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From the
Received for publication, November 26, 2001
Caveolae are flask-shaped invaginations at the
plasma membrane that constitute a subclass of detergent-resistant
membrane domains enriched in cholesterol and sphingolipids and that
express caveolin, a caveolar coat protein. Autocrine motility factor
receptor (AMF-R) is stably localized to caveolae, and the cholesterol
extracting reagent, methyl- Endocytosis via clathrin-coated vesicles represents the best
characterized endocytic pathway, however, other clathrin-independent endocytic mechanisms also exist (1-4). The large GTPase dynamin has
been shown to regulate the fission of clathrin-coated pits, and
expression of the dynamin K44A
(dynK44A)1 mutant inhibits
clathrin-mediated endocytosis (5-7). The dynK44A mutant does not
affect fluid phase endocytosis or the clathrin-independent endocytic
pathway defined by ricin endocytosis indicating that non-clathrin-coated cell surface invaginations can detach from the
plasma membrane in the apparent absence of dynamin-mediated membrane
fission (8-11). However, introduction of inhibitory antibodies to
dynamin into hepatocytes resulted in the accumulation of both clathrin-coated vesicles and smooth caveolar invaginations and inhibited the endocytosis of cholera toxin (12). In endothelial cells,
caveolae budding from isolated membranes was shown to be dynamin-dependent, and caveolae were shown to contain the
molecular machinery necessary for vesicle budding (13, 14). Regulation of caveolae budding by dynamin identifies caveolae as
endocytosis-competent cell surface invaginations.
Caveolae or smooth plasmalemmal vesicles were first identified in
endothelial cells and are morphologically identifiable as smooth flask
shaped invaginations of the plasma membrane (15-18). Caveolae are rich
in cholesterol and sphingolipids, disrupted by cholesterol extracting
agents, and insoluble in Triton X-100 and are therefore considered to
form a subclass of cholesterol-rich detergent-resistant membrane
domains or glycolipid rafts (19-22). The caveolins (caveolin-1, -2, and -3) are cholesterol binding proteins that form a spiral coat on the
cytoplasmic surface of caveolar invaginations and represent caveolae
markers (23-25). Caveolar invaginations are not present in cells that
express little or no caveolin, and the reintroduction of caveolin-1
into such cells has been shown to induce the formation of caveolae
implicating caveolin in the invagination of glycolipid raft
microdomains (26-28). Caveolin-1 expression is inversely proportional
to cell transformation, and caveolin-1 has been characterized as a
tumor suppressor gene (27, 29-31).
Caveolae have long been proposed to be involved in transcytosis
across the endothelial cell (18, 32-34). Caveolae- or
raft-mediated endocytosis has been reported for cholera toxin-bound
GM1 ganglioside, sphingolipids,
glycosylphosphatidylinositol-anchored proteins, SV40, and bacteria, as
well as the endothelin, growth hormone, interleukin-2, and autocrine
motility factor (AMF) receptors (35-46). Autocrine motility
factor receptor (AMF-R) is a seven-transmembrane domain receptor
localized at steady state to caveolae and the smooth endoplasmic
reticulum (ER) that follows an endocytic pathway sensi- tive to
cholesterol extraction with methyl- Antibodies, Reagents, and Cells--
Monoclonal rat antibody
against AMF-R was used in the form of concentrated hybridoma
supernatant (50). Rabbit anti-caveolin antibody was purchased from
Transduction Laboratories (Mississauga, Ontario, Canada), mouse
anti-c-Myc from Santa Cruz Biotechnologies (Santa Cruz, CA), and
mouse anti-hemagglutinin (HA) was a gift from Luc Desgroseillers
(Department of Biochemistry, Université de Montréal).
Horseradish peroxidase, fluorescein isothiocyanate, and gold-conjugated
secondary antibodies were obtained from Jackson ImmunoResearch
Laboratories (West Grove, PA). Rabbit phosphohexose isomerase (referred
to as AMF) was purchased from Sigma Chemical Co. (Oakville, Ontario,
Canada) and biotinylated with long chain N-hydroxyl-succinimido-biotin (Pierce, Rockford, IL)
according to the manufacturer's instructions. Nanogold-streptavidin
and the HQ-Silver Enhancement kit were purchased from Nanoprobes, Inc.
