1 Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310
Oslo, Norway
2 Structural Cell Biology Unit, Department of Medical Anatomy, The Panum
Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark
* Author for correspondence (e-mail: ksandvig{at}radium.uio.no )
Accepted 6 May 2002
![]() |
Summary |
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Key words: Ruffling, Macropinocytosis, Rac1, Cholesterol, Ricin
![]() |
Introduction |
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In the present study we have investigated the importance of cholesterol for
macropinocytosis in A431 cells. Macropinocytosis can be induced by growth
factors such as the epidermal growth factor (EGF) or by the phorbol ester
12-O-tetradecanoylphorbol 13-acetate (TPA). A prerequisite for
macropinocytosis is the formation of plasma membrane ruffles that subsequently
close to form macropinosomes. This process requires reorganization of the
actin filament network to the cell periphery
(Swanson and Watts, 1995;
Ridley, 1994
). TPA, known as a
potent activator of protein kinase C (PKC)
(Keller, 1990
), induces
macropinocytosis via Rac1, a Ras-related GTP-binding protein
(Ridley et al., 1992
).
Activated Rac1 localizes to the plasma membrane where it stimulates actin
filament reorganization and membrane ruffling
(Swanson and Watts, 1995
;
Ridley, 1994
;
Ridley et al., 1992
;
Kraynov et al., 2000
).
To study the effect of decreased and increased cellular cholesterol content on macropinocytosis, mßCD was used to extract cholesterol from the plasma membrane and a complex of mßCD and cholesterol (mßCD/chol) was used to insert cholesterol. Here we demonstrate that membrane ruffling and macropinocytosis are sensitive to a decreased cholesterol content of the plasma membrane.
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Materials and Methods |
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Rhodamine-labeled phalloidin was obtained from Molecular Probes (Eugene,
OR). A mouse anti-human Rac1 from Transduction Laboratories (Lexington, KY)
was used for immunofluorescence localization of endogenous Rac1. For
localization of Rac1 constructs we used a mouse antibody against the six amino
acid epitope tag EYMPME (termed Glu-Glu) from Covance (Princeton, NJ).
Detection of ARF6 for immunofluorescence was performed using a rabbit
polyclonal antibody, which was a generous gift from J. G. Donaldson (National
Institutes of Health, Bethesda, MD). Fluorescein isothiocyanate (FITC)-labeled
donkey anti-mouse, FITC-labeled goat anti-rabbit and rhodamine-labeled goat
anti-mouse were from Jackson Immunoresearch (West Grove, PA).
Methyl-ß-cyclodextrin (average degree of substitution: 10.5-14.7 methyl
groups per molecule), cholesterol, Hepes, 12-O-tetradecanoylphorbol
13-acetate (TPA), Pipes (dipotassium salt), saponin, horseradish peroxidase
(HRP), and ricin were obtained from Sigma (St Louis, MO). Na[125I]
was purchased from DuPont (Brussels, Belgium). Ricin was
125I-labeled as descibed (Fraker
and Speck, 1978) to a specific activity of
(2-6)x104 cpm/ng. When protease inhibitors were required,
CompleteTM EDTA-free from Roche Diagnostics (GmbH) was used. The TPA
concentrations used in this study to stimulate ruffling and macropinocytosis
range from 0.1 to 1 µM. Different concentrations were used because the
response at a given concentration seemed to vary. It is not clear whether this
is due to changes in the cells (perhaps due to the serum batch) or due to
differences in the TPA-batches.
Preparation of mßCD saturated with cholesterol
The saturated complex was prepared mainly as previously described
(Klein et al., 1995). 30 mg
cholesterol was added to 1 g of mßCD dissolved in 20 ml H2O.
The mixture was rotated overnight at 37°C, and the resulting clear
solution freeze-dried. The complex was stored at room temperature.
Cholesterol determination
Cell monolayers were washed carefully with PBS, lysed in a buffer
containing 0.1% SDS, 1 mM Na2EDTA and 0.1 M Tris-HCl, pH 7.4, and
homogenised using a 19 gauge needle attached to a 1 ml syringe. The
cholesterol content was determined enzymatically by the use of a cholesterol
assay kit (Sigma).
