Download Endocytosis Via Caveolae

Traffic 2002; 3: 311–320
Munksgaard International Publishers
Copyright C Munksgaard 2002
ISSN 1398-9219
Review
Endocytosis Via Caveolae
Lucas Pelkmans1,* and Ari Helenius1,*
Swiss Federal Institute of Technology Zürich (ETHZ), HPM1,
ETH Hoenggerberg, CH-8093 Zürich, Switzerland
* Corresponding authors: Lucas Pelkmans,
[email protected] and Ari Helenius,
[email protected]
Caveolae are flask-shaped invaginations present in the
plasma membrane of many cell types. They have long
been implicated in endocytosis, transcytosis, and cell
signaling. Recent work has confirmed that caveolae are
directly involved in the internalization of membrane
components (glycosphingolipids and glycosylphosphatidylinositol-anchored proteins), extracellular ligands
(folic acid, albumin, autocrine motility factor), bacterial
toxins (cholera toxin, tetanus toxin), and several nonenveloped viruses (Simian virus 40, Polyoma virus). Unlike
clathrin-mediated endocytosis, internalization through
caveolae is a triggered event that involves complex signaling. The mechanism of internalization and the subsequent intracellular pathways that the internalized
substances take are starting to emerge.
Key words: caveolae, caveosomes, endocytosis, GPIanchored proteins, glycosphingolipids, lipid rafts, Simian Virus 40
Received 8 February 2002, accepted for publication 11
February 2002
While clathrin-mediated endocytosis constitutes the main
pathway for internalization of extracellular ligands and plasma
membrane components in most cell types, it has been recognized for some time that alternative, parallel uptake mechanisms also exist. These ‘clathrin-independent pathways’ have
been more difficult to study; hence, detailed information is
still scarce [for recent reviews see (1–3)]. In this review, we
focus on one of these alternatives: endocytosis via caveolae.
Our own interest in this field was evoked by the observation
that certain non-enveloped viruses such as Simian Virus 40
(SV40) use caveolae-mediated endocytosis to enter cells
(4–6). Similar uptake processes are likely to be involved in
many interesting endogenous processes including cholesterol homeostasis, recycling of glycosylphosphatidylinositol
(GPI)-anchored proteins, glycosphingolipid transport, and
transcytosis of serum components (7–12).
Caveolae
Caveolae were first identified in the 1950s by Palade (13) and
Yamada (14) due to their characteristic morphology observed
by electron microscopy of thin sections. They typically appear
as rounded plasma membrane invaginations of 50–80 nm in
diameter. Their composition, appearance and function are
cell-type dependent. In endothelial cells, caveolae can be
more constricted at the mouth, or they may contain a diaphragm that restricts diffusion (15,16). In muscle cells, caveolae are often observed in the form of composite clusters or
linear rows of multiple flask-shaped units involved in the formation of T-tubules (17,18). In epithelial tissue culture cells,
caveolae are open to the extracellular medium, do not contain a diaphragm, appear as single indentations or grape-like
structures, and are on average a bit smaller (19,20). Recent
video microscopy and fluorescence recovery after photobleaching (FRAP) analysis has shown that in these cells caveolae are stationary and held in place by the cortical actin
cytoskeleton underlying the plasma membrane (21,22). As
discussed below, only upon specific signals do they detach
from the membrane as an endocytic vesicle.
Although caveolae do not show an electron-dense layer on
their cytosolic surface in thin-section electron micrographs,
they do have a protein ‘coat’ composed primarily of a protein
called caveolin-1 (or caveolin-3 in muscle cells) (20). Caveolins are integral membrane proteins of 21 kDa. They have an
unusual topology in that the cytosolic N- and C-terminal domains are cytosolic connected by a hydrophobic sequence
that is buried in the membrane but does not span the bilayer
(23,24). Caveolins are palmitoylated in the C-terminal segment (25), they can be phosphorylated on tyrosine residues
(26), they bind cholesterol (27), and they form dimers and
higher oligomers (24). On the cytosolic surface they can be
visualized by electron microscopy as part of parallel, shallow
ridges in replicas obtained after shadowing (20,28).