(Stony Brook, NY). M
An NIH-3T3 fibroblast clone (43) and H-ras- and
v-abl-transformed NIH-3T3 cells (29) were grown in complete
medium consisting of Dulbecco's modified Eagle's medium supplemented
with 10% calf serum, non-essential amino acids, vitamins, glutamine,
and a penicillin-streptomycin antibiotic mixture (Invitrogen Canada
Inc., Burlington, Ontario, Canada). Treatment of cells
with 5 mM m Viral Infection--
Recombinant adenoviruses expressing the
tetracycline-regulatable chimeric transcription activator (tTA),
HA-tagged, and dynK44A, and myc-tagged caveolin-1 under the control of
the tetracycline-regulated promoter were as previously described
(51-53). To enhance infection rates, viral stocks of the tTA and
dynK44A or caveolin-1 adenoviruses were diluted in 320 µl of sterile
PBS and preincubated with 72 µl of 1 µg/ml polylysine for 30 min at
room temperature. Infection with only the tTA adenovirus was used as a
control. 2.5 × 105 untransformed,
ras-transformed or abl-transformed NIH-3T3 cells were plated onto 10-cm dishes for 10 h and rinsed once with PBS before addition of the adenovirus polylysine mixture in 3 ml of serum-free media for 1 h at 37 °C. After removal of the
adenovirus mixture, the cells were rinsed twice with serum-free media
and then incubated for 36 h in regular culture media. For the EM
studies, infection rates of the three cell lines were determined by
immunofluorescence labeling with anti-HA or anti-MYC antibodies, and
viral titers were used that resulted in greater than 75% infection
rates. The rate of infection was determined in parallel for each
experiment and infection with only the tTA adenovirus was used as a
control. Where indicated, cells coinfected with tTA and dynK44A
adenoviruses were then treated with 5 mM m Electron Microscopy--
All three cell types were pulsed with
bAMF for the indicated times and processed for electron microscopy, and
bAMF was revealed with nanogold-streptavidin followed by silver
enhancement as previously described (44). For the double labeling with
anti-AMF-R or polyclonal anti-caveolin antibodies, bAMF was first
revealed with nanogold-streptavidin and silver amplification,
and then the sections were labeled with the primary antibodies
following by the appropriate gold-conjugated secondary antibodies. The
sections were contrasted with uranyl acetate and lead citrate and
examined in a Zeiss CEM902 electron microscope. In the absence of
nanogold-streptavidin labeling, silver particles due to nonspecific
silver enhancement were not observed.
For the quantification of internalized bAMF, the number of silver
particles localized to ER, endosomes, and mitochondria were counted,
and the surface area of the indicated organelles was measured using a
Sigma Scan measurement system. ER labeling included both ribosomes
studded rough ER profiles as well as morphologically identified smooth
ER (43, 47-49). To ensure that the smooth membranous organelles were
not early endosomes, only smooth membrane-bound structures wider than
75 nm and longer than 200 nm were considered to be ER tubules.
Similarly, smooth caveolar invaginations and clathrin-coated vesicles
within 100 nm of the plasma membrane were counted per unit membrane
(43). The average and standard error from 36 images obtained from two
separate experiments are presented. Alternatively, bAMF expression at
the plasma membrane, in endosomes and in the ER, and caveolin
expression at the plasma membrane (including membrane invaginations)
and in morphologically identifiable smooth caveolar invaginations were
quantified from cells incubated with bAMF and labeled with
streptavidin-nanogold and silver amplification followed by
anti-caveolin antibodies and 12-nm gold-conjugated anti-rabbit
secondary antibodies by postembedding immunoelectron microscopy. For
each experiment, 25 intact cell profiles were counted.
FACS Analysis--
Cells were detached from the dish with EDTA
and resuspended in bicarbonate-free medium supplemented with 25 mM Hepes and 5% calf serum. The cells were then incubated
with anti-AMF-R antibodies at 4 °C for 1 h, washed three times
with cold media, incubated with fluorescein isothiocyanate-conjugated
anti-rat IgM at 4 °C for 1 h, and then washed three more times
with cold media and twice with PBS. The cells were then incubated with
0.5 µg/ml propidium iodide for 10 min at 4 °C. Cell-associated
fluorescence intensity was analyzed on a Beckman FACScan. Cells stained
for propidium iodide were discarded from the analysis, and cell surface
AMF-R expression was determined only on intact cells.
Immunoblot--
Cells cultured at ~70% confluency were
scraped, lysed, and sonicated in lysis buffer containing 1%
SDS, 5 mM EDTA, and protease inhibitors. Protein content
was assayed using the BCA protein assay (Pierce, Rockford, IL), and 40 µg of protein was separated by SDS-PAGE and blotted onto
nitrocellulose paper. The blots were blocked with 5% milk in PBS,
incubated with rabbit anti-caveolin antibody together with mouse
anti-HA, and then with horseradish peroxidase-conjugated anti-rabbit
and anti-mouse secondary antibodies. The labeled bands were revealed by
chemiluminescence and exposed to preflashed Kodak XRP-1 film.
Caveolae-mediated Endocytosis of AMF-R to the ER--
In NIH-3T3
cells, AMF-R is localized to smooth caveolar invaginations and is
endocytosed via a m
Quantification of bAMF labeling of the ER, endosomes, and mitochondria
(see "Experimental Procedures" for details) showed increased bAMF
internalization to the ER in ras- and
abl-transformed cells compared with control NIH-3T3 cells
(Fig. 3A).
Clathrin-dependent endocytosis to endosomal structures (44)
was detected at similar levels between the three cell types.
Nonspecific labeling of mitochondria and control labeling performed in
the absence of endocytosed bAMF are also presented. The significantly
reduced expression of caveolae and caveolin in the transformed NIH-3T3
cell lines does not therefore prevent endocytosis of bAMF to the
ER.