Protein determination
The protein content of the homogenised cells was measured using the micro
bicinchoninic acid method (Pierce, Rockford, IL) according to the
manufacturer's instructions.
Measurement of TPA-induced macropinocytosis
TPA-induced macropinocytosis was measured as the amount of
125I-labeled ricin endocytosed during 15 minutes after 4 hours of
serumstarvation. The cells were incubated with 1 µM TPA for 10 minutes at
37°C before addition of 125I-labeled ricin, and endocytosed
ricin was measured after 15 minutes at 37°C as the amount of toxin that
could not be removed with lactose as previously described
(Sandvig and Olsnes, 1979). To
look at the effect of changes in the cholesterol level, the cells were
incubated with 5 mM mßCD, mßCD/chol or a 1:1 mixture of mßCD
and mßCD/chol for 30 minutes at 37°C prior to addition of TPA, and
then together with TPA for the duration of the experiment.
Measurement of PKC activity
Cells were seeded onto 10 cm petri dishes (5x105 cells per
dish) 2 days in advance. They were then washed twice with DMEM medium,
serum-starved for 4 hours and preincubated with or without 5 mM mßCD or
mßCD/chol for 30 minutes at 37°C before addition of 1 µM TPA.
After incubating further for 10 minutes, the PKC activity was measured using
the Protein Kinase C Assay System from Life Technologies according to the
manufacturer's instructions.
Electron microscopy
The cells were washed twice with Hepes medium, serum-starved for 4 hours
and preincubated with or without 5 mM mßCD, mßCD/chol or a 1:1
mixture of mßCD and mßCD/chol for 30 minutes at 37°C before
addition of 1 µM TPA and HRP (10 mg/ml). After incubating the cells further
for 15 minutes at 37°C, they were washed with PBS and fixed in monolayer
with 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 60 minutes at
room temperature. The cells were then carefully washed with PBS (five times)
and incubated in PBS containing 0.5 mg/ml diaminobenzidine and 0.5 µl/ml of
a 30% H2O2 solution for 60 minutes at room temperature.
The cells were then washed, scraped off the flasks, pelleted, post-fixed with
OsO4, contrasted en block with 1% uranyl acetate, dehydrated in a
graded series of ethanols and embedded in Epon. Sections were further
contrasted with lead citrate and uranyl acetate and examined in a Phillips CM
100 electron microscope (Phillips, Eindhoven, The Netherlands).
Immunofluorescence
Cells grown on glass coverslips were serum-starved for 4 hours before
incubation with or without 5 mM mßCD, mßCD/chol or a 1:1 mixture of
mßCD and mßCD/chol for 30 minutes at 37°C, followed by
incubation with TPA (concentrations as indicated in figure legends) in the
absence or presence of mßCD, mßCD/chol or a 1:1 mixture of mßCD
and mßCD/chol for the indicated times at 37°C. After a brief wash
with PBS, the cells were processed accordingly.
For vizualisation of actin the cells were fixed and permeabilized with 3% paraformaldehyde in PBS for 1 hour at room temperature, and then incubated with 50 mM NH4Cl in PBS for 10 minutes. The cells were labeled with rhodamine-labeled phalloidin diluted in PBS containing 0.5% FCS for 30 minutes at room temperature.
For localization of endogenous Rac1 or epitope-tagged Rac1, the cells were permeabilized with 0.05% saponin in Pipes buffer (80 mM Pipes (dipotassium salt), 5 mM EGTA, 1 mM MgCl2, pH 6.8) for 5 minutes at room temperature both before and after fixation with 3% paraformaldehyde for 15 minutes, and then incubated with 50 mM NH4Cl in PBS for 10 minutes. The cells were labeled with primary and secondary antibodies diluted in PBS containing 0.05% saponin for 30 minutes at room temperature.
