Platelet-derived growth factor (PDGF) is a chemotactic factor for fibroblasts that triggers actin cytoskeleton reorganization by increasing the level of GTP-Rac, the activated form of a small Rho family GTPase. GTP-Rac induces membrane ruffling and lamellipodium formation that are required for adhesion, migration and macropinocytosis, among other functions. We have shown that WIP interacts with members of the Wiskott-Aldrich syndrome protein family and is essential for filopodium formation regulated by Cdc42 GTPase. In this report, we show that WIP participates in the actin reorganization that leads to ruffle formation. WIP overexpression in murine fibroblasts (3T3 cells)enhances ruffle formation in response to PDGF stimulation, as shown by immunofluorescence and electron and video microscopy. More importantly,microinjection of anti-WIP antibody or absence of WIP in murine fibroblasts results in decreased ruffle formation in response to PDGF treatment. Finally,overexpression of a modified form of WIP lacking the actin-binding site blocks PDGF-induced membrane ruffling. These data suggest a role for WIP in actin reorganization to form PDGF-induced ruffles. This is the first in vivo evidence in mammalian cells for a function of WIP dependent on its ability to bind actin.

Introduction

Actin cytoskeleton reorganization mediates a wide range of cellular events that include polarization, phagocytosis, motility, metastasis, chemotaxis,proliferation and cytokinesis (Penninger and Crabtree, 1999). Many efforts have been directed at linking the flow of signals from transmembrane receptors to downstream effectors of the actin cytoskeleton and have revealed the Rho GTPases Cdc42, Rac and Rho as principal targets that promote distinct cytoskeletal changes leading to the formation of filopodia, lamellipodia or stress fibers, respectively(Hall, 1998; Higgs and Pollard, 1999). Some of the specific downstream effectors of each Rho GTPase belong to the Wiskott-Aldrich syndrome protein (WASP) family. For instance, N-WASP mediates filopodium formation induced by Cdc42(Miki et al., 1998a) and WAVE2/Scar (WASP family verprolin-homologous protein) mediates membrane ruffling by Rac (Miki et al.,1998b). WASP family members exert their effect through activation of the Arp2/3 (actin-related protein) complex, which contains seven polypeptides with an essential role in actin nucleation and dendritic branching of actin filaments (Higgs and Pollard, 1999; Machesky and Insall, 1998; Svitkina and Borisy, 1999).

Actin reorganization mediates changes in cell shape associated with cell motility, migration and chemotaxis in response to platelet-derived growth factor (PDGF) in a range of cell types including 3T3 fibroblasts(Hooshmand-Rad et al., 1997; Wennstrom et al., 1994b; Westermark et al., 1990). PDGF binds to the PDGF receptor (PDGFR) and induces receptor dimerization,stimulation of its receptor tyrosine kinase activity and rapid autophosphorylation. The phosphorylated residues recruit phosphatidylinositol 3-kinase (PI3K), Nck-1, and Nck-2 through Src-homology domain 2 (SH2). Activation of PI3K leads to Rac1 activation and actin polymerization at the plasma membrane to produce edge ruffles and lamellipodia(Chen et al., 2000; Kazlauskas, 1994; Ridley et al., 1992; Wennstrom et al., 1994a).

The WASP-interacting protein, WIP, is broadly expressed(Ramesh et al., 1997; Vetterkind et al., 2002) and regulates actin polymerization and spatial organization in different cell types. WIP interacts in vitro with globular and filamentous actin (G- and F-actin, respectively) and stabilizes actin filaments(Martinez-Quiles et al.,2001). WIP also interacts with profilin, a regulator of actin polymerization and depolymerization(Ramesh et al., 1997). WIP overexpression in human B cell lines causes an increase in cellular F-actin content and induces the formation of subcortical actin patches(Ramesh et al., 1997). WIP microinjection to fibroblasts induces filopodia. This process depends on Cdc42(Martinez-Quiles et al.,2001), a GTPase that (in its active GTP-loaded form) binds to the hematopoietic-cell-specific WASP(Aspenstrom et al., 1996; Kolluri et al., 1996; Symons et al., 1996) and its more ubiquitously expressed homolog N-WASP(Miki et al., 1998a), causing a conformational change that allows WASP and N-WASP to interact with the Arp2/3 complex and initiate actin polymerization(Kim et al., 2000; Miki et al., 1996; Rohatgi et al., 2000). Because WIP regulates N-WASP-induced actin nucleation and is important for induction of actin-containing microspikes by bradykinin and Cdc42 in 3T3 fibroblasts(Martinez-Quiles et al.,2001), we set out to analyse its role in PDGF-stimulated actin cytoskeletal rearrangement in 3T3 cells. We found that WIP is involved in PDGF-induced the formation of dorsal circular ruffles, and that WIP binding to actin is essential for this function.

Materials and Methods

Antibodies

Anti-WIP antibody was raised by immunizing rabbits with a 14-amino-acid C-terminal peptide common to human and murine WIP sequences(483-ESRSGSNRRERGGP-496). Anti-WIP specificity was confirmed using WIP null cells derived from WIP-/- mice in western blots(Anton et al., 2002).

3T3 fibroblast culture and PDGF stimulation

NIH 3T3 or Swiss 3T3 murine fibroblasts were transfected with control plasmid (pcDNA), with pcDNA containing human WIP coding sequence (pcDNA-WIP)or with pcDNA containing a deletion mutant coding sequence(pcDNA-WIPΔ43-54) using the standard calcium phosphate protocol. The vector used as a control or as backbone for cloning was a modified pcDNA3 vector that expresses cloned cDNA as a N-terminal FLAG fusion protein(Ramesh et al., 1997). pcDNA-WIPΔ43-54 was constructed using the Qickchange®Site-Directed mutagenesis kit (Stratagene) with the oligo 5′-GCTCTCCTTTCTGATATCGACAGAAGTGCACC-3′.

