2955
Journal of Cell Science 113, 2955-2961 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
JCS1380
Membrane recruitment of Rac1 triggers phagocytosis
Flavia Castellano1,*, Philippe Montcourrier2 and Philippe Chavrier1,‡
1Centre d’Immunologie INSERM-CNRS de Marseille-Luminy, 13288 Marseille Cedex
2CNRS UMR 5539, Université Montpellier II, 34095 Montpellier Cedex 5, France
9, France
*Present address: CNRS UMR144, Institut Curie, Section Recherche, 26 rue d’Ulm, 75241 Paris Cedex 5, France
‡Author for correspondence (e-mail: philippe.chavrier@curie.fr)
Accepted 14 June; published on WWW 9 August 2000
SUMMARY
Rac1 is a Rho-family GTP-binding protein that controls
lamellipodia formation and membrane ruffling in
fibroblasts. Recently, Rac1 and Cdc42, another member of
the Rho-family, have been shown to regulate Fc receptormediated phagocytosis in macrophages by controlling
different steps of membrane and actin dynamics leading to
particle engulfment. Here, we investigated the function of
Rac1 using a membrane recruitment system that mimics
phagocytosis. Recruitment of an activated Rac1 protein to
the cytoplasmic domain of an engineered membrane
receptor by using rapamycin as a bridge induces ingestion
of latex beads bound to the receptor. Rac1-mediated bead
uptake depends on actin polymerisation since actin
filaments accumulate at the bead/membrane binding
sites and internalisation is inhibited by cytochalasin D.
Internalisation is also abolished upon substitution of Phe37
to Leu in the Rac1 effector region. Our results indicate that
by promoting actin polymerisation at particle attachment
sites, Rac1 by acting through specific downstream effectors
induces plasma membrane remodeling that allows particle
internalisation in a membrane-enclosed phagosome.
INTRODUCTION
engaged FcRs and Rho GTPases may involve specific guanine
nucleotide exchange factors, such as Vav, which promotes GTP
exchange in a protein tyrosine kinase- and PI3-kinasedependent manner (Crespo et al., 1997; Han et al., 1998). In
addition, local actin cytoskeletal reorganisation is required
during cell invasion by various bacterial pathogens and in some
cases a role for Rho GTP-binding proteins has been
demonstrated during the entry process (Finlay and Cossart,
1997).
We have recently developed a new approach in order to
induce the translocation of activated Rho proteins to the
cytoplasmic domain of a membrane receptor, bypassing
upstream signalling events (Castellano et al., 1999). This
system is based on the ability of rapamycin to act as an adaptor
to join FKBP and FRB protein domains (Rivera et al., 1996).
In our experimental set-up, phagocytic RBL-2H3 cells express
a membrane receptor that includes FKBP domains within its
cytoplasmic region. These cells also express a constitutively
activated Rho GTPase fused with an FRB domain. In the
absence of rapamycin these two chimeric proteins cannot join
and the Rho/FRB chimera accumulates as an inactive protein
in the cytosol, whereas upon addition of rapamycin to the
culture medium, the activated Rho GTPase is recruited to the
FKBP receptor. Moreover, local enrichment of the active
GTPase can be achieved by clustering FKBP receptors with
antibody-coated latex beads (Castellano et al., 1999). Using
this system we have found that Cdc42 orchestrates multiple
pathways leading to filopodium formation (Castellano et al.,
1999).
We have now used the rapamycin system to test Rac1 and
Phagocytosis is the process which results in the uptake of large
particles (≥1 µm) by an actin-based mechanism. It is initiated
by the circumferential zippering of particle and phagocytic cell
surface mediated by the serial attachment of particle-bound
ligands to phagocytic cell membrane receptors (Griffin et al.,
1975). In the case of Fc receptors (FcRs), binding of Igopsonized particles triggers the recruitment of various
signalling proteins to the particle binding site and results in
actin filament polymerisation (Greenberg, 1995). Actin
assembly provides the driving force for particle engulfment by
allowing the extension of membrane pseudopods that wrap the
particle and eventually close to form a phagosome.
