ARTICLE |
Correspondence to: Wiebke Möbius, Dept. of Cell Biology, University Center Utrecht and Center for Biogenetics, AZU G02.525, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail: w.moebius@lab.azu.nl
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Summary |
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We used a proteolytically modified and biotinylated derivative of the cholesterol-binding -toxin (perfringolysin O) to localize cholesterol-rich membranes in cryosections of cultured human lymphoblastoid cells (RN) by electron microscopy. We developed a fixation and immunolabeling procedure to improve the preservation of membranes and minimize the extraction and dislocalization of cholesterol on thin sections. We also labeled the surface of living cells and applied high-pressure freezing and subsequent fixation of cryosections during thawing. Cholesterol labeling was found at the plasma membrane, with strongest labeling on filopodium-like processes. Strong labeling was also associated with internal vesicles of multivesicular bodies (MVBs) and similar vesicles at the cell surface after secretion (exosomes). Tubulovesicular elements in close vicinity of endosomes and the Golgi complex were often positive as well, but the surrounding membrane of MVBs and the Golgi cisternae appeared mostly negative. Treatment of cells with methyl-ß-cyclodextrin completely abolished the labeling for cholesterol. Our results show that the
-toxin derivative, when used in combination with improved fixation and high-pressure freezing, represents a useful tool for the localization of membrane cholesterol in ultrathin cryosections. (J Histochem Cytochem 50:4355, 2002)
Key Words: membrane cholesterol, cryosections, electron microscopy, exosomes, cholesterol-enriched microdomains
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Introduction |
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CHOLESTEROL is the most abundant sterol in mammalian cells and an essential component of cell membranes. The major pool of membrane cholesterol is present at the plasma membrane (
Apart from caveolae, membrane cholesterol appears to be involved in the formation of microdomains at the cell surface by preferential association with glycosphingolipids and sphingomyelin to form the so-called liquid ordered phase (reviewed by
The polyene antibiotic filipin (
We have studied the usefulness of a biotinylated and non-cytolytic form of -toxin (perfringolysin O) from Clostridium perfringens (termed BC
hereafter) to localize cholesterol at the subcellular level. The specificity of cholesterol binding by BC
was demonstrated previously (
was introduced as a cytochemical probe for detection of plasma membrane cholesterol by
was used to specifically detect cholesterol-rich microdomains in the plasma membrane of cells (
is a valuable tool to analyze the distribution of membrane cholesterol on thin cryosections of cells.
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Materials and Methods |
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Reagents
Perfringolysin O (-toxin) was prepared and digested with subtilisin Carlsberg as reported previously (
-toxin (BC
) was produced from the nicked
-toxin as described (
Cells
The EBV-transformed human B-cell line RN (HLA-DR15+) was maintained as described (
Cell Surface Labeling
For detection of plasma membrane cholesterol with BC in living cells, RN cells were washed several times with ice-cold PBS, pelleted, and kept on ice for 15 min. For surface labeling of fixed cells, RN cells were fixed with 2% formaldehyde, 0.2% glutaraldehyde in 0.1 M PHEM buffer for 30 min on ice and washed several times with ice-cold 0.1% glycine in PBS. Then the cells were resuspended in PBS containing BC
at a concentration of 15 µg/ml and incubated for 30 min on ice with constant shaking. Next, the cells were washed thoroughly with ice-cold PBS and fixed with 2% formaldehyde, 0.2% glutaraldehyde in 0.1 M PHEM buffer (60 mM PIPES, 25 mM HEPES, 2 mM MgCl2, 10 mM EGTA, pH 6.9) overnight at 4C and infiltrated with 2.3 M sucrose in 0.1 M PHEM buffer for 2 hr on ice. Then droplets of cells in sucrose were mounted on pins and frozen in liquid nitrogen. Cryosections were labeled with anti-biotin antibodies and protein Agold (see below).
Flow Cytometric Analysis of BC Binding to Intact Cells
Living or prefixed RN cells were incubated with BC as described above. Living cells incubated with buffer only served as negative control. To confirm specificity of BC
binding, plasma membrane cholesterol was extracted by treatment of cells with 10 mM cyclodextrin for 30 min at 37C in complete medium before incubation with BC
. After washing with PBS, the samples were fixed with 2% formaldehyde and 0.2% glutaraldehyde in 0.1 M PHEM buffer for 30 min on ice. After washing with 0.1% glycine in PBS, the samples were incubated with anti-biotin antibodies (4 µg/ml PBS with 1% BSA), followed by an incubation with anti-rabbitFITC (1:50 in PBS with 1% BSA). Cell-associated fluorescence was analyzed by flow cytometry using FACS Calibur (Becton Dickinson; Franklin Lakes, NJ).