Transfected cells were selected in the presence of G418 (1 mg ml-1) and maintained in Iscove's medium supplemented with 10% fetal bovine serum (FBS) and G418. Confluent 3T3 cells were split onto glass coverslips (1:10 for 3T3pcDNA and 1:20 for 3T3pcDNA-WIP) in Iscove's 10% FBS containing 1 mg ml-1 G418, grown for 24 hours and then serum-starved for 3-4 days in Iscove's 0.5% FBS with G418. Cells were washed twice with warm Iscove's medium and then stimulated or not with human recombinant PDGF-bb (Intergen) (50 ng ml-1) for 8 minutes at 37°C. In some experiments, cells were pretreated with DMSO as a control or with 12 nM wortmannin for 30 minutes at 37°C and then washed and stimulated with PDGF-bb as described above.

Microinjection

Confluent NIH 3T3 transfected cells were split onto glass coverslips in Iscove's 10% FBS containing G418 (1 mg ml-1), grown for 24 hours and then serum-starved for 3-4 days in Iscove's 0.5% FBS with G418. Microinjection was carried out using an Eppendorf automatic microinjector(Transjector 5246 and InjectMan Micromanipulator 5179) set at 50-60 hPa(hectopascals) for 0.3 seconds. Following microinjection, cells were incubated for 20 minutes prior to stimulation. Dextran cascade blue (Molecular Probes)was used as a tracer for microinjected cells mixed with affinity-purified anti-WIP IgG rabbit antibody or control purified rabbit IgG (Cappel) at 500μg ml-1. At least 150 cells were injected for each reagent. A total of 200 noninjected cells in the areas surrounding the injected cells were count for each cover slip.

Immunofluorescence

Stably transfected NIH or Swiss 3T3 fibroblasts, microinjected NIH 3T3 fibroblasts, or primary murine fibroblasts grown and stimulated as described above were fixed with 4% formalin solution in PBS (Sigma) for 10 minutes at room temperature, washed twice with PBS and permeabilized with 0.2% Triton X-100 in PBS at room temperature for 5 minutes. Cells were rinsed twice with PBS and non-specific binding was blocked with 10% bovine serum albumin (BSA)in PBS for 20 minutes at room temperature. Rabbit antiserum specific for WIP was diluted 1/250 in PBS containing 1% BSA and added to the cells for 1 hour at room temperature. Cells were rinsed twice with PBS and incubated with Alexa488-conjugated goat anti-rabbit (Molecular Probes; 1/1000) and TRITC(rhodamine)-conjugated phalloidin (Sigma; 1 μg ml-1) in PBS containing 1% BSA for 1 hour at room temperature. Cells were rinsed three times with PBS and air dried, and the coverslips were then mounted with antifade reagent (Molecular Probes) and visualized with a Nikon Eclipse E800 microscope. Photographs were taken using a CCD-300-RC camera and images were processed using Adobe Photoshop and Microsoft PowerPoint software. Software program NIH Image 1.62 was used to quantify fluorescence intensity.

Confocal microscopy was performed on a Bio-Rad MRC600. The CM program was used throughout. For double-labeling visualization, K1 and K2 filters were used. Software programs NIH Image 1.57 and Photoshop were used to analyse and construct images.

Electron microscopy

WIP-transfected and vector-transfected NIH 3T3 fibroblasts were grown and plated on coverslips as described above and starved for 4 days in Iscove's medium 0.5% FBS with G418. Starved cells were stimulated for 8 minutes with 50 ng ml-1 PDGF and fixed with PHEM buffer (60 mM PIPES, 25 mM HEPES,10 mM MgCl2 and 10 mM EGTA) containing 0.75% Triton X-100, 1 μM phallacidin and 0.05% glutaraldehyde for 2 minutes. Permeabilized cells were washed in PHEM buffer without fixative and then fixed with 1% glutaraldehyde in PHEM buffer for 10 minutes. The cytoskeletons were extensively washed into water, rapidly frozen on a helium-cooled copper block, freeze-dried in a Cressington CFE-50 apparatus at –90°C and rotary coated with 1.4 nm of platinum and 4 nm carbon without rotation. The cells were examined in a JEOL 1200 EX electron microscope using a 100 kV accelerating voltage.

Actin-binding assay

The expression and purification of the recombinant proteins glutathione-S-transferase/WIP (GST-WIP) 1-127 and GST-WIP 1-127Δ43-54, which lacks the actin-binding domain, were performed as previously described (Martinez-Quiles et al., 2001). The ability of these fusion proteins to bind G-actin was tested using a GST pull-down assay as previously described(Martinez-Quiles et al.,2001).

Video microscopy

Stably transfected NIH 3T3 fibroblasts were grown on glass coverslips,serum-starved for 4 days in the presence of 0.5% FBS, washed twice with warm Iscove's medium and then stimulated with PDGF-bb (50 ng ml-1) at 37°C using a warm stage. Frames were taken at 2-second intervals starting at 5 minutes using NIH Image 1.62 software at 400× magnification on a Nikon Eclipse TE200 microscope with a CCD-300-RC charge-coupled device camera. Images were processed using Microsoft PowerPoint software.

