1101
Journal of Cell Science 112, 1101-1110 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS9832
Direct demonstration of the endocytic function of caveolae by a cell-free
assay
Anne Gilbert, Jean-Pierre Paccaud, Michelangelo Foti, Geneviève Porcheron, Jacqueline Balz
and Jean-Louis Carpentier*
Department of Morphology, CMU, 1 rue Michel Servet, CH-1211 Geneva 4, Switzerland
*Author for correspondence (e-mail: [email protected])
Accepted 12 October 1998; published on WWW 10 March
SUMMARY
The endocytic function of caveolae was challenged by
taking advantage of a cell-free assay directly measuring the
detachment of receptor-containing vesicles from isolated
plasma membranes. Plasma membranes from cultured
cells surface-labeled with 125I-cholera toxin (segregating in
caveolae) were isolated as described previously. Following
incubation of these labeled membranes in the presence of
nucleotide(s) and cytosol, a significant proportion of the
initially membrane-associated radioactivity was released
into the incubation medium in sedimentable form (14×106
g). Results of biochemical, morphological, and
fractionation analysis of the material containing the
released radioactivity directly demonstrated that caveolae
are plasma membrane domains involved in an endocytic
process and resulting in the formation of caveolae-derived
vesicles.
In addition, these studies allowed a direct comparison of
caveolae- and clathrin-coated pit-mediated endocytosis and
reveal that these two processes diverge in terms of kinetics,
cytosol and nucleotide requirements as well as in terms of
the density and size of the endocytic vesicles formed.
INTRODUCTION
chemotactic peptides and phorbol esters (Haigler et al., 1979;
Swanson, 1989; Bar-Sagi and Feramisco, 1986; Keller, 1990;
Carpentier et al., 1991).
However, while the clathrin-coated pit and macropinocytotic
pathways could account for the uptake of a wide variety of
receptors and a large volume of fluid, some molecules excluded
from these internalization processes are nevertheless
internalized (Huet et al., 1980; Montesano et al., 1982; Tran et
al., 1987; Hansen et al., 1991; Sandvig and van Deurs, 1994;
Parton et al., 1994). The nature of the structures involved in
these non-clathrin-coated pit, non-macropinocytotic uptake
processes remains highly controversial. Caveolae, the flaskshaped invaginations of the plasma membrane with a diameter
of ±60 nm, enriched in cholesterol and containing the
transmembrane protein caveolin (Montesano et al., 1982;
Rothberg et al., 1990, 1992; Parton, 1994), have for many years
represented an attractive candidate for such an alternative
endocytic pathway. In endothelial cells, these structures were
proposed to play a role not only in endocytosis but also in
subsequent transcytotic processes (Palade, 1953; Bruns and
Palade, 1968; Milici et al., 1987; Predescu et al., 1997;
Schnitzer et al., 1996). In other cell types, in spite of
observations favoring endocytosis of GPI-anchored receptors
and gangliosides via caveolae (Montesano et al., 1982; Tran et
al., 1987; Raposo et al., 1989; Keller et al., 1992), the budding
of these invaginations into primary endocytic vesicles is
refuted by other studies assigning them either a potocytotic
Internalization of cell surface receptors occurs via a complex,
multistep process termed receptor-mediated endocytosis.
Classically, this process is mediated by internalization gates
decorated on their cytoplasmic leaflet with clathrin: the
clathrin-coated pits (Goldstein et al., 1985). On the basis of
quantitations revealing that the rate of clathrin-dependent
endocytosis can account for total fluid phase uptake by cells,
it was proposed that the function of clathrin-coated pits was
not limited to the uptake of surface associated receptors but
that these plasma membrane domains were also responsible for
total bulk fluid and plasma membrane endocytosis (Marsh and
Helenius, 1980; Griffiths et al., 1989). This proposal has,
however, been questioned based on evidence supporting the
existence of alternative clathrin-independent endocytic
pathways. Among these, macropinocytosis, which involves the
formation of cytoplasmic projections rich in cytoskeleton
elements and results in the formation of large intracellular
vacuoles (for review see Swanson and Watts, 1995), was the
first pinocytotic process to be described (Lewis, 1931).
Macropinocytosis is not only responsible for the constitutive
uptake of a large volume of fluid and for receptor-mediated
endocytic events in specialized cell types (e.g. neutrophils and
macrophages) (Swanson, 1989; Swanson and Watts, 1995;
Carpentier et al., 1991) but, in addition, it can be induced in
different cell types via stimulation by growth factors,
Key words: Caveolae, Clathrin, Endocytosis
1102 A. Gilbert and others
function or no dynamic function at all (for reviews see Severs,
1988; van Deurs et al., 1993; Anderson, 1993; Sandvig and van
Deurs, 1994). In addition to (or alternative to) caveolae,
another endocytic pathway involving structures with a diameter
undistinguishable from that of clathrin-coated pits but lacking
clathrin was recently proposed. These structures were initially
observed in conditions with severe perturbations of the
clathrin-coated pit pathway not accompanied by a major effect
on bulk fluid endocytosis or specific adsorptive endocytic
processes (Daukas and Zigmond, 1985; Hansen et al., 1991;
Madshus et al., 1987; Sandvig et al., 1987; Oka et al., 1989).
