Department of Pathology and Cell Biology, Université de Montréal, Montréal, Québec, Canada
* Author for correspondence (e-mail: ivan.robert.nabi{at}umontreal.ca)
Accepted 17 December 2002
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Summary |
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Key words: Endocytosis, Caveolae, Glycolipid rafts, Cholera toxin, Endoplasmic reticulum, Autocrine motility factor
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Introduction |
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However, in hippocampal neurons, CTX bound to GM1 is found in
detergent-insoluble membranes at the cell surface, but CTX endocytosis was
blocked by inhibitors of clathrin-dependent endocytosis and not by filipin or
mßCD (Shogomori and Futerman,
2001a). MßCD rather blocks the cholesterol-dependent delivery
of CTX from endosomes to the Golgi apparatus
(Shogomori and Futerman,
2001b
), as previously reported for GPI-anchored folate receptor
(Mayor et al., 1998
). These
studies therefore describe a clathrin-dependent, endosome-mediated pathway for
CTX to the Golgi. They further indicate that cholesterol sensitivity is not
necessarily an indicator of caveolae- or raft-mediated internalization to the
Golgi.
Both clathrin- and caveolae/raft-dependent endocytosis are dynamin
dependent (Damke et al., 1994;
Henley et al., 1998
;
Herskovits et al., 1993
;
Le et al., 2002
;
Oh et al., 1998
;
van der Bliek et al., 1993
).
The fact that expression of mutant dynK44A inhibited only 40-50% of CTX uptake
in HeLa cells and that neither filipin nor caveolin-1 overexpression inhibited
CTX internalization in confluent CaCo-2 cells led to the suggestion that CTX
also follows the dynamin-insensitive non-clathrin pathway
(Torgersen et al., 2001
).
However, in human skin fibroblasts and in subconfluent CaCo-2 cells,
cholesterol-disrupting agents inhibit CTX internalization whereas agents that
inhibit clathrin-dependent endocytosis, such as chlorpromazine and potassium
depletion, do not affect CTX endocytosis
(Orlandi and Fishman, 1998
;
Puri et al., 2001
). Depletion
of membrane cholesterol prevents CTX entry into the cells, transport to the
Golgi and induction of chloride secretion
(Wolf et al., 2002
).
Furthermore, inhibition of dynamin function by either dynK44A expression or
microinjection of anti-dynamin antibodies has been shown to qualitatively
inhibit CTX endocytosis (Henley et al.,
1998
; Oh et al.,
1998
). These data from multiple studies in multiple cell types
argue strongly for a role of caveolae in the internalization of CTX. However,
a single study in a single cell type demonstrating the clathrin-independent,
dynamin-dependent, cholesterol-sensitive, caveolae-mediated endocytosis of CTX
has yet to be performed.
CTX delivery to the Golgi apparatus in Cos-7 cells is filipin sensitive but
is not blocked by inhibition of clathrin-dependent endocytosis with the eps15
mutant or inhibition of endosome function with a rab5 mutant. Therefore, a
cholesterol-sensitive non-endosomal pathway is used for CTX delivery to the
Golgi apparatus (Nichols et al.,
2001). CTX is delivered instead to a caveolin-1GFP-positive
transferrin receptor (TfR)-negative endocytic intermediate prior to delivery
to the Golgi, implicating caveolae in this pathway
(Nichols, 2002
). Similarly,
SV40 has previously been shown to be internalized to the ER via a
caveolin-1GFP-positive intermediate, named the caveosome
(Pelkmans et al., 2001
). SV40
delivery via caveolae to the caveosome is associated with tyrosine kinase
activation, the breakdown of actin filaments and the recruitment of dynamin
II, whereas passage from the caveosome to the ER is blocked by treatment of
the cells with nocodazole and is therefore microtubule dependent
(Pelkmans et al., 2001
;
Pelkmans et al., 2002
). The
recent demonstration that BFA (brefeldin A) and a 20°C temperature block,
as well as mutants of arf1 and sar1 and antibodies to ß-cop, block SV40
internalization and infectivity as well as the internalization of CTX suggests
that both caveolar ligands follow a similar internalization route
(Norkin et al., 2002
;
Richards et al., 2002
).