Rho GTPases regulate actin cytoskeleton organisation in
various cell types, including macrophages, by cycling between
GDP-bound inactive and GTP-bound active conformations
(Van Aelst and D’Souza-Schorey, 1997; Allen et al., 1997; Cox
et al., 1997). Recently, we and others have shown that members
of the Rho family control actin organisation during FcRmediated phagocytosis (Hackam et al., 1997; Cox et al., 1997;
Massol et al., 1998; Caron and Hall, 1998). Our morphological
analyses have revealed that Cdc42 and Rac1 act at different
stages during phagosome assembly by promoting pseudopod
extension and phagosome closure, respectively (Massol et al.,
1998). RhoA, the third well characterised member of the Rho
family, is also recruited to FcγR phagosomes but whether it
directly participates or not in phagosome formation remains
controversial (Hackam et al., 1997; Caron and Hall, 1998).
Although not precisely defined, the connection between
Key words: Phagocytosis, Rac1, Actin, Rapamycin
2956 F. Castellano, P. Montcourrier and P. Chavrier
report that recruitment of Rac1 to the cytoplasmic domain of
the FKBP receptor triggers the internalisation of receptorbound latex beads in a process that is inhibited by cytochalasin
D and resembles phagocytosis. A F37L substitution in the Rac1
effector site that is known to prevent interaction with
downstream targets including POR1, abolishes Rac1-mediated
phagocytosis. This is the first demonstration that Rac-1
recruitment to the receptor site is sufficient to allow particle
internalisation. We propose that Rac1, by recruiting specific
effectors to the site of particle binding, is responsible for
plasma membrane zippering around the particle surface that
eventually results in phagosome closure and particle
internalisation.
MATERIALS AND METHODS
Chimera construction and transfection
Rac1V12-FRB was generated by PCR by replacing the carboxyterminal CVLL membrane anchoring motif of Rac1 with the FRB
domain of human FRAP (from plasmid pCGNN-FRB(B); Ariad,
Boston, USA) that was tagged with a myc epitope at the carboxy
terminus. The double mutants Rac1V12L37-FRB and Rac1V12H40FRB have been generated by PCR following the megaprimer method
with the mutagenizing oligos 5′-cccactgtcCTTgataat-3′ and 5′tttgacaatCACtctgcc-3′, respectively. The cell line 15B, expressing
CD25-FKBP2, was transfected as previously described (Guillemot et
al., 1997) with constructs encoding RacV12-FRB (clone 15BE22),
RacV12L37-FRB (clone 15BE1L37) or Rac1V12H40-FRB (clones
15BE7H40 and 15BE10H40).
Latex bead phagocytosis assay
RBL-2H3-derived stable cell lines were grown on coverslips (2×105
cells/well in 24-well plates) and incubated with 100 nM rapamycin in
dimethylsulfoxide (DMSO) or DMSO only for 16 hours, followed by
incubation with 5 µg/ml biotinylated mouse anti-CD25 antibody
(clone B1.49.9, Immunotech, Marseille, France) for 1 hour. After
washes in cold DMEM, 1 µm diameter green fluorescent or non
colored streptavidin-labeled latex beads (Sigma) were added (10
beads/cell), sedimented on the cells by centrifugation at 400 g for 2
minutes, and incubated on ice for 20 minutes. When indicated,
cytochalasin D (1 µg/ml) was added 5 minutes before addition of the
beads and maintained throughout the experiment. Excess beads were
washed off and pre-warmed 37°C medium was added. Incubations
were carried out for 20 minutes to 2 hours at 37°C and the cells were
fixed with 3% paraformaldehyde. For quantification of phagocytosis,
after 2 hours at 37°C, cells were fixed and processed for
immunofluorescence without permeabilisation. Rabbit anti-mouse
antibodies followed by Texas-red-conjugated anti-rabbit antibodies
were used to detect uningested anti-CD25 coated (red) beads. Cells
were scored for the presence of attached, but uningested (red) beads
and total beads, visualised by phase contrast. Internalised beads
represent the difference between total and attached, uningested beads
(i.e. total minus red). At least 50 cells were scored for each clone and
each condition and data from at least three independent experiments
were averaged. Data are presented as average percentage of
internalised beads to total number of beads per cell. Significance of
data was calculated by the Student’s t-test.