Derivation of murine primary fibroblasts

Lung pieces from wild-type or WIP-/- mice were washed with PBS,minced and cultured in Iscove's medium supplemented with 10% FBS, penicillin and streptomycin (50 U ml-1) for several days. After removal of unattached debris, adherent cells were trypsinized and maintained in culture. Trypsinized cells were lysed in SDS gel-loading buffer and analysed by western blot with rabbit anti-WIP serum as previously described(Anton et al., 1998).

Results

Overexpression of WIP increases ruffle formation in response to PDGF stimulation

We have previously identified an important role for WIP in the induction of Cdc42-dependent filopodia by bradykinin(Martinez-Quiles et al.,2001). To determine whether WIP is also involved in Rac-dependent ruffle formation by PDGF, we tested the effect of WIP overexpression on PDGF induction of ruffles in NIH 3T3 fibroblasts. NIH 3T3 cells were stably transfected with pcDNA plasmid containing the WIP coding sequence or with empty vector as control. The WIP transfected cells expressed three times more WIP than the parental (data not shown). Fig. 1Ashows that, in control pcDNA-transfected cells serum-starved for 4 days, most polymerized F-actin is present in the periphery of the cell, as seen by phalloidin staining(Fig. 1A, top left); these quiescent cells displayed few other actin structures such as stress fibers and ruffles. NIH 3T3 fibroblasts stably transfected with WIP were smaller and did not reach a completely quiescent state even after 4 days of serum starvation and they retained their stress fibers (Fig. 1A, bottom left). PDGF stimulation of control serum-starved cells resulted in the formation of dorsal or circular membrane ruffles of variable size and actin content rather than classical lamellipodia(Fig. 1A, top center). This phenotype is possibly the result of the long period of serum starvation that was necessary to obtain maximum quiescence of WIP-transfected cells and resembles the effect of overstimulation with agonists(Hawkins et al., 1995). PDGF treatment of serum-starved WIP-transfected cells induced very prominent ruffles that stained more intensely for actin than ruffles in control cells(Fig. 1A, bottom center). Quantitative analysis showed a 2.4 times increase in the F-actin content of ruffles from WIP-transfected cells versus control ones (53±24 units versus 121±20 units, P<10-8). In addition, PDGF treatment induced actin clusters and finger-like actin-containing projections in WIP-transfected cells that were not detected in control cells. The time course of ruffle formation and disappearance was similar in both cell lines:maximal ruffle formation was achieved 8 minutes after PDGF stimulation and all ruffling disappeared by 15 minutes (data not shown). The enhanced effect of PDGF in WIP-transfected cells was not due to a secreted factor because supernatants from these cells failed to enhance the ruffling response of control cells to PDGF (data not shown).

Fig. 1.

WIP overexpression increases PDGF-induced dorsal ruffle formation. (A)Fluorescence micrographs of TRITC-phalloidin stained NIH 3T3 fibroblasts transfected with control plasmid (3T3pcDNA) or with WIP coding sequence(3T3pcDNA-WIP) before and after PDGF challenge. Cells were grown on glass coverslips, serum starved for 4 days in the presence of 0.5% FBS, stimulated or not with PDGF-bb (50 ng ml-1) for 8 minutes and then fixed and labeled with TRITC-phalloidin to visualize actin filaments. Indicated cells(Wort+PDGF) were pretreated with 12 nM wortmannin for 30 minutes at 37°C and then stimulated with PDGF and stained as described. Magnification 400×. (B) Electron micrographs of cortical actin network of NIH 3T3pcDNA-WIP (left) and NIH 3T3pcDNA (right) fibroblasts stimulated as described above. (C) Frames of phase-contrast microscopy capture of NIH 3T3pcDNA and NIH 3T3pcDNA-WIP fibroblasts grown on glass coverslips and stimulated at 37°C with PDGF for the indicated times. Frames were processed using Microsoft PowerPoint software. Arrows point to membrane ruffles. Magnification 400×.

Fig. 1.

WIP overexpression increases PDGF-induced dorsal ruffle formation. (A)Fluorescence micrographs of TRITC-phalloidin stained NIH 3T3 fibroblasts transfected with control plasmid (3T3pcDNA) or with WIP coding sequence(3T3pcDNA-WIP) before and after PDGF challenge. Cells were grown on glass coverslips, serum starved for 4 days in the presence of 0.5% FBS, stimulated or not with PDGF-bb (50 ng ml-1) for 8 minutes and then fixed and labeled with TRITC-phalloidin to visualize actin filaments. Indicated cells(Wort+PDGF) were pretreated with 12 nM wortmannin for 30 minutes at 37°C and then stimulated with PDGF and stained as described. Magnification 400×. (B) Electron micrographs of cortical actin network of NIH 3T3pcDNA-WIP (left) and NIH 3T3pcDNA (right) fibroblasts stimulated as described above. (C) Frames of phase-contrast microscopy capture of NIH 3T3pcDNA and NIH 3T3pcDNA-WIP fibroblasts grown on glass coverslips and stimulated at 37°C with PDGF for the indicated times. Frames were processed using Microsoft PowerPoint software. Arrows point to membrane ruffles. Magnification 400×.

Induction of ruffles by PDGF depends on PI3K-mediated activation of Rac(Wennstrom et al., 1994a). However, PDGF also activates other signaling pathways that include phospholipase C-γ and Ras-GTPase-activating protein(Olivera and Spiegel, 1993). WIP overexpression might enhance PI3K-dependent ruffling or, alternatively,might synergize with a PI3K-independent pathway to induce dorsal ruffles. To distinguish between these two possibilities, we examined the effect of the PI3K inhibitor wortmannin on the PDGF response of control and WIP-transfected cell lines. Pretreatment with wortmannin completely inhibited PDGF-induced membrane ruffling in both control and WIP-transfected NIH 3T3 cells(Fig. 1A, right). This suggests that WIP exerts its effect on PI3K-dependent ruffle formation.

