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Vol. 14, Issue 8, 3254-3265, August 2003
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* Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation,
Rochester, Minnesota 55905;
Department of Chemistry and Biochemistry, Queens College, The City University
of New York, Flushing, New York 11367
Submitted December 11, 2002;
Revised March 14, 2003;
Accepted April 11, 2003
Monitoring Editor: Vivek Malhotra
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGMENTSREFERENCES |
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; Gleizes et al.,
1996; Anderson,
1998; Lobie et al.,
1999; Okamoto et al.,
2000; Shin et al.,
2000; Pelkmans et
al., 2001; Shubert et
al., 2001; Marjomaki
et al., 2002). Caveolar endocytosis is a
clathrin-independent, dynamin-dependent process
(Henley et al., 1998;
Oh et al., 1998);
however, the molecular machinery that differentiates caveolar endocytosis from
other forms of clathrin-independent endocytosis is not yet fully defined.
Seminal studies by Orlandi and Fishman
(1998) demonstrated that the
cholera toxin B subunit (CtxB), which binds to GM1 at the plasma
membrane (PM), is internalized by caveolae in several cell types. However,
more recent studies have shown that CtxB can be significantly internalized by
clathrin-dependent endocytosis in some cell types
(Shogomori and Futerman, 2001;
Torgersen et al.,
2001). Another potential marker for caveolar uptake is labeled
albumin, which is reported to be internalized primarily by caveolae
(Schnitzer et al.,
1994; Shubert et al.,
2001).
One well-characterized marker for caveolar internalization is SV40 virus
that colocalizes with GFP-tagged caveolin-1 (cav-1) during endocytosis
(Pelkmans et al.,
2001); however, the intracellular itinerary and slow rate of
transport of SV40 virus differs from that of some other cargo internalized via
caveolae (Gleizes et al.,
1996; Orlandi and Fishman,
1998; Lobie et al.,
1999; Sharma et al.,
2003). These and other observations suggest the possibility that
several forms of caveolar endocytosis may exist
(Mineo and Anderson, 2001;
Le and Nabi, 2003).
We previously showed that fluorescent (BODIPY-labeled) glycosphingolipid
(GSL) analogs (lactosylceramide [LacCer] and globoside) are selectively
internalized by a dynamin-dependent, clathrin-independent mechanism in human
skin fibroblasts (HSFs) and on the basis of multiple criteria suggested that
these lipids were internalized by a "caveolarrelated" process
(Puri et al., 2001).
This study raised several important questions concerning caveolar endocytosis.
First, is selective endocytosis via caveolae a property of other GSLs with
different carbohydrate headgroups? Second, what are the biological or
biophysical mechanisms (Anderson and
Jacobson, 2002; Brown and
London, 2000; Edidin,
2003) involved in the selective entry of GSLs into caveolarderived
smooth vesicles but not clathrin-coated vesicles? Finally, does selective
caveolar endocytosis of GSLs only occur in some cell types (e.g., HSFs) or is
it a widely occurring process in mammalian cells? The mechanism for CtxB
internalization has been reported to be variable (i.e., clathrin-independent
and/or -dependent) depending on cell type and cav-1 expression levels
(Shogomori and Futerman, 2001;
Torgersen et al.,
2001; Sandvig and van Deurs,
2002). This raises the possibility that some GSLs (e.g.,
GM1) are not internalized primarily via caveolae in some cell
types, or alternatively that CtxB perturbs the internalization mechanism of
endogenous GM1, and that fluorescent GSL analogs may better reflect
the movement of endogenous GSLs. In the present article we sought to resolve
these uncertainties by carrying out a detailed study of GSL analog
internalization in multiple cell types and by comparing those results to
parallel studies using fluorescent CtxB. Our data provide strong evidence that
fluorescent GSL analogs of widely varying structure are internalized primarily
by a clathrin-independent, cav-1–dependent process. We further show that
the mechanism of fluorescent GM1 internalization is altered when
bound by CtxB in HeLa cells where cav-1 expression is low. These data suggest
that caveolar endocytosis plays a significant role in plasma membrane GSL
internalization in multiple cell types.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGMENTSREFERENCES |
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30–50% confluency on acid-etched glass
or gridded coverslips for microscopy or in 60-mm dishes for biochemical
experiments.
Fluorescent Lipids, Toxins, and Other Reagents
The structures of the various fluorescent lipid analogs are shown in
Figure 1.