Scanning and transmission electron microscopy
SEM analyses were performed as described previously (Castellano et
al., 1999). For transmission EM, cells on glass coverslips were fixed
with 2.5% glutaraldehyde, 1.8% paraformaldehyde in 0.1 M
cacodylate buffer, pH 7.4, for 1 hour at room temperature. Cells were
then scraped into the fixative, centrifuged for 10 minutes at 13000 rpm
and left in 0.1 M cacodylate buffer overnight. After washing in the
same buffer, the cell pellet was post-fixed with 2% osmium tetroxide
and stained ‘en bloc’ with 1% aqueous uranyle acetate, dehydrated
and embedded in Epon 812. Lead citrate contrasted ultra-thin sections
were observed with a Hitachi 7100 electron microscope.
Surface co-immunoprecipitation assay
15BE22 cells treated with 100 nM rapamycin (or DMSO as a control)
were incubated with rat anti-human CD25 antibody (clone 33B3.1,
Immunotech) for 1 hour at 37°C. Excess antibody was washed out
and cells were incubated in the presence of anti-rat IgG-coated
magnetic beads (M-450, Dynal) for 20 minutes on ice followed by 30
minutes at 37°C. The cells were subsequently lysed in 0.2% Triton
X-100 in BRB buffer (10 mM PIPES, pH 7.4, 3 mM NaCl, 100 mM
KCl, 3.5 mM MgCl2, 1.25 mM EGTA). The magnetic beads were
washed 3 times in BRB buffer by magnetic separation and bound
proteins were analysed by SDS-PAGE and immunoblotting with antimyc (clone 9E10) or anti-HA (clone 3F10, Boehringer Mannheim)
antibodies.
RESULTS
We have recently described a cell line (called 15B) derived
from Rat basophilic leukemia (RBL-2H3) cells that stably
express a chimeric surface receptor (Castellano et al., 1999).
This receptor, termed CD25-FKBP2, consists of the
extracellular and trans-membrane regions of human CD25,
fused with two copies of the rapamycin-binding FKBP12
polypeptide as a cytoplasmic domain (see Fig. 1a). 15B cells
were further transfected with a construct expressing Rac1V12FRB consisting of constitutively activated Rac1V12 deleted of
its carboxy terminal membrane-anchoring motif (CAAX box)
that was replaced by the FKBP/rapamycin-binding (FRB)
domain of human FRAP (Chen et al., 1995) (Fig. 1a). One of
the selected doubly-transfected cell lines was used throughout
this study (clone 15BE22). As shown in Fig. 1b, association of
Rac1V12-FRB to the cytoplasmic FKBP domains of the
chimeric membrane receptor in 15BE22 cells is induced by
rapamycin that acts as an adaptor to join FKBP to FRB (see
Castellano et al., 1999).