Caveolins are essential for the formation and stability of caveolae: in their absence no caveolae are seen, and, when expressed in cells that lack caveolae, they induce caveolar formation (29). In addition to the plasma membrane, caveolins
are present in the trans-Golgi network (TGN) (23,30) and in
a newly discovered organelle called the ‘caveosome’ (21),
which will be discussed below. Upon extraction or oxidation
of plasma membrane cholesterol, caveolins re-localize to intracellular structures that can be endosomes, the Golgi complex, or the ER (18,31). When caveolins are over-expressed or
retained in the ER, they can also localize to intracellular lipid
droplets (32–34). Immunofluorescence with conformationspecific anti-caveolin antibodies suggests that the conformation of caveolin-1 on the plasma membrane and in caveosomes is similar, but different from that in the Golgi complex
(21,23).
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During endocytosis of caveolae, caveolin-1 moves along with
the vesicles into the cytosol with no visible remnant left in
the plasma membrane (6, 21, 35–37). Whether caveolin has
a direct role in the endocytic process is not clear. On the one
hand, it has been shown that N-terminally truncated or Nterminally GFP-tagged caveolin constructs serve as dominant
negative inhibitors of caveolar endocytosis during SV40 entry
(21,38). On the other hand, recent data indicate that the presence of caveolin-1 can actually slow down the endocytic process via caveolae (39,40). This implies that the role of caveolin may be to stabilize caveolae and thus to counteract an
underlying raft-dependent endocytic process. The finding
that certain ligands internalize via a lipid raft-dependent but
clathrin-independent mechanism in cells that lack caveolae,
has led to the postulation that lipid rafts can internalize independently of caveolae (41). In this context, it is significant that
caveolin knockout mice survive surprisingly well, although
their cells do not have detectable caveolae (42–44).
In addition to caveolins, caveolae are known to contain dynamin (45,46), a GTPase involved in the formation of clathrincoated vesicles (47). This molecule has been localized to the
neck of flask-shaped caveolar indentations (45,46), and is
therefore most likely involved in pinching off the caveolar vesicle in a way similar to its role in coated vesicle fission
(48,49). Recent video imaging of the dynamics of GFPtagged dynamin2 during SV40 entry showed that it is only
transiently recruited to virus-loaded caveolae (50). It appears
as small spots on the site of caveolae with a residence time
of about 8 s. Thus, dynamin is not a permanent component
of caveolae but rather belongs to a group of factors recruited
in response to specific signals. Also, caveolae contain the
molecular machinery for vesicle docking and fusion (51).
Caveolae are, moreover, rich in GPI-anchored proteins and
several receptor and nonreceptor protein tyrosine kinases
(52,53). Since these kinases are involved in signal transduction events, caveolae are thought to constitute especially active sites for signal transmission (9,54,55). Furthermore,
some of the kinases might be involved in regulating the internalization of caveolae, as will be discussed below.
The lipid composition of caveolae corresponds to that of lipid
rafts, i.e. caveolae are rich in cholesterol and sphingolipids
[extensively reviewed in (54,56)]. These are, in fact, essential
for the formation and stability of caveolae. If cholesterol is
removed from the plasma membrane, caveolae disappear
(20). Consistent with their high content of raft lipids, caveolae
resist solubilization by nonionic detergents at 4 æC. One may
thus define caveolae as caveolin-containing plasma membrane invaginations rich in raft lipids.
Caveolar Entry of Simian Virus 40
We will first concentrate on one of the most extensively
studied ligands for caveolar endocytosis, SV40, which uses
this pathway for infectious entry into the cell (4–6, 21, 38,
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50, 57–67). The entry process of this non-enveloped DNA
virus, analyzed in several laboratories including ours, is
emerging as a useful paradigm in the field.