BODIPY-maltosylceramide (MalCer) was synthesized by boron trifluoride mediated
glycosylation of D-erythro-2-azido-3-benzoylsphingosine
(prepared from D-erythro-C18-sphingosine;
Matreya, Pleasant Gap, PA) with per-O-acetylmaltosyl
trichloroacetimidate in dichloromethane in the presence of molecular sieves.
Hepta-O-acetylmaltosyl trichloroacetimidate was prepared from
octa-O-acetylmaltose (Sigma-Aldrich, St. Louis, MO) in two steps: 1)
treatment with hydrazine acetate in dimethylformamide, and 2) treatment with
trichloroacetonitrile and sodium in dichloromethane. After deprotection of the
hepta-O-acetylated GSL with sodium methoxide and reduction of the
azide with triphenylphosphine in aqueous tetrahydrofuran, the final product
was obtained by N-acylation with the N-hydroxysuccinimidoyl
(NHS) ester of
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic
acid (BODIPY-FL-C5, SE; Molecular Probes, Eugene, OR) and purified
by column chromatography on silica gel (elution with chloroform/methanol/water
65:35:4 vol/vol/vol), followed by preparative TLC (elution with
chloroform/methanol/water 65:25:4 vol/vol/vol). Suspended silica gel was
removed by filtration through a Cameo filter (Fisher Scientific, Pittsburgh,
PA).
|
C3-BODIPY-LacCer was synthesized as described
(Martin and Pagano, 1994),
using lyso-LacCer (Matreya) and the NHS ester of
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid (BODIPY-FL, SE; Molecular Probes). C5-BODIPY-LacCer analogs
having shorter (C12 or C16) or longer (C20)
sphingoid backbones than C3-BODIPY-LacCer (C18) were
synthesized from D-erythro-2-azido-3-benzoylsphingosine
analogs as described above for BODIPY-MalCer using C12,
C16, or C20 D-erythro-sphingosines, custom
synthesized by Matreya and BODIPY-FL-C5, SE (Molecular Probes).
C5-BODIPY-fatty acid labeled analogs of galactosylceramide
(GalCer), LacCer, globoside, GM1, and sulfatide, and
6-[(7-nitro-benz-2-oxa-1,3-diazol-4-yl)amino]caproic acid labeled-LacCer
(NBD-LacCer) were synthesized and purified as described
(Martin and Pagano, 1994;
Watanabe et al.,
1999; Puri et al.,
2001). The D-isomer of NBD-phosphatidylcholine
(NBD-D-PC) was prepared as described
(Martin and Pagano, 1987).
The concentrations of the lipid analogs were determined by measurements of
fluorescence intensity relative to known standards. Complexes of the
fluorescent lipids with defatted bovine serum albumin (DF-BSA; Sigma-Aldrich)
were prepared as described (Martin and
Pagano, 1994) and diluted to a final working concentration using
10 mM HEPES-buffered minimal essential medium (pH 7.4) (HMEM). AlexaFluor 488
and 594 (AF488 and AF594)labeled CtxB, transferrin (Tfn), and albumin, and
Cascade blue dextran (MW 10 kDa) were from Molecular Probes. Other reagents
were from Sigma-Aldrich unless otherwise noted.
Incubation of Cells with Fluorescent Lipids and Various Markers
Cell cultures were washed with ice-cold HMEM (10 mM HEPES-buffered MEM),
transferred to a water bath at 10°C, and then incubated with fluorescent
lipid/DF-BSA for 30 min to label the PM
(Chen et al., 1997).
(In one series of experiments, a 0.5 mM ethanolic stock solution of
BODIPY-LacCer was prepared, diluted to 2 µM in HMEM, and incubated with the
cells as above.) The cells were then washed with cold HMEM and warmed to
37°C for various times to induce endocytosis. After this incubation, the
medium was replaced with ice-cold HMEM without glucose containing the
inhibitors, 5 mM NaN3 and 50 mM 2-deoxyglucose (HMEM-G+I) and the
culture dishes were transferred to a 10°C bath. Fluorescent lipid present
at the cell surface was removed by incubating the cells (six times, 10 min
each) with 5% DF-BSA in HMEM-G+I at 10°C
(Martin and Pagano, 1994;
Chen et al., 1997).
For CtxB labeling, cells were incubated with 7.5 µg/ml AF594 CtxB for 45
min at 10°C, washed, and further incubated at 37°C for the indicated
times. For Tfn labeling, cultures were preincubated in serum-free culture
medium for 2 h at 37°C to upregulate the Tfn receptor. Samples were then
washed, incubated with 30 µg/ml AF594 Tfn for 45 min at 10°C, washed,
and further incubated at 37°C for the indicated times. Excess CtxB or Tfn
at the cell surface was removed by acid stripping (30 s at 10°C with HMEM,
pH 3.5).