After staining, the coverslips were mounted in Mowiol (Calbiochem, San Diego, CA). Confocal microscopy was performed by using a Leica (Wetzlar, Germany) confocal microscope. Images were taken atx63 magnification and captured as images at 1024x1024 pixels. Montages of images were prepared with the use of PhotoShop 4.0 (Adobe, Mountain View, CA).
Transient transfection of cells
The wild-type and T17N mutant Rac1 constructs containing an N-terminal
epitope tag (MEYMPMEHM; termed EE) in the modified pCDL-SR expression
vector (pXS) (Takebe et al.,
1988
) and untagged wild-type and T27N mutant ARF6 constructs also
in the pXS expression vector were a generous gift from J.G. Donaldson
(National Institutes of Health, Bethesda, MD).
Cells grown on glass coverslips were transfected using Fugene 6 (Roche) according to the manufacturer's instructions. 40 hours after transfection the cells were serum-starved for 4 hours before being stimulated with 0.1 µM TPA for 10 minutes and processed for indirect immunofluorescence.
Determination of Rac1 activation state
Cells grown to confluence in 3 cm dishes were washed twice with
phosphate-free DMEM (Life Technologies), and serum-starved for 4 hours at
37°C with CO2 in the same medium supplemented with 0.5 mCi/ml
[32P]orthophosphate. The cells were then incubated with or without
5 mM mßCD for 30 minutes at 37°C with CO2 before
stimulation with 0.5 µM TPA for 10 minutes. After being washed three times
with ice-cold PBS, the cells were lysed in the lysis buffer (50 mM Hepes, pH
7.4, 1% Triton X-100, 100 mM NaCl, 5 mM MgCl2, 0.1 mM GTP, 1 mM
ATP, 10 mM Na-phosphate, protease inhibitors) for 5 minutes on ice. Nuclei and
cell debris were removed by centrifugation at 15,000 g for 2
minutes at 4°C, and the soluble fraction was subjected to
immunoprecipitation for 1.5 hours at 4°C with 5 µg/ml of anti-Rac1
coupled to protein A-sepharose per sample. The beads were then washed three
times with buffer (50 mM Hepes, pH 7.4, 500 mM NaCl, 5 MgCl2)
containing 1% Triton X-100, and three times with the same buffer containing
0.1% Triton X-100 and 0.005% SDS. The bound nucleotides were eluted in 8 µl
elution buffer (2 mM EDTA, 2 mM DTT, 0.2% SDS, 5 mM GDP, 5 mM GTP) for 15
minutes at 70°C, spotted onto a 0.1 mm PEI-cellulose TLC plate (Aldrich,
Milwaukee, WI), and developed for 40 minutes in 0.6 M Na-phosphate, pH 3.4 for
separation. Separated [32P]GDP and [32P]GTP were
quantified with the PhosphorImager (Applied Biosystems, Foster City, CA).
Binding of Rac1 to liposomes
Liposomes were prepared as earlier described
(Patki et al., 1997) by mixing
50% phosphatidylserine and 50% phosphatidylethanolamine or 50%
phosphatidylserine, 45% phosphatidylethanolamine and 5% cholesterol. The
mixtures were dried under nitrogen, and resuspended to a final concentration
of 1 mg/ml of total phospholipid in a Hepes buffer (50 mM Hepes, pH 7.2, 100
mM NaCl, 0.5 mM EDTA). The resuspended lipids were sonicated on ice for 5
minutes to obtain a homogeneous suspension. Liposomes were collected by
centrifugation at 140,000 g for 10 minutes and resuspended in
lysis buffer (20 mM Hepes, pH 7.2, 100 mM KCl, 2 mM MgCl2, 1 mM
DTT, protease inhibitors) to 3.75 mg/ml of total lipid.
