Membrane microdomains, caveolae, and caveolar endocytosisof sphingolipids (Review)

ZHI-JIE CHENG, RAMAN DEEP SINGH, DAVID L. MARKS & RICHARD E. PAGANO

Department of Biochemistry and Molecular Biology, Thoracic Diseases Research Unit, Mayo Clinic and Foundation,

Rochester, Minnesota, USA

(Received 29 September 2005; in revised form 4 November 2005)

AbstractCaveolae are flask-shape membrane invaginations of the plasma membrane that have been implicated in endocytosis,transcytosis, and cell signaling. Recent years have witnessed the resurgence of studies on caveolae because they have beenfound to be involved in the uptake of some membrane components such as glycosphingolipids and integrins, as well asviruses, bacteria, and bacterial toxins. Accumulating evidence shows that endocytosis mediated by caveolae requires uniquestructural and signaling machinery (caveolin-1, src kinase), which indicates that caveolar endocytosis occurs through amechanism which is distinct from other forms of lipid microdomain-associated, clathrin-independent endocytosis.Furthermore, a balance of glycosphingolipids, cholesterol, and caveolin-1 has been shown to be important in regulatingcaveolae endocytosis.

Keywords: Lactosylceramide, gangliosides, rho proteins

Introduction

Multiple clathrin-independent mechanisms of endo-

cytosis have recently been described and character-

ized. These internalization mechanisms are distinct

from clathrin-dependent endocytosis which has

been more extensively studied [1,2]. Clathrin-inde-

pendent uptake mechanisms include caveolae-

mediated endocytosis, rhoA-dependent endocytosis

of the interleukin 2 receptor ß subunit (IL-2R ß),

and cdc42-regulated endocytosis of GPI-anchored

proteins and fluid phase markers [1�/7]. Each of

these endocytic mechanisms has distinct biochem-

ical sensitivities and specific requirements for certain

adaptor and signaling proteins that are summarized

in Table I. For example, caveolae-mediated endocy-

tosis is dependent on dynamin 2 (Dyn2) and src

kinase but is insensitive to Clostridium difficile Toxin

B, which inhibits Rho family GTPases such as RhoA

and cdc42. In contrast, the endocytosis of IL-2R and

fluorescent dextran is sensitive to Toxin B because of

their dependence on RhoA and cdc42, respectively,

but is insensitive to specific src kinase inhibitors such

as PP2. A further distinction between the RhoA and

cdc42-regulated mechanisms is that only the latter is

independent of Dyn2. It has been proposed that

caveolae and other cholesterol-dependent microdo-

mains are internalized via a single, common pathway

[8]; however, several studies have shown the persis-

tence of separate non-clathrin endocytic pathways

with different cargo, protein machinery and phar-

macologic sensitivities within a single cell type [5�/7].

In the present review, we focus on endocytosis via

caveolae. Our laboratory has used fluorescent sphin-

golipid (SL) analogues and the SL-binding toxin,

cholera toxin subunit B (CtxB) to study the mechan-

ism by which SLs are internalized and subsequently

sorted and transported to various intracellular com-

partments [4,9]. Glycosphingolipids (GSLs), a sub-

group of SLs, have been found to be internalized

predominantly by a clathrin-independent, caveolar

mechanism in most cell types studied [4,6,7,10].

Here, we discuss recent studies on the caveolar

endocytosis of GSLs and other caveolar markers

(albumin, CtxB, SV40) and highlight current studies

of the molecular mechanisms regulating this endo-

cytic process. The controversial role of caveolin-1

(cav1) in caveolae-mediated endocytosis is also

discussed.