Pharmacological Inhibitors of Endocytosis
Cells were treated with various inhibitors to differentiate
clathrin-dependent from clathrin-independent endocytosis as described
(Puri et al., 2001).
For inhibition of clathrin-dependent endocytosis, samples were pretreated with
8 µg/ml chlorpromazine (CPZ; Gustavsson
et al., 1999; Okamoto
et al., 2000) or were potassium depleted
(Larkin et al., 1983;
Hansen et al., 1993);
for disruption of caveolar endocytosis, cells were pretreated with 25 µg/ml
nystatin (Rothberg et al.,
1992) or 200 µM genistein
(Aoki et al., 1999;
Chen and Norkin, 1999;
Liu and Anderson, 1999). The
specificity of each inhibitor treatment was evaluated by monitoring the
internalization of fluorescent CtxB, albumin, and Tfn as endocytic markers
(see Results). Cell viability was >90% for each inhibitor treatment as
judged by trypan blue staining.
Transfection Studies
GFP constructs of Eps15 (D3
2 [control] and the EH21 mutant
E95/295 [dominant negative Eps15]) were from Drs. A. Benmerah and A.
Dautry-Varsat (Inserm; Paris); GFP constructs of dynamin 2 (Dyn2ab [control]
and Dyn2ab K44A [dominant negative]) were from Dr. M. McNiven (Mayo Clinic).
DsRed1-cav-1 was generated from cav-1-GFP (a generous gift from Dr. A.
Helenius, Swiss Federal Institute of Technology, Zurich) by removing a
BamH1-HindIII fragment containing the cav-1 gene from the
cav-1-GFP construct and inserting it into a pDsRed1-C1 expression vector (BD
Biosciences Clontech, Palo Alto, CA). Cells were treated with FuGENE 6
transfection reagent (Roche Applied Science, Indianapolis, IN) and 2 µg/ml
DNA using the manufacturer's protocol. After a 4–6-h treatment, the
cells were washed and subsequently cultured for 24–48 h in DMEM
containing 10% FBS before treatment with the fluorescent lipids or toxins as
above. Transfected cells were detected by GFP- or DsRed fluorescence and the
effect on lipid internalization was evaluated as described
(Puri et al., 2001;
Choudhury et al.,
2002).
Colocalization Studies
BODIPY-LacCer with Fluorescent Albumin or Dextran. RFs were
incubated for 30 min at 10°C with 2 µM BODIPY-LacCer to label the PM.
Cells were then washed and incubated for 2 or 5 min at 37°C in the
presence of 30 µg/ml AF594 albumin or 1 mg/ml Cascade blue Dextran. Cells
were then washed, back-exchanged, and observed under the fluorescence
microscope. In control experiments using cells in which only AF594 albumin (or
Cascade blue dextran) was present, no fluorescence was detected in the green
microscope channel used to visualize BODIPY-fluorescence.
DsRed-cav-1 with BODIPY-LacCer, Fluorescent Albumin, or Tfn. RFs were transfected with the DsRed-cav-1 construct and 48 h later were pulse-labeled with BODIPY-LacCer, AF488 albumin, or AF488 Tfn as above. Transfected cells were identified by DsRed fluorescence. In control experiments using singly labeled specimens, no overlap between DsRed and either LacCer or AF488 fluorescence was detected under the experimental conditions used.
Colocalization values were calculated using Metamorph image processing software (v4.6; Universal Imaging Corp., Dowingtown, PA) and are based on the overlapping area of green puncta (BODIPY-LacCer, AF488 albumin, or AF488 Tfn) with red puncta (DsRed-cav-1) in doubly labeled specimens.
BODIPY-GM1 Internalization with and without CtxB
HeLa cells were pretreated with nystatin (25 µg/ml) or CPZ (8 µg/ml)
in HMEM for 30 min at 37°C. Cells were then washed with ice-cold HMEM,
incubated with 1 µM BODIPY-GM1 for 35 min at 10°C, washed,
and further incubated for 1 h at 10°C in HMEM with or without 1 µg/ml
AF594 CtxB. The cells were then washed, incubated for 5 min at 37°C, acid
stripped (see above), and back-exchanged with 5% DF-BSA in HMEM-G+I (2x
2 min at 37°C, followed by 4x 10 min at 10°C). Samples were then
observed under the fluorescence microscope. In control experiments using cells
in which only AF594 CtxB was present, no fluorescence was detected with the
"green" microscope filters under the same exposure conditions used
to visualize BODIPY-fluorescence.
