The relationship between cholesterol and caveolae has been examined in many studies, as cited in the Introduction (
An early cytochemical result showed that the filipin-cholesterol complex is formed densely at the orifices of caveolae (
On the other hand, by biochemical analysis, a relatively high content of cholesterol was recovered in the detergent-insoluble complex (
In the present study, binding of BC did not occur preferentially in the vicinity of caveolae in fixed and in unfixed specimens incubated on ice. Only after crosslinking with avidin D or streptavidin and incubation at 37C was bound BC
sequestered to the caveola-rich region. Because of the problems discussed above, whether cholesterol is enriched in or around caveolae in native cells cannot be concluded from the BC
experiment. However, the pattern of redistribution after crosslinking was the same as for GPI-anchored proteins (
Summing up, the present study introduced a new cytochemical probe for membrane cholesterol. It can be applied to living cells with minimal damage to cellular integrity. This probe bound to the plasma membrane of living cells without marked concentration, but was sequestered to the caveola-rich region when crosslinked with the secondary reagent. The result showed that not only GPI-anchored proteins and sphingolipids (
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Acknowledgments |
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Supported by Grants-in-Aid for Scientific Research (B) (no. 08457001) and for Exploratory Research (no. 08878121) of the Ministry of Education, Science, Sports, and Culture of the Japanese Government and by a research grant from the Ciba-Geigy Foundation (Japan) for the Promotion of Science.
We are grateful to Ms Fujie Miyata and Ms Yukiko Takahashi for their excellent technical and secretarial assistance.
Received for publication August 8, 1996; accepted March 14, 1997.
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