Kinetic analysis of bAMF endocytosis to the ER in the three
cell lines showed that the rate of accumulation of bAMF in the ER was
equivalent in the ras- and abl-transformed
NIH-3T3 cells and approximately 2-fold greater than that in
untransformed NIH-3T3 cells (Fig. 3B). Over the 2-h time
course of the experiment, delivery of bAMF to the ER was maintained and
not saturable in the three cell lines. FACS analysis showed that cell
surface expression of AMF-R is reduced following transformation of
NIH-3T3 cells (Fig. 3C), although total AMF-R expression as
determined by immunoblot was equivalent or increased in ras-
and abl-transformed NIH-3T3 cells, respectively,
compared with untransformed NIH-3T3 cells (data not shown). The
increased rate of delivery of bAMF to the ER is therefore not a
consequence of increased receptor expression at the plasma membrane but
rather due to rapid receptor recycling.
Incubation of NIH-3T3 cells with 5 mM m
The caveolar distribution of AMF-R is based on the EM localization of
AMF-R to smooth plasmalemmal invaginations and its partial colocalization with caveolin by immunofluorescence labeling (43). To
ensure that the smooth invaginations to which AMF-R is localized are
indeed caveolin-positive and therefore correspond to accepted definitions of caveolae, NIH-3T3 cells were double-labeled by postembedding EM for bAMF (nanogold-streptavidin and silver
amplification) and caveolin (12-nm gold particles) (Fig.
4A) or for AMF-R (12-nm gold
particles) and caveolin (18-nm gold particles) (Fig. 4B). Both bAMF- and AMF-R-positive invaginations are labeled for
caveolin.
Adenoviral Expression of dynK44A Induces Caveolae and Inhibits AMF
Endocytosis--
Infection of NIH-3T3 cells with an adenovirus
expressing the dynK44A mutant enhanced our ability to identify
double-labeled caveolae. NIH-3T3 cells expressing this mutant show
numerous caveolin-positive caveolae at the plasma membrane (Fig. 4,
C-H), including those exhibiting the typical long neck
associated with dynamin inhibition (Fig. 4G) (12). Caveolae
double-labeled for caveolin and either bAMF (Fig. 4, C and
E) or AMF-R (Fig. 4, D, F, and
H) are readily detected. bAMF is therefore localized with
its receptor to caveolae in NIH-3T3 cells.
ras- and abl-transformed NIH-3T3 cells exhibit
significantly fewer caveolae relative to NIH-3T3 cells (Fig.
5, A and B), as reported previously (29), and introduction of the dynK44A mutant by
adenoviral infection (51, 52) into ras- and
abl-transformed NIH-3T3 cells induced the expression of
numerous smooth invaginations morphologically similar to caveolae (Fig.
5, C-I). Treatment of dynK44A-infected ras- and
abl-transformed NIH-3T3 cells with m
Adenoviral expression of dynK44A blocked both the
clathrin-dependent endocytosis of bAMF to endosomes and the
caveolae-like pathway to the smooth ER (Fig.
7A). Expression of the tTA
adenovirus alone did not influence either of the AMF endocytic pathways
indicating that inhibition of AMF endocytosis is specifically due to
expression of the dynK44A mutant and not to adenoviral infection (Fig.
7B). Dynamin-mediated budding of caveolar vesicles from the
plasma membrane therefore regulates AMF-R endocytosis to the ER.
To ensure that dynK44A expression is not inducing caveolin expression
and thereby affecting the expression of caveolae, we quantified plasma
membrane-associated caveolin labeling by postembedding immunoelectron
microscopy of whole cell profiles. As presented per micron of membrane
in Fig. 6, the number of caveolar invaginations per cell is
dramatically reduced in ras- and abl-transformed
cells, and dynK44A expression induces the stable expression of a large number of smooth caveolar invaginations (Fig.
8A). Caveolin labeling associated with the plasma membrane, including caveolae (Fig. 8B) or specifically with caveolae (Fig. 8C) is
significantly reduced in ras- and abl-transformed
NIH-3T3 cells relative to untransformed NIH-3T3 cells. Expression of
dynK44A does not affect total plasma membrane-associated caveolin
expression (Fig. 8B) indicating that increased expression of
caveolin or its increased recruitment to the plasma membrane is not
responsible for the dynK44A-mediated induction of smooth caveolar
invaginations. A slight increase in caveolin labeling of caveolar
invaginations is observed in all three cell lines (Fig. 8C)
but is minimal relative to the increased number of caveolae expressed
(Fig. 8A). Immunoblot analysis reveals that caveolin
expression in the ras and abl transformants remains significantly below that in NIH-3T3 cells even after adenoviral expression of HA-tagged dynK44A (Fig. 9).
Expression of dynK44A has not therefore induced the formation of
caveolar invaginations by increasing caveolin expression levels.
Adenoviral Expression of Caveolin-1 Negatively Regulates AMF
Endocytosis to the ER--
Infection of the three cell types with tTA
and caveolin-1 adenoviruses induces increased levels of caveolin-1
expression significantly above those in uninfected NIH-3T3 cells (Fig.