Caveolae and other lipid microdomains

Caveolae were first described in the 1950s by

Palade and Yamada based on their characteristic

Correspondence: Richard E. Pagano, Department of Biochemistry and Molecular Biology, Thoracic Diseases Research Unit, Mayo Clinic

and Foundation, 200 First Street, SW, Rochester, Minnesota, 55905, USA. Tel: �/1 507 284 8754. E-mail: pagano.richard@mayo.edu

Molecular Membrane Biology, January�/February 2006; 23(1): 101�/110

ISSN 0968-7688 print/ISSN 1464-5203 online # 2006 Taylor & Francis

DOI: 10.1080/09687860500460041

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morphology as 50�/80 nm diameter flask-shape

invaginations observed by electron microscopy of

thin sections [11,12]. In the 1990s, a series of

studies provided evidence for the existence of plasma

membrane (PM) microdomains which were en-

riched in GSLs, cholesterol, GPI-linked proteins

and certain intracellular signaling proteins (e.g., src

family kinases) [13]. Many of these studies relied on

the characteristic of insolubility in cold (48C)

detergent (Triton X-100 or CHAPS) solutions for

the isolation of these microdomains [14�/17]. A key

study in 1992 identified a major protein found in

these detergent-insoluble complexes, VIP21 [18],

which was found to be identical to cav1, a major coat

protein of caveolae [19]. Cav1 was soon found to

directly bind to cholesterol [20]. Because early

studies focused on the use of detergent insolubility

to isolate lipid microdomains, it was initially sug-

gested that these isolated microdomains were synon-

ymous with caveolae. Additional confusion was

caused by observations that GPI-linked proteins

visualized using antibodies were present in caveolae

[16,21]. Later studies have demonstrated that cells

without cav1 also possess detergent-insoluble micro-

domains [22,23], and that most GPI-linked proteins

probably reside in non-caveolar microdomains but

can be sequested into caveolae when living cells are

treated with crosslinking antibodies [24,25]. In

contrast, GM1 ganglioside and cholesterol have

been shown to be concentrated in caveolae by a

variety of methods [26�/29]. Based on a variety of

studies and technologies, it has become evident that

there are probably several different types of PM

cholesterol-enriched microdomains besides caveolae

[2,30�/32]. As noted above, caveolar and non-

caveolar lipid microdomains are associated with

distinct forms of clathrin-independent endocytosis.

Evidence from video microscopy and fluorescence

recovery after photobleaching (FRAP) analysis has

shown that cav1-GFP is relatively immobile at the

PM and that only a minority of cav1-positive vesicles

actually internalize, suggesting that caveolae are

rather stable structures that do not have a high basal

turnover at the PM [33,34]. These studies give the

impression that endocytosis via caveolae is a rare

event. However, much evidence has suggested that

caveolar endocytosis is a regulated process that

can be induced or stimulated [6,35�/37]. Caveolar

uptake is greatly accelerated by phosphatase inhibi-

tors and inhibited by certain kinase inhibitors

[4,33,35,37,38]. Also, caveolae appear to be an-

chored to the actin cytoskeleton, and disruption of

this network leads to increased lateral mobility of

cav1 and to clustering of caveolae in the plane of the

PM [39,40]. Finally, cargo to be internalized via

caveolae has been shown to stimulate caveolar

uptake. For example, SV40 virus has been shown

to enter host cells by inducing its own endocytosis

via caveolae, whereas caveolae devoid of the virus

particles remain static at the PM in the same cells

[41]. Once bound to the cell surface, SV40 activates

local tyrosine phosphorylation, disrupts the local

actin cytoskeleton and recruits dynamin 2 [40].

These signaling events subsequently trigger the

internalization of the virus-containing caveolae

[40]. Binding of albumin to its cell surface receptor,

gp60, also triggers caveolar endocytosis via a

Gi-coupled src kinase-mediated pathway [42]. As

described below, GSLs also stimulate endocytosis

via caveolae [6].

Caveolae-mediated endocytosis of GSLs

As already mentioned, SLs are enriched, along with

cholesterol, in membrane microdomains at the PM.