SV40 has several advantages as a model ligand. The particle
itself is well characterized in terms of composition and structure. The X-ray structure of this non-enveloped DNA virus
has been determined at a resolution of 3.1Å and shows an
icosahedral shell with 360 subunits of the major coat protein
VP1 (62,66). The diameter of the particle is 50 nm. Although
replication of the virus is restricted to monkey cells, the entry
process and initial infection can be followed in most cell
types.
Uptake of SV40 occurs specifically by the caveolar pathway.
Even when cells are incubated with amounts of virus particles
by far exceeding receptor saturation, less than 5% of internalized particles is found to pass through clathrin-coated pits
and vesicles (4,21,57,61). When clathrin-dependent endocytosis was inhibited by expressing a dominant negative mutant of the EGF receptor pathway substrate 15 (Eps15), SV40
endocytosis and infection were not affected (50). In contrast,
when dominant negative mutants of caveolin were expressed, internalization or infection was not observed (21,38).
The interaction of the incoming virus particles with cells has
been studied by morphological techniques (electron microscopy and light microscopy), by biochemical techniques
(quantitative endocytosis assays) and by virological methods
(infection assays). Most of the studies have been performed
in tissue culture cells from green African monkeys. A variety
of inhibitors have been tested, as well as expression of dominant negative mutant proteins. Extensive use has recently
been made of video microscopy in live cells with fluorescently labeled virus particles and GFP-labeled cellular proteins. This has made it possible to follow single virus particles
during their journey into the cell. The stepwise process of
SV40 entry is today known in some detail.
Sequestration and internalization (Figure 1)
After binding to the plasma membrane via major histocompatibility (MHC) class I antigens (64), virus particles diffuse
laterally along the membrane, until trapped in caveolae (21).
Virus-containing caveolae are slightly smaller in diameter
than the virus-free caveolae (6). The reason may be the tight
interaction between the virus and the caveolar membrane; in
electron micrographs it almost looks like the viruses would
be ‘budding’ into the cell.
Virus particles stay in the caveolae for 20 min or more, after
which the caveolae pinch off and move as caveolin-coated
endocytic vesicles into the cytoplasm. Caveolae devoid of virus particles do not internalize (21). Interestingly, virus uptake
does not involve endocytosis of MHC class I antigens (68).
This implies the presence of a secondary receptor that mediates the tight binding of the virus to the caveolar membrane, and possibly triggers the signal needed to induce the
endocytic process (see below).
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Endocytosis via Caveolae
Figure 1: Initial stages of SV40 internalization via caveolae. After binding to the membrane, virus particles are mobile until trapped in
caveolae, which are linked to the actin cytoskeleton (step 1). In the caveolae, SV40 particles trigger a signal transduction cascade that leads
to local protein tyrosine phosphorylation and depolymerization of the cortical actin cytoskeleton (step 2). Actin monomers are recruited to
the virus-loaded caveolae and an actin patch is formed (step 3). Concomitantly, Dynamin is recruited to the virus-loaded caveolae and a
burst of actin polymerization occurs on the actin patch (step 4). Virus-loaded caveolae vesicles are now released from the membrane and
can move into the cytosol (step 5). After internalization, the cortical actin cytoskeleton returns to its normal pattern (step 6).
During the lag period preceding internalization, a complex
series of virus-induced events takes place. The arrival of the
SV40 particle in the caveolae triggers phosphorylation of
tyrosine residues in proteins associated with the caveolae
(50). However, the kinases and substrates involved remain to
be identified.
Transport to caveosomes
The caveolar vesicles have a diameter of 60–70 nm and contain single virus particles surrounded by a tight-fitting membrane (4). The local depolymerization of cortical actin seems
to be necessary for their passage deeper into the cytosol (50)
and the actin tail is not needed once the vesicle is released.