The actin networks in the cortical cytoplasm of PDGF-stimulated NIH 3T3 cells were examined electron microscopically after detergent extraction of the membrane. As observed by light microscopy, large ruffles within the cortex were readily visible as bands of dense actin lattice in WIP-transfected cells(Fig. 1B, left, inset ruffles at low magnification). By contrast, PDGF-stimulation of vector transfected cells shows only small ruffles at the cell edge(Fig. 1B, right, inset low magnification).

To determine the dynamics of ruffle formation, we videotaped serum-starved control and WIP-transfected fibroblasts starting 5 minutes after addition of PDGF and continuing for 5-10 minutes. Fig. 1C and see movie online(Supplementary Information)show that control cells developed few ruffles on the cell surface that, in phase-contrast microscopy, are visualized as black curves (arrows in Fig. 1C upper frames; see movie online). WIP-transfected fibroblasts showed more ruffles that were more motile than the ruffles of control cells (arrows in Fig. 1C lower frames; see movie online). WIP-transfected cells form 1.8 times more dorsal ruffles than pcDNA-transfected cells (11±2 versus 6±1 ruffles per field). Video microscopy confirmed that the time course of ruffle formation was similar in both cells lines (data not shown). These data suggest that overexpression of WIP enhances ruffle formation in response to PDGF stimulation.

WIP partially redistributes to membrane ruffles after PDGF stimulation

We next investigated whether WIP localizes to ruffles in PDGF-stimulated fibroblasts. Immunofluorescence analysis using rabbit anti-WIP serum revealed that endogenous WIP in quiescent control fibroblasts is primarily located in the cytoplasm with a punctate distribution that has a higher density in the perinuclear area (Fig. 2A, top left). PDGF stimulation induced redistribution of a small amount of WIP towards the membrane ruffle area (Fig. 2A, white arrows) while the strong staining in the perinuclear area persisted. Redistribution of WIP to membrane ruffles was more prominent in WIP-transfected cells (Fig. 2B, white arrows).

Fig. 2.

Subcellular distribution of WIP in the presence or absence of PDGF. Fluorescence micrographs of NIH 3T3 fibroblasts transfected with control plasmid (3T3pcDNA) (A) or with plasmid containing WIP coding sequence(3T3pcDNA-WIP) (B). Cells were grown and fixed as described in Fig. 1A and then labeled with rabbit serum specific for WIP followed by anti-rabbit Alexa488 (anti-WIP) plus TRITC-phalloidin to visualize actin filaments (Actin). Arrows point to dorsal/circular ruffles areas. Magnification 600×.

Fig. 2.

Subcellular distribution of WIP in the presence or absence of PDGF. Fluorescence micrographs of NIH 3T3 fibroblasts transfected with control plasmid (3T3pcDNA) (A) or with plasmid containing WIP coding sequence(3T3pcDNA-WIP) (B). Cells were grown and fixed as described in Fig. 1A and then labeled with rabbit serum specific for WIP followed by anti-rabbit Alexa488 (anti-WIP) plus TRITC-phalloidin to visualize actin filaments (Actin). Arrows point to dorsal/circular ruffles areas. Magnification 600×.

To determine the localization of WIP in ruffles, we performed confocal microcopy. WIP-transfected fibroblasts stimulated with PDGF were double stained for WIP and actin, and a total of 11 stacks from the substrate to the highest level with detectable fluorescence signal were recorded. Weak but specific anti-WIP staining was observed in the middle sections, with maximal intensity in stack 6 (Fig. 3,Section 6), but not in the lowermost or uppermost sections(Fig. 3, Sections 1 and 11). This signal was specific because no signal was detected using preimmune rabbit serum (data not shown).

Fig. 3.

Distribution of WIP along the membrane ruffle structure by confocal microscopy. NIH 3T3pcDNA-WIP fibroblasts were grown, stimulated with PDGF, and stained as described in Fig. 2. Immunocytochemical analysis was carried out using a polyclonal antibody against a WIP peptide (anti-WIP) and F-actin was stained with TRITC-phalloidin(Actin). 11 equidistant stacks were recorded with a confocal microscope system. The bottom, middle and top stacks of a representative field of cells containing ruffles (arrows) are shown. Magnification 1000×.

Fig. 3.

Distribution of WIP along the membrane ruffle structure by confocal microscopy. NIH 3T3pcDNA-WIP fibroblasts were grown, stimulated with PDGF, and stained as described in Fig. 2. Immunocytochemical analysis was carried out using a polyclonal antibody against a WIP peptide (anti-WIP) and F-actin was stained with TRITC-phalloidin(Actin). 11 equidistant stacks were recorded with a confocal microscope system. The bottom, middle and top stacks of a representative field of cells containing ruffles (arrows) are shown. Magnification 1000×.

The above results raised the possibility that WIP associates with the actin cytoskeleton in ruffles. We were, however, unable to detect an increase of WIP in the Triton-insoluble cytoskeletal fraction after PDGF stimulation using western blot analysis (data not shown). This might have been because only a small proportion of WIP localized to ruffles as assessed by immunofluorescence.