Although a role for the Golgi apparatus in SV40 delivery to the ER remains to
be established (Kartenbeck et al.,
1989
; Norkin et al.,
2002
; Pelkmans et al.,
2001
; Richards et al.,
2002
), SV40 and CTX appear to follow a similar, if not identical,
pathway via the caveosome to the ER.
We have previously proposed the caveolae-mediated endocytosis of AMF to the
ER on the basis of its sensitivity to mßCD, its inhibition by the
dynamin-1 K44A mutant (dynK44A) and its negative regulation by overexpression
of caveolin-1 (Benlimame et al.,
1998; Le et al.,
2000
; Le et al.,
2002
). AMF-R is localized to a smooth ER subdomain, and we have no
evidence from multiple EM studies that AMF-R is localized to the Golgi or that
endocytosed AMF traverses the Golgi (Accola
et al., 2002
; Benlimame et al.,
1998
; Benlimame et al.,
1995
; Le et al.,
2002
; Wang et al.,
1997
; Wang et al.,
2000
). In order to compare the caveolae-mediated endocytosis of
AMF and the retrograde pathway of CTX via the Golgi to the ER, we first
undertook to define the endocytic pathway of CTX in NIH-3T3 fibroblasts. We
show here that in NIH-3T3 cells, 80% of CTX uptake is blocked by adenoviral
expression of the dynK44A mutant, but inhibition of clathrin-dependent
endocytosis with the dominant-negative hub fragment of the clathrin heavy
chain has only a limited effect on CTX endocytosis. MßCD treatment and
overexpression of caveolin-1 selectively reduce CTX endocytosis to the Golgi
apparatus but not to endosomes. In NIH-3T3 fibroblasts, the majority of CTX is
therefore internalized via a caveolae-dependent mechanism to the Golgi with a
minor part targeted via a clathrin-dependent mechanism to the endosome. Of
particular interest is the fact that although BFA, nocodazole or a 20°C
temperature block inhibit CTX delivery to the Golgi apparatus, they do not
affect AMF delivery to the ER. Furthermore, although genistein inhibits the
caveolae/raft-mediated endocytosis of both CTX and AMF to the Golgi and smooth
ER, respectively, after five minutes of endocytosis in the presence of the
clathrin hub the two ligands do not cointernalize and AMF is targeted,
apparently directly, to the AMF-R-labeled smooth ER. The caveolae-mediated
endocytic pathway of AMF is therefore distinct from that of CTX and SV40. Two
caveolae-mediated endocytic pathways therefore exist that target either the
Golgi or the ER from the plasma membrane.
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Materials and Methods |
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An NIH-3T3 fibroblast clone (Benlimame
et al., 1998) was grown in complete medium consisting of DME
supplemented with 10% calf serum, non-essential amino acids, vitamins,
glutamine and a penicillin-streptomycin antibiotic mixture (Invitrogen Canada
Inc., Burlington, ON).
Viral infection
Recombinant adenoviruses expressing the tetracycline-regulated chimeric
transcription activator (tTA), HA-tagged wild-type dynamin-1, HA-tagged
dynK44A mutant, T7-tagged clathrin hub and myc-tagged caveolin-1 under the
control of the tetracycline-regulated promoter were as previously described
(Altschuler et al., 1998;
Altschuler et al., 1999
;
Le et al., 2002
;
Zhang et al., 2000
). To
enhance infection rates, viral stocks of the tTA and the indicated
adenoviruses were diluted in 100 µl sterile PBS and pre-incubated with 18
µl of 1 µg/ml polylysine for 30 minutes at room temperature. 50,000
NIH-3T3 cells were plated on 35 mm dishes for 10 hours and rinsed once with
PBS before the addition of the adenovirus/polylysine mixture in 0.7 ml
serum-free media. The cells were then incubated with the adenoviruses for 1
hour at 37°C. After removal of the adenovirus mixture, the cells were
rinsed twice with serum-free media and then incubated for 36 hours in regular
culture media. For the immunofluorescence studies, viral titres were used such
that 25-50% of the cells were infected. For some experiments, the degree of
infection with the clathrin hub adenovirus was assessed by labeling
paraformaldehyde fixed cells on parallel coverslips with monoclonal anti-T7
tag antibodies to ensure that at least 90% of the cells expressed the clathrin
hub.