One effect of the activated signal transduction cascade is the
disassembly of nearby actin stress fibers. Subsequently, actin
is recruited to the virus-loaded caveolae as a small actin
patch, followed by bursts of actin polymerization resulting in
the transient appearance of actin ‘tails’ (average length about
1.5 mm) emanating from the virus-loaded caveolae. Dynamin
is also recruited to the sites of virus internalization but, as
already mentioned, it stays there only for a short period of
time (50). Another effect is the up-regulation of the primary
response genes c-myc, c-jun and c-sis (69).
These primary endocytic vesicles transfer their viral cargo,
presumably by membrane fusion, to larger, more complex
tubular membrane organelles that we have termed ‘caveosomes’ (21) (Figure 2). Since the uptake of virus particles is
relatively slow (half-time 90 min and maximal uptake after
3 h), accumulation of virus particles in caveosomes becomes
visible after about 40–60 min and continues for the following
3 h.
Studies with inhibitors and dominant negative mutants show
that the association of SV40 with caveolae, the tyrosine
phosphorylation, the recruitment of actin, the formation of
actin tails, and the association of dynamin are all necessary
for efficient closure of caveolar vesicles and ultimately for
infection of the cell (50,67). Most of the changes seem to be
transient; once the virus particles are internalized, the phosphotyrosines disappear, and the actin cytoskeleton returns to
the normal pattern.
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Caveosomes are pre-existing, caveolin-1-containing membrane organelles distributed throughout the cytoplasm. The
pH in caveosomes is neutral, and the membrane is rich in
cholesterol and glycosphingolipids ((21,70) (L. Pelkmans, C.
Buser, A. Helenius, unpublished results). Caveosomes do not
accumulate ligands endocytosed via clathrin-coated pits, or
components such as transferrin that cycle between endosomes and the plasma membrane. Nor do they accumulate
detectable amounts of fluid phase markers such as FITC-dextran or horseradish peroxidase, even when these are added
to cells together with SV40 (21,61). Antibodies against
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Figure 2: Subcellular distribution and morphology of caveosomes in green African monkey kidney epithelial cells. Left: Laser
scanning confocal micrograph (intracellular plane) of a CVª1 cell expressing caveolin-1-GFP (cav1-GFP, green) and Texas Red-labeled
Simian Virus 40 (TRX-SV40, red). Virus particles have been allowed to accumulate in caveosomes for 3 h. Most caveosomes are now filled
with virus particles (yellow), but some are still empty (green). Scale-bar, 10 mm Middle: Thin-section electron micrograph of an intracellular
region of a CVª1 cell having internalized virus particles for 2 h. Caveosomes appear as heterogeneous organelles containing tightly packed
SV40 particles. Vacuolar endosomes do not accumulate virus particles. (CCV: clathrin-coated vesicle, CV: caveolar vesicle). Scale-bar,
500 nm. Right: Higher-magnification thin-section electron micrograph shows the heterogeneous morphology of caveosomes, with virus
particles appearing as ‘peas in a pod’. Scale-bar, 100 nm.
markers of endosomes, lysosomes, TGN, Golgi complex, or
the ER do not stain the caveosome. Ultra-structural analysis
shows tubulovesicular structures, heterogeneous in size and
shape, with the virus particles usually present in narrow tubules like peas in a pod (21,61).
Molecular sorting and transport to the ER
During the accumulation of virus particles, the caveosomes
become increasingly dynamic. Longer tubular extensions
filled with virus particles but devoid of caveolin-1 emerge and
detach from the caveosomes leaving caveolin-1 behind (21).
These vesicles are transported along microtubules to perinuclear membrane organelles identified as the smooth ER.
The receiving compartment can grow in size when more virus
particles are added, forming an anastomosing, tubular expansion of the ER (57,61). Here, the majority of virus particles
remain undegraded for up to 16 h or longer. How the viral
genome is transported from the ER to the nucleus is unclear,
but it seems to passage through the cytosol and the nuclear
pore complex (71).