Microinjection of anti-WIP antibody decreases ruffle formation induced by PDGF

To determine the role of WIP in PDGF induction of ruffles, we examined the effect of microinjection of anti-WIP antibody on the cellular response to PDGF. The antibody we used has previously been shown to inhibit filopodium induction by bradykinin (Martinez-Quiles et al., 2001). NIH 3T3pcDNA fibroblasts were injected with rabbit anti-WIP antibody. The proportion of anti-WIP-injected cells with ruffles was 54%, compared with 78% for uninjected cells and 72% for cells injected with control IgG (Fig. 4). Similar inhibition levels were obtained after injection of anti-WIP IgG in WIP-transfected fibroblasts (data not shown). These results suggest that WIP plays an important role in ruffle formation induced by PDGF.

Fig. 4.

Microinjection of anti-WIP IgG inhibits PDGF-induced ruffle formation. The proportion of NIH 3T3pcDNA cells developing ruffles after PDGF stimulation is plotted against antibody injection. Columns represent the proportion of cells with ruffles among noninjected cells (–, 78%), cells injected with control rabbit IgG (IgG, 72%) and cells injected with anti-WIP rabbit IgG(α-WIP, 54%). Results are representative of three independent experiments. Similar results were obtained after injection of anti-WIP IgG in WIP-transfected fibroblasts (data not shown).

Fig. 4.

Microinjection of anti-WIP IgG inhibits PDGF-induced ruffle formation. The proportion of NIH 3T3pcDNA cells developing ruffles after PDGF stimulation is plotted against antibody injection. Columns represent the proportion of cells with ruffles among noninjected cells (–, 78%), cells injected with control rabbit IgG (IgG, 72%) and cells injected with anti-WIP rabbit IgG(α-WIP, 54%). Results are representative of three independent experiments. Similar results were obtained after injection of anti-WIP IgG in WIP-transfected fibroblasts (data not shown).

PDGF-induced ruffle formation is impaired in WIP-deficient fibroblasts

To establish a definitive role for WIP in ruffle formation by PDGF, we examined the effect of PDGF on lung fibroblasts derived from WIP-deficient mice. First, we confirmed, by western blot, WIP expression in the fibroblast cell lines derived from wild-type (+/+) mice and lack of WIP expression in fibroblasts derived from knockout (–/–) mice(Fig. 5A). Later, we treated serum-starved primary murine lung fibroblasts derived from wild-type(WIP+/+) or WIP-/- mice(Anton et al., 2002) with PDGF and stained for F-actin with TRITC-phalloidin. Actin distribution in serum-starved fibroblasts from WIP-/- mice was similar to control fibroblasts from WIP+/+ mice(Fig. 5B, left). Most of the actin accumulated at the periphery of the cell (cortical actin), as previously observed for starved 3T3 fibroblasts. PDGF treatment of WIP+/+cells caused the formation of many circular or dorsal ruffles heavily enriched in actin, peaking at 8 minutes and mostly disappearing by 15 minutes(Fig. 5B, top middle and top right, respectively). By contrast, ruffle formation was virtually absent in WIP-/- fibroblasts after 8 minutes of PDGF treatment(Fig. 5B, bottom middle). A very few WIP-/- cells exhibited fewer ruffles 15 minutes after PDGF treatment, at a time point when the ruffles had disappeared from most WIP+/+ fibroblasts (Fig. 5B, bottom right). These results are highly indicative that WIP plays an important role in ruffle formation induced by PDGF.

Fig. 5.

PDGF-induced actin reorganization is altered in WIP-/- primary fibroblasts. (A) Lysates of lung-derived fibroblasts (Fib) from WIP+/+ and WIP-/- mice were subjected to western blot analysis using anti-WIP rabbit serum as probe. (B) WIP+/+ and WIP-/- primary murine fibroblasts were serum starved (Medium) or serum starved and stimulated with PDGF for different times (PDGF). TRITC-phalloidin-stained cells were visualized on a Nikon Eclipse E800 microscope using 40× or 20× objectives. Lower magnification fields are depicted at 15 minutes to include more cells, which represent a more accurate proportion of ruffling cells. Arrows indicate ruffle formation.

Fig. 5.

PDGF-induced actin reorganization is altered in WIP-/- primary fibroblasts. (A) Lysates of lung-derived fibroblasts (Fib) from WIP+/+ and WIP-/- mice were subjected to western blot analysis using anti-WIP rabbit serum as probe. (B) WIP+/+ and WIP-/- primary murine fibroblasts were serum starved (Medium) or serum starved and stimulated with PDGF for different times (PDGF). TRITC-phalloidin-stained cells were visualized on a Nikon Eclipse E800 microscope using 40× or 20× objectives. Lower magnification fields are depicted at 15 minutes to include more cells, which represent a more accurate proportion of ruffling cells. Arrows indicate ruffle formation.

WIP binding to actin is essential for ruffle formation by PDGF

WIP has three functional domains: an N-terminal verprolin-homology domain(VH; residues 1-116) that binds actin; the central proline-rich region that interacts with Src-homology domain 3 (SH3); and a C-terminal domain that binds WASP and N-WASP. To test the hypothesis that WIP binding to actin is important for its effect on PDGF-induced ruffle formation, we examined the consequences of overexpressing a WIP mutant that lacks the ability to bind actin on PDGF ruffling.

We constructed a WIP deletion mutant that lacks a 12 amino acid sequence,including the putative actin-binding motif45KLKK48 in the VH domain (WIPΔ43-54). Because expression of recombinant full length(FL) GST-WIP protein is poor(Martinez-Quiles et al.,2001), we compared the capacity of GST-WIP 1-127Δ43-54 with that of GST-WIP 1-127 to bind actin in a pull-down assay. As previously shown,WIP1-127 bound G-actin (Martinez-Quiles et al., 2001). By contrast, there was no detectable binding of G-actin to GST-WIP 1-127Δ43-54 (Fig. 6A), indicating that the 12 deleted amino acids are crucial for actin binding. WIPΔ43-54 retained the ability to bind Nck and N-WASP in the yeast two hybrid system (S.P.S. and R.S.G., unpublished).