To measure the binding of Rac1, a cell lysate was prepared of cells grown to confluence in 10 cm dishes, washed briefly with ice-cold PBS and scraped into 0.5 ml of lysis buffer (see above) on ice. The cells were lysed by passage through a 27-gauge needle attached to a 1 ml syringe five times at 4°C, and nuclei and cell debris were removed from the homogenates by spinning at 2500 g for 5 minutes at 4°C. Aliquots of lysate were mixed with 200 µl of each liposome mixture, vortexed once, and after 15 minutes at room temperature centrifuged at 140,000 g for 10 minutes. The supernatants and liposome pellets were separated on 12% SDS-PAGE and transferred to a PVDF membrane (Millipore Corporation, Bedford, MA) for detection of Rac1.
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Results |
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TPA-induced macropinocytosis can be measured as increased uptake of the
plant toxin ricin (Sandvig and van Deurs,
1990). We therefore investigated whether a decreased or increased
cellular cholesterol level affected this stimulation of endocytosis. A431
cells were serum-starved for 4 hours and incubated with TPA for 10 minutes to
stimulate macropinocytosis before the endocytosis of ricin was measured. To
remove or insert cholesterol, the cells were incubated with 5 mM mßCD or
mßCD/chol for 30 minutes prior to addition of TPA. As shown in
Fig. 1, TPA treatment increased
ricin endocytosis by nearly 60%. This stimulation of ricin endocytosis was
inhibited following treatment with mßCD, while treatment with
mßCD/chol had no effect on the TPA-stimulated increase in ricin
endocytosis (Fig. 1). As
previously shown (Rodal et al.,
1999
), extraction of cholesterol had essentially no effect on the
basal ricin endocytosis in this cell line
(Fig. 1). Control experiments
showed that when a 1:1 mixture of mßCD and mßCD/chol (total
concentration of 5 mM) was added, a mixture without any significant effect on
the cholesterol level (98.4%±3.6, n=3), the TPA-induced
endocytosis of ricin was unaffected (Fig.
1). In addition, as observed for TPA, EGF-stimulated
macropinocytosis was also inhibited following cholesterol depletion (data not
shown).
|
TPA is a potent activator of PKC
(Keller, 1990), and formation
of ruffles at the plasma membrane can in some cells be stimulated by PKC
(Swanson and Watts, 1995
;
Miyata et al., 1989
).
Therefore, we wanted to investigate whether the PKC activity was affected by
changes in the cellular cholesterol content. However, neither treatment with
mßCD nor mßCD/chol had any effect on the TPA-stimulated PKC activity
(Fig. 2A,B). This was supported
by the finding that mßCD inhibited the stimulated increase in ricin
endocytosis even when added after TPA (data not shown), while mßCD/chol
still had no effect on the TPA-induced increase in endocytosed ricin. Thus,
cholesterol depletion inhibits the TPA-stimulated increase in ricin
endocytosis without affecting PKC-activity.
|
Cholesterol depletion inhibits TPA-stimulated macropinocytosis at the
cell membrane
A prerequisite for macropinocytosis is formation of plasma membrane ruffles
and closure of these to form macropinosomes. To investigate whether this
process was affected by changes in the cholesterol content, serum-starved
cells incubated in the absence or presence of 5 mM mßCD or mßCD/chol
prior to and along with TPA treatment were studied by electron microscopy. In
addition, to check that mßCD in itself did not affect the processes
studied, the effect of a 1:1 mixture of mßCD and mßCD/chol (total
concentration of 5 mM) was investigated. As described above, the 1:1 mixture
of mßCD and mßCD/chol has no significant effect on the cholesterol
level nor on the TPA-induced increase of ricin endocytosis
(Fig. 1).
Control cells had irregular membrane protrusions at the free (`apical') surface and a few microvilli-like structures at the intercellular space (Fig. 3A,B). There was no macropinocytosis; only moderate endocytosis of HRP into endosomes had taken place (Fig. 3A). In addition, caveolea were present at the lateral membrane facing the intercellular space (Fig. 3B, arrowheads). Stimulation with TPA resulted in closure of ruffles (Fig. 3C,D, arrows), and subsequent formation of a large number of HRP-containing macropinosomes at the free surface (Fig. 3C,D). Furthermore, TPA treatment led to an increased complexity of lateral membranes at the intercellular space (Fig. 3E,F). As in control cells, caveolea were present along the intercellular space (Fig. 3F, arrowheads). Following treatment with mßCD, TPA-stimulated macropinocytosis of HRP at the free surface and formation of complex membrane structures at the lateral membranes facing the intercellular space were strongly inhibited (Fig. 4A,B). Moreover, the number of caveolea at the lateral membrane facing the intercellular space were strongly reduced (Fig. 4B).