Capacity of clustered Rac1 to mediate phagocytosis
Treatment with rapamycin did not induce dramatic
morphological perturbations of the cell surface nor did it cause
a visible reorganisation of the actin cytoskeleton in 15BE22
cells (data not shown). This observation suggests that
membrane recruitment of activated Rac1 to diffusely
distributed receptors at the plasma membrane was not
sufficient to induce reorganisation of the cortical actin
cytoskeleton and plasma membrane remodelling. We therefore
investigated whether increasing the concentration of Rac1V12
at discrete sites of the plasma membrane would result in local
cell surface changes. Enrichment was achieved by clustering
CD25-FKBP2 receptors with latex beads (Castellano et al.,
1999). Rapamycin-treated 15BE22 cells were incubated in the
presence of biotinylated anti-CD25 antibodies followed by
incubation with streptavidin-coated beads, fixed and processed
for scanning electron microscopy analysis. Surprisingly,
recruitment of activated Rac1 at bead binding sites was
sufficient to trigger internalisation of beads after 20 minutes at
37°C (Fig. 2b). Bead internalisation was not observed with
streptavidin beads when biotinylated anti-CD25 antibodies
Rac1-mediated phagocytosis 2957
a
CD25
(extracellular)
CD25
(TM)
FKBP
CD25-FKBP2
G12V
Rac1-V12-FRB
FRB myc
HA
filaments with some running parallel to the beadmembrane interface (Fig. 4). These microfilaments
have an average diameter of 8.2±0.5 nm, the
expected size of actin filaments although we
cannot exclude that they might be intermediate
filaments. These filamentous structures are likely
to be actin filaments accumulating upon local
recruitment of activated Rac1 at bead-membrane
contact sites, suggesting that bead internalisation
was accompanied by and perhaps dependent on
highly localised cytoskeletal reorganisation.
Rac1-mediated phagocytosis is an actinbased process
To determine whether Rac1-mediated bead
internalisation was dependent on changes in the
actin cytoskeleton, 15BE22 cells were treated with
cytochalasin D, an inhibitor of actin filament
polymerisation at barbed ends and a classical
inhibitor of phagocytosis (Zigmond and Hirsch,
1972). The percentage of internalised beads was
evaluated by indirect immunofluorescence
according to Braun et al. (1998). This method
Fig. 1. Regulation of Rac1V12 membrane translocation by rapamycin.
allows staining of the beads that are outside non
(a) Schematic representation of CD25-FKBP2 with the CD25 extracellular and
permeabilised cells using anti-CD25 antibodies,
transmembrane domains fused to two tandemly repeated copies of the rapamycinwhile the total number of beads is evaluated by
binding protein FKBP12 (FKBP) as a cytoplasmic region. In Rac1V12-FRB, the
contrast phase microscopy. Internalised beads
carboxy-terminal membrane anchoring motif (CAAX box) of Rac1 is replaced by
were evaluated as difference between the total
the FKBP/rapamycin-binding domain, FRB. (b) Intracellular association of
Rac1V12-FRB with CD25-FKBP2 was tested by an anti-CD25 surface conumber of bead per cell and the external red beads
immunoprecipitation assay (see Materials and Methods) of stably transformed
(Fig. 5). Rapamycin-treatment of control 15B cells
15BE22 RBL-2H3-derived cells cultured in the absence (−) or presence (+) of
did not cause a significant increase of bead
rapamycin. Immunoprecipitates were analysed by immunoblotting with anti-HA
internalisation over the background level (15B
and anti-myc antibodies to detect HA-tagged CD25-FKBP2 and myc-tagged
cells ingested approx. 10% of cell associated beads
Rac1V12-FRB, respectively.
irrespective of rapamycin addition, Fig. 5a). In
contrast, bead internalisation was shown to
were omitted (beads did not bind to the cell surface, not
increase from a background value of approx. 10% in the
shown). Furthermore, neither anti-CD25 dependent binding of
absence of rapamycin up to 40% upon rapamycin-treatment of
streptavidin coated beads to 15BE22 cells in the absence of
15BE22 cells (Fig. 5a). Treatment of 15BE22 cells by
rapamycin, nor to rapamycin-treated 15B cells that express the
cytochalasin
D
blocked
rapamycin-induced
bead
CD25-FKBP2 receptor alone was sufficient to induce bead
internalisation (Fig. 5a) without affecting binding of the beads
internalisation (Figs 2a and 5a). CD25-bound beads were seen
(not shown), demonstrating that actin polymerisation is
to sink directly into the cytoplasm of rapamycin-treated
required during Rac1-mediated phagocytosis. These findings
15BE22 cells (Fig. 2b, arrows). Small cytoplasmic extensions
strongly suggest that recruitment of activated Rac1 at beadwere observed at the internalisation sites, but the membrane
membrane binding sites induces actin polymerisation leading
never rose over the particles as with Fc receptor-(FcR)
to actin cytoskeleton remodelling that triggers phagocytosis.