Other Procedures
Fluorescence microscopy using an Olympus IX70 fluorescence microscope and
quantitative image analysis were performed as described
(Puri et al., 2001;
Choudhury et al.,
2002). For Western blotting, aliquots of cell lysates were
solubilized in sample buffer, run on 15% SDS-PAGE gels, and transferred to
polyvinylidene difluoride membranes in transfer buffer with 20% methanol.
Blots were probed with monoclonal anticav-1 antibody (BD Biosciences
PharMingen, San Diego, CA) followed by antimouse horseradish peroxidase
secondary antibody (Amersham Life Sciences, Piscataway, NJ).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGMENTSREFERENCES |
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). Thus, it was of interest to further study the
internalization of GSL analogs to learn if this was a general phenomenon. We
first extensively studied LacCer internalization in RFs. The uptake of
BODIPY-LacCer was compared with that of fluorescent Tfn and CtxB, which are
selective endocytic markers for clathrin-dependent vs. caveolar endocytosis,
respectively. Using a series of pathway-specific pharmacological inhibitors
and a DN construct of Eps15, we demonstrated that LacCer internalization in
RFs was inhibited 80% by nystatin and genistein, but only 10% by
CPZ, K+-depletion, or DN Eps15 expression, similar to the behavior
of CtxB (Figure 2A). In
contrast, internalization of fluorescent Tfn was inhibited by CPZ,
K+ depletion, or expression of DN Eps15, but was not significantly
affected by nystatin or genistein (Figure
2A). Examples of the selective inhibition of Tfn uptake by DN
Eps15 and LacCer internalization by nystatin are shown in
Figures 2, B and C, respectively. In addition, we found that internalization of Tfn, CtxB, and
LacCer were each inhibited by 90% in RFs transfected with a DN construct
of dynamin (McNiven et al.,
2000; Figure 2A).
Together, these results demonstrate that in RFs, LacCer internalization
occurred by a clathrin-independent mechanism, consistent with our previous
studies in HSFs (Puri et al.,
2001).
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To further characterize the endocytosis of LacCer, we carried out
colocalization experiments using labeled albumin, which internalizes via
caveolae in some cell types (Shubert
et al., 2001). In preliminary studies, we found that
internalization (5 min at 37°C) of fluorescent albumin was inhibited by
88.9 ± 6.8% in cells pretreated with nystatin, similar to that seen
using LacCer and CtxB (Figure
2A), but was inhibited by only 7.2 ± 1.7% following
pretreatment with CPZ. Extensive overlap (>93%) of BODIPY-LacCer and
fluorescent albumin was seen after 3 min of internalization at 37°C
(Figure 3). We then carried out
a parallel experiment using BODIPY-LacCer and fluorescent dextran, which at
low concentrations (1 mg/ml) acts as a marker for cdc42-dependent pinocytosis
(Sabharanjak et al.,
2002). In preliminary studies we found that dextran
internalization was virtually unaffected by pretreatment of cells with CPZ
(
2% inhibition) or nystatin (5% inhibition), demonstrating that
dextran internalization occurred by a mechanism which was distinct from those
utilized for endocytosis of Tfn or CtxB. In double-label experiments there was
<10% colocalization of LacCer with fluorescent dextran after 5 min of
internalization (Figure 3). We
also pretreated cells with Clostridium difficile toxin B, an
inhibitor of the Rho family GTPases
(Aktories et al.,
2000), and found that this treatment blocked dextran uptake but
had no effect on LacCer internalization (unpublished data).
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Finally, we carried out colocalization studies in RFs transfected with the DsRed-cav-1 construct. BODIPY-LacCer was internalized for 2 min at 37°C, and images of DsRed-cav-1 (red) and LacCer (green) were acquired. Punctate LacCer fluorescence (green) extensively overlapped with DsRedcav-1 puncta as seen in Figure 4, A and B. Importantly, in adjacent nontransfected cells no red fluorescence from LacCer was detected at these exposure settings. Furthermore, in control samples expressing DsRed-cav-1 but not incubated with BODIPY-LacCer, no fluorescence was detected at green wavelengths (Figure 4A, bottom right panel). Similarly, endocytosed AF488 albumin extensively colocalized with DsRed-cav-1 (Figure 4C), whereas no overlap of DsRedcav-1 was seen in cells incubated with AF488 Tfn.