9). As previously reported (27), the reintroduction of caveolin-1 into
ras- and abl-transformed NIH-3T3 cells induces
numerous caveolae at the plasma membrane (Fig.
10) that are morphologically
indistinguishable from the caveolae induced by dynK44A infection (Fig.
5). Quantitatively, a dramatic increase in caveolae expression and in
anti-caveolin labeling at both the plasma membrane and in caveolae was
observed in ras- and abl-infected cells such that
caveolae and caveolin levels were equivalent to or greater than those
of uninfected NIH-3T3 cells (Fig. 11,
A-C). Infection of NIH-3T3 cells with the caveolin-1
adenovirus induced lesser (1.5- to 2-fold) increases in the number of
caveolae and in caveolin expression at the cell surface (Fig. 11,
A-C). Caveolin-1 overexpression in the transformed cells
reduced bAMF endocytosis to the ER to levels comparable to uninfected
NIH-3T3 cells but did not affect bAMF internalization to endosomes; the
increased expression of caveolin-1 in NIH-3T3 cells also selectively
decreased bAMF endocytosis to the ER (Fig. 11, D and
E). Of particular interest, the reintroduction of caveolin-1 into ras- and abl-transformed NIH-3T3 cells was
associated with the accumulation of bAMF within cell surface caveolae
(Fig. 11F). Overexpression of caveolin-1 therefore reduces
caveolae-mediated internalization of bAMF identifying caveolin-1 as a
negative regulator of caveolae-mediated endocytosis.
Caveolae-mediated Endocytosis to the ER--
The AMF/AMF-R
endocytic pathway to the ER represents the first identified
receptor-mediated endocytic pathway that delivers its ligand to an ER
subdomain via caveolae. Inhibition of AMF endocytosis to the ER by both
m Caveolin Is Not Necessary for Caveolar Invagination and Budding of
Caveolar Vesicles--
The caveolae-mediated endocytic pathway of
AMF-R to the ER is still present in ras- and
abl-transformed NIH-3T3 cells that exhibit reduced levels of
caveolin and caveolae. Overexpression in these cells of the dynK44A
dominant negative mutant using an adenoviral expression system induces
the expression of morphologically identifiable caveolae and inhibits
AMF-R endocytosis to the ER, confirming previous reports that
dynamin regulates the budding and endocytic function of caveolae (12,
13). The smooth caveolar invaginations formed in dynK44A-infected
ras- and abl-transformed NIH-3T3 cells are not
enriched for caveolin yet are still sensitive to cholesterol depletion
with m
The induction of caveolae by caveolin expression in
caveolin-minus cells, as described here and in other reports (26-28),
is due to the stabilization of caveolae by caveolin at the plasma membrane, permitting their visualization by electron microscopy of
fixed samples, as previously suggested (43). Caveolin association with
rafts may modify their functional properties by regulating the protein
and lipid composition of individual plasma membrane microdomains (19,
21). Distinct dominant negative caveolin mutants differentially affect
SV40 endocytosis and RAS signaling and suggest that caveolin may act to
regulate caveolae function and endocytosis by controlling the
cholesterol content of glycolipid rafts and perhaps caveolar vesicles
(58).
Our study therefore demonstrates that morphological flask-shaped
caveolae form independently of caveolin-1 expression. Indeed, the term
caveolae was invoked long before the identification of caveolin (15).
Nevertheless, because few caveolae are visualized at the plasma
membrane in the absence of caveolin, stably expressed caveolae are
necessarily caveolin-associated, and caveolin is therefore a reliable
marker for caveolae expression. The caveolin-1 knockout mouse is
viable, and the phenotype is relatively minor suggesting that if
caveolae function is essential for development and survival of the
organism, it is not dependent on caveolin-1 expression (60, 61).
Furthermore, although caveolae expression was dramatically reduced in
the caveolin-1 knockout mice, a few caveolar invaginations were still
identified (61).
Caveolin Is a Negative Regulator of Caveolae
Internalization--
Similar to the AMF endocytosis to the ER reported
here in ras- and abl-transformed NIH-3T3 cells,
prior studies have also reported the internalization of cholera toxin,
glycosylphosphatidylinositol-anchored proteins, or the interleukin 2 receptor via non-clathrin cholesterol-dependent pathways in
cells that do not express caveolin (42, 45, 62). The significant
overexpression of caveolin-1 obtained using adenoviral infection
significantly reduced but did not completely inhibit AMF
internalization (Fig. 11D) indicating that caveolin-1
stabilization of caveolae at the plasma membrane slows but does not
prevent caveolae-mediated endocytosis.
Caveolae-mediated endocytosis in endothelial cells that express
significant amounts of caveolin-1 is well documented (63). Although
caveolin-1 knockout mice did not exhibit altered serum albumin levels
(61), lung endothelial cells of caveolin-1 knockout mice exhibit
reduced albumin uptake (64), and reintroduction of caveolin-1 into
caveolin-1 knockout fibroblasts induced albumin internalization (60).