In summary, the entry pathway of SV40 has revealed several
key features that seem to define the caveolar endocytic pathway and make it distinct from clathrin-mediated endocytosis.
First, SV40 uptake through caveolae is ligand triggered. Triggering involves one or more tyrosine kinases that initiate a
phosphorylation-dependent signaling cascade. Second, the
pathway taken by the virus inside the cell bypasses the organelles involved in clathrin-coated vesicle endocytosis.
Moreover, along the route, the pH is maintained at a neutral
level and no degradative end-station is reached. Instead, the
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cargo is delivered to the ER. Thus, this pathway provides a
direct route from the plasma membrane to the ER.
Remarkable also is the role of the cytoskeleton. Actin plays a
central role in the formation of the caveolar vesicles, and in
the initial transfer of the newly formed vesicles through the
cortex of the plasma membrane. The ability to locally depolymerize, and also the ability to polymerize actin are important. This is in contrast to the internalization of clathrincoated pits, which appears to be enhanced by, but is not
strictly dependent on, a functional actin cytoskeleton (72,73).
After internalization, the microtubule cytoskeleton takes over.
Initial transport to caveosomes does not seem to be dependent on microtubules, but video microscopy shows that early
vesicles can move along them. Later, microtubules play an
essential role in the sorting of SV40 from caveosomes and
transport to the smooth ER.
Caveolar Endocytosis of Other Ligands and
Membrane Constituents
Other ligands or membrane constituents that can be internalized via caveolae are cholera toxin (19,35), folic acid (74,75),
serum albumin (76), autocrine motility factor (AMF) (77), alkaline phosphatase (35), GPI-anchored green fluorescent
protein (GPI-GFP) (78), and the bodipy-labeled
glycosphingolipid Lactosyl Ceramide (LacCer) (70). (See
Table 1 for more details.) Certain FimH-expressing bacteria
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Table 1: Materials endocytosed via caveolae or lipid rafts
Ligand
or consitituent
Ligands
Folic acid
Albumin
Autocrine motility factor
(AMF)
Interleukin-2
(IL2)
Membrane constituents
Alkaline phosphatase
GPI-GFP
Lactosyl ceramide
Toxins
Cholera toxin
Tetanus toxin
Viruses
Simian virus 40
Polyoma virus
Receptor or
membrane
moiety
Induction
Folic acid receptor
(GPI anchor)
gp60 (TM),
others
AMF receptor
(TM)
IL2 receptor
(TM)
Affinity
References
Caveolae/
Lipid rafts
Clathrin
Yes
ππ
π
(74,75,96)
Yes
ππ
π
(39,76)
ND
ππ
π
(77,97)
ND
ππ
–
(41)
GPI-anchor
GPI-anchor
Glycosphingolipid
Yes
ND
Yes
ππ
ππ
ππ
–
–
–
(35)
(78)
(70)
GM1 gangliosides
GPI-anchored
molecules
ND
ND
ππ
ππ
π
–
(19,35,94,95)
(19,98,99)
Yes
ND
ππ
ππ
–
–
(4–6, 64)
(80, 100, 101)
ND
ND
π
π
ND
ND
(82)
(83)
Yes
ππ
–
(79)
MHC1 (TM), ND
Sialic acid, GM1
gangliosides
Echovirus 1
a2b1-Integrin
Respiratory syncytial virus
ND
Bacteria (caveolae-mediated phagocytosis)
FimH expressing E. coli
CD48 (GPI anchor)
TM, transmembrane insertion; ND, not determined; E. coli, Escherichia coli.
are also internalized in a caveolae-dependent manner in mast
cells (79).