In principle, SLs at the PM may be internalized by

one or more endocytic mechanisms and targeted to

specific intracellular destinations (e.g., lysosomes or

the Golgi complex). We set out to determine the

mechanism of SL internalization using a series of

fluorescent SL analogues in which the naturally

occurring fatty acid moiety of the SL is replaced

with a short chain fatty acid labeled with boron

dipyrromethene difluoride (BODIPY) [43�/45]. The

Table I. Distinguishing characteristics of endocytic mechanisms. Data are compiled from [3�/7] and our unpublished data.

Endocytic mechanism

Caveolar RhoA-dependent Cdc42-dependent Clathrin-dependent

Cargo CtxB, LacCer, SV40 IL2R GPI-linked proteins,

fluid phase markers

Transferrin, LDL, EGF

Treatment:

Dynamin 2 DN Inhibits Inhibits Inhibits

PP2 (src inhibitor) Inhibits

Clostridium toxin B

(Rho inhibitor)

Inhibits Inhibits

AP180 DN Inhibits

Chlorpromazine Inhibits

102 Z-J. Cheng et al.

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BODIPY fluorophore exhibits a concentration-de-

pendent shift in its fluorescence emission from green

to red wavelengths as a result of excimer formation

[45,46]. Thus, BODIPY-labeled lipids are especially

helpful in determining the rapid and dynamic

changes in SL concentrations in specific intracellular

locations.

The endocytosis of BODIPY labeled GSLs [e.g.,

BODIPY-lactosylceramide (BODIPY-LacCer) or

BODIPY-globoside] was first studied in human

skin fibroblast (HSFs), but similar results were later

reported for rat fibroblasts (RFs) and other cell

types [4,7]. The uptake of these GSL analogues is

insensitive to treatments that specifically inhibit

clathrin-dependent endocytosis [chlorpromazine

(CPZ), K�/depletion, or expression of a dominant

negative (DN) mutant form of EGF receptor path-

way substrate 15 (Eps15)]. When HSFs are trans-

fected with DN Rab5, which specifically inhibits the

transport of clathrin-derived endocytic vesicles from

the PM to sorting endosomes, the endocytosis of

Tfn is inhibited 80% but BODIPY-LacCer uptake is

not affected [10]. These results suggest that the

endocytosis of these GSL analogues is independent

of clathrin.

Expression of the dynamin 2 mutant, Dyn

2 K44A, blocks BODIPY-LacCer internalization

[4,6,7]. Dynamin 2 has been shown to be involved in

clathrin-dependent endocytosis, caveolae-mediated

endocytosis and RhoA-regulated IL-2 receptor in-

ternalization, but not in cdc42-regulated pinocytosis

[3,5]. Also, BODIPY-LacCer shows little co-locali-

zation with fluorescent dextran (a marker for cdc42-

regulated pinocytosis) [7]. Furthermore, Clostridium

difficile toxin B (Toxin B), a general inhibitor of the

Rho family GTPases, blocks dextran uptake but has

no effect on LacCer internalization [6]. Together,

these results suggest that LacCer is internalized via

an endocytic mechanism distinct from that utilized

by dextran.

Further evidence indicates that LacCer is inter-

nalized via a caveolae-related mechanism in HSFs

and RFs. First, LacCer uptake is significantly

inhibited by the cholesterol binding or extracting

agents, nystatin and methyl-ß-cyclodextrin, respec-

tively [4,6,7,36], suggesting the involvement of lipid

microdomains. Second, at early time points of

endocytosis (B/5 min), BODIPY-LacCer exhibits

extensive overlap (�/80%) with either fluorescent

albumin or CtxB, but little co-localization with DiI-

LDL and transferrin (Tfn), two markers for clathrin-

dependent endocytosis [4,7,10,36]. Although the

endocytic mechanism of certain markers (such as

CtxB) has been shown to be cell type-dependent,

albumin and CtxB extensively overlap (�/50%) with

cav1-GFP or DsRed-cav1 in HSFs [4,7], indicating

that both are valid markers for caveolae in HSFs.