Internalization of albumin by gp60 or albondin requires gp60
activation and interaction with caveolin-1; albumin endocytosis was
disrupted by caveolin-1 overexpression, which resulted in the
sequestration of G
It is possible that all glycolipid raft domains, currently defined
biochemically, can invaginate to form caveolar vesicles. However, it is
more likely that different classes of rafts exist with differential
abilities to invaginate and bud from the plasma membrane and to form
functionally distinct caveolar vesicles. For instance, caveolae and
raft domains mediate both endocytosis to the ER (41, 43, 44, 46) and to
endosomes and the Golgi (35-37, 59, 65, 66) and in endothelial cells,
distinct caveolar vesicle populations have been shown to mediate
transcytosis of albumin and insulin (67).
Caveolin-1 is shown here to be a negative regulator of caveolar
endocytosis that acts to slow detachment of caveolar vesicles from the
plasma membrane. Reduced caveolin-1 expression is associated with
different forms of cancer in vivo, and decreased caveolin-1 expression in vitro is associated with cell transformation
and tumorigenicity identifying caveolin-1 as a tumor suppressor gene (27, 29, 30, 53, 68-71). The increased rate of internalization of AMF-R to the ER in transformed NIH-3T3 cells corresponds to decreased cell surface expression of AMF-R suggesting that, in cells
lacking caveolin-1, AMF-R rapidly transits the plasma membrane. Similarly, FACS analysis of B16 melanoma and K1735 fibrosarcoma metastatic variants reported decreased cell surface AMF-R expression in
the high metastatic variants (72). It is therefore conceivable that
decreased expression of caveolin-1 results in the destabilization of
AMF-R cell surface expression and the deregulation of AMF-R traffic.
The well-characterized association of AMF-R expression with tumor
malignancy (73-79) implicates this caveolae-mediated endocytic pathway
in AMF-R function in tumor cell motility and metastasis.
We thank Michael Lisanti and Philippe Frank
for kindly providing the ras- and abl-transformed
NIH-3T3 cells and caveolin-1 adenovirus as well as for their helpful
suggestions. We thankfully acknowledge the precious help of Anne
Guénette for the EM quantification and Jean Léveillé
for the preparation of the figures.
*
This study was supported in part by a grant from the
Canadian Institutes for Health Research (CIHR).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Recipient of a Graduate Student Award from the Cancer Research
Society Inc.
Published, JBC Papers in Press, November 27, 2001, DOI 10.1074/jbc.M111240200
The abbreviations used are:
dynK44A, dynamin-1
K44A mutant;
AMF, autocrine motility factor;
AMF-R, autocrine motility
factor receptor;
bAMF, biotinylated autocrine motility factor;
ER, endoplasmic reticulum;
m
Caveolin-1 Is a Negative Regulator of Caveolae-mediated
Endocytosis to the Endoplasmic Reticulum*
§,,
Department of Pathology and Cell Biology,
Université de Montréal, Montreal, Quebec H3C 3J7, Canada
and the ¶ Department of Pharmacology, Hebrew University of
Jerusalem, Jerusalem 91120, Israel
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyclodextrin, inhibits its
internalization to the endoplasmic reticulum implicating caveolae
in this distinct receptor-mediated endocytic pathway. Curiously, the
rate of methyl--cyclodextrin-sensitive endocytosis of AMF-R to the
endoplasmic reticulum is increased in ras- and
abl-transformed NIH-3T3 cells that express
significantly reduced levels of caveolin and few caveolae.
Overexpression of the dynamin K44A dominant negative mutant via an
adenovirus expression system induces caveolar invaginations sensitive
to methyl--cyclodextrin extraction in the transformed cells without
increasing caveolin expression. Dynamin K44A expression further
inhibits AMF-R-mediated endocytosis to the endoplasmic reticulum in
untransformed and transformed NIH-3T3 cells. Adenoviral expression of
caveolin-1 also induces caveolae in the transformed NIH-3T3 cells and
reduces AMF-R-mediated endocytosis to the endoplasmic reticulum to
levels observed in untransformed NIH-3T3 cells. Cholesterol-rich
detergent-resistant membrane domains or glycolipid rafts therefore
invaginate independently of caveolin-1 expression to form
endocytosis-competent caveolar vesicles via rapid
dynamin-dependent detachment from the plasma membrane.
Caveolin-1 stabilizes the plasma membrane association of caveolae and
thereby acts as a negative regulator of the caveolae-mediated endocytosis of AMF-R to the endoplasmic reticulum.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyclodextrin (mCD) via
caveolae to the smooth ER (43, 44, 47-49). Using AMF as a marker for
this caveolae-mediated endocytic pathway, we show that caveolar
invaginations and caveolar vesicles mediate AMF-R endocytosis in
ras- and abl-transformed NIH-3T3 cells that express little caveolin and few caveolae. Adenoviral expression of the
dominant negative dynK44A mutant or of caveolin-1 has allowed us
to demonstrate that: 1) even when caveolin levels are significantly reduced or absent, caveolae form and rapidly bud from the plasma membrane to form caveolar vesicles that target the ER; and 2) caveolin-1 regulates this endocytic pathway by stabilizing caveolae expression at the plasma membrane thereby slowing down the
internalization of caveolar vesicles.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CD, poly-L-lysine, and propidium
iodide were purchased from Sigma.