Immunofluorescence
NIH-3T3 cells were incubated with 5 µg/ml FITC-CTX alone or coincubated
with 5 µg/ml FITC-CTX and with 15 µg/ml Rh-Tf for 30 minutes at
37°C, washed five times with culture medium and fixed with 3%
paraformaldehyde and permeabilized with 0.2% Triton X-100. Alternatively,
cells were incubated with 50 µg/ml of AMF-FITC alone or coincubated with 5
µg/ml Alexa 594-CTX at 37°C for the indicated time, washed five times
with culture medium and then fixed with precooled (80°C)
methanol/acetone (Le et al.,
2000). The cells were then labeled for GM130, TfR, AMF-R,
caveolin, T7, Myc or HA tags with appropriate primary antibodies and
Alexa-568- or -647-conjugated secondary antibodies, as indicated. Cell-surface
FITC-CTX labeling was performed by incubating the cells at 4°C with 5
µg/ml FITC-CTX in bicarbonate-free DMEM-HEPES containing 0.5% BSA (cold
DMEM) for 30 minutes (Benlimame et al.,
1998
). The cells were then rinsed three times with cold
DMEM/HEPES/BSA, fixed with 3% paraformaldehyde and permeabilized with 0.2%
Triton X-100 prior to labeling for the appropriate tags as described
previously. Where indicated, cells were pretreated for 30 minutes at 37°C
with 5 mM mßCD (Le et al.,
2002
), 10 µg/ml BFA, 10 µM nocodazole or 100 µg/ml
genistein, and the drugs were maintained during incubation with the endocytic
ligands. AMF-FITC endocytosis at 20°C was performed in bicarbonate-free
DMEM-HEPES containing 0.5% BSA. Fluorescently labeled cells were visualized
with a Leica TCS-SP1 confocal microscope using 63x or 100x
planapochromat objectives.
To quantify endocytosis of FITC-CTX and Rh-Tf, the fluorescence intensity
within the perinuclear region of the cell was quantified from confocal images
with Northern Eclipse imaging software (Empix Imaging, Mississuaga, Ontario).
Cell-surface FITC-CTX was quantified by measuring the fluorescence intensity
of the entire cell. Specific FITC-CTX internalization to the Golgi or
endosomes and AMF-FITC internalization to smooth ER tubules were quantified
using Northern Eclipse mask overlay software (Empix Imaging) as previously
described (Wang et al., 2000).
The intensity of FITC-CTX-labeled pixels within a mask region defined by
either the GM130-labeled Golgi or TfR-labeled endosomes was determined
relative to total cellular FITC-CTX labeling. Owing to the complex pattern of
endocytosed AMF, including multivesicular bodies and fibronectin fibrils, in
addition to the AMF-R-labeled smooth ER
(Le et al., 2000
), the
absolute intensity of AMF-FITC-labeled pixels within a mask region defined by
smooth ER AMF-R labeling was measured. Each measurement represents the
quantification of at least 30 cells from three distinct experiments.
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Results |
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Quantitative analysis shows that dynK44A mutant expression significantly inhibits (by >80%) both FITC-CTX and Rh-Tf endocytosis into the perinuclear region, whereas the dynamin wildtype exhibits no effect (Fig. 2A). Clathrin hub expression inhibits >75% of Rh-Tf endocytosis and <25% of FITC-CTX endocytosis, whereas caveolin-1 overexpression does not affect Rh-Tf endocytosis but inhibits >60% of the endocytosis of FITC-CTX. To ensure that infection of NIH-3T3 cells with the various adenoviruses did not affect FITC-CTX binding to cell-surface GM1, cell-surface binding of FITC-CTX at 4°C was quantified in non-infected and infected cells. As shown in Fig. 2B, cells infected with the various adenoviruses did not present a reduction of cell-surface FITC-CTX labeling (Fig. 2B), indicating that infection with the various adenoviruses did not affect accessibility of exogenous FITC-CTX to cell-surface GM1. A significant increase (P<0.05) in cell-surface labeling was observed following infection with dynK44A, which is consistent with its ability to inhibit both the caveolae- and clathrin-mediated endocytosis of FITC-CTX. In NIH-3T3 cells, the majority of FITC-CTX is therefore internalized via a caveolin-dependent pathway and a minor portion via a clathrin-dependent pathway.
|
Caveolae-mediated endocytosis of CTX selectively targets the Golgi
and not endosomes
Treatment of NIH-3T3 cells with 5 mM mßCD resulted in the
significantly increased cell-surface binding of FITC-CTX, owing to the release
of sequestered GM1 from closed caveolae, but did not inhibit the endocytosis
of FITC-CTX or Rh-Tf to endosomes (Fig.