Fig. 6.

WIP binding to actin is essential for PDGF-induced ruffle formation. (A)GST pull-down assay. The indicated GST-WIP constructs (5 μg per 10 μl beads) were incubated with 0.05 μM G-actin. After SDS-PAGE, samples were blotted with anti-actin monoclonal antibody. (B) Fluorescence micrographs of TRITC-phalloidin-stained Swiss 3T3 fibroblasts transfected with control plasmid (3T3pcDNA), WIP coding sequence (3T3pcDNA-WIP) or the deletion mutant WIPΔ43-54 (3T3pcDNA-WIPΔ43-54), before and after PDGF challenge. Cells were grown on glass coverslips, serum starved for 3 days in the presence of 0.5% FBS, stimulated or not with PDGF (50 ng ml-1) for 8 minutes and then fixed and labeled with TRITC-phalloidin to visualize actin filaments. Magnification 400×.

Fig. 6.

WIP binding to actin is essential for PDGF-induced ruffle formation. (A)GST pull-down assay. The indicated GST-WIP constructs (5 μg per 10 μl beads) were incubated with 0.05 μM G-actin. After SDS-PAGE, samples were blotted with anti-actin monoclonal antibody. (B) Fluorescence micrographs of TRITC-phalloidin-stained Swiss 3T3 fibroblasts transfected with control plasmid (3T3pcDNA), WIP coding sequence (3T3pcDNA-WIP) or the deletion mutant WIPΔ43-54 (3T3pcDNA-WIPΔ43-54), before and after PDGF challenge. Cells were grown on glass coverslips, serum starved for 3 days in the presence of 0.5% FBS, stimulated or not with PDGF (50 ng ml-1) for 8 minutes and then fixed and labeled with TRITC-phalloidin to visualize actin filaments. Magnification 400×.

We then examined the PDGF response of Swiss 3T3 fibroblasts transfected with EL WIPΔ43-54 mutant in pcDNA vector. Transfection with WIPΔ43-54 by itself did not alter the morphology or actin distribution of the cells. Following PDGF treatment, there was a complete absence of circular ruffles in WIPΔ43-54-transfected cells at all time points examined (0-15 minutes) (Fig. 6B). However, PDGF did induce actin clusters in WIPΔ43-54-transfected fibroblasts. These results suggest that pcDNA-WIPΔ43-54 acts as a dominant negative inhibitor of ruffle formation by PDGF. More importantly, they also suggest that WIP interaction with actin is crucial for PDGF ruffle formation.

Discussion

In this report, we present data that support a role for WIP in the PDGF-mediated reorganization of the actin cytoskeleton that leads to circular ruffle formation in fibroblasts. WIP overexpression increases circular ruffle formation in response to PDGF, whereas the absence of WIP delays and reduces this response. These results suggest that WIP is involved in PDGF-induced ruffle organization.

After the long period of serum starvation that was necessary to obtain maximum quiescence of WIP-transfected cells, PDGF stimulation resulted mainly in the formation of dorsal and circular membrane ruffles rather than classical lamellipodia (Figs 1, 2, 6). Circular ruffle formation has been described in response to growth factors such as PDGF(Plattner et al., 1999) and hepatocyte growth factor [HCF (Warn et al., 1993)]. HCF-stimulated Madin-Darby canine kidney (MDCK) cells in confluent islands show very similar structures(Warn et al., 1993) to those described here. Circular ruffle formation in MDCK cells and fibroblasts correlates with pinocytic activity(Veithen et al., 1996; Warn et al., 1993), a process that occurs prior to cell movement. The decreased formation of circular ruffles in anti-WIP-IgG-injected cells and in WIP-/- fibroblasts suggests a potential role for WIP in extracellular compound intake and cell movement, and will be a focus of future analysis. In addition to circular ruffles, PDGF treatment induced finger-like actin-containing projections and loss of stress fibers in WIP-transfected cells. This result is consistent with previous reports that PDGF activates Cdc42 and N-WASP to mediate filopodium formation and actin stress fiber disassembly(Jimenez et al., 2000), and that WIP plays a role in Cdc42-mediated filopodium formation(Martinez-Quiles et al.,2001). Moreover, a recent report described the involvement of a novel WIP-related (WIRE) protein in the formation of filopodia and lamellipodia following PDGF treatment(Aspenstrom, 2002).

Activation of PI3K and of the downstream effector Rac is required for PDGF-stimulated membrane ruffling (Chung et al., 1994; Wennstrom et al.,1994a). The effect of WIP overexpression on PDGF-induced ruffle formation depended on PI3K because ruffle formation in PDGF-treated WIP-overexpressing fibroblasts was inhibited by wortmannin, a fungal metabolite that inhibits the catalytic subunit of PI3K(Thelen et al., 1994). Pretreatment of WIP-transfected cells with wortmannin completely inhibited PDGF-induced membrane ruffling, suggesting that WIP participates in the PI3K/Rac-mediated ruffle formation. Moreover, PDGF stimulation of WIP-transfected cells pretreated with wortmannin induced an increase in stress fiber formation. This result suggests a secondary role for WIP in stress fiber formation that is revealed when Rac activation by PI3K is blocked.