CD was performed as previously described
(44).
CD for 90 min
prior to fixation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CD-sensitive caveolae-mediated pathway to the
smooth ER tubules defined by AMF-R expression (43, 44). To specifically
assess the specific role of caveolae in this endocytic route,
endocytosis of biotinylated AMF (bAMF) was followed in ras-
and abl-transformed NIH-3T3 cells that exhibit significantly
reduced expression of caveolae and caveolin (29). Following a 60-min
bAMF pulse at 37 °C, endocytosed bAMF detected by nanogold labeling
and silver amplification was localized to smooth and rough ER tubules
of NIH-3T3 and ras- and abl-transformed NIH-3T3
cells (Fig. 1). Endocytosis of bAMF to
multivesicular bodies (MVBs) was also detected in all three cell lines
(Fig. 1) as previously reported in NIH-3T3 cells (44). Double labeling of the cells for bAMF (nanogold and silver amplification) and for AMF-R
(12-nm gold) by electron microscopy confirmed that bAMF is delivered to
AMF-R-positive ER tubules in all three cell types (Fig.
2). AMF-R labeling of rough ER tubules
appears to be qualitatively increased in the ras- and
abl-transformed NIH-3T3 cells relative to untransformed
NIH-3T3 cells. Previous quantitative studies have shown that the
predominant distribution of AMF-R at steady state is to smooth ER
tubules in Madin-Darby canine kidney cells, NIH-3T3, and HeLa cells,
although significant labeling of rough ER tubules was observed in
Madin-Darby canine kidney cells (43, 47). For the purpose of this study
the rough and smooth ER were not morphologically distinguished.
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Fig. 1.
AMF is internalized to the ER in ras-
and abl-transformed NIH-3T3 cells. NIH-3T3 (A),
NIH-ras (B), and NIH-abl (C) cells
were pulse labeled with biotinylated AMF (bAMF) for 60 min at 37 °C.
Postembedding labeling with nanogold-streptavidin and silver
amplification revealed bAMF localization to ER tubules as well as to
MVBs. Arrowheads, ER tubules; arrows, MVBs.
Bar = 0.2 µm.
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Fig. 2.
Endocytosed bAMF colocalizes with AMF-R in ER
tubules. NIH-3T3 cells (A, B) as well as
ras (C, D)-transformed and
abl (E, F)-transformed NIH-3T3 cells
were pulse-labeled with bAMF for 60 min at 37 °C. bAMF was first
revealed by nanogold-streptavidin and silver amplification following by
the labeling of AMF-R tubules with anti-AMF-R antibodies and 12-nm
gold-conjugated anti-rat IgM secondary antibodies.
Arrowheads, bAMF labeling; arrows, AMF-R
labeling. Bar = 0.2 µm.
View larger version (40K):
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Fig. 3.
Increased rate of AMF-R endocytosis to the ER
in ras- and abl-transformed cells.
A, NIH-3T3 (white bars) or ras
(gray bars)- and abl (black
bars)-transformed NIH-3T3 cells were pulsed with bAMF for 60 min
at 37 °C in the absence or presence of m CD. bAMF was revealed by
postembedding labeling with nanogold-streptavidin and silver
amplification. The numerical density of silver particles associated
with the ER, endosomes, and mitochondria was determined from 36 images
(×12,000) from two different experiments for each condition. Control
values in the absence of a bAMF pulse are presented. The results are
presented as a ratio of the number of silver particles to the surface
area of each organelle (±S.E.). The data shows that bAMF targeting to
the ER is selectively inhibited by mCD. B, quantitative
analysis of AMF delivery to the ER after 30-, 60-, and 120-min
incubation with bAMF at 37 °C in NIH-3T3 (circles),
NIH-ras (squares), and NIH-abl
(triangles) cells. ER-associated silver particles were
counted from 25 intact cell profiles, and the data represent the
average per cell profile. C, FACS analysis of AMF-R cell
surface expression in untransformed and ras- and
abl-transformed NIH-3T3 cells shows that AMF-R cell surface
expression is decreased following cell transformation. The data are
presented as relative fluorescence intensity in percentage compared
with NIH-3T3 cells.
CD
selectively blocks bAMF endocytosis to the ER but not the
clathrin-dependent endocytosis of bAMF to multivesicular
endosomes (44). As can be seen in Fig. 3A, mCD also
blocks bAMF delivery to the ER in both untransformed and
ras- and abl-transformed NIH-3T3 cells without
significantly affecting bAMF endocytosis to endosomes. High
concentrations (10 mM) of mCD have been shown to block
clathrin-dependent endocytosis (54, 55); however, the lack
of an effect on the clathrin-dependent endocytosis of bAMF
to endosomes serves as an internal control demonstrating that at the 5 mM concentration used, mCD is selectively inhibiting the
caveolae-like pathway of bAMF to the ER. The ability of mCD to
inhibit bAMF endocytosis to the ER in ras- and
abl-transformed NIH-3T3 cells confirms the similar nature of
this pathway in the three cell lines.