Importantly, BODIPY-LacCer also shows extensive

co-localization with expressed fluorescent cav1

fusion proteins [6,7].

Interestingly, time course studies of the endocy-

tosis of BODIPY-LacCer and AF594-Tfn in doubly

labeled cells show that although LacCer and Tfn are

internalized by distinct endocytic mechanisms (little

co-localization between LacCer and Tfn after 1 min

internalization), these markers rapidly converge

and co-localize in EEA1-positive early endosomes

after 5 min internalization before further sorting

to different intracellular locations [10]. Similarly,

fluorescent albumin, another marker for caveolar

endocytosis in HSFs, also merges with Tfn-positive

early endosomes [10]. These results are consistent

with the earlier observation of Pol et al [47,48] who

report the detection of cav1 in early endosomes.

Recently, Pelkmans et al. [49] reported cav1-positive

vesicles containing SV40 or CtxB (referred to as

caveosomes) also transiently interact with early

endosomes to form subdomains, suggesting that

merging with early endosomes might be a general

phenomenon for cargo internalized via caveolae.

Similarly, IL2-R alpha subunit and MHC I endocy-

tosed by clathrin-independent endocytosis are initi-

ally internalized into vesicles which are devoid of

LDL, a marker for the clathrin pathway, but at later

times (e.g., 20 min) are delivered to early and then

late endosomes [50]. In contrast, GPI-APs inter-

nalized via the cdc42-dependent pathway are not

delivered to early endosomes but are held in discrete

structures (GPI-enriched early endosomal compart-

ments; GEECs), and are later delivered to the

recycling endosome [5].

Structural determinants for GSL

internalization via caveolae

Structurally, GSLs consist of a large lipid family in

which the composition of the carbohydrate head-

group varies. We were interested in determining

whether GSLs with different head groups are inter-

nalized via a similar mechanism to that utilized by

BODIPY-LacCer.

We systematically varied the carbohydrate head-

group of the fluorescent GSL analogues as shown in

Figure 1A and examined the effect of these varia-

tions on the mechanism of analogue internalization.

Surprisingly, we found that BODIPY-labeled Gal-

Cer, MalCer, globoside, GM1, and sulfatide were

each internalized identically to BODIPY-LacCer

[7]. Second, since lipid hydrophobicity is presumed

to affect its ability to partition into microdomains,

we prepared a series of BODIPY-LacCer analogues

in which the chain length of the sphingosine base

Caveolar endocytosis of sphingolipids 103

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(e.g., C12 vs. C20 sphingosine), or fluorescent fatty

acid was varied (see Figure 1). However, these

variations had no effect on the LacCer internaliza-

tion mechanism [7]. Also, replacing the BODIPY-

fluorophore with NBD did not influence the caveo-

lar internalization of the LacCer analogues [7].

Thus, GSL uptake via caveolae is not selective for

a specific carbohydrate headgroup, acyl chain hydro-

phobicity, or fluorophore substitution. We also

examined the uptake of a fluorescent glyceropho-

spholipid analogue, NBD-phosphatidylcholine (PC)

(Figure 1B), which has a different lipid backbone

from LacCer (glycerol vs. ceramide). The endocy-

tosis of NBD-PC in RFs is predominantly CPZ-

inhibitable, suggesting that its uptake occurs largely

by a clathrin-dependent mechanism [7]. These

studies suggest that the sphingoid backbone of the

GSLs (but not the headgroup or overall hydropho-

bicity) may play an important role in caveolar

endocytosis of GSLs.

Stimulation and regulation of caveolar

endocytosis by GSLs and cholesterol

The regulation of endocytosis at caveolae is just

beginning to be understood. Cholesterol depletion

or sequestration (e.g., with filipin or nystatin) have

been demonstrated to disrupt caveolae integrity and

inhibit the endocytosis of SV40, CtxB, BODIPY-

LacCer and albumin [4,6,7,40,51,52].