Whether this pathway is compensatory to the loss of the
clathrin-associated pathway or whether it is constitutive
remains a subject of debate (Cupers et al., 1994; Damke et al.,
1995).
In view of these controversies, the aim of the present work
was to directly test the endocytic function of caveolae in nonendothelial cells. To that end, we took advantage of a recently
developed cell-free assay allowing the direct comparison of
receptors concentrated in caveolae with those selectively
associated with clathrin-coated pits. Our results demonstrate
that caveolae are endocytic structures leading to the formation
of vesicles detached from the plasma membrane and provide
information on the requirements for caveolae-derived vesicle
formation.
MATERIALS AND METHODS
Materials
Culture dishes were from Becton Dickinson and Thermanox
coverslips (15 mm round) were from NUNC (Naperville IL). The
following
culture
reagents:
DMEM
medium,
FCS,
penicillin/streptomycin, non essential amino acids and EDTA were
purchased from Gibco laboratories (Grand Island, NY). Sephadex G25 was from Pharmacia (Uppsala, Sweden). 125Iodine was from
Amersham (Buckinghamshire, UK) and IODO-GENTM from Pierce
(Rockford, IL); α2 macroglobulin and cholera toxin B-subunit (CT)
were from Calbiochem (La Jolla, CA). The following reagents were
obtained from Sigma (St Louis, MO): nucleotides and analogues,
Nycodenz, poly-L-lysine, Hepes, EDTA, DTT, BSA fraction V.
Glutaraldehyde was from Fluka Chemie (Buchs, Switzerland), agar
from Difco Laboratories (Detroit Michigan, USA). Cytosol was
extracted from calf brain as described by Lin et al. (1991).
Cell culture
3T3 L1 fibroblasts were grown to confluence in DMEM medium
supplemented with 10% FCS, penicillin/streptomycin and non
essential amino acids. Fibroblasts required for α2-macroglobulin in
vitro experiments were incubated overnight in FCS depleted medium
to remove most cellular bound α2-macroglobulin derived from serum.
Iodination
The B-subunit of cholera toxin (CT) and α2-macroglobulin were
radiolabeled with 125Iodine using IODO-GEN and according to the
manufacturer’s indications. The labeled ligands were separated from
125Iodine by gel filtration on a PD10 Sephadex G-25 column using
0.1 M sodium phosphate buffer (pH 7.4) as eluent.
125I-α -macroglobulin-trypsin
complexes (125I-α2-M) were
2
prepared as described previously (Gliemann et al., 1983; Gilbert et
al., 1997).
Preparation of 125I-CT and 125I-α2-M labeled membranes
Purified plasma membrane preparations were prepared according to
the method of Gilbert et al. (1997). Incubations with 125I-CT were
done in serum free culture medium containing 2% of albumin. When
125I-α -M was used as a ligand, the buffer was made of 124 mM NaCl,
2
4.7 mM KCl, 2.5 mM CaCl2, 1.25 mM MgCl2, 2.5 mM H2PO4−/
HPO42−, 1.25 mM Na2SO4, 25 mM Hepes and 5% albumin (pH 7.6)
at 4°C (Gliemann and Davidsen, 1986). Fibroblasts in suspension
were incubated 2 hours at 4°C in the presence of 125I-ligand and
unbound 125I-ligand was removed by centrifugation (5 minutes, 1000
rpm). All operations were performed at 4°C. Incubated cells were
sedimented for 1 hour onto poly-L-lysine (1 mg/ml)-coated coverslips
(15 mm diameter) stuck to the bottom of 35 mm Petri dishes. Medium
was aspirated and 1 ml of binding buffer (containing at least 2% BSA)
was added for 30 minutes. Adherent cells were then washed twice in
buffer A (50 mM Hepes, pH 7.4, 100 mM NaCl) and twice in buffer
B (25 mM Hepes-KOH, 25 mM KCl, 2.5 mM Mg acetate, 0.2 mM
DTT, pH 7.0) before being disrupted in buffer B by gentle sonication
(Branson, sonifier 250). To remove cellular debris, coverslips were
immediately rinsed in buffer B.
Plasma membrane budding
Five coverslips with adherent plasma membranes were fixed with
double-sided tape to a plastic support. The support was next turned
upside down in a Petri dish sitting on ice and containing 3.5 ml of
buffer B supplemented or not with factors to be tested. Petri dishes
containing the sets of 5 coverslips were incubated for various periods
of time at 37°C or on ice (which results in a temperature of 8-10°C
at the membrane level). Two sets of 5 coverslips were used per
condition. At the end of the incubation, the coverslips were cooled by
returning the Petri dishes to ice when needed. Coverslips were next
rinsed in buffer B at 4°C. Incubation mixtures and rinsing buffer were
pooled and centrifuged at 10×103 g, then the supernatants were
collected and centrifuged at 14×106 g in an 70.1 Ti rotor (Beckman
ultracentrifuge L8-M, instrument Co, Fullerton, CA). Supernatants of
this second centrifugation were collected and pellets of the two
centrifugations as well as coverslip adherent membranes were
solubilized in 0.5 M NaOH. The radioactive content of each fraction
was measured in a Beckman Gamma 5500 counter. Radioactivity
present in each fraction (collected and pooled from the two sets of
five coverslips) was expressed as a percentage of the total radioactivity
associated with these ten coverslips before incubation.