Cryosections After Cryofixation
This method was briefly introduced earlier (
After five brief rinses with 0.1 M PHEM buffer, sections were incubated with BC at a concentration of 15 µg/ml in 0.1 M PHEM buffer for 30 min at RT, washed, and fixed with 1% glutaraldehyde for 15 min at RT. After several washes with 0.1% glycine in 0.1 M PHEM buffer, sections were incubated for 30 min at RT with anti-biotin antibodies (4 µg/ml in 0.1 M PHEM buffer with 1% BSA), followed by 20-min incubation with protein Agold in 0.1 M PHEM buffer with 1% BSA. After fixation with 1% glutaraldehyde for 3 min, sections were washed with water, contrasted, and dried as described (
Cryosections After Chemical Fixation
RN cells were washed with 0.1 M PHEM buffer. According to the conventional protocol of for 3 hr at 4C. To confirm the specificity of cholesterol labeling, sections were incubated with 10 mM methyl-ß-cyclodextrin for 20 min and washed with 0.1 M PHEM buffer before labeling to also remove intracellular membrane cholesterol.
Quantification
To determine the labeling density of the plasma membrane cholesterol, living RN cells were incubated with BC as described above. After washing the cells were fixed with 2% formaldehyde and 0.2% glutaraldehyde in 0.1 M PHEM buffer and processed for cryosectioning. To make sure that three independent samples were used for quantification, 15 µm of the block was trimmed away between cutting sections. After labeling of sections with anti-biotin and protein Agold, 10 random pictures were taken from a single section of each of the three grids and printed at a final magnification of x28,000. We determined the labeling density (gold/µm membrane) by using point and intersection counting with a line lattice (10-mm distance) overlay as described by
To determine the density of cholesterol labeling on endosomes and the Golgi complex, sections were cut as described above and incubated with BC for 30 min at RT, followed by labeling with anti-biotin antibodies and 10-nm protein Agold. For quantification of labeling on the Golgi complex, five pictures of different Golgi areas with clearly discernible stacks were taken from a single section of each of the three grids at a magnification of x20,000. The labeling densities on Golgi cisternae and TGN associated vesicles/tubules were determined on prints with a final magnification of x47,000 using a curvilinear lattice with 5-mm distance between lines (isotopic test system according to
For quantification of labeling on endosomes, pictures of seven to nine endosomes (Type 3 in
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Results |
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Search for Optimal Cholesterol Labeling
Labeling of Cholesterol on the Cell Surface.
It is well known that the major fraction of the cell's membrane cholesterol resides in the plasma membrane. Accordingly, we found significant labeling of the cell surface when we incubated living RN cells with BC at 4C (Table 1), fixed the cells, and processed them for cryosectioning and immunolabeling with anti-biotin and protein Agold (Fig 1A). Removal of cholesterol by pretreatment with 10 mM methyl-ß-cyclodextrin completely abolished plasma membrane labeling. If we changed the sequence of steps in the cell surface labeling procedure so that cells were first fixed and then incubated with BC
, the plasma membrane labeling was significantly reduced (Fig 1B). This was not due to cholesterol extraction during the fixation procedure because we could still achieve BC
binding to cryosections of these preparations (see below). A possible explanation for the failure to label intact cells after fixation is that a coat of crosslinked surface proteins makes the membrane cholesterol inaccessible to the probe. To confirm these morphological data, we performed a quantitative analysis using flow cytometry and compared the extent of surface labeling in living cells to that of fixed cells. As shown in Fig 2, indeed less BC
bound to fixed cells (Curve 3) than to living cells (Curve 4). The binding was even more reduced after cholesterol extraction with 10 mM cyclodextrin (Curve 2).
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Labeling of Plasma Membrane Cholesterol in Conventional Cryosections.
Because we wanted to study the intracellular cholesterol distribution, we explored the possibility of using cryosections for BC binding and subsequent immunogold labeling. First, cryosections were prepared in the conventional way as described in Materials and Methods, incubated with BC
and immunolabeled with anti-biotin and protein Agold (Table 1). The pattern of labeling on these sections showed that the distribution of cholesterol was seriously disturbed (Fig 1C). The plasma membrane itself was usually not labeled, but a wide zone of gold particles was present at the extracellular side, indicating extraction of cholesterol.