|
|
In contrast to the observations described above, treatment with a 1:1 mixture of mßCD and mßCD/chol did not affect the TPA-induced macropinocytic activity at the free surface (Fig. 4C, arrows). In addition, complex membrane structures were formed at the lateral membranes at the intercellular space, and caveolea were also present at these membranes (Fig. 4D). Also, increasing the cholesterol content by treatment with mßCD/chol had no effect on the TPA-induced formation of macropinosomes at the free surface nor on the presence of caveolea and complex membrane structures at the intracellular space. An overview of the findings obtained by electron microscopy is presented in Table 2.
|
TPA-induced actin reorganization at the cell periphery is inhibited
following extraction of cholesterol
Since formation of membrane ruffles is dependent on the reorganization of
filamentous actin at the plasma membrane
(Swanson and Watts, 1995;
Ridley, 1994
), we investigated
whether any changes in the TPA-stimulated actin reorganization were
discernible following changes in the cholesterol level. Serum-starved cells
were incubated in the absence or presence of 5 mM mßCD, mßCD/chol or
a 1:1 mixture of mßCD and mßCD/chol prior to and along with TPA
treatment and studied in the confocal microscope using rhodamine-labeled
phalloidin to visualize filamentous actin.
In control cells the actin cytoskeleton was seen mainly as distinct filament bundles at the free surface of the cell, running both parallel and perpendicular to the surface (Fig. 5A-C). The latter orientation probably corresponds to the irregular protrusions seen at the free surface in control cells by EM. The actin staining along the intercellular space showed great variation, from hardly visible to quite pronounced (Fig. 5A-C). Treatment with TPA primarily caused a marked reorganization of the actin cytoskeleton at the cell periphery (Fig. 5D-I). Within 2-4 minutes ruffling activity was observed at the free surface (Fig. 5E-G). After 8-10 minutes this activity was somewhat reduced (Fig. 5H,I). Also, TPA treatment led within minutes to an increased and consistent actin staining at the lateral borders of the cells (Fig. 5D-I), presumably corresponding to the increased frequency of complex membrane structures at the lateral intercellular space seen by EM after TPA treatment. Importantly, the actin reorganization at the free surface observed after TPA treatment was prevented by cholesterol depletion (Fig. 6A,B). Neither an increased cholesterol level nor treatment with a 1:1 mixture of mßCD and mßCD/chol that does not change the cholesterol content had any effect on the TPA-induced reorganization of the actin filaments to the cell periphery (Fig. 6C,D). Thus, cholesterol depletion seems to inhibit TPA-stimulated ruffling at the plasma membrane by inhibiting the reorganization of the filamentous actin network at the cell periphery.
|
|
The localization of Rac1 to the plasma membrane is dependent on
cholesterol
Earlier studies have shown that TPA-stimulation can localize the activated
GTP-binding protein Rac 1 to the plasma membrane where it induces actin
reorganization necessary for formation of membrane ruffles
(Ridley, 1994;
Ridley et al., 1992
). Thus,
lack of actin filament reorganization and formation of ruffles following
cholesterol depletion might be due to prevention of Rac 1 activation or
localization of activated Rac 1 to the plasma membrane. We first investigated
whether the TPA-induced activation of Rac 1 was affected by cholesterol
depletion by determining the GTP:GDP ratio of Rac 1. For this purpose the
cells were incubated with 32PO43- during
serum-starvation to label endogenous pools of GTP and GDP. The cells were then
lysed, Rac 1 was immunoprecipitated and 32P-labelled nucleotides
bound to the immunoprecipitated protein were analyzed by thin-layer
chromatography. As shown in Fig.