Pang et al. [53] report that CtxB internalization is

sensitive to cholesterol depletion in high-GM1-

expressing cells, but not in low-GM1 cells, and

addition of exogenous GM1 to the PM enhances

the cholesterol-dependent delivery of CtxB to the

Golgi apparatus. As mentioned above, there is

evidence that endocytosis via caveolae can be stimu-

lated in various ways. First, endocytosis of caveolae

can be stimulated by phosphatase inhibitors such as

okadaic acid and sodium vanadate [33,35]. In

addition, caveolar endocytosis can be stimulated by

the presence of cargo (e.g., SV40 virus, albumin)

[37,41]. The stimulation of caveolar endocytosis is

accompanied by increased activation of src and

phosphorylation of cav1 and dynamin [37,40,54],

suggesting that stimulation occurs via a triggering of

increased kinase activities.

In a preliminary experiment in our laboratory,

CtxB internalization was found to be very robust in

cells co-labeled with BODIPY-LacCer, but was

much lower in the absence of BODIPY-LacCer.

Further study showed that acute treatment of HSFs

with non-fluorescent natural LacCer or GM1 gang-

lioside, or with a synthetic short-chain C8-LacCer

selectively stimulates the caveolar uptake of BOD-

IPY-LacCer, whereas the internalization of markers

internalized by fluid phase or clathrin-dependent

endocytosis are not affected [6]. The caveolar

internalization of albumin and ß1-integrin is also

greatly enhanced in cells pretreated with non-fluor-

escent GSLs or cholesterol [6,36]. These results

provide a partial interpretation to our previous

observation that BODIPY-LacCer is internalized

through caveolae within seconds [6,10], which is in

apparent contradiction to studies of some other

caveolar markers in which an hour or more is

required for significant internalization to occur

[40,41,52]. Thus, it is likely that BODIPY-LacCer

may stimulate its own internalization. Furthermore,

when the cellular cholesterol level of HSFs is

increased (by culturing in the presence of excess of

A

RO

OH

HN

O

( ) n

R'( )n'

N NB

Me

Me

F FR' =

n' = 1 or 3

n = 1, 5, 7, or 9

R

Gal- GalCer

Gal-Glc (β(1→4))- LacCer

Glc-Glc (α(1→4))- MalCer

Gal-Gal-Glc- Globoside

SO4-Gal- Sulfatide

Gal-GalNAc-Gal-Glc- GM1

NeuAc

B

O

OCC15H31

P

O

OCH2CH2N+Me3

O-

O

H

ON

ON

NO2N

H

R =OC(CH2)5-R

Figure 1. Structures of fluorescent lipid analogues used to

evaluate the features critical for caveolar uptake of the lipids.

(A) Various headgroups (R) were attached to BODIPY-ceramide,

resulting in BODIPY-GalCer, -LacCer, -MalCer, -globoside,

-sulfatide, or -GM1. BODIPY-LacCer analogues were also

synthesized using various chain length (C12, C16, C18, or C20)

sphingosines or BODIPY-fatty acids (C3 vs C5 spacer). Fluor-

escent LacCer bearing an NBD-fatty acid (see panel B) in place of

the BODIPY-fatty acid was also synthesized. (B) Structure of the

D-isomer of NBD-labeled PC, a glycerolipid. Reproduced from

[7] with permission from the American Society for Cell Biology.

104 Z-J. Cheng et al.

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LDL, or acute treatment with a methyl-b-cyclodex-

trin (mb-CD)/cholesterol complex, the internaliza-

tion of albumin and BODIPY-LacCer is significantly

increased, but no effect is seen on the endocytosis of

Tfn [6], suggesting that addition of exogenous

cholesterol also can selectively stimulate caveolar

uptake.