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Together the results in Figures 2, 3, 4 provide strong evidence that LacCer was internalized via caveolae in RFs, rather than by pinocytosis or other Rho-dependent, nonclathrin endocytic mechanisms.
To investigate the structural determinants of GSLs that result in selective
internalization via caveolae, we systematically varied the structure of the
fluorescent GSL analogs (see Figure
1) and examined the effect of these variations on the mechanism of
analog internalization. To examine the significance of the carbohydrate
head group, we used fluorescent analogs of GalCer, globoside,
GM1, LacCer, MalCer, and sulfatide. (Fluorescent glucosylceramide
was not used because this lipid is internalized from the PM by a combination
of endocytic and nonendocytic mechanisms;
Martin and Pagano, 1994.) For
each of these fluorescent GSL analogs, the fluorescent fatty acid and
sphingosine base were identical (n' = 3, n = 7;
Figure 1A). RFs were incubated
with each analog in the presence or absence of various inhibitors to
differentiate clathrin-dependent from clathrin-independent endocytosis as in
Figure 2, and the amount of
internalization (at 5 min) was quantified by image analysis. The
internalization of each GSL analog (GalCer, MalCer, globoside, sulfatide, and
GM1) was substantially inhibited by nystatin (but not by CPZ),
similar to BODIPY-LacCer (Table
1), indicating that the specific carbohydrate headgroup structure
or the stereochemistry of the glycosidic linkage in the disaccharide moiety of
the GSL does not play a signifi-cant role in selective internalization of
these GSLs by the clathrin-independent, caveolar mechanism.
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Finally, we carried out a control experiment exactly as above, except that BODIPY-LacCer was introduced into cells from an ethanolic solution rather than from a BSA complex (see MATERIALS AND METHODS). As seen in Table 1, virtually identical results were obtained when the LacCer analog was delivered to RFs using BSA vs. ethanol injection, indicating that the BSA carrier did not influence the LacCer uptake mechanism.
We also examined the importance of hydrophobicity on the mechanism of GSL endocytosis. For these studies, we used BODIPY-LacCer analogs in which the chain length of the sphingosine base (C12 to C20), or fatty acid (C3 vs. C5 spacer) was varied (n = 1,5,7, or 9; n' = 1 or 3; Figure 1A). Endocytosis of the series of LacCer analogs was studied in RFs as above, but surprisingly none of the modifications in chain length affected the mechanism of LacCer internalization (Table 2). To investigate the possible influence of the BODIPY fluorophore, we studied the internalization of NBD-labeled LacCer and found that its internalization was nystatin-inhibitable and CPZ-insensitive (Table 1), similar to our findings for BODIPY-LacCer (and other BODIPY-GSLs). This demonstrated that the fluorophore (NBD vs. BODIPY) had no apparent influence on the internalization mechanism.
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Finally, we compared NBD-LacCer with NBD-D-PC to study the
possible influence of the lipid backbone (ceramide vs. glycerol) on the
internalization mechanism. For these studies, it was necessary to use the
nonhydrolyzable D-stereoisomer of NBD-PC, because the natural
L-isomer is rapidly degraded by cells
(Sleight and Pagano, 1984),
precluding its use in our endocytosis studies. As shown in
Table 1, the endocytosis of
NBD-D-PC was found to be predominantly CPZ-inhibitable, suggesting
that unlike LacCer, its uptake occurred largely by clathrin-dependent
endocytosis. In summary, these structural studies suggest that GSL uptake via
caveolae is not selective for a specific carbohydrate headgroup, acyl chain
hydrophobicity, or fluorophore substitution; however, comparison with the
uptake of NBD-D-PC suggests that the ceramide core of GSLs may play
an important role in caveolar endocytosis of GSLs.
LacCer Internalization in Other Cell Types
We next extended our studies of BODIPY-LacCer endocytosis to six other cell
types in addition to RFs: HSFs, CHO, MDCK, Calu-1, Calu-6, and HeLa cells. In
preliminary experiments we incubated each cell type with various
concentrations (1–3 µM) of BODIPY-LacCer at 4°C, washed the
cells, and quantified the PM fluorescence. This allowed us to adjust the
incubation conditions such that the initial labeling of the PM was nearly
identical (±10%) for each cell type (e.g., see
Figure 5). Cells were then
warmed for 5 min at 37°C, and the amount of LacCer internalization from
the PM was quantified. Approximately 30–40% of the lipid analog was
internalized from the PM in each cell type except in HeLa and Calu-6 cells,
where internalization was only 5–6% of the initial lipid present at
the PM (Figure 6C). An example
of this reduced internalization is shown in
Figure 5 for HeLa cells vs.