mediated phagocytosis (see Massol et al., 1998). Many
Immunofluorescence analysis of phalloidin-labelled cells
ingested beads were also visible underneath the plasma
membrane (arrowheads).
Table 1. Distribution of beads by location
Upon comparative analyses by transmission EM and
Number of beads per 100 sectioned cells
Total number
scanning EM of beads at different stages of internalisation we
of
confirmed that CD25-bound beads were internalised by sinking
15BE22
Sinking*
Ingested‡
sectioned cells
into the cytoplasm upon rapamycin treatment (Fig. 3a-c; Table
− Rapamycin
5.7
5.7
616
1). In addition, we definitively confirmed the presence of latex
+ Rapamycin
34.4
47.1
433
beads that were present in membrane-enclosed phagosomes in
15BE22 cells treated or not with rapamycin were incubated with
rapamycin-treated 15BE22 cells (Fig. 3d; Table 1). Beads in
biotinylated anti-CD25 antibodies followed by incubation with streptavidincoated latex beads at 37°C for 2 hours. Then cells were fixed and processed
the process of internalisation were often associated with small
for electron microscopy. Location of the beads is defined by two categories:
extensions of the membrane, resembling microvilli (Fig. 3b-c).
*Sinking: the plasma membrane underneath the bead is curved and partially
We also noticed an accumulation of invaginated coated pits at
surrounds the bead that is not completely internalised. This location
the bead-membrane interface that were also visible on the
corresponds to any of the stages documented in Fig. 3a-c.
‡Ingested: the bead is inside the cell in a membrane-closed phagosome as
phagosome membrane (Fig. 3, arrows). Higher magnification
in Fig. 3d.
examination revealed the presence of a loose meshwork of
2958 F. Castellano, P. Montcourrier and P. Chavrier
Fig. 2. Scanning electron microscopy of Rac1-mediated
phagocytosis. 15B cells expressing the CD25-FKBP2 receptor alone
or 15BE22 cells expressing both CD25-FKBP2 and Rac1V12-FRB
were treated with rapamycin and incubated with biotinylated antiCD25 antibodies followed by streptavidin-coated latex beads (1 µm
diameter) for 20 minutes. (a) CD25-bound beads at the surface of
rapamycin-treated 15B cells. (b) Rapamycin-treated 15BE22 cells.
This micrograph shows partially (arrows) and fully ingested CD25bound beads (arrowheads) upon rapamycin-mediated recruitment of
Rac1V12 to the bead-membrane binding site. Bar, 3.6 µm.
revealed a weak accumulation of F-actin (not shown),
confirming that cytoskeletal changes accompanying bead
internalisation were local.
Effect of effector domain mutations on Rac1mediated phagocytosis
Selected amino acid substitutions in the effector loop of Rac1
have been shown to destroy interaction with specific
downstream targets. One such mutant, Rac1V12L37 (F37L
substitution) binds serine/threonine kinases of the PAK family
while it fails to bind POR1, a Rac1-interacting protein
functioning in membrane ruffling (Van Aelst et al., 1996).
Indeed, Rac1V12L37 was shown to activate PAK but did not
induce membrane ruffle formation (Joneson et al., 1996;
Lamarche et al., 1996). In contrast, Rac1V12H40 (Y40H
Fig. 3. Transmission and
scanning electron microscopy of
rapamycin-treated 15BE22 cells
incubated with latex beads. (a)
Micrographs showing CD25bound beads adhering to the
plasma membrane. (b and c)
Beads during the internalisation
process. (d) Fully ingested beads
in membrane-bound vacuoles.