Labeling of Plasma Membrane Cholesterol in Cryosections of Cryofixed Cells.
Next, we circumvented chemical prefixation and used high-pressure frozen cells (Table 1). Cryosections were cut at -170C on a dry diamond knife. To achieve maximal preservation of ultrastructure, it was important that the cryosections were cut flat without compression, picked up, thawed without stretching, and transferred flat to a grid. The pick-up solution contained a mixture of sucrose, methylcellulose, formaldehyde, glutaraldehyde, acrolein, and uranyl acetate. Including methylcellulose and uranyl acetate in the pick-up solution was crucial for good preservation of the ultrastructure. As shown previously (, prominent plasma membrane labeling was observed (Fig 1D). Although extraction of cholesterol was still apparent in some plasma membrane areas, this result clearly demonstrated that under these conditions localization of plasma membrane cholesterol is feasible.
Effect of Fixation Procedures on Labeling of Plasma Membrane Cholesterol.
The results with the high-pressure frozen samples showed that the tissue processing of chemically fixed material and the way of section retrieval are critical for the maintenance of cholesterol within the membrane. In the initial experiments with fixed cells, we used fixatives containing formaldehyde and glutaraldehyde. These fixatives bind to proteins without any known direct interaction with membrane lipids. Therefore, we next investigated whether fixatives such as acrolein, uranyl acetate, and OsO4 which act on membrane lipids (
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Influence of Low Temperature During Cholesterol Labeling.
Cholesterol-rich microdomains, or rafts, are insoluble in Triton X-100 at low temperature ( in the cold (Table 1). Surprisingly, labeling at 4C did not enhance the labeling of cholesterol or prevent cholesterol extraction from sections of samples fixed with formaldehyde, glutaraldehyde, and acrolein followed by infusion with sucrose containing the same fixative or OsO4 and uranyl acetate. However, on sections of samples fixed with formaldehyde only and infiltrated with sucrose in the presence of formaldehyde, plasma membrane cholesterol was detectable when labeling was performed in the cold (Fig 3C) but not after labeling at RT.
Subcellular Distribution of Cholesterol
The Plasma Membrane.
Cholesterol-enriched membrane areas were heterogeneously distributed and often found on filopodium-like processes of the cell surface. To analyze this finding quantitatively, we determined the labeling density in three different domains of the plasma membrane: flat plasma membrane, large extensions/invaginations, and filopodia after labeling the surface of living cells with BC at 4C (Table 2; Fig 1A). Filopodia showed 2.9 times more labeling than the flat areas of the plasma membrane. Large plasma membrane extensions and invaginations were 2.4 times more labeled than flat membrane. On cryosections of cryofixed cells (Fig 1D) or chemically fixed cells (Fig 3A and Fig 3B), this pattern was also frequently observed but was less reproducible due to cholesterol extraction from the sections. Removal of plasma membrane cholesterol by incubating cells with 10 mM cyclodextrin before fixation abolished plasma membrane labeling completely, confirming the specificity of labeling. The consistency of the heterogeneous distribution of cholesterol obtained by different approaches indicates that this pattern of plasma membrane labeling is not due to relocation artifacts during tissue processing.
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Intracellular Membranes.
The modified fixation and infiltration procedure was also suitable to localize intracellular cholesterol. Cholesterol in intracellular membranes appeared less susceptible to extraction than plasma membrane cholesterol. With all different approaches we observed essential similar cholesterol labeling. The results could also be reproduced with cryosections of high-pressure frozen cells that were fixed after sectioning (data not shown). We used sections prepared according to Table 1, section 5 for detection of intracellular membrane cholesterol because this fixation resulted in a very well-preserved ultrastructure. Cholesterol was not detectable in the ER. Labeling of cholesterol was observed on vesicles and tubules associated with the TGN, whereas the Golgi cisternae were mostly unlabeled (Fig 4A). As shown in Table 3, the labeling density on the vesicles and tubules was 25 times higher than the labeling associated with the Golgi cisternae. Along the endocytic pathway, cholesterol was regularly localized to small cytoplasmic vesicles associated with MVBs (Fig 4B). MVBs often showed strong cholesterol labeling of their vesicular content, indicating cholesterol-rich membranes, whereas the perimeter membrane remained mostly unlabeled (Fig 4C). For a quantitative analysis of this observation, we selected early endosomes according to
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Sometimes labeled particles in the lumen of MVBs were observed that were smaller than the internal vesicles and probably represented LDL particles. Interestingly, lysosomes with a multilaminar content next to the MVBs were almost devoid of labeling (Fig 4C). RN cells are able to secrete the vesicular content of MVBs by exocytosis (Fig 4D). This process leads to the production of small extracellular vesicles, so-called exosomes ( abolished labeling for cholesterol.