Electron microscopy
Thin section electron microscopy of intact cells
Fibroblasts incubated in the presence of CT-gold (10 nm) prepared as
previously described (Dickson et al., 1981; Tran et al., 1987), were
fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for
30 minutes at room temperature. They were next washed several times
in the cacodylate buffer and further processed for electron
microsocopy as previously described (Tran et al., 1987).
Quick frozen rotatory replicas of membrane
3T3-L1 fibroblasts surface-labeled at 4°C with CT-gold were
sedimented on poly-L-lysine (1 mg/ml) coated nickel grids. Purified
plasma membranes were prepared as described before (Lin et al.,
1991; Carpentier et al., 1993). Replicas were prepared by the quickfreeze rotary shadowed technique of Heuser (1980).
Vesicles
To visualize the content of the pellet obtained through plasma
membrane budding, coverslips with adherent membranes (12× 5
coverslips) were incubated with 1 mg/ml of cytosol and 1 mM of ATP
for 10 minutes at 37°C and the incubation medium was submitted to
high speed centrifugation as described above. The pellet was then
fixed with 0.5% glutaraldehyde in 0.1 M cacodylate, pH 7.0,
embedded in agar, stained by Kellenberger tannic acid and embedded
in Epon. Thin sections of pellet obtained from the cytosol fraction
were used as control.
In vitro formation of caveolae-derived vesicles 1103
Nycodenz density gradient centrifugation
Pellets resulting from high-speed centrifugation of the incubation
medium (see above) were resuspended by Dounce homogenization in
1 ml of buffer B. Suspensions from 125I-CΤ were deposited in the
bottom of the tubes and overlaid with a discontinuous gradient
consisting of 0.9 ml of 1.25/1.17/1.13/1.02 g/ml Nycodenz. After
centrifugation at 39000 rpm for 16 hours, the gradient was cut and the
fractions were collected. The radioactivity present in each fraction
was measured.
EDTA washes and acid washes
Fibroblasts were detached from culture flasks and resuspended in
binding buffer as described above. Duplicate samples of 1.5×106
cells/ml were incubated in the presence of 125I-α2-M or 125I-CT for 2
hours at 4°C at the end of which the medium was removed by
centrifugation (5 minutes, 1000 rpm) and replaced by fresh incubation
medium in which a second spin was carried out for various periods
of time at 37°C in the absence of 125I-α2-M or 125I-CT. Incubation
was terminated by diluting samples in ice-cold PBS and centrifuging
at 4°C. For 125I-α2-M internalization, pellets were resuspended three
times in cold 50 mM EDTA (in PBS solution) for 20 minutes to
remove cell-surface bound ligand. Pellet (internalized 125I-α2M) and
the three supernatants were counted. Internalization was expressed as
a percentage of the cells bound plus the EDTA washes.
125I-CT internalization was estimated in 3T3L1 fibroblasts as
described for 125I-α2-M internalization except that the pellets were
resuspended three times in cold PBS, pH 1.5, for 20 minutes to
remove cell-surface-bound ligand.
RESULTS
CT is segregated in caveolae on isolated plasma
membrane
Previous studies carried out on thin section and freezefracture replicas have shown that α2-macroglobulin almost
exclusively associated with clathrin-coated pits while
cholera toxin was preferentially segregated in caveolae on
the surface of target cells (Dickson et al., 1981; Montesano
et al., 1982; Carpentier et al., 1985; Tran et al., 1987; Parton,
1994; Parton et al., 1994) (Fig. 1). To establish whether these
different surface localizations corresponded to different
internalization pathways we took advantage of a cell-free
system which we recently developed to track in vitro
formation of clathrin-coated vesicles (Gilbert et al., 1997).
The experimental set up was derived from the one of Lin et
al. (1991): 3T3-L1 fibroblasts were surface-labeled in
suspension with CT tagged with colloidal gold for 2 hours
at 4°C, sedimented on poly-L-lysine-coated grids or
coverslips, sonicated and extensively washed so that only
plasma membranes remained adhered to the coverslips or
grids. As shown on rapid freeze deep-etched replicas, the
inner surfaces of plasma membranes were cleaned of
cytoplasmic organelles except for the cytoskeletal elements.
Clathrin-coated pits and flat clathrin lattices as well as
caveolae with their typical onion-like ridges (Peters et al.,
1985; Rothberg et al., 1992; Moore et al., 1987) remained
well preserved (Fig. 1). As previously demonstrated (Tran et
al., 1987; Montesano et al., 1982; Carpentier et al., 1985;
Parton, 1994; Parton et al., 1994), following a 2 hour
incubation at 4°C, CT tagged with colloidal gold was
preferentially associated with caveolae (Fig. 1).