3A-F). In confocal sections obtained with a reduced pinhole (0.6
Airy units) to exclude the increased cell-surface FITC-CTX labeling from the
image, internalized FITC-CTX exhibited extensive colocalization with
Rh-Tf-positive endosomes (Fig.
3G,H,K). Within the crowded perinuclear region, the GM130-labeled
FITC-CTX-unlabeled Golgi could be clearly seen to intercalate between
FITC-CTX- and Rh-Tf-positive endosomes
(Fig. 3J,L). Golgi delivery of
CTX is therefore significantly reduced in the presence of mßCD.
|
We subsequently infected cells with the caveolin-1 adenovirus and assessed
FITC-CTX delivery to either the GM130-labeled Golgi or TfR-labeled endosomes.
In caveolin-1-overexpressing cells, identified with a polyclonal anti-caveolin
antibody, delivery of FITC-CTX to the Golgi was significantly reduced
(Fig. 4A-D). Perinuclear
FITC-CTX was localized predominantly to TfR-positive endosomes that were not
labeled for caveolin, whereas peripheral FITC-CTX labeling colocalized
extensively with caveolin, apparently at the cell surface
(Fig. 4E-J). Using mask overlay
image analysis software (Wang et al.,
2000), we quantified the delivery of FITC-CTX to either
GM130-positive Golgi or TfR-positive endosomes in caveolin-1-infected and in
-uninfected cells. Caveolin-1 overexpression resulted in a significant
reduction (
50%) in the delivery of FITC-CTX to the Golgi but did not
affect delivery of FITC-CTX to endosomes
(Fig. 4K,L). The fact that
caveolin-1 overexpression selectively reduces CTX delivery to the Golgi but
not transferrin-positive endosomes provides direct evidence for a
caveolae-mediated endocytic pathway to the Golgi apparatus.
|
To confirm that CTX internalization to the Golgi is caveolae-mediated, FITC-CTX was added to NIH-3T3 cells infected with the clathrin hub adenovirus (Fig. 5). Five minutes after its addition, FITC-CTX colocalizes extensively with caveolin and after 30 minutes, FITC-CTX is associated predominantly with the Golgi apparatus. The fact that the clathrin hub only blocks a minor portion of CTX endocytosis (Fig. 2) together with its inability to inhibit CTX delivery to the Golgi demonstrates clearly that in NIH-3T3 cells, the majority of CTX is targeted via a clathrin-independent, caveolin-regulated pathway to the Golgi apparatus.
|
Caveolae mediate distinct endocytic pathways to the Golgi and ER
AMF-R is a marker for a mitochondria-associated smooth ER subdomain
(Benlimame et al., 1998;
Benlimame et al., 1995
;
Wang et al., 1997
;
Wang et al., 2000
), and in
NIH-3T3 cells, AMF-R is specifically localized to smooth and not rough ER
tubules (Benlimame et al.,
1998
). We have previously reported, using quantitative electron
microscopy, that caveolin-1 overexpression reduces the caveolae-mediated
endocytosis of AMF to the ER in NIH-3T3 fibroblasts as well as in ras and
abl-transformed NIH-3T3 fibroblasts that express little caveolin-1
(Le et al., 2002
).
Quantification using a mask overlay assay revealed a 42.0±2.4%
reduction in AMF-FITC delivery to the AMF-R-labeled smooth ER in NIH-3T3
fibroblasts that overexpress Myc-tagged caveolin-1
(Fig. 6). These results are
equivalent to those previously obtained by quantitative EM
(Le et al., 2002
). The tubular
distribution of the AMF-R-labeled smooth ER, a reflection of its interaction
with mitochondria, can be disrupted by ilimaquinone or low cytosolic calcium
(Wang et al., 1997
;
Wang et al., 2000
). In
caveolin-1-infected cells, the AMF-R-labeled smooth ER exhibits a more diffuse
distribution (Fig. 6B, arrow),
suggesting that regulation of caveolae-mediated endocytosis to this ER
subdomain may influence the extent of its association with mitochondria. The
fact that overexpression of caveolin-1 reduces the internalization of AMF to
the ER and of CTX to the Golgi demonstrates that they are both mediated by a
similar caveolae-based endocytic mechanism.