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Discussion |
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BC, a non-cytolytic and biotinylated derivative of perfringolysin O (
for subcellular localization of cholesterol by labeling thin cryosections with the probe and then visualizing probe-binding sites with anti-biotin antibodies and protein Agold. In principle, this approach allows the localization of intracellular cholesterol in situ with high-resolution electron microscopy.
As an important prerequisite, novel procedures and techniques had to be worked out to preserve the cholesterol distribution in cell membranes. Apart from the preservation of cholesterol, the properties of BC itself may influence the results. An ideal cytochemical probe should detect cholesterol in any membrane in which it is present and the amount of labeling should represent the concentration of cholesterol in the labeled area. The specificity of cholesterol binding by
-toxin and its derivatives was confirmed previously by detection of cholesterol on TLC plates and by using liposomal assays and cells (
-toxin (C
) binds membrane cholesterol with two different affinities depending on the amount of cholesterol and the lipid composition in a given membrane (
. By using cholesterol depletion,
to intact cells requires a high concentration of membrane cholesterol. Therefore, BC
is a useful tool for selective detection of cholesterol-rich membranes rather than for quantitative in situ determination of membrane cholesterol.
The access of a relatively large protein such as BC to a small molecule such as cholesterol in a membrane can also be restricted by steric hindrance. As depicted in the model (Fig 5), our observations on intact living cells show that BC
could bind plasma membrane cholesterol (Fig 5A) but that prefixation prevented this (Fig 5B). This was probably due to a penetration barrier formed by crosslinking of membrane proteins. In particular, cholesterol-binding proteins could mask membrane cholesterol. The accessibility of cholesterol to binding of the probe in sectioned membranes (Fig 5C) differs from that in intact cells, which may influence labeling efficiency.
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In cryosections, a large proportion of the plasma membrane cholesterol is extracted and dislocated, most likely during section pick-up and thawing. It is conceivable that, during the process of thawing, the membrane lipids diffuse and re-seal the hydrophobic interior of the cross-sectioned membrane (Fig 5C). This would be especially relevant for the localization of lipids because these are mostly unfixed and easily displaced. Therefore, it is crucial to adjust the composition of the pick-up solution in such a way that damage and lipid extraction are minimized. It was shown previously that introduction of methylcellulose into the sucrose pick-up solution improves the integrity of cryosections (
In this study we developed an improved protocol for the preparation of chemically fixed samples for intracellular cholesterol localization. Fixation and temperature during sample preparation and incubation with BC were crucial for the detection of membrane cholesterol on cryosections. As an important modification of the conventional protocol, fixatives were included in the infusion sucrose. The continued fixation during sucrose infiltration, in particular using fixatives reacting with membrane lipids such as acrolein, OsO4, and uranyl acetate, minimized cholesterol extraction. When a weak fixation with formaldehyde alone was used, low temperature during incubation of cryosections with BC
reduced cholesterol extraction. Compared to the plasma membrane, cholesterol in intracellular membranes of chemically fixed cells appeared more resistant to extraction, and therefore the detection was less dependent on the processing conditions. In particular, MVBs and exosomes showed strong labeling with BC
independent of the fixation protocol. Because exosomes from RN cells have a special protein composition that differs from that of other cell membranes (
In agreement with
Intracellular cholesterol was specifically detectable in small cytoplasmic vesicles and tubules associated with the TGN and endosomes. These results support the hypothesis that cholesterol-enriched microdomains or rafts function in sorting processes in the biosynthetic route in polarized cells (
In conclusion, we have shown that, under well-defined conditions, membrane cholesterol can be demonstrated at the ultrastructural level. Our results indicate relative high concentrations of cholesterol in highly curved membrane structures. Further studies are required to elucidate the functional aspects of such a restricted localization.
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Acknowledgments |
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WM is a CBG (Centre of Biomedical Genetics) postdoctoral fellow at the UMC Utrecht, The Netherlands. This work was supported by a short-term fellowship of the Human Frontier Science Program Organization (Strasbourg, France).
We thank R.M.C. Scriwanek and M. van Peski for excellent photographic work.
Received for publication January 24, 2001; accepted August 6, 2001.
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