Arrows at thin sections point to
coated pits at the beadmembrane interface. Left panels,
transmission EM of thin
sections. N, nucleus; mvb,
multivesicular body. Right
panels, SEM. All micrographs
were taken at the same
magnification. Bar, 1 µm.
Rac1-mediated phagocytosis 2959
Fig. 4. Higher magnification view of bead-membrane interface. The
thin section shows filamentous structures accumulating at bead
attachment site with some filaments running parallel to the plasma
membrane (arrows).
substitution) binds POR1 but not PAK (Joneson et al., 1996).
These mutations were introduced in the Rac1V12-FRB coding
sequence and the mutants were stably transfected into 15B
cells. Rac1V12L37 was expressed and associated with CD25FKBP2 to similar level than the Rac1V12 chimera (Fig. 5b and
c). In contrast, in two independent clones expressing a Rac1V12H40 mutant protein, the level of expression and association
with CD25-FKBP2 was reduced as compared to Rac1V12-FRB
(Fig. 5b-c). Rapamycin-mediated membrane recruitment of
Rac1V12L37 did not trigger bead uptake (Fig. 5a). In V12H40expressing cells rapamycin-mediated bead internalisation was
modest and not significantly different as compared to non
treated control cells (Fig. 5a). However, the lower level of
expression of the V12H40-FRB chimera in these clones does
not allow to conclude that this mutation affects bead
internalisation. Our results indicate that F37-dependent
interactions are essential for Rac1-mediated uptake.
DISCUSSION
Phagocytosis is a process initiated by the ligation of phagocytic
cell surface receptors with ligand-bound particles in a zipperlike fashion (Griffin et al., 1975). Receptor aggregation triggers
the recruitment of various signalling proteins at the particle
binding site and results in actin cytoskeleton reorganisation
required for particle ingestion (Greenberg, 1995). In the case
of FcRs, the principal receptors involved in bacterial clearance
during infection, aggregation induces activation of Src-family
PTKs that phosphorylate tyrosine residues present in
cytoplasmic motifs of the receptor subunits and referred to as
ITAMs (immunoreceptor tyrosine-based activation motif)
(Ghazizadeh et al., 1994). The phosphorylated ITAMs
subsequently serve as docking sites for the SH2 domains of the
cytosolic PTK p72Syk that coordinates early signalling events
leading to actin cytoskeleton reorganisation (Durden and Liu,
1994; Greenberg et al., 1994, 1996; Cox et al., 1996). A variety
of observations suggest that PI 3-kinase lies downstream of
p72Syk and is required for completion of particle internalisation
(Araki et al., 1996; Crowley et al., 1997). Interestingly,
particle-mediated aggregation of chimeric receptors composed
of an FcγR extracellular domain fused with a cytoplasmic tail
consisting of either p72Syk or PI 3-kinase (p85 subunit) was
sufficient to trigger actin cytoskeleton reorganisation and
particle internalisation (Greenberg et al., 1996; Lowry et al.,
1998). Even though the molecular mechanisms coupling
p72Syk and PI 3-kinase activities with phagocytosis are largely
unknown, recent observations indicate that FcR signalling
pathways converge to activate Rho GTP-binding proteins, and
in particular Cdc42 and Rac1, both essential for FcR-mediated
phagocytosis (Hackam et al., 1997; Cox et al., 1997; Massol
et al., 1998; Caron and Hall, 1998).