RFs. Although the amount of LacCer internalization varied among the different
cell types, its internalization was sensitive to nystatin but not to CPZ in
each case (Figure 6A),
suggesting that LacCer endocytosis was clathrin independent for all the cell
types studied. In addition, we carried out colocalization experiments using
BODIPY-LacCer and fluorescent albumin, analogous to the experiment shown in
Figure 3. Extensive
colocalization of the lipid analog and fluorescent albumin was found in all
the cell types tested after 3 min of internalization. These experiments were
not carried out using HeLa or Calu-6 cells because internalization of LacCer
was very low in those cell types.
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Previous studies have demonstrated that Calu-6 and HeLa cells have low
levels of cav-1 mRNA or cav-1 expression relative to other cell types
(Racine et al., 1999;
Skretting et al.,
1999). In the present study we examined cav-1 expression and found
that of the seven cell types used, only Calu-6 and HeLa cells had low levels
of cav-1, suggesting that the extent of LacCer internalization correlates with
cav-1 expression (Figure 6, B and
C). To extend this study, Calu-6 and HeLa cells were transfected
for 48 h with a construct for DsRedcav-1. The effect on LacCer internalization
was then evaluated. Cav-1–transfected cells exhibited dramatically
enhanced uptake of LacCer (5 min internalization at 37°C) relative to
adjacent nontransfected cells for both HeLa
(Figure 7) and Calu-6 cells. In
parallel experiments, electron microscopy of DsRed-cav-1–transfected
HeLa cells showed numerous uncoated invaginations at the PM and uncoated
vesicles in the cytoplasm, whereas almost no such structures were seen in
nontransfected cells (unpublished data). These results demonstrate that cav-1
overexpression in HeLa cells stimulates LacCer internalization and promotes
the formation of uncoated vesicles.
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Mechanism of CtxB Internalization Varies with Cell Type
We used similar techniques to those described for LacCer to examine the
internalization mechanism of CtxB in the cell types used above. (CHO cells
were not used because they lack GM1, which is required for CtxB
binding.) Cells were incubated with fluorescent CtxB at 4°C to bind
endogenous GM1 at the PM, warmed for 5 min at 37°C, washed,
acid stripped, and viewed under the fluorescence microscope. Internalization
was quantified by image analysis. Nystatin and CPZ pretreatments were used to
evaluate, respectively, the relative contributions of clathrin-independent and
clathrin-dependent endocytosis to the uptake of CtxB. As shown in
Figure 6D, inhibition of CtxB
uptake by nystatin or CPZ was variable. Nystatin (but not CPZ) pretreatment
significantly inhibited CtxB internalization in RFs, HSFs, MDCK, and Calu-1
cells, whereas CPZ had a significant inhibitory effect in HeLa and Calu-6
cells. This result for CtxB is in striking contrast to that obtained using
LacCer whose internalization was inhibited by nystatin (but not CPZ),
regardless of the cell type (Figure
6A). Thus, in cell types with low cav-1 expression, CtxB
internalization occurred predominantly via clathrin-dependent endocytosis,
whereas LacCer internalization remained clathrin independent.
CtxB Alters the Internalization Mechanism of
BODIPY-GM1
Finally, we tested the possibility that CtxB binding might alter the
mechanism of internalization of exogenously added GM1. HeLa cells
were incubated with BODIPY-GM1 at 10°C to label the PM, washed,
and further incubated for 1 h at 10°C with or without AF594-CtxB. Samples
were then warmed at 37°C for 5 min, and the amount of internalization was
assessed after acid stripping and back exchange (see MATERIALS AND METHODS).