125I-CT
is released into the incubation medium in a
sedimentable form via the formation of vesicles
distinct from clathrin-coated vesicles
When coverslips with attached 125I-CT-labeled membranes
were incubated for various periods of time at 37°C, in the
presence of cytosol (for unfractionated cytosol 1 mg/ml of
protein was used according to the method of Lin et al., 1991)
and/or nucleotides, radioactivity appeared in the incubation
medium in a time- and nucleotide-dependent fashion (Fig. 2).
Similarly to what was previously described in the case of 125Iα2-M, a minimal amount (<3%) of the membrane-associated
125I-CT was released into the incubation medium in a soluble
form, while less than 3% was sedimentable by low speed
centrifugation (Fig. 2). High speed sedimentable radioactivity
recovered from membranes labeled with 125I-CT ranged from
≈2% to 12% depending on the incubation conditions, in sharp
contrast with the range of ≈3% to 60% observed in the case of
125I-α2-M-labeled membranes (Fig. 2 and Gilbert et al., 1997).
Following centrifugation through a Nycodenz gradient,
>75% of the sedimentable radioactivity was recovered in the
light fraction (1.02-1.13) (Fig. 3) which corresponds to the
density of caveolae in sucrose gradients (Chang et al., 1994;
Lisanti et al., 1994). Similar results were obtained whether the
incubation medium was deposited in the bottom of the
centrifugation tube (flotation gradient) or laid over the gradient
(Fig. 3). This distribution was clearly different from that of
125I-α2-M and 125I-transferrin-containing structures in a
similar gradient (Fig. 3 and Gilbert et al., 1997). Moreover, a
marker of the plasma membrane (alkaline phosphodiesterase)
which was detected in a microsomal fraction of the same
density as the ones where 125I-CT was recovered (Gilbert et
al., 1997), was not detected in the pellet (data not shown).
Based on the recently described lack of this plasma membrane
enzyme in caveolae fractions (Gafencu et al., 1998), these
observations favor a specific detachment of caveolae from the
plasma membrane.
Direct observation, at the EM level, of the pellet collected
at the end of an incubation in the presence of 1 mM ATP and
cytosol indicated that it was almost exclusively composed of
vesicles, most of which were not clathrin-coated (Fig. 4).
Incubations carried out in the absence of cytosol and/or
nucleotide did not result in the formation of any visible pellet
and vesicles were absent from a pellet obtained by
ultracentrifugation of cytosol alone. Vesicles had a mean
diameter of 71.3±9.9 nm consistent with the diameter of
vesicles derived from caveolae whereas the mean diameter of
surface-associated caveolae reached a value of 69.4±8.7 nm
(Fig. 5) (van Deurs et al., 1989). Following surface labeling
with CT-colloidal gold (Tran et al., 1987), the mean size of
colloidal gold labeled vesicles showed a mean diameter of
69.0±7.3 nm again consistant with the size expected from
caveolae-derived vesicles (Fig. 4).
Previous attempts to reveal the presence of clathrin in the
vesicular pellet by biochemical approaches were
unproductive due to the insufficient quantities of membrane
being available which hampered any protein detection
(Gilbert et al., 1997). For the same reasons, biochemical
attempts to reveal the presence of caveolin in the vesicular
pellet by immunoblotting reactions were not successful.
Such failure cannot, however, be attributed to the absence of
1104 A. Gilbert and others
the two types of coats which, as directly shown
morphologically, are present on the cytoplasmic side of the
respective invaginations on isolated membrane preparations
(Fig. 1).
Taken together, these data demonstrate that sedimentable
was released into the incubation medium via the
formation of vesicles whose size and density are compatible
with that of caveolae-derived vesicles.
125I-CT
Fig. 1. Electron microscopic localization of CT in caveolae. Cultured 3T3 L1 fibroblasts were incubated (A and C) or not (B) for 2 hours at 4°C
in the presence of CT-gold (10 nm) before being prepared for either thin section electron microscopy (A) or quick freeze, rotary shadowing (B
and C) as described in Materials and Methods. (A and B) Gold particles are associated with caveolae (arrows) seen either on thin sections (A)
or on a three-dimensional view of the inner aspect of the plasma membrane where caveolae (arrows) show their typical ridges. In this latter
case, colloidal gold particles are seen by transparency and appear white because the negative was reversed. (C) Comparison of the general
organisation of caveolae (arrows) seen from the inside of the cell with that of the typical honeycomb structure of the clathrin coat (cp).
In vitro formation of caveolae-derived vesicles 1105
10
15
15
Fig. 2. Release of 125I-CT from adherent plasma membranes.
Coverslips coated with 125I-CT membranes were prepared as
described in Materials and Methods and incubated in buffer B
containing CaCl2 (1 mM) and supplemented or not with cytosol (1
mg/ml), ATP (1 mM) or GTP (1 mM) for the indicated periods of
time at 37°C. Incubation medium was first submitted to a low speed
centrifugation (10×103 g) and the radioactive content of the pellet
was measured. Second, the supernatant resulting from the first
centrifugation was submitted to high speed centrifugation (14×106 g)
and radioactivity present in the supernatant and in the pellet were
measured. Sedimentable (low speed and high speed) and soluble
radioactivity are expressed as a percentage of the total radioactivity
initially associated with plasma membranes (= total sedimentable
radioactivity + soluble radioactivity + radioactivity remaining
associated with the membranes at the end of the incubation period).