View larger version (181K):
[in a new window]
Fig. 4.
bAMF and AMF-R are localized to
caveolin-positive caveolae. NIH-3T3 (A, B)
or NIH-3T3 cells infected with dynK44A (C-H) were
pulse-labeled with bAMF for 60 min at 37 °C. Postembedding labeling
with nanogold-streptavidin and silver amplification first revealed the
localization of bAMF to caveolae, and then caveolin distribution was
determined using polyclonal anti-caveolin antibodies and 12-nm
gold-conjugated anti-rabbit secondary antibodies (A,
C, E, and G). Double labeling for
AMF-R and caveolin was performed by first adding anti-AMF-R and
anti-caveolin primary antibodies following by 12-nm gold-conjugated
anti-rat IgM and 18-nm gold-conjugated anti-rabbit secondary
antibodies, respectively (B, D, F, and
H). In A, C, E, and
G, the arrows indicate caveolin and the
arrowheads bAMF. In B, D,
F, and H, the arrows indicate AMF-R
and the arrowheads caveolin. Bars = 0.2 µm.
CD prior to fixation
resulted in the complete absence of smooth caveolar invaginations (Fig.
5, K and L), as observed for uninfected cells (Fig. 5, A and B). The caveolar invaginations
induced by dynK44A are, therefore, sensitive to cholesterol depletion
and represent a cholesterol-rich membrane domain or class of glycolipid
rafts. Quantification of the expression of caveolae and clathrin-coated pits in uninfected and dynK44A-infected cells demonstrated the significantly increased expression per micron of membrane of
morphological caveolae but not of clathrin-coated pits in all three
cell types (Fig. 6).
View larger version (189K):
[in a new window]
Fig. 5.
Expression of dynK44A induces caveolae in
ras- and abl-transformed NIH-3T3 cells.
Ras (A, C, F, G,
I, K)- and abl (B,
D, E, H, J,
L)-transformed NIH-3T3 cells, either uninfected
(A, B) or expressing dynK44A via adenoviral
infection (C-L) were pulse-labeled with bAMF for 60 min at
37 °C. Plasma membrane profiles show the dramatically increased
expression of smooth caveolar invaginations in the dynK44A infected
cells (C-I) relative to uninfected cells (A,
B). DynK44A-infected ras- and
abl-transformed NIH-3T3 cells pretreated with 5 mM m CD for 90 min prior to fixation exhibited no
caveolar invaginations (K, L). Postembedding
labeling with nanogold-streptavidin and silver amplification revealed
bAMF localization to caveolar invaginations (E,
F, G, H) and to clathrin-coated
vesicles (H, J). Bar = 0.2 µm.
View larger version (18K):
[in a new window]
Fig. 6.
Quantitative analysis of caveolae and
clathrin-coated vesicles at the plasma membrane following dynK44A
infection. The number of morphologically identifiable caveolae
(A) and clathrin-coated vesicles (B) per micron
of plasma membrane length was determined for untransformed and
ras- and abl-transformed NIH-3T3 cells either
uninfected (white bars) or expressing dynK44A via adenoviral
infection (black bars). The increase in the number of
caveolae expressed in dynK44A-infected NIH-3T3 cells was significant
(p < 10 
3).
View larger version (27K):
[in a new window]
Fig. 7.
DynK44A blocks endocytosis of biotinylated
AMF to both the ER and endosomes. Untransformed NIH-3T3
(white bars) and ras (gray bars)- or
abl (black bars)-transformed NIH-3T3 cells were
infected with the tTA and dynK44A adenoviruses and then pulsed with
bAMF for 60 min at 37 °C (A). bAMF was revealed by
postembedding labeling with nanogold-streptavidin and silver
amplification. The numerical density of silver particles and the
surface area of the ER, endosomes, and mitochondria were determined
from 36 images (×12,000) from two different experiments for each
condition. Control labeling was measured in the absence of added bAMF.
Infection with the tTA adenovirus alone did not affect the
internalization of bAMF (B). The results are presented as
the ratio of the number of silver particles to the surface area of each
organelle (±S.E.).
View larger version (15K):
[in a new window]
Fig. 8.
Expression of caveolin at the plasma membrane
is not increased following dynK44A infection. Quantification of
the number of morphologically identifiable caveolae (A),
caveolin labeling at the plasma membrane (including smooth caveolar
invaginations) (B), and caveolin labeling specific to smooth
caveolar invaginations (C) was determined for 50 complete
cell profiles from anti-caveolin-labeled EM grids for untransformed and
ras- and abl-transformed NIH-3T3 cells either
uninfected (white bars) or expressing dynK44A via adenoviral
infection (black bars). The data were obtained from two
distinct experiments and represent the average per cell profile.