In the absence of bound CtxB, internalization of BODIPY-GM1 was
significantly inhibited by pretreatment with nystatin, whereas CPZ treatment
had little effect (Figure 8A,
top panels, and C). In the presence of bound CtxB, however, two effects were
seen. First, the amount of BODIPY-GM1 internalized during 5 min at
37°C was significantly increased over that seen in the absence of CtxB
(Figure 8, A and C). In this
experiment, the presence of bound CtxB was confirmed by visualization of AF594
fluorescence which was seen to colocalize with the BODIPY-fluorescence
(Figure 8B). Second, in the
presence of bound CtxB, the relative amount of GM1 uptake which was
inhibited by nystatin decreased from 79 to 40%, whereas the fraction which was
inhibited by CPZ increased from 18 to 64%
(Figure 8C). These results
suggest that in HeLa cells, CtxB stimulated the internalization of
BODIPY-GM1 mainly by increasing the extent of its uptake via
clathrin-dependent endocytosis.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGMENTSREFERENCES |
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BODIPY-LacCer Is Internalized by Caveolae
Several pieces of evidence indicate that the BODIPY-LacCer analog was
endocytosed through caveolae in RFs. First, we demonstrated that LacCer
internalization occurred by a clathrin-independent mechanism using multiple
pathway-specific inhibitors as well as a DN construct of Eps15 that blocks
clathrin-dependent endocytosis (Figure
2), consistent with our previous results in HSFs
(Puri et al., 2001).
Second, we showed that the fluorescent LacCer analog colocalized with
fluorescently labeled albumin (Figure
3), which is reported to be internalized via caveolae in other
cell types (Shubert et al.,
2001). Furthermore, both the LacCer analog and fluorescent albumin
extensively colocalized with DsRed-cav-1 in RFs at an early stage (2 min) of
internalization (Figure 4).
Third, the internalized LacCer did not colocalize with fluorescent dextran
(Figure 3), a marker for
cdc42-dependent pinocytosis. In addition, unlike LacCer uptake, dextran uptake
was not inhibitable by nystatin. We also found that C. difficile toxin
B, an inhibitor of rho-dependent endocytic mechanisms including the cdc42
pathway (Aktories et al.,
2000; Sabharanjak et
al., 2002), had no effect on LacCer internalization. In
addition, recent studies by Mayor and colleagues have shown that dextran
uptake occurs by a dynamin-independent mechanism
(Sabharanjak et al.,
2002), in contrast to LacCer internalization which was dynamin
dependent (Figure 2A). Taken
together, our results provide strong evidence that internalization of GSL
analogs in RFs occurs via caveolar endocytosis rather than another
clathrin-independent endocytic mechanism.
In addition to our studies with RFs, we examined LacCer internalization in
HSFs, HeLa, CHO, MDCK, Calu-1, and Calu-6 cells. In each case, LacCer
internalization appeared to occur by a similar mechanism to that found for RFs
when we examined the effects of biochemical inhibitors (i.e., nystatin vs.
CPZ; Figure 6A) or
colocalization with labeled albumin. Thus, it seems reasonable to suggest that
the mechanism of LacCer internalization does not vary among these cell types
and was predominantly via caveolae in each case. However, the amount
of LacCer internalization was dramatically reduced in cell types with low
levels of cav-1 (HeLa and Calu-6) and could be stimulated in those cell types
by overexpression of this protein. These results strongly suggest that the
extent of LacCer internalization is closely related to the expression of cav-1
in multiple cell types. Our results are in apparent contrast to those of Nabi
and coworkers who found that the overexpression of cav-1 negatively regulates
the caveolar-mediated endocytosis of the autocrine motility factor receptor in
NIH-3T3 cells (Le et al.,
2002). One possible explanation is that there are distinct
caveolar-mediated endocytic pathways with different characteristics
(Le and Nabi, 2003). In this
context, we recently found that BODIPY-LacCer internalized via
caveolar-related endocytosis in HSFs is rapidly delivered to early endosomes
where it merges with Tfn, a marker for the clathrin pathway
(Sharma et al.,
2003). In contrast, SV40 virus internalized via caveolae is
reported to be transported intracellularly by a slow process that does not
include passage through early endosomes
(Pelkmans et al.,
2001).
Structural Requirements for GSL Internalization
We also attempted to define the molecular features of GSL analogs that
result in their selective internalization by caveolae in RFs. To do so, we
systematically varied the structure of the fluorescent lipid by modifying its
carbohydrate headgroup, chain length of the sphingosine base, or chain length
of the fluorescent fatty acid and examined their initial internalization and
sensitivity to inhibitor treatments. Fluorescent GalCer, LacCer, MalCer,
globoside, GM1, and sulfatide were internalized identically to
BODIPY-LacCer (Table 1). Each
of these molecules had an identical BODIPY-ceramide backbone and differed only
in the saccharide moieties present in the polar headgroup. Of particular
interest is the finding that BODIPY-MalCer, whose headgroup is
Glc(
1
4)Glc, and LacCer, whose headgroup is
Gal(
14)Glc, behaved identically despite their different
conformations in the vicinity of the carbohydrate/sphingolipid interface,
suggesting that the presence of galactose is not required for caveolar
endocytosis of the various GSL analogs. To evaluate the possibility that the
hydrophobicity of the analog might influence its internalization, we used a
series of BODIPY-LacCer analogs in which the chain length of the sphingosine
base or fluorescent fatty acid was varied (refer to
Figure 1). Alterations in lipid
chain length are presumed to affect the hydrophobicity of the analogs (e.g.,
C12 vs. C20 sphingosine); however, there was no effect
on the LacCer internalization mechanism
(Table 2). These results
suggest that the hydrophobicity of the GSL analogs is not a major determinant
for their selective internalization via caveolae.