Sedimentable 125I-CT and 125I-α2-M are released into
the incubation medium with different kinetics and
different susceptibility to filipin
Under conditions of sustained release of the vesicles containing
the radioactive ligand, a maximum of less than 12% of the
initially membrane associated 125I-CT was released into the
incubation medium. The kinetics of release of this ligand were
superposable with those of 125I-CT internalization in intact
3T3-L1 cells but were very different from those of 125I-α2-M
release (Fig. 6) (Tran et al., 1987). Indeed, the release of
sedimentable 125I-α2-M occurred very rapidly and included
more than 50% of the initially bound radioactivity (Fig. 6).
Here again, the percentage of 125I-α2-M released was
superposable with that of 125I-α2-M internalized in intact 3T3Fig. 3. Density distribution in a Nycodenz gradient of the
sedimentable radioactivity resulting from the budding reaction.
Fibroblasts were incubated with radiolabeled CT or α2-M at 4°C.
Plasma membrane coated coverslips were prepared and incubated for
15 minutes at 37° in buffer B containing containing CaCl2 (1 mM),
cytosol and ATP. Pellet resulting from high speed centrifugation of
incubation medium was resuspended by Dounce homogenization in 1
ml of buffer B and (A) was deposited in the bottom of the tube and
overlaid with the gradient as described in Materials and Methods or
(B) loaded on a Nycodenz discontinous gradient (Gilbert et al.,
1997). Data show the percentage of radiolabeled ligand recovered in
each fraction of the gradient at the end of the centrifugation. (A) n=3,
(B) n=4. C is derived from Gilbert et al. (1997).
20
0
1.25/load
20
density (g/ml)
60
B
CT
40
20
0
1.21/1.30
15
density (g/ml)
100
C
α2M
80
60
40
20
0
density (mg/ml)
1.25/1.30
10
1.17/1.25
5
40
1.17/1.21
+
+
+
1.17/1.25
-
1.13/1.17
+
+
-
1.13/1.17
+
+
-
60
1.07/1.13
+
+
-
CT
1.13/1.17
+
+
-
A
1.02/1.13
cytosol
ATP
GTP
min at 37°C
Recovered radioactivity (%)
0
Recovered radioactivity (%)
5
Recovered radioactivity (%)
80
load/1.07
15
load/1.13
20
Released radioactivity
(% of total)
L1 cells estimated either by quantitative EM analysis (Tran et
al., 1987) or by the EDTA-wash technique (Gliemann and
Davidsen, 1986) (Fig. 6).
Filipin is a sterol-binding drug which on the one hand
flattens invaginated caveolae and unravels their striated coat,
sed (14 106 gmin)
sed (10 103 gmin)
soluble
1106 A. Gilbert and others
Fig. 4. View at the electron microscopic
level of the pellet obtained by high speed
sedimentation of the incubation medium.
(A and B) Cultured 3T3 L1 fibroblasts
were incubated for 2 hours at 4°C in the
presence of CT (A) or CT-gold (10 nm)
(B). Plasma membrane coated coverslips
were incubated for 10 minutes at 37°C in
buffer B containing cytosol (1 mg/ml),
CaCl2 (1 mM) and ATP (1 mM).
Incubation medium was then submitted to
high speed centrifugation (14×106 g). The
pellet was fixed with glutaraldehyde 2.5%
in cacodylate buffer (pH 7.0) and
processed for thin section electron
microscopy as described in Materials and
Methods. The pellet consists essentially of
vesicles among which several are gold
labelled (B). The general appearance was
similar whether or not the cells were
preincubated with CT.
and inhibits the uptake of folate by cells (Rothberg et al., 1992;
Chang et al., 1992), but on the other hand perturbs neither
clathrin-coated pit organisation nor the uptake of ligands
A
50
Non-clathrin-coated vesicles
mean: 71.3 +/- 9.9 nm
Percent vesicles
40
30
20
10
0
30 41 53 67 80 94 107 121 133
Size (nm)
B
50
Caveolae
mean: 69.4 +/- 8.7 nm
Percent vesicles
40
30
20
10
0
30 41 53 67 80 94 107 121 133
Size (nm)
through this pathway (Schnitzer et al., 1994). When applied to
isolated membranes exposed to cytosol + ATP, filipin (0.25
µg/ml) only slightly affected 125I-α2-M release in a
sedimentable form in the incubation medium (14.3%
inhibition) while it reduced by more than 50% the 125I-CT
released in sedimentable form (63.9% inhibition) (Fig. 7).
Thus, taken together with the previously described initial
preferential association of α2-M and CT with clathrin-coated
pits and caveolae, respectively, and the above-mentioned
structural evidence that 125I-α2-M and 125I-CT are released via
two different populations of vesicles, the respective kinetics of
the two events and their different susceptibility to filipin
demonstrate directly the existence of two parallel but different
internalization pathways involving clathrin-coated pits and
caveolae.