Several lines of evidence suggested that WIP plays a role in the physiological induction of ruffles by PDGF. Microinjection of anti-WIP antibody inhibited PDGF ruffle formation(Fig. 4). More importantly,PDGF-induced ruffle formation was severely impaired in WIP-deficient fibroblasts derived from WIP-/- mice(Fig. 5B). Finally, a WIP deletion mutant that fails to bind actin (WIPΔ43-54) acted as a dominant negative inhibitor of ruffle formation by PDGF(Fig. 6). This last result strongly suggests that the role of WIP in ruffle formation depends, at least in part, on its ability to bind actin. In this regard, WIP has been shown to stabilize actin filaments in vitro(Martinez-Quiles et al.,2001). Binding of WIP to F-actin might also explain the localization of WIP to ruffles in cells treated with PDGF (Figs 2, 3).

WIP might exert an effect on PDGF-induced ruffle formation at levels other than actin binding. WIP binds to SH3-domain-containing adaptor proteins such as Nck-1 (Anton et al., 1998),which also binds to phosphorylated PDGFR(Chen et al., 2000). A recent report excludes a role for Nck-1 in ruffle formation following PDGF stimulation but shows that Nck-2 is involved(Chen et al., 2000). Given the high amino acid identity between Nck-1 and Nck-2 (68%), their similar domain structure, their common partners and their co-expression in 3T3 fibroblasts(Braverman and Quilliam, 1999; Chen et al., 1998; Chen et al., 2000), the possibility that WIP binds to Nck-2 and that Nck-2 is involved in the effect of WIP on ruffle formation should be tested.

WIP also binds to the SH3 domain of cortactin (N.M.Q. and R.S.G.,unpublished). Cortactin binds actin via its N-terminal domain, activates Arp2/3-dependent actin polymerization and localizes to ruffles. A cortactin-WIP complex might promote ruffle formation by both inducing F-actin formation and stabilizing actin filaments. WIP binding to N-WASP seems unlikely to play an important role in ruffle formation, because N-WASP-deficient fibroblasts still form lamellipodia after PDGF stimulation(Snapper et al., 2001). Actin polymerization might also be promoted by WIP binding to the SH3 domain of myosin, because it has been shown that myosin-I-induced actin polymerization in yeast is regulated through interactions with both Las17p, a homolog of mammalian WASP, and verprolin, a homolog of WIP(Mochida et al., 2002).

PDGF is thought transiently to promote, through Rac activation, the assembly of an actin-based signal transduction unit at sites of actin remodeling that results in the movement of a range of proteins [including protein kinase A, Abl and WAVE2, the principal WAVE family member expressed in fibroblasts (Miki et al.,2000)] to sites of cytoskeletal reorganization(Westphal et al., 2000). The tyrosine kinase c-Abl not only binds to Nck(Miyoshi-Akiyama et al.,2001), a WIP partner, but also contains an SH3 domain that could bind directly to the proline-rich WIP. WAVE belongs to the WASP family of proteins that includes WASP and N-WASP, and stimulates actin polymerization mediated by the Arp2/3 complex (Miki et al., 2000). Because WIP binds directly to WASP and N-WASP, it would be interesting to determine whether it also binds to WAVE. It is tempting to hypothesize that WIP-mediated enhanced membrane ruffling might result from synergy between WAVE's ability to activate actin polymerization and WIP's ability to stabilize nascent actin filaments. Further work is needed to test this hypothesis.

Note in proof

There is a recent manuscript describing the presence of WIP in lamellipodia at the cell periphery and showing a role for WIP on membrane protusions(Kinley et al., 2003).

Acknowledgements

We thank G. Forni for his support, S. Lanzardo for assistance, L. Torroja for help with confocal microscopy and J. da Silva and A. Ceccarelli for computer assistance. I.M.A. is a recipient of a Lady Tata Memorial Trust Award. C.C. is a recipient of a Fondazione Angela Bossolasco Fellowship. This work was supported by USPHS grants 59561 and by grants from Baxter, Aventis and Gentiva Corporations, the Jeffrey Modell Foundation and the March of Dimes.