Biochemical inhibitors and dominant negative

proteins (see Table I) were used to determine that

exogenous GSLs or cholesterol did not induce

endocytosis by an alternate mechanism rather than

via caveolae [6]. In addition, the endocytosis of both

BODIPY-LacCer and albumin was significantly

inhibited by cav1 siRNA under both stimulated

and unstimulated conditions ([36], and our unpub-

lished studies). Finally, at very short times (30 sec)

of BODIPY-LacCer internalization, this lipid was

shown to co-localize with expressed cav1-mRed in

stimulated and unstimulated HSFs [6]. Thus, the

endocytic pathway stimulated by exogenous GSLs

and cholesterol retained identical properties to those

of caveolar endocytosis in unstimulated cells. Im-

portantly, addition of C8-LacCer or mß-CD/choles-

terol to cells results in an 8�/10 fold increase in src

kinase activity and transient cav1 phosphorylation,

similar to findings for stimulation of albumin in

endothelial cells [37,54]. Thus, different stimuli

evoke caveolar uptake via the same signaling cas-

cade.

Two models have been proposed concerning the

mechanism by which addition of GSLs to the PM

stimulates caveolar uptake [6]. In the first model,

addition of GSLs to the outer leaflet of the PM may

cause a specific interaction of the GSL with a

particular PM protein, which in turn could initiate

a signaling cascade resulting in stimulation of

caveolar endocytosis. However, since exogenous

cholesterol can elicit similar effects on caveolar

endocytosis, this model seems unlikely. Another

model is that GSLs (or cholesterol) added to the

outer leaflet of the PM bilayer could change the

organization of lipids in the PM, thereby inducing

the clustering and activation of a transmembrane

protein and/or signaling proteins on the inner leaflet

of the PM (Figure 2). Support for this hypothesis

comes from recent work in our laboratory which

takes advantage of the concentration-dependent

spectral properties of BODIPY fluorescence [36].

We examined the PM distribution and organization

of BODIPY-LacCer incubated with HSFs at low

temperature (108C). In control HSFs, the PM is

uniformly labeled with BODIPY-LacCer and

emitted only green fluorescence. However when

cells were treated with C8-LacCer or mb-CD/

cholesterol, small ‘‘patches’’ of BODIPY-LacCer

with increased red emission (shown as yellow/orange

areas in green/red overlay) were observed at the PM

(Figure 3A), suggesting the formation or coalescence

of GSL (BODIPY-LacCer)-enriched membrane mi-

crodomains.

One possible class of proteins that might mediate

the stimulatory effect of GSL and cholesterol on

caveolar uptake is the integrins. Integrins are a

family of ab heterodimeric integral membrane pro-

teins at the PM that are responsible for many types

of cell adhesion and migration events [55,56].

Binding of extracellular matrix (ECM) proteins

and extracellular ligands to integrins induces a series

of signaling cascades including activation of src

kinase, phosphorylation of focal adhesion kinase,

and elevation of intracellular calcium [55,57]. Upon

binding of ligands or crosslinking antibodies, some

integrins are activated and redistributed into lipid

microdomains [58�/60]. GSLs have been shown to

modulate integrin-based cell attachment. For exam-

ple, gangliosides (sialic acid-terminated GSLs) are

reported to enhance binding of integrins to the ECM

or collagen in a variety of cell lines [61,62].

We recently found that the addition of C8-LacCer

or cholesterol to HSFs at low temperature causes the

clustering and activation of b1-integrin within GSL-

enriched PM microdomains (Figure 3B and [36]).

The same effect was observed by crosslinking of

integrins with integrin antibodies which is an estab-

lished method for integrin activation [60,63].

Furthermore, b1-integrins are rapidly internalized

via caveolae upon warming to 378C in cells treated

with C8-LacCer or cholesterol, whereas little en-

docytosis of b1-integrin is seen in untreated cells

[36]. This is consistent with the previous observation

that GSLs and cholesterol stimulate caveolar uptake

[6]. A series of integrin-associated, downstream

signaling events are also observed upon integrin

clustering, including src activation, increased cav1

phosphorylation and reorganization of the actin

cytoskeleton which precedes integrin internalization,

RhoA movement away from the PM and transient

cell detachment after integrin internalization [36].