7, treatment with TPA increased the GTP:GDP ratio of Rac 1 to
about 150%. Interestingly, treatment with mßCD prior to TPA-stimulation
increased the GTP:GDP ratio of Rac 1 even further, to about 218% of control
levels. Thus, cholesterol depletion does not prevent the TPA-induced Rac 1
activation, but rather increases it.
|
We then investigated whether the membrane localization of activated Rac 1 was affected by decreased cellular cholesterol levels. Serum-starved cells incubated in the absence or presence of 5 mM mßCD prior to and along with TPA treatment were labeled with antibody against Rac 1 and studied in the confocal microscope. As shown in Fig. 8Aa, Rac 1 could not be observed at the plasma membrane in control cells. Following TPA-stimulation Rac 1 was clearly visible at the free surface of the cells where membrane ruffling and macropinocytosis take place (Fig. 8Ab). However, after treatment with mßCD, Rac 1 was no longer localized to the cell periphery following TPA-stimulation (Fig. 8Ac). Also, there was no longer staining of the membrane between the cells. Furthermore, when TPA-stimulated cells were subjected to a 5 minutes pulse with 15 mM mßCD to remove cholesterol, Rac 1 was (in contrast to cells not exposed to mßCD) no longer detected at the cell periphery (Fig. 8Ad). Thus, Rac 1 already localized to the plasma membrane is also sensitive to cholesterol depletion. By contrast, treatment with mßCD/chol did not affect the TPA-stimulated localization of Rac 1 to the cell periphery (data not shown). To verify that Rac 1 is important for TPA-induced ruffling and macropinocytosis in A431 cells, the effect of TPA-stimulation on cells transiently transfected with wild-type or a dominant-negative mutant form of Rac 1 was investigated. Following TPA-stimulation wtRac 1 was able to localize to the free surface of the cells and also actin reorganization was induced (Fig. 8Ba,b). In contrast, the dominant-negative Rac 1T17N did not localize to the free surface and the TPA-induced actin reorganization otherwise seen was inhibited (Fig. 8Bc,d). Consequently, Rac 1 is required for ruffling and macropinocytosis in A431 cells.
|
Since membrane cholesterol is important for localization of Rac 1 to the
free surface, we investigated whether one could measure a
cholesterol-dependent binding of Rac 1 to liposomes. However, this was not the
case (data not shown). Hence, although cholesterol is required for the
localization of activated Rac 1 to the plasma membrane, additional components
may be necessary for this localization. It has previously been shown that the
ARF proteins function as regulators of membrane traffic
(Moss and Vaughan, 1998), and
it has been suggested that ARF6 is required for localization of activated Rac
1 to the plasma membrane (Radhakrishna et
al., 1999
; Zhang et al.,
1999
). Thus, the possibility that ARF6 colocalized with Rac 1 at
the cell periphery in TPA-stimulated cells was investigated. As shown
(Fig. 9), TPA treatment changed
the distribution also of ARF6, and ARF6 localized to Rac 1-containing regions
at the free surface in TPA-stimulated cells. This TPA-induced localization was
inhibited following treatment with mßCD to remove cholesterol
(Fig. 9). Consequently,
cholesterol depletion may affect the ability of ARF6 to localize activated Rac
1 to the plasma membrane. We also transfected A431 cells with a
dominant-negative ARF6 mutant to investigate whether this would affect the
TPA-induced Rac 1 localization and actin distribution. However, these
experiments were not conclusive: in some transfected cells mutant ARF6 seemed
to inhibit downstream effects whereas in others this was not clear, a finding
that may be due to the high endogenous level of ARF6 in A431 cells (J. G.