We now further extend these observations by showing that
localisation of Rac1 activity to the site of particle attachment
triggers particle internalisation in a process that requires
rearrangement of the actin cytoskeleton as uptake is impaired
by cytochalasin D. In contrast, Rac1-mediated uptake is
insensitive to the Src-family PTK specific inhibitor PP2 (Hanke
et al., 1996; data not shown), demonstrating that in this system,
early signalling events that normally precede Rac1 activation
(such as ITAM phosphorylation) may be bypassed. In addition,
inhibition of PI 3-kinase activity by wortmannin or LY294002
led only to a slight and non significant reduction of the level
of particle ingestion (wortmannin: 22±4.8%, LY294002:
22.3±5.8%), indicating that this process very likely occurs in
the absence of PI 3-kinase activation. Therefore, our results
suggest that PI 3-kinase enzymatic products may not exert their
main role in phagosome closure and/or actin cytoskeleton
remodelling during phagocytosis, but rather as second
messengers by promoting the localisation/activation of PH
domain-containing proteins such as GEF(s) upstream of Rho
GTP-binding proteins (Han et al., 1998).
Interestingly, our electron microscopy data show that Rac1
recruitment induces particles to sink into the cytoplasm and
does not trigger the formation of pseudopods that accompany
FcR-mediated phagocytosis (Massol et al., 1998). One possible
explanation is that, here, internalisation proceeds very likely in
the absence of Cdc42 activation. We have recently reported that
Cdc42 is essential during FcR-mediated phagocytosis by
controlling pseudopod extension (Massol et al., 1998), and
using the rapamycin-based approach we have shown that
membrane recruitment of activated Cdc42 leads to the
formation of actin-rich membrane protrusions (Castellano et
al., 1999). Altogether these findings indicate that Cdc42
regulates the formation of actin-driven protrusive structures. In
the absence of Cdc42 activation, Rac1 may be sufficient to
allow internalisation of the relatively small sized beads (1 µm
diameter) used during this study which are known to require a
moderate level of cytoskeletal reorganisation for ingestion
(Koval et al., 1998). Rac1-mediated entry appears
morphologically to be similar to complement receptor (CR)mediated phagocytosis (Allen and Aderem, 1996) or bacterial
invasion by Listeria or Yersinia that enter host cells via closefitting ‘zipper-like’ phagosomes (Dramsi and Cossart, 1998).
However, CR-mediated phagocytosis does not seem to require
2960 F. Castellano, P. Montcourrier and P. Chavrier
Fig. 5. Role of actin polymerisation and effects of the
Rac effector mutations L37 and H40 on Rac1
mediated phagocytosis. (a) Control cells expressing
the CD25-FKBP2 receptor alone (clone 15B), or cells
co-expressing the CD25-FKBP2 receptor together
with the FRB chimeras: Rac1V12 (clone 15BE22),
Rac1V12L37 (clone 15BEIL37), or Rac1V12H40
(clones 15BE10H40 and 15BE7H40) were treated or
not with rapamycin and the bead internalisation assay
was performed as described in Materials and Methods,
except that cells were fixed after 2 hours in the
presence of streptavidin beads. When indicated, cells
were treated with cytochalasin D (1 µg/ml) 5 minutes
before adding the beads. Cytochalasin D did not
interfere with binding of the beads (not shown). The
percentage of internalised beads was measured as
described in Materials and Methods. At least 50 cells
were scored for each condition per experiment. Data
represent mean percentages of ingested beads ±
s.e.m., n=3 independent experiments. The percentage
of internalised beads of samples marked with an
asterisk are significantly different from that of
DMSO-treated 15BE22 cells (P<0.001).
(b) Expression level of the different FRB chimeras.
Equal amount of total protein lysates were
immunoblotted with anti-myc antibody. Rac1V12FRB (clone 15BE22); Rac1V12L37-FRB (clone
15BEIL37); Rac1V12H40-FRB (clones 15BE10H40
and 15BE7H40). (c) Intracellular association of the
different Rac-FRB chimeras with CD25-FKBP2 was
tested by co-immunoprecipitation with anti-HA
tagged antibody from cells cultured in the absence (−)
or the presence (+) of rapamycin. Immunoprecipitates
were analysed by immunoblotting with anti-HA and
anti-myc antibodies to detect HA-tagged CD25FKBP2 and Myc-tagged Rac-FRB chimeras,
respectively.