Results from these studies suggest the possibility that

aberrant levels of GSLs found in cancer cells may

influence cell attachment by modulating integrin

clustering and internalization.

Cav1 and caveolae endocytosis

There are three members of the caveolin family,

cav1, cav2 and cav3. Cav1 and cav2 are co-expressed

in most cell types, whereas cav3 is expressed only in

muscle cells [64]. Cav1 is the defining protein

component of caveolae, and is essential for caveolae

biogenesis in most cells, although cav3 plays a similar

role in muscle cells [64,65]. In nonmuscle cells

Caveolar endocytosis of sphingolipids 105

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Figure 2. Model for GSL-initiated clustering of plasma membrane microdomains. (1) In untreated cells, PM microdomains are too small

or transient to be visualized. Certain transmembrane proteins (e.g., ß1-integrins) are dispersed in the membrane. (2) Addition of exogenous

GSL or cholesterol to the membrane causes the formation or coalescence of GSL-enriched microdomains. (3) Certain transmembrane

proteins and intracellular signaling proteins (e.g., src kinases) become clustered in GSL-enriched microdomains. Clustering leads to protein

activation and intracellular signaling.

A

B

Figure 3. C8-LacCer and cholesterol induce clustering of b1-integrins within GSL-enriched microdomains. (A) Visualization of PM

domains after induction by various treatments. Human skin fibroblasts were incubated with BODIPY-LacCer for 30 min at 108C, washed,

and then incubated in buffer alone (untreated) or with C8-LacCer, Mb-CD/cholesterol complex, or ß1-integrin IgG for 30 min at 108C.

In the far right panel, cells were preincubated with 5 mM mß-CD to deplete cholesterol and then treated with BODIPY-LacCer and ß1-

integrin IgG. The samples were then washed and observed by fluorescence microscopy at green and red BODIPY emission wavelengths.

Samples were maintained at 108C at all times to prevent endocytosis. Micrographs shown are overlays of red and green images. Areas

outlined with white rectangles are further magnified in insets. Yellow orange patches indicate regions with the highest red signal, indicating

enrichment of BODIPY-LacCer in these regions of the PM. Bar, 5 mm. (B) b1-integrin clusters are localized to PM lipid domains. Cells

were co-labeled with BODIPY-LacCer and Alexa647-anti-b1-integrin Fab fragments for 30 min at 108C. Samples were then further

incubated for 30 min at 108C9/C8-LacCer, washed, and viewed by fluorescence microscopy. Images were acquired at red and green

(for BODIPYas above) and at far red wavelengths for AF647-ß1-integrin Fab labeling. Note the overlap of integrin staining (shown in blue)

with the enriched (red/orange) PM domains of BODIPY-LacCer. Bar, 2 mm. Reproduced from [36] with permission from the American

Association for Cancer Research.

106 Z-J. Cheng et al.

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which do not express cav-1, such as normal lympho-

cytes, some transformed cell lines, and cells from

cav1 knockout mice, there are no morphologically

identifiable caveolae [22,23,66�/68]. Conversely,

overexpression of cav-1 in caveolin-deficient cell

lines results in the formation of recombinant caveo-

lae vesicles [68,69].

Despite the need for cav1 expression in the

formation of caveolae, the role of cav1 in caveolae-

mediated endocytosis has been controversial. A

number of studies have shown that cav1 knockdown

or expression of dominant negative cav1 decreases

endocytosis via caveolae [36,41,70,71]. In addition,

we have shown that BODIPY-LacCer internalization

is dramatically reduced in cell types with low levels

of cav1, and could be stimulated in cell types with

low cav1 levels by overexpression of cav1 [6,7].