Donaldson, personal communication)
(Donaldson and Radhakrishna,
2001
).
|
![]() |
Discussion |
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Cholesterol has been found to be required for the membrane localization of
several proteins (Oliferenko et al.,
1999; Maekawa et al.,
1999
; Thiele et al.,
2000
). Interestingly, confocal microscopy showed that the
TPA-stimulated localization of Rac 1 to the cell periphery was inhibited in
cholesterol-depleted cells. Furthermore, localization of membrane-associated
Rac1 to the plasma membrane (following TPA-stimulation) was also changed by
cholesterol depletion. These results are in agreement with recent findings
showing that Rac1 is localized in raft domains
(Michaely et al., 1999
;
Kumanogoh et al., 2001
), and
can be partially solubilized following treatment with mßCD
(Kumanogoh et al., 2001
).
Following stimulation with phorbol esters the small GTPase Rac1 is normally
activated and localized to the plasma membrane where it induces actin
reorganization and subsequent membrane ruffling
(Ridley et al., 1992
). As
shown here, the TPA-stimulated activation of Rac1 was not inhibited by
decreased cellular cholesterol levels. Rather, treatment with mßCD
increased the TPA-stimulated activation of Rac1 by nearly 70%. The reason for
this increase is not obvious, but could be due to the lack of membrane
localization and a membrane-dependent or membrane-localized GTPase activity.
Thus, the inhibition of membrane ruffling following cholesterol depletion was
not due to prevention of Rac1 activation, but seems to be due to lack of
membrane localization of GTP-bound Rac1.
Cholesterol depletion might lead to a redistribution of other lipids
required for the formation of ruffles. Phosphatidylinositol 4,5-bisphosphate
[PtdIns(4,5)P2] has been detected in cholesterol-enriched
microdomains (Pike and Casey,
1996), and such a compartmentalization would be lost following
treatment with mßCD (Pike and Miller,
1998
). Accumulation of PtdIns(4,5)P2 at the
plasma membrane was found to precede the formation of ruffles prior to
macropinocytosis (Tall et al.,
2000
), and it has been suggested that the local concentration of
this lipid promotes the recruitment of proteins necessary for actin anchorage
and reorganization (Tall et al.,
2000
; Botelho et al.,
2000
). However, accumulation of PtdIns(4,5)P2
seems to occur downstream of membrane recruitment of Rac1
(Tolias et al., 2000
). It was
recently shown that activated Rac1 stimulates
PtdIns(4,5)P2 synthesis through binding to
phosphatidylinositol-4-phosphate-5-kinase
[PtdIns(4)P5-kinase
] resulting in actin reorganization
(Tolias et al., 1995
;
Tolias et al., 2000
). As
cholesterol depletion inhibits the localization of activated Rac1 to the
plasma membrane this might prevent stimulation of
PtdIns(4,5)P2 synthesis required for actin reorganization
and subsequent membrane ruffling at the plasma membrane. This is in agreement
with our findings that actin reorganization at the cell periphery following
TPA-stimulation was perturbed in cholesterol-depleted cells.
Although cholesterol-rich membrane domains might be required for the
localization of activated Rac1 at the plasma membrane, such domains may not be
sufficient. Cholesterol could be required for an indirect binding of Rac1 to
the plasma membrane through protein-protein interactions. Several proteins
have been found to bind directly to the activated Rac1 and participate in the
ruffling response (Van Aelst et al.,
1996; Di Cesare et al.,
2000
; Hansen and Nelson,
2001
). One protein that might be important for Rac1 function is
ARF6. ARF6 belongs to a protein family that function as regulators of membrane
traffic (Moss and Vaughan,
1998
). It has been suggested that ARF6 is required for the
localization of activated Rac1 to the plasma membrane
(Radhakrishna et al., 1999
;
Zhang et al., 1999
).
Interestingly, we found that cholesterol depletion inhibited the TPA-induced
colocalization of ARF6 and Rac1 at the free surface of the cells. Although
this does not demonstrate that ARF6 is involved in the response studied here,
cholesterol-dependent binding of ARF6 might be required for localization of
activated Rac1 to the plasma membrane. This could explain why we were unable
to demonstrate a cholesterol-dependent binding of Rac1 to liposomes. Together
our results suggest that cholesterol is necessary for the membrane association
of the small GTPase Rac1 required for membrane ruffling and
macropinocytosis.
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Acknowledgments |
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References |
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