View larger version (24K):
[in a new window]
Fig. 9.
Caveolin expression levels following dynK44A
and caveolin-1 adenovirus infection. Cell lysates from NIH-3T3,
and ras- and abl-transformed NIH-3T3 cells were
separated by SDS-PAGE, and the blots were probed with antibodies to
either caveolin or the HA tag of dynK44A, as indicated. Caveolin
expression levels are significantly reduced in ras- and
abl-transformed NIH-3T3 relative to untransformed NIH-3T3
cells and are not affected in dynK44A-expressing cells. However, in
caveolin-1-infected cells, caveolin expression is significantly
increased in the three cell lines.
View larger version (111K):
[in a new window]
Fig. 10.
Expression of caveolin-1 induces the
formation of caveolae in ras- and
abl-transformed NIH-3T3 cells. NIH-3T3 (A,
B) and ras (C, D)- and
abl (E, F)-transformed NIH-3T3 cells,
either uninfected (A, C, E) or
expressing caveolin-1 via adenoviral infection (B,
D, F) were pulse-labeled with bAMF for 60 min at
37 °C. Postembedding labeling with nanogold-streptavidin and silver
amplification first revealed the localization of bAMF to caveolae, and
then caveolin distribution was determined using anti-caveolin
polyclonal antibodies and 12-nm gold-conjugated anti-rabbit secondary
antibodies. Plasma membrane profiles of ras- and
abl-transformed NIH-3T3 cells show the dramatically
increased expression of caveolae in the caveolin-1 infected cells
(D, F) relative to uninfected cells
(C, E). Bar = 0.2 µm.
View larger version (30K):
[in a new window]
Fig. 11.
Caveolin-1 expression induces caveolae
formation and down-regulates bAMF endocytosis to the ER.
Untransformed and ras- or abl-transformed NIH-3T3
cells were either not infected (white bars) or infected with
the tTA (gray bars) or with the tTA and caveolin-1
adenoviruses (black bars) and then pulsed with bAMF for 60 min at 37 °C. Control cells were not pulsed with bAMF (dotted
bars). Postembedding labeling of bAMF was first revealed with
nanogold-streptavidin and silver amplification, and then caveolin was
revealed using anti-caveolin polyclonal antibodies and 12-nm
gold-conjugated anti-rabbit secondary antibodies. Quantification of the
number of caveolae at the plasma membrane (A), caveolin
labeling at the plasma membrane (including caveolae) (B),
caveolae-associated caveolin labeling (C), bAMF endocytosis
to the ER (D) or to endosomes (E), and bAMF
labeling of caveolae (F) was determined for 50 complete cell
profiles obtained from two distinct experiments. The data presented
represents the average per cell profile (±S.E.).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CD and dynK44A distinguishes this pathway from the
clathrin-independent pathway (9, 10, 54) and defines at least two
distinct non-clathrin endocytic pathways. SV40 is also internalized via
cell surface caveolae to the smooth ER (41, 46, 56-58).
Internalization of SV40 to the ER is significantly slower (4-6 h) (41)
than that observed for AMF-R, and the identity between the two pathways
remains to be established. If identical, the fact that a cellular
receptor, AMF-R, is delivered via caveolae to the ER suggests that SV40
has not induced a novel endocytic pathway but has rather co-opted a
pre-existing one.
CD. In cells that express limited amounts of caveolin,
cholesterol-rich and detergent-resistant membrane domains or glycolipid
rafts invaginate to form caveolae that rapidly give rise to
endocytosis-competent vesicles such that caveolae are visible only when
budding is inhibited. Caveolin is not essential for caveolae
invagination or endocytosis, and, indeed, the endocytic potential of
cholesterol-rich and detergent-resistant membrane domains or glycolipid
rafts in the absence of caveolin is quite significant. Glycolipid rafts
are therefore dynamic endocytic structures (59) that, upon invagination
and budding from the plasma membrane, are equivalent, if only
transiently, to the morphological definition of caveolae. The absence
of caveolae at the plasma membrane does not preclude the presence of a
caveolar endocytic pathway.
i preventing gp60 activation (34). Caveolin-1 expression and association with caveolar domains may
regulate not only their rate of internalization but also select the
cargo that follows this endocytic route.
ACKNOWLEDGEMENTS
FOOTNOTES
Recipient of a CIHR Investigator award. To whom correspondence
should be addressed: Dépt. de Pathologie et Biologie Cellulaire, Université de Montréal, C. P. 6128, Succursale A,
Montréal, Québec H3C 3J7, Canada. Tel.: 514-343-6291; Fax:
514-343-2459; E-mail: ivan.robert.nabi@umontreal.ca.
ABBREVIATIONS
CD, methyl--cyclodextrin;
tTA, tetracycline-regulatable chimeric transcription activator;
HA, hemagglutinin;
PBS, phosphate-buffered saline;
FACS, fluorescence-activated cell sorting;
MVB, multivesicular bodies;
EM, electron microscopy.
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