Although some studies have demonstrated an in-

crease in endocytosis via caveolae upon over-expres-

sion of wild type cav1 [71,72], other groups have

reported that over-expression of cav1 negatively

regulates caveolar endocytosis of albumin, CtxB,

and autocrine motility factor receptors [42,73]. This

apparent discrepancy might be a result of cell type

differences in the levels of cav1 and caveolar lipids

(GSLs and cholesterol) that regulate caveolar en-

docytosis. For example, albumin uptake in HeLa

cells is inhibited by cav1 over-expression, but when

cav1 over-expressing cells are also treated with C8-

LacCer, albumin uptake is stimulated [6]. Thus, the

balance between cav1, GSLs, and cholesterol may

affect whether cav1 expression has a positive or

negative impact on endocytosis via caveolae.

Another confounding factor in the study of the

role of cav1 in endocytosis is the possibility that

the same endocytic cargo may be internalized by

different mechanisms in different cell types or even

switch pathways in a single cell type under different

experimental conditions. For example, CtxB is

internalized via caveolae in some cells which express

abundant cav1 and exhibit morphological caveolae,

but is internalized through other endocytic mechan-

isms in cells with little or no cav1 [7,52,74,75]. A

possible limitation of some of these studies is that the

criteria used to determine endocytosis via caveolae

have not always excluded the possibility of inter-

nalization via other non-clathrin, non-caveolar me-

chanisms. For example, the use of cholesterol

depletion or sequestration could affect other me-

chanisms of internalization apart from caveolar

uptake [7,52,74,75].

Studies of cells from cav1 knockout mice have only

partially addressed the question of the role of cav1 in

endocytosis. Endothelial cells from cav1 knock-out

mice show defects in the uptake and transport of

albumin in vivo, confirming the role of caveolae in

the transcytosis of albumin [76]. However, two

recent studies carried out using murine embryonic

fibroblasts (MEFs) from cav1 knockout and wild

type mice, have shown that CtxB and SV40, which

internalize via caveolae in many cell types, could be

internalized via cholesterol-dependent, but clathrin-

and cav1-independent mechanisms, in both the

cav1 knockout and the wild type MEFs [77,78].

These studies are in agreement with others which

showed that CtxB can be internalized by non-

caveolar mechanisms in cells with little or no cav1

[52,75,79]. However, since the process of caveolar

endocytosis could not be demonstrated (using either

SV40 or CtxB) in wild type MEFs which do express

cav1, no conclusion can be made concerning the role

of cav1 in endocytosis in this cell type.

Conclusions

Studies from our laboratory and others have begun

to characterize the process of caveolar endocytosis

and shown it to be distinct from other clathrin-

independent endocytic mechanisms. GSLs are se-

lectively internalized via caveolae and this selectivity

may occur because of the capability of GSLs to

enhance microdomain formation at the PM. How-

ever, the precise mechanism by which GSLs stimu-

late domain formation, and the possible role of

transmembrane proteins, requires further study.

Although GSLs and other materials (e.g., SV40

virus) are internalized via caveolae, it is not clear if

these occur by identical process since they differ in

their kinetics and the intracellular compartments

that are eventually targeted. For example, endocy-

tosed BODIPY-LacCer is first visualized in small

endocytic vesicles and then rapidly moves to EEs,

without obvious occurrence in caveosomes, such as

those seen upon SV40 uptake. Furthermore, the

possibility that there are multiple forms of caveolar

endocytosis has not been ruled out. While it is clear

that cav1 expression is a prime requirement for the

existence of morphological caveolae, the role of cav1

in the endocytic process is still in question. Is cav1 a

stabilizer of the caveolae structure and thus an

inhibitor of internalization from the membrane?

Do stimulatory lipids such as GSLs and cholesterol

initiate the phosphorylation of cav1 and thus its

movement away from caveolae, allowing for a

destabilization of the caveolae coat? The answers to

these questions will require multiple cell biological,

biochemical, and molecular approaches.

Acknowledgements

This work was supported by USPHS grant

GM-22942 to REP and an NRSA award to ZJC.

Caveolar endocytosis of sphingolipids 107

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