Cytosol, nucleotide and Ca2+ requirements for the
formation of 125I-CT-containing vesicles
In contrast to what was observed in the case of 125I-α2-Mcontaining vesicles, the formation of 125I-CT-containing
vesicles did not occur in the sole presence of ATP or GTP (Fig.
8 and Gilbert et al., 1997) and non-hydrolysable analogues of
GTP (GTPγS and GMP-PNP) were similarly inactive (Fig. 8).
Caveolae-derived vesicle formation required cytosol and the
most efficient stimulation was observed when cytosol, ATP and
GTP were all present in the incubation medium or in the
presence of cytosol and GTPγS (Fig. 8). These results are
therefore different from those obtained when 125I-α2-M was
Fig. 5. Size distribution of surface-connected caveolae (B) and
vesicles recovered in the incubation medium (A). (B) The diameter
of 189 surface-connected caveolae photographed from 50 cell
sections were measured. (A) Plasma membrane coated coverslips
were incubated for 10 minutes at 37°C in buffer B containing cytosol
(1 mg/ml), CaCl2 (1 mM), and ATP (1 mM). The incubation medium
was then submitted to high speed centrifugation (14×106 g). The
pellet was fixed with glutaraldehyde 2.5% in cacodylate buffer (pH
7.0) and processed for thin section electron microscopy as described
in Materials and Methods. Measurements were carried out on a total
of 203 non-clathrin-coated vesicles.
In vitro formation of caveolae-derived vesicles 1107
25
75
75
Control
0
0
0
2
4
6
8
Time (min at 37°C)
10
Sedimentable radioactivity (%)
25
25
Sedimentable radioactivity
(Percent of total) ( )
( EM; EDTA wash)
50
50
125
Ι−α2 M internalized (%)
A
Filipin (0.25 µg/ml)
20
15
10
5
0
20
125
acid wash)
15
15
10
10
5
5
(
I-CT internalized (%)
B
0
Sedimentable radioactivity
(Percent of total) (䉭)
20
α2M
CT
Fig. 7. Effect of filipin on clathrin-coated vesicle and caveolae
formation. 125I-α2-M and 125I-CT labeled plasma membranes
adherent to coverslips were incubated or not (control) for 30 minutes
at 4°C in buffer B supplemented with 0.25 µg/ml of filipin and then
incubated with cytosol + ATP for 10 minutes at 37°C. At the end of
the incubation period, medium was centrifuged (14×106 g) and the
radioactivity present in the pellet was measured and expressed in
terms of the percentage radioactivity initially associated with plasma
membranes.
0
0
5
10
15
Time (min at 37°C)
20
Fig. 6. Kinetics of 125I-α2-M and 125I-CΤ release in the incubation
medium. (A) The kinetics of 125I-α2-M release (sedimentable at
14×106 g) from isolated plasma membrane exposed for various
periods of time at 37°C to ATP (1 mM) + GTP (1 mM) + cytosol (1
mg/ml protein) (cell-free assay) was compared with the kinetics of
125I-α M internalization in intact cells as measured morphologically
2by quantitative EM autoradiography (calculated from Tran et al.,
1987) or biochemically by the EDTA-wash technique. (B) The
kinetics of 125I-CΤ release (sedimentable at 14×106 g) from isolated
plasma membrane exposed for various periods of time at 37°C to
ATP (1 mM) and cytosol (1 mg/ml protein) was compared with the
kinetics of 125I-CΤ internalization in intact cells as measured
biochemically by the acid-wash technique (n=2).
used as a ligand and when the formation of clathrin-coated
vesicles was under study (Gilbert et al., 1997). By contrast,
similarly to what was observed in the case of 125I-α2-M
containing vesicles, the addition of 1 mM EDTA to the
incubation medium did not affect the release of 125I-CT,
indicating that Ca2+ is not required for the formation of
caveolae-derived vesicles (data not shown).
DISCUSSION
One way of addressing the controversial question of whether
caveolae represent structures involved in receptor-mediated
endocytic processes in non-endothelial cells involves making
use of cell-free systems allowing the formation of endocytic
vesicles from isolated membranes to be studied. We recently
developed such an assay, which directly measures the
formation of clathrin-coated vesicles and allows detailed
characterization of the endocytic machinery governing this
process (Gilbert et al., 1997). It consists of: (1) surface-labeled
cultured fibroblasts (3T3-L1) with a radioactive ligand
preferentially segregated in clathrin-coated pits (125I-α2-M);
(2) preparing plasma membranes by sonication of the cells
attached to a poly-L-lysine-coated substratum (Lin et al.,
1991); (3) incubating the membrane in medium containing
nucleotides and cytosol which allows the detachment of
endocytic vesicles in the medium; and (4) ultracentrifugation
of the medium to recuperate a pellet of 125I-α2-M-containing
endocytic vesicles. This assay has the advantage of directly
measuring the formation of endocytic vesicles rather than the
loss of a substance as in Lin’s assay. By tracking different
radioactive ligands segregated in distinct domains of the
plasma membrane, our assay allows a specific analysis of the
implication of these respective surface domains in endocytosis.