We also compared the internalization of NBD- vs. BODIPY-labeled LacCer and
found that replacing the BODIPY-fluorophore with NBD did not influence the
caveolar internalization of the LacCer analogs
(Table 1). Finally, we compared
the internalization of NBD-LacCer with NBD-D-PC. Unlike the GSL
analogs, NBD-D-PC was internalized primarily via
clathrin-dependent endocytosis (Table
1). Taken together, these studies suggest that GSL analogs are
internalized mainly via caveolae with no specificity toward particular
carbohydrate headgroups. However, the sphingoid backbone of the GSLs appeared
to be important for selective caveolar endocytosis because the
glycerophospholipid, NBD-D-PC, was not internalized by this
mechanism. Interestingly, BODIPY-sphingomyelin, which has both a ceramide core
(as in GSLs) and a phosphocholine headgroup (as in PC), is internalized
approximately equally by both caveolar- and clathrin-mediated endocytosis
(Puri et al.,
2001).
CtxB (but not LacCer) Internalization Mechanism Varies with Cell
Type
We compared the mechanism of internalization of CtxB and LacCer in six
different cell types (Figure 6)
and found that although LacCer was internalized by a clathrin-independent
mechanism in each case, both clathrin-independent (nystatin-sensitive) and
clathrin-dependent (CPZ-sensitive) mechanisms could contribute to CtxB uptake
to varying extents depending on the cell type. One possible explanation for
this is that the internalization mechanism of fluorescent LacCer mimics that
of endogenous PM GM1 and that binding of CtxB to GM1
alters its internalization. Alternatively, the presence of the BODIPY- (or
NBD-) fluorophore on LacCer could bias its mechanism of internalization as
cell type is varied. To test these possibilities, we compared the
internalization of BODIPY-GM1 in HeLa cells, with and without bound
CtxB (Figure 8). In the absence
of bound CtxB, internalization of BODIPY-GM1 was largely
nystatin-sensitive (i.e., clathrin-independent and caveolar-related), whereas
CPZ (which inhibits clathrin-dependent internalization) had little effect. In
contrast, when the same experiment was performed after binding CtxB to the PM,
the fraction of GM1 uptake that was inhibited by nystatin
decreased, whereas the fraction that was inhibited by CPZ increased
(Figure 8). Furthermore, CtxB
treatment also increased the total amount of BODIPY-GM1
internalization about fourfold. These results demonstrate that the
internalization of the fluorescent GM1 analog was perturbed by CtxB
binding, and raise the possibility that internalization of endogenous
GM1 might also be affected by the bound toxin in some cell types.
We speculate that this perturbation occurs because CtxB binding (five
molecules of GM1 bound per molecule of CtxB;
Lencer et al., 1999)
disrupts the normal interactions of GM1 with neighboring lipids,
proteins, and cholesterol that are required for clathrin-independent
internalization.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: AF, AlexaFluor; BODIPY, boron dipyrromethenedifluoride; Cav-1, caveolin-1; CPZ, chlorpromazine; CtxB, cholera toxin, B-subunit; DF-BSA, defatted BSA; Eps15, EGFR pathway substrate clone 15; GalCer, galactosylceramide; GM1, ganglioside GM1; GSL, glycosphingolipid; HMEM, 10 mM HEPES-buffered minimal essential medium (pH 7.4); HMEMG+I, HMEM without glucose containing 5 mM NaN3 and 50 mM 2-deoxyglucose; HSFs, human skin fibroblasts; LacCer, lactosylceramide; MalCer, maltosylceramide; NBD, 7-nitrobenz-2-oxa-1,3-diazol-4-yl; PC, phosphatidylcholine; PM, plasma membrane; RFs, rat fibroblasts; SL, sphingolipid; SM, sphingomyelin; So, sphingosine; Tfn, transferrin.
Corresponding author. E-mail address:
pagano.richard{at}mayo.edu.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGMENTSREFERENCES |
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