|
Entry of CTX is sensitive to BFA (Donta
et al., 1993; Lencer et al.,
1993
; Morinaga et al.,
2001
; Nambiar et al.,
1993
; Orlandi et al.,
1993
), and BFA has recently been shown to prevent delivery of CTX
to the Golgi but not to endosomes
(Richards et al., 2002
). In
cells treated with nocodazole, SV40 entry is blocked at the level of the
caveosome, and it is not delivered to the ER
(Pelkmans et al., 2001
).
Furthermore, a 20°C temperature block and BFA treatment inhibit early
entry steps of SV40, preventing its delivery to the ER
(Norkin et al., 2002
;
Richards et al., 2002
). As
seen in Fig. 7, BFA treatment
of NIH-3T3 fibroblasts inhibits FITC-CTX delivery to the fragmented Golgi but
not to endosomes (Fig. 7A-F).
By contrast, BFA does not affect AMF-FITC delivery to the AMF-R-labeled smooth
ER (Fig. 7G-I). In the presence
of nocodazole, FITC-CTX is not present in the Golgi apparatus although
extensive colocalization is observed with caveolin and with TfR
(Fig. 8A-H). By contrast,
AMF-FITC is delivered efficiently to the smooth ER in the presence of
nocodazole as well as at 20°C (Fig.
8I-N).
|
|
The ability of BFA, nocodazole and a 20°C block to inhibit the
targeting of CTX to the Golgi but not of AMF to the ER indicates that
caveolae-internalized ligands can follow distinct intracellular targeting
pathways. To determine whether the sorting of caveolae-internalized CTX and
AMF occurred at the plasma membrane or intracellularly, clathrin-hub-infected
NIH-3T3 cells were incubated with AMF-FITC and Alexa-594CTX for only 5
minutes (Fig. 9A-F).
Essentially no colocalization between the two caveolar ligands could be
detected and, interestingly, internalized AMF colocalized with AMF-R-labeled
smooth ER tubules. After 30 minutes, although CTX is colocalized predominantly
to the Golgi apparatus, internalized AMF remains colocalized with the smooth
ER and is excluded from the Golgi (Fig.
9G-J). AMF and CTX would therefore appear to be segregated at the
plasma membrane into different caveolae-mediated internalization pathways. To
reconfirm that caveolae truly mediate delivery of both CTX to the Golgi and
AMF to the smooth ER, we treated the cells with genistein, a tyrosine kinase
inhibitor that inhibits the caveolae-mediated endocytosis of SV40 and the
transcytosis of albumin across the endothelial cell
(Pelkmans et al., 2002;
Tiruppathi et al., 1997
). As
seen in Fig. 10, in the
presence of genistein, CTX delivery to the Golgi but not to TfR positive
endosomes was inhibited, confirming that CTX delivery to the Golgi is caveolae
mediated. Genistein also inhibited the delivery, detected after 5 minutes of
incubation, of AMF-FITC to the smooth ER
(Fig. 10G-L).
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Discussion |
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Treatment of NIH-3T3 cells with mßCD selectively inhibited CTX
delivery to the Golgi but not to transferrin-positive endosomes
(Fig. 3). Although these
results could be explained by intracellular mßCD inhibition of CTX
delivery to the Golgi from the endosome
(Mayor et al., 1998;
Shogomori and Futerman,
2001b
), overexpression of caveolin-1
(Fig. 4) and treatment with
genistein (Fig. 10), an
inhibitor of caveolae-mediated endocytosis
(Pelkmans et al., 2002
;
Tiruppathi et al., 1997
), also
selectively inhibit Golgi delivery but not endosomal delivery of CTX.
Caveolin-1 overexpression affects neither the cell-surface expression of GM1
nor the endocytosis of CTX to endosomes and therefore does not reduce Golgi
delivery of CTX by reducing clathrin-mediated endocytosis to the endosome.