As previous studies have extensively documented the
preferential segregation of cholera toxin in caveolae
(Montesano et al., 1982; Tran et al., 1987), 125I-CT was used
as a reporter molecule for caveolae in this cell-free assay. The
results obtained indicate that caveolae are endocytic structures
whose pinching off from the plasma membrane results in the
formation of vesicles. Our results also demonstrate that
caveolae- and clathrin-coated pit-mediated endocytic processes
are distinct processes which can be differentiated in terms of
kinetics, cytosol and nucleotide requirements as well as in
terms of the density and size of the endocytic vesicles formed.
Following incubation of 125I-CT-labeled membranes in the
Fig. 8. Effect of cytosol, ATP, GTP, GTPγS or GMPPNP on caveolae-derived vesicle formation. 125I-CΤlabeled plasma membrane were incubated for 15
minutes at 37°C in buffer B containing CaCl2 (1 mM)
and supplemented or not with cytosol, ATP (1 mM),
GTP (1 mM), GMP-PNP (1 mM) or GTPγS (1 mM)
as indicated. At the end of the incubation period,
medium was centrifuged (14×106 g) and the
radioactivity present in the pellet was measured and
expressed in terms of the percentage 125I-CT initially
associated with the plasma membranes (n=3).
Sedimentable radioactivity (%)
1108 A. Gilbert and others
15
10
5
0
cytosol
+
+
+
+
-
-
-
-
-
-
+
ATP (1mM)
-
+
-
+
-
+
-
+
-
-
-
GTP (1mM)
-
-
+
+
-
-
+
+
-
-
-
-
-
-
-
-
-
-
-
GTP γ S (1mM)
+
+
-
-
-
-
-
-
-
-
+
-
-
GMP PNP (1mM)
presence of cytosol and nucleotides, radioactivity released into
the incubation medium represents intact 125I-CT contained in
sealed vesicles for the following reasons: (a) it is sedimentable
at 14×106 g demonstrating it is not soluble but associated with
membranous material; (b) it is not sedimentable at 10×103 g
indicating that it is not associated with cell fragments or large
membranous segments; (c) morphological analysis of the pellet
reveals that it is composed essentially of vesicles; (d) when
experiments are conducted in the presence of CT-gold, gold
particles are recovered inside vesicles forming the pellet at the
end of the ultracentrifugation of the incubation medium.
Not only is 125I-CT released into the incubation medium via
the formation of vesicles but, in addition, these vesicles are
distinct from those derived from clathrin-coated pits. Indeed:
(a) in Nycodenz gradients 125I-CT-containing structures are
recovered at densities distinct from those where clathrin-coated
vesicles (tagged with either 125I-α2-M or 125I-transferrin) are
concentrated; (b) the diameter of CT-containing vesicles
recovered in the incubation medium is smaller (70 nm) than
that of clathrin-coated vesicles (90 nm) (Gilbert et al., 1997);
(c) the respective release of 125I-CT and 125I-α2-M differ not
only in terms of kinetics, but also in terms of nucleotide and
cytosolic requirements.
Finally, 125I-CT-containing vesicles are clearly derived from
caveolae since: (a) the diameter of the vesicles is homogenous
and consistent with the size of vesicles that result from
caveolae pinching off, which argues against a non-specific
vesiculation; (b) when centrifuged through a Nycodenz
gradient, more than 85% of the radioactivity released
equilibrates at a density consistent with that of caveolaederived vesicles; (c) the formation of 125I-CT-containing
vesicles is sensitive to treatment with the sterol-binding drug
filipin, known to flatten caveolae, to disintegrate their coat and
to inhibit potocytosis (Rothberg et al., 1992; Chang et al.,
1992), while by contrast, the formation of 125I-α2-Mcontaining vesicles is poorly affected by the drug; (d) the
kinetics of release of 125I-CT in the medium are superposable
with those of 125I-CT uptake in intact cells and previously
shown to occur via the formation of caveolae-derived vesicles
(Montesano et al., 1982; Tran et al., 1987; Parton et al., 1994);
(e) alkaline phosphodiesterase, absent from caveolae (Gafencu
et al., 1998), was detected in fractions containing the plasma
membrane but not in the vesicular pellet.
The potential implication(s) of caveolae in cellular
trafficking in general and in endocytosis in particular remains
highly controversial. In endothelial cells, these structures have
been proposed to play a role not only in endocytosis but also
in subsequent transcytotic processes (Palade, 1953; Bruns and
Palade, 1968; Milici et al., 1987; Predescu et al., 1997).
Similarly, in a series of other cell types, we and others have
provided morphological evidence for a role of caveolae in
endocytosis (Huet et al., 1980; Montesano et al., 1982; Tran et
al., 1987; Raposo et al., 1989; Parton, 1994; Parton et al., 1994;
Carpentier et al., 1989; Keller et al., 1992, Kiss and Geuze,
1997). Such a function has been questioned, however, by others
who considered caveolae as (semi-)permanent differentiations
of the cell surface (Bundgaard et al., 1983; Frokjaer-Jensen et
al., 1988; Frokjaer-Jensen, 1991; van Deurs et al., 1989, 1993).