SV40 is not the only virus that enters via caveolae. The
closely related polyoma virus was recently found to enter
mouse cells by the caveolar route and to have a similar dissociating effect on actin stress fibers (80). Interestingly, polyoma virus appears to specifically bind to branched sialic acid
groups present in glycoproteins with O-linked carbohydrates
and in GM1 gangliosides (81). Echovirus 1, a member of the
picorna virus family, which binds to a2b1-integrin, also internalizes via caveolae (82). In addition, respiratory syncytial virus
has also been reported to associate with caveolae (83). Finally, there is evidence that HIV-1 uses caveolae for transcytosis across endothelia (84). It is not unlikely that the caveolar pathway is important for a variety of viruses for which
the entry mechanism has remained obscure.
How Does It Work?
The systems studied so far provide a rather heterogeneous
picture of caveolar endocytosis, and it is not easy to define a
common denominator. However, caveolae seem to be used
by cells to internalize membrane components that are enriched in rafts, such as cholesterol, glycosphingolipids, GPI-anTraffic 2002; 3: 311–320
chored proteins, and any ligands that bind to them. However,
to be internalized, affinity for rafts or raft components is, in
general, not sufficient; some form of induction is in addition
is needed.
One way to induce caveolar uptake is by cross-linking caveolar components. This has been shown for several GPI-anchored proteins (35,85). Many of the ligands in Table 1 are
multivalent and therefore capable of cross-linking their receptors. Such clustering ability may explain why multivalent
ligands such as virus particles and bacteria are sequestered
into caveolae. They may also induce the formation of new
caveolae (86,87).
In several cases internalization of caveolae is induced upon
activation of a phosphorylation cascade (9,39,50). It is also
known that phosphatase inhibitors can induce internalization
of caveolae (22,35). How extracellular ligands activate phosphorylation is unclear. One way to transmit a signal is by
cross-linking transmembrane tyrosine kinase receptors, of
which several are enriched in caveolae. Another, more recently postulated mechanism suggests that clustering of
GPI-anchored proteins or glycosphingolipids in the extracellular leaflet may stabilize lipid rafts, which in turn may lead to
the recruitment of proteins with high affinity for rafts, such
as caveolin-1 and Src family protein tyrosine kinases, on the
cytosolic leaflet (54,87).
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Figure 3: Proposed pathway for intracellular trafficking of ligands and constituents internalized via caveolae or lipid rafts. Depicted is a model for internalization via caveolae and lipid rafts. After internalization, caveolae- or lipid raft-derived vesicles travel to caveosomes, which are distinct from endosomes in content and pH. In caveosomes, internalized ligands or membrane constituents could reside, be
sorted to the Golgi complex, or to the endoplasmic reticulum (ER). Membrane constituents that are sorted to the Golgi complex are GPIGFP and Lactosyl Ceramide (LacCer). Ligands that are sorted to the ER are Simian Virus 40 (SV40) and autocrine motility factor (AMF).
Whether ligands or constituents can cycle from caveosomes directly back to the plasma membrane has not yet been studied. Caveolin-1
partly resides in caveosomes. Examples of ligands internalized via clathrin-coated pits that travel to endosomes are also depicted. From
endosomes, the envelope of Semliki Forest Virus (SFV) and low-density lipoprotein (LDL) are sorted to lysosomes, Shiga toxin is sorted to
the Golgi complex and transferrin (Tfn) is recycled back to the plasma membrane (1,102,103). Whether ligands or membrane constituents
can travel between endosomes and caveosomes has not been studied.
Direct signaling via raft-enriched lipids such as ceramide or
phosphatidyl inositols could also be involved. For instance,
internalization of folic acid via caveolae is regulated by the
serine/threonine kinase Protein Kinase Ca (PKCa) (9), which
is activated by diacylglycerol, a hydrolysis product of phosphatidyl inositol 4,5-bisphosphate (PIP2). It has also been
suggested that PKCa regulates the internalization or receptor recycling of SV40, since the phorbol ester PMA, which
mimics diacylglycerol, reduces SV40 uptake (5).