These data provide direct evidence for the existence of a caveolae-mediated
endocytic route to the Golgi and are consistent with and support previous
studies describing the caveolae-mediated endocytosis of CTX to the Golgi
(Nichols, 2002
;
Nichols et al., 2001
). CTX can
therefore be endocytosed via both caveolae- and clathrin-mediated pathways.
Differential rates of clathrin- or caveolae-mediated endocytosis and
differential affinities of GM1 for endocytosis-competent raft domains may
influence the choice of endocytic pathway of CTX in different cell types.
Caveolin-1- and caveolae/raft-mediated endocytosis
In cells expressing caveolin-1GFP, a caveolin-positive endocytic
intermediate, the caveosome, has been implicated in caveolae-mediated
endocytosis, including that of both CTX and SV40
(Mundy et al., 2002;
Nichols, 2002
;
Parton et al., 1994
;
Pelkmans et al., 2001
). In our
study, expression of perinuclear caveolin-positive, TfR-negative vesicular
structures was observed, particularly after caveolin-1 overexpression, and
some were positive for internalized CTX. Cell-associated CTX exhibited
significant colocalization with caveolin-1, primarily to peripheral
cell-surface regions but also to some perinuclear structures (Figs
4,
5). It was, however, difficult
to ascertain whether these caveolin-positive structures are indeed
intracellular endocytic vesicles and equivalent to caveosomes.
The reduced endocytosis of CTX in caveolin-1-expressing cells corroborates
our previous report of the negative regulation by caveolin-1 of the
caveolae-mediated delivery of AMF to the ER
(Le et al., 2002). As reported
for the ER delivery of AMF in ras- and abl-transformed NIH-3T3 cells
expressing little caveolin (Le et al.,
2002
), CTX is internalized via a cholesterol-sensitive raft
pathway in CaCo-2 cells that do not express caveolin
(Orlandi and Fishman, 1998
).
Furthermore, reduction of caveolin-1 levels using RNAi did not affect the
internalization of CTX to the Golgi
(Nichols, 2002
). The fact that
the caveolae-mediated endocytosis of AMF and CTX to the smooth ER and Golgi,
respectively, occurs independently of caveolin-1 expression argues that
caveolae- and raft-mediated endocytosis are essentially equivalent processes
defined by cholesterol sensitivity, tyrosine kinase activation,
dynamin-dependence and regulation by caveolin-1.
The role of caveolin-1 as a negative regulator of caveolae/raft-mediated
endocytosis is consistent with recent reports describing the immobilization of
caveolin-1 at the cell surface (Pelkmans
et al., 2002; Thomsen et al.,
2002
). Caveolin-1 expression may serve to stabilize the mobility
of raft domains in and out of the plane of the membrane. The role of
caveolin-1 as a regulator and not as an essential component of the
caveolae/raft endocytic machinery does not question its importance in defining
this endocytic pathway. Caveolin-1 remains a critical determinant of
caveolae/raft-mediated endocytosis as a negative regulator of caveolae budding
from the plasma membrane, a recruiter of cargo to endocytic caveolar domains
or as a component of caveolae-specific endosomal intermediates.
Existence of two distinct caveolae/raft-mediated pathways
BFA and a 20°C incubation, classic inhibitors of CTX delivery to the
Golgi (Donta et al., 1993;
Lencer et al., 1993
;
Morinaga et al., 2001
;
Nambiar et al., 1993
;
Nichols et al., 2001
;
Orlandi et al., 1993
;
Richards et al., 2002
), also
inhibit SV40 endocytosis (Norkin et al.,
2002
; Richards et al.,
2002
). We further show here that nocodazole treatment, shown to
prevent SV40 endocytosis past the caveosome
(Pelkmans et al., 2001
), also
prevents CTX delivery to the Golgi. SV40 targeting to the ER may follow a
similar retrograde pathway to that of CTX via the caveosome and potentially
the Golgi apparatus before delivery to the ER.