Rac1 but has been shown to depend on the function of RhoA
(Caron and Hall, 1998). Inhibition of RhoA activity by
Clostridium botulinum exoenzyme C3 did not affect Rac1mediated bead internalisation induced upon rapamycin
treatment, indicating that this process does not involve RhoA
(data not shown). Therefore, the mechanisms for CR- and
Rac1-mediated phagocytosis are probably different. In
contrast, our observations may be more relevant to bacterial
invasion since internalin B-mediated invasion of epithelial cells
by Listeria requires the activity of PI-3 kinase (Ireton et al.,
1996), suggesting that the zipper-type entry of Listeria may be
mediated by PI-3 kinase-dependent Rac1 activation. Recently,
Reddien and Horvitz (2000) have shown that Ced10/Rac1 in
C. elegans controls engulfement of apoptotic cells, together
with the product of the Ced2 and Ced5 genes. Phagocytosis of
apoptotic bodies seems to occur without major membrane
remodelling (G. Chimini, personal communication). Our
observations of Rac1-dependent entry of particles could be
relevant also to this type of phagocytosis.
The machinery that acts downstream of Rac1 to promote
phagocytosis is presently unknown. Our results clearly
demonstrate that particle internalisation following Rac1
recruitment and activation requires actin polymerisation even
though it occurs in the absence of membrane ruffling. Rac1
activation has been shown to induce actin polymerisation in
vivo (Hartwig et al., 1995; Machesky and Hall, 1997) but fails
to do so in vitro (Zigmond et al., 1997). Interestingly, Rac1mediated internalisation is impaired by substitution of Phe37
to Leu in the Rac1 effector region, a mutation shown to abolish
the ability of Rac1 to mediate membrane ruffling and to
interact with POR-1 (Joneson et al., 1996). Therefore, POR-1
may be one of the effectors involved in Rac1-mediated uptake
we described here. However, there are no experimental data
supporting a direct role for POR-1 in actin polymerisation. In
platelets, Rac1 activation has been shown to stimulate the
production of PI 4,5 P2 to create free barbed ends by filament
uncapping and, hence, to promote actin polymerisation
(Hartwig et al., 1995). More recent observations suggest that
Rac1 may also be involved in formation of free barbed ends by
actin nucleation. Machesky and Insall have recently shown that
WASP/Scar/WAVE family members interact with and activates
the Arp2/3 complex, an actin filament nucleator and crosslinker (Machesky and Insall, 1998; Machesky et al., 1999).
Scar-1/WAVE that acts downstream of Rac1 may therefore link
Rac1 signalling to actin polymerisation (Miki et al., 1998).
How the cascades linking Rac1 to PI 4P-5 kinase or to
Scar/WAVE are affected by the Phe37 to Leu substitution is
presently unknown. We postulate that recruitment and local
Rac1-mediated phagocytosis 2961
activation of Rac1 at particle attachment sites trigger actin
polymerisation by filament uncapping and/or de novo
nucleation. These filaments would promote or stabilise the
zipper-type interaction between phagocytic receptors and
particle-bound ligands inducing a progressive engulfment of
the particle and eventually the internalisation of the particle in
a membrane closed phagosome.
We are indebted to Dr V. M. Rivera and ARIAD Pharmaceuticals,
Inc for providing the FKBP and FRB encoding cDNAs and to Dr M.
Popoff for the gift of C. botulinum ezoenzyme C3. We thank L.
Lepecuchel for help during the selection of the transfected cell lines.
We also thank Dr W. M. Hempel for critical reading of the manuscript.
F.C. was a recipient of a NATO fellowship. This work was supported
by INSERM and CNRS institutional fundings and a grant from the
Association pour la Recherche sur le Cancer (ARC No. 5449) to P.C.
We also thank the ‘Centre Régional d’Imagerie Cellulaire de
Montpellier’ for electron microscopy facilities.
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