The actin cytoskeleton seems to play an essential role in the
internalization of caveolae. First of all, local recruitment of an
actin patch to caveolae, similar to that observed in clustered
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lipid rafts (88), is necessary and might function as a scaffold
for the build-up of the internalization machinery (50). Temporary depolymerization of the cortical actin cytoskeleton is
necessary to allow proper closure of caveolae and transit to
the cytosol (22,50) (P. Verkade and K. Simons, personal communication). The actin patches are likely formed by actin–
protein or actin–lipid interactions. At least during SV40 entry,
the patches are formed in conditions where actin depolymerization is temporarily favored. The mechanism of actin recruitment is not known, but it may involve enrichment of PIP2 on
the cytosolic leaflet of caveolae, which can recruit the
necessary machinery to locally polymerize actin (89,90). It
likely also involves several SH2 and SH3 adapter proteins
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that link the actin cytoskeleton to tyrosine phosphorylation.
Furthermore, filamin, an actin-binding protein that binds caveolin-1, might play a role (104).
Dynamin is also involved in the internalization of caveolae.
Over-expression of a mutant dynamin, defective in GTP hydrolysis, inhibits release of caveolae from plasma membranes
in an in vitro assay (45). EM images showed that caveolae
have extended necks in the presence of this mutant (45,46).
Functional data come from the observation that this dynamin
mutant inhibits uptake of cholera toxin and muscarinic cholinergic receptors via caveolae and uptake of interleukin-2 (IL2)
via lipid rafts (41,45,46,91). It now appears that, as a result
of the ligand-induced kinase activity, dynamin is temporarily
recruited to the internalization site to perform its function in
the fission of caveolar vesicles.
Once internalized, caveolar vesicles seem to follow an intracellular route distinct from the classical endocytic pathway
(Figure 3). They enter a caveolin-1-rich sorting compartment,
the caveosome, which, as already discussed, is distinct from
endosomes. From caveosomes, the internalized substances
are distributed to the ER, to the Golgi complex, and possibly
to other compartments. The pathway is best analyzed for
SV40, GPI-GFP, and LacCer (21,70,78). Available information
regarding internalization of cholera and tetanus toxins, albumin, folic acid, and FimH expressing Escherichia coli is consistent with this picture (70,75,78,9,92,93). The issue is
somewhat complicated, however, by the observation that
some of the ligands (cholera toxin, albumin and the folic acid
receptor) are also internalized via clathrin-coated pits, and
can therefore be found in endosomes as well (94–96).
Perspectives
Endocytosis through caveolae provides a true alternative to
the clathrin-mediated pathway. Being ligand-triggered, it provides an intrinsically more selective way for uptake of specified substances, and it allows tighter control by cellular regulation. It can be used to route incoming ligands and membrane components to organelles that are not easily accessed
by other endocytic mechanisms such as the ER. This advantage could, for example, be important in the homeostasis of
cholesterol for which most of the regulatory sensors reside
in the ER (12). A direct connection between the plasma membrane and the ER could also be important in other processes
such as the immune response and in the regulation of secretion. Moreover, the pathway bypasses low pH and digestive
compartments, which may be important for some of the internalized ligands.
The analysis of caveolar endocytosis is still in its early stages.
Many conceptual and mechanistic issues remain to be
solved. It is important to address the differences and similarities between caveolae and other non-clathrin-mediated uptake systems. Is caveolar uptake just a special form of raftmediated endocytosis? The mild phenotype of knockout
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mice suggests that, while required for formation of caveolae,
caveolin-1 may not be essential for endocytosis nor for crucial signaling functions. The molecular mechanisms of endocytosis and the connection with the cytoskeleton also need
to be addressed in detail. Here, complicated signaling and
regulatory cascades are involved, of which we now only see
the tip of the iceberg.
Acknowledgments
The authors would like to thank all members of the laboratory for helpful
suggestions and valuable comments. Work is supported by grants from
the Swiss National Science Foundation and the Swiss Federal Institute of
Technology.
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