In contrast to CTX endocytosis to the Golgi and SV40 delivery to the ER,
BFA, nocodazole and a 20°C incubation did not prevent the
caveolae-mediated delivery of AMF to the ER (Figs
7,
8). Furthermore, following
incubations of both 5 and 30 minutes in the presence of the clathrin hub, the
vast majority of AMF and CTX did not colocalize
(Fig. 9). AMF is therefore
internalized via a caveolae-mediated endocytic route distinct from that of
either CTX or SV40. The fact that AMF could be detected in AMF-R-positive
smooth ER tubules after only 5 minutes argues strongly that this pathway is a
direct pathway to the ER. We have no evidence for the involvement of a
caveolin-positive vesicular intermediate in the endocytosis of AMF. Since
nocodazole does not inhibit AMF delivery to the ER, any such endosomal
intermediate would necessarily be distinct from the caveosome that mediates
SV40 endocytosis (Pelkmans et al.,
2001). Distinct endosomal populations are apparently targeted by
clathrin, non-clathrin and caveolar vesicles
(Nichols, 2002
;
Pelkmans et al., 2001
;
Sabharanjak et al., 2002
). The
demonstration here that caveolae-derived vesicles can target different
organelles shows that intracellular targeting in endocytosis is more complex
than `one vesicle one endosome'.
The functional significance of an alternative, apparently direct, endocytic
pathway to the ER remains to be determined. Such a pathway could be involved
in the recovery of Golgi or ER proteins mistargeted to the plasma membrane or
in the targeting of proteins for degradation in the ER. Caveolin redistributes
to the ER in the presence of cholesterol oxidase
(Conrad et al., 1995), and
cholesteryl ester is transported to the ER from the plasma membrane
(Uittenbogaard et al., 2002
);
however, these pathways appear to be non-vesicular and distinct from the
endocytic pathway of AMF-R. AMF-R is localized to a mitochondria-associated
subdomain of the smooth ER, whose association with mitochondria is calcium
dependent (Wang et al., 2000
).
A direct endocytic pathway to this smooth ER subdomain may function to
maintain its integrity and functionality. The IP3R is also localized to
caveolae and smooth ER (Fujimoto et al.,
1992
; Ross et al.,
1989
; Sharp et al.,
1992
), implicating these two organelles and any interaction
between them in the regulation of calcium homeostasis.
The existence of two caveolae-mediated endocytic pathways implies the
existence of distinct endocytosis-competent caveolae populations at the cell
surface (Maxfield, 2002). AMF
and CTX may be either segregated in different caveolae/raft domains at the
plasma membrane or their segregation may occur upon internalization of these
domains. In endothelial cells of the rete mirabel, albumin and insulin were
localized to distinct caveolae populations, which demonstrates the segregation
of caveolar endocytic cargo (Bendayan and
Rasio, 1996
). The existence of distinct rafts and segregation of
raft components has been demonstrated by the distribution of GM1 to the uropod
and GM3 to the lamellipodia of migrating T lymphocytes
(Gomez-Mouton et al., 2001
).
Furthermore, immunoisolation techniques have allowed the separation of
caveolin-1-positive rafts from caveolin-1-negative rafts, which suggests the
existence of at least two, and potentially multiple, distinct classes of rafts
at the plasma membrane (Badizadegan et al.,
2000
; Matveev and Smart,
2002
; Riddell et al.,
2001
; Stan et al.,
1997
).
The composition of raft domains is a determinant of their endocytic
potential. As shown here for CTX and previously for AMF
(Le et al., 2002), caveolin-1
is a regulator of caveolae/raft endocytosis that may act to segregate
endocytic and non-endocytic raft domains. However, the caveolin-1-independent
internalization of CTX (Nichols,
2002
; Orlandi and Fishman,
1998
), AMF (Le et al.,
2002
) and the IL2 receptor in lymphocytes
(Lamaze et al., 2001
) argues
that regulation of caveolae/raft-mediated endocytosis is more complex than
just caveolin. The ability of SV40 to induce a signaling cascade that
regulates its internalization demonstrates a role for ligand binding and
receptor-mediated signal transduction in the induction of caveolae
invagination and internalization (Pelkmans
et al., 2002
). Although common denominators of
caveolae/raft-mediated endocytosis include cholesterol sensitivity,
dynamin-mediated internalization, tyrosine kinase activation and regulation by
caveolin-1, other factors may sort cargo to diverse plasma membrane
caveolae/raft domains, segregate endocytic cargo within caveolae/raft domains
and regulate the invagination and intracellular targeting of
caveolae/raft-derived caveolar vesicles.
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Acknowledgments |
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References |
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