A third alternative function for caveolae was proposed recently,
in which caveolae are able to close transiently without moving
away from the plasma membrane via a process named
potocytosis (Anderson, 1993). The present data, demonstrating
directly the ability of caveolae to fission and form vesicles
detached from the plasma membrane of cultured fibroblasts,
support a role for these membrane invaginations in
endocytosis. These results are in agreement with those
obtained by a cell-free assay that makes use of a preparation
of caveolae-enriched membrane fractions and demonstrates
that, in endothelial cells, an endocytic function can be devoted
to these structures (Schnitzer et al., 1996). Such an endocytic
function does not exclude the existence of a potocytotic process
although in the course of potocytosis the fission allowing the
detachment of the endocytic vesicle ought not to occur.
Formation of caveolae-derived vesicles is a very slow
process, significantly less efficient than clathrin-coated vesicle
formation. In this respect, the present data confirm our previous
observations since a maximum of ≈15% of the initially bound
125I-CT can be recovered in the medium after 20 minutes of
In vitro formation of caveolae-derived vesicles 1109
incubation at 37°C, in contrast to up to 60% after <5 minutes
of incubation when 125I-α2-M is used as a ligand. Even if this
process is relatively slow it may nevertheless contribute to the
uptake of a large amount of material internalized by cells if a
large proportion of the cell surface is occupied by caveolae as
is the case in specific cell types, e.g. endothelial cells,
adipocytes and muscle cells. Finally, the relative contribution
of caveolae in endocytosis can be modulated in response to
stimuli since, for example, the number of caveolae increases
by ninefold upon differentiation of 3T3-L1 fibroblasts to
adipocytes with a corresponding increase in caveolin
expression (Fan et al., 1983; Scherer et al., 1994).
The subsequent fate of caveolae-derived vesicles also
remains a subject of debate: on the one hand GPI-anchored
folate receptor and VIP21/caveolin could not be observed
within endosomal elements (Rothberg et al., 1990, 1992; van
der Goot, 1997) while on the other hand CT-gold was
recovered in the same endosomes as those containing α2-Mgold (Tran et al., 1987). In addition, GPI-anchored proteins
have been shown to internalize into endosomes (Hjelle et al.,
1991; Birn et al., 1993; Turek et al., 1993). Further studies are
required to resolve this issue. The physiological relevance of
the endocytic function of caveolae also remains an open
question. It is tributary to the elucidation of the function of
caveolae themselves which have been proposed to play a role
in various cellular events including signal transduction (Lisanti
et al., 1994), potocytosis (Anderson, 1993), calcium regulation
and signaling (Fujimoto, 1993; Fujimoto et al., 1993).
The exposure of the cytosolic side of the isolated membranes
allowed us to assay the respective influence of ATP, GTP and
cytosol on caveolae-derived vesicle formation. While neither
ATP nor GTP potentiated the effect of cytosol, the conjunction
of both nucleotides enhanced the release of caveolae-derived
vesicles implying a complementary role for the two nucleotides
in creating optimal conditions for the formation of these
vesicles. The presence of cytosol was essential for this optimal
release: ATP and GTP alone or in combination were either
ineffective or only weakly stimulatory. These results confirm
the involvement of GTP in caveolae-derived vesicle formation
recently proposed by Schnitzer et al. (1996) and in addition
they reveal a role for ATP and cytosol in the process. However,
in contrast to previous observations (Schnitzer et al. 1996),
GTP hydrolysis does not seem to be required for the process
to be completed in our experimental set up. Since similar
discrepancies in terms of GTP hydrolysis requirements were
noted between results obtained with our assay and other assays
in the case of clathrin-coated vesicle formation (Gilbert et al.,
1997), the extensive cleaning of the membranes before use in
our experimental set up could be responsible for this
observation. Nevertheless, it must be stressed that our
observations agree with those obtained in studies of COPcoated vesicle formation. Here, addition of GTPγS to in vitro
intra-Golgi transport assays caused accumulation of COPIcoated vesicles (Malhotra et al., 1989) and addition of the nonhydrolyzable GTP analogue GMP-PNP to in vitro ER budding
assays resulted in the accumulation of COPII-coated vesicles
(Barlow et al., 1994).
Nucleotide and cytosolic requirements are distinct from
those recorded in the case of clathrin-coated vesicle formation
where GTP or ATP alone (GTP>ATP) were additive to the
effect of cytosol. In addition, the specific role of cytosol
appeared to be minor in the case of clathrin-coated vesicle
formation since, in contrast to what was observed in the case
of caveolae-derived vesicle formation, both ATP and GTP
could efficiently promote vesicle formation in the absence of
added cytosol (Gilbert et al., 1997).
In conclusion, the application of a recently developed cellfree endocytic assay to molecules segregated in caveolae, has
allowed the direct demonstration of the endocytic function of
these invaginated surface domains, the characterization of the
basic requirements for this function and the direct comparison
between this alternative endocytic pathway and the more
classical clathrin-coated pit pathway. Further studies are in
progress to take advantage of this assay to identify the cytosolic
factors involved and to dissect the machinery driving this
endocytic function.
We thank Ms L. Burkhardt for her skilled secretarial assistance.
This work has been supported by grant: 31-53686.98/1 from the Swiss
National Science Foundation.
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