References

Anton, I., Lu, W., Mayer, B. J., Ramesh, N. and Geha, R. S.(
1998
). The Wiskott-Aldrich Syndrome protein interacting protein(WIP) binds to the adaptor protein Nck.
J. Biol. Chem.
273
,
20992
-20995.
Anton, I. M., de la Fuente, M. A., Sims, T. N., Freeman, S.,Ramesh, N., Hartwig, J. H., Dustin, M. L. and Geha, R. S.(
2002
). WIP deficiency reveals a differential role for WIP and the actin cytoskeleton in T and B cell activation.
Immunity
16
,
193
-204.
Aspenstrom, P. (
2002
). The WASP-binding protein WIRE has a role in the regulation of the actin filament system downstream of the platelet-derived growth factor receptor.
Exp. Cell Res.
279
,
21
-33.
Aspenstrom, P., Lindberg, U. and Hall, A.(
1996
). Two GTPases Cdc42, and Rac bind directly to a protein implicated in the immunodeficiency disorder Wiskott-Aldrich syndrome.
Curr. Biol.
6
,
70
-75.
Braverman, L. E. and Quilliam, L. A. (
1999
). Identification of Grb4/Nckbeta, a Src homology 2 and 3 domain-containing adapter protein having similar binding and biological properties to Nck.
J. Biol. Chem.
274
,
5542
-5549.
Chen, M., She, H., Davis, E. M., Spicer, C. M., Kim, L., Ren,R., le Beau, M. M. and Li, W. (
1998
). Identification of Nck family genes, chromosomal localization, expression, and signaling specificity.
J. Biol. Chem.
273
,
25171
-25178.
Chen, M., She, H., Kim, A., Woodley, D. T. and Li, W.(
2000
). Nckbeta adapter regulates actin polymerization in NIH 3T3 fibroblasts in response to platelet-derived growth factor bb.
Mol. Cell. Biol.
20
,
7867
-7880.
Chung, J., Grammer, T. C., Lemon, K. P., Kazlauskas, A. and Blenis, J. (
1994
). PDGF- and insulin-dependent pp70S6k activation mediated by phosphatidylinositol-3-OH kinase.
Nature
370
,
71
-75.
Hall, A. (
1998
). Rho GTPases and the actin cytoskeleton.
Science
279
,
509
-514.
Hawkins, P. T., Eguinoa, A., Qiu, R. G., Stokoe, D., Cooke, F. T., Walters, R., Wennstrom, S., Claesson-Welsh, L., Evans, T., Symons, M. et al. (
1995
). PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide 3-kinase.
Curr. Biol.
5
,
393
-403.
Higgs, H. N. and Pollard, T. D. (
1999
). Regulation of actin polymerization by Arp2/3 complex and Wasp/SCAR proteins.
J. Biol. Chem.
274
,
32531
-32534.
Hooshmand-Rad, R., Claesson-Welsh, L., Wennstrom, S., Yokote,K., Siegbahn, A. and Heldin, C. H. (
1997
). Involvement of phosphatidylinositide 3′-kinase and Rac in platelet-derived growth factor-induced actin reorganization and chemotaxis.
Exp. Cell Res.
234
,
434
-441.
Jimenez, C., Portela, R. A., Mellado, M., Rodriguez-Frade, J. M., Collard, J., Serrano, A., Martinez, A. C., Avila, J. and Carrera, A. C. (
2000
). Role of the PI3K regulatory subunit in the control of actin organization and cell migration.
J. Cell Biol.
151
,
249
-262.
Kazlauskas, A. (
1994
). Receptor tyrosine kinases and their targets.
Curr. Opin. Genet. Dev.
4
,
5
-14.
Kim, A. S., Kakalis, L. T., Abdul-Manan, N., Liu, G. A. and Rosen, M. K. (
2000
). Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein.
Nature
404
,
151
-158.
Kinley, A. W., Weed, S. A., Weaver, A. M., Karginov, A. V.,Bissonette, E., Cooper, J. A. and Parsons, J. T. (
2003
). Cortactin interacts with WIP in regulating Arp2/3 activation and membrane protrusion.
Curr. Biol.
13
,
384
-393.
Kolluri, R., Tolias, K. F., Carpenter, C. L., Rosen, F. S. and Kirchhausen, T. (
1996
). Direct interaction of the Wiskott-Aldrich syndrome protein with GTPase Cdc42.
Proc. Natl. Acad. Sci. USA
93
,
5615
-5618.
Machesky, L. and Insall, R. (
1998
). Scar1 and the related Wiskott-Aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex.
Curr. Biol.
8
,
1347
-1356.
Martinez-Quiles, N., Rohatgi, R., Anton, I. M., Medina, M.,Saville, S. P., Miki, H., Yamaguchi, H., Takenawa, T., Hartwing, J., Geha, R. S. et al. (
2001
). WIP regulates N-WASP-mediated actin polymerization and filopodium formation.
Nat. Cell Biol.
3
,
484
-491.
Miki, H., Miura, K. and Takenawa, T. (
1996
). N-WASP, a novel actin-depolymerizing protein, regulates the cortical cytoskeletal rearrangement in a PIP2-dependent manner downstream of tyrosine kinases.
EMBO J.
15
,
5326
-5335.
Miki, H., Sasaki, T., Takai, Y. and Takenawa, T.(
1998a
). Induction of filopodium formation by a WASP-related actin-depolymerizing protein N-WASP.
Nature
391
,
93
-96.
Miki, H., Suetsugu, S. and Takenawa, T.(
1998b
). WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac.
EMBO J.
17
,
6932
-6941.
Miki, H., Yamaguchi, H., Suetsugu, S. and Takenawa, T.(
2000
). IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling.
Nature
408
,
732
-735.
Miyoshi-Akiyama, T., Aleman, L. M., Smith, J. M., Adler, C. E. and Mayer, B. J. (
2001
). Regulation of Cbl phosphorylation by the Abl tyrosine kinase and the Nck SH2/SH3 adaptor.
Oncogene
20
,
4058
-4069.
Mochida, J., Yamamoto, T., Fujimura-Kamada, K. and Tanaka,K. (
2002
). The novel adaptor protein, Mti1p, and Vrp1p, a homolog of Wiskott-Aldrich syndrome protein-interacting protein (WIP), may antagonistically regulate type I myosins in
Saccharomyces cerevisiae. Genetics
160
,
923
-934.
Olivera, A. and Spiegel, S. (
1993
). Sphingosine-1-phosphate as second messenger in cell proliferation induced by PDGF and FCS mitogens.
Nature
365
,
557
-560.
Penninger, J. M. and Crabtree, G. R. (
1999
). The actin cytoskeleton and lymphocyte activation.
Cell
96
,
9
-12.
Plattner, R., Kadlec, L., DeMali, K. A., Kazlauskas, A. and Pendergast, A. M. (
1999
). c-Abl is activated by growth factors and Src family kinases and has a role in the cellular response to PDGF.
Genes Dev.
13
,
2400
-2411.
Ramesh, N., Anton, I. M., Hartwig, J. H. and Geha, R. S.(
1997
). WIP, a protein associated with the Wiskott-Aldrich syndrome protein, induces actin polymerization and redistribution in lymphoid cells.
Proc. Natl. Acad. Sci. USA
94
,
14671
-14676.
Ridley, A. J., Paterson, H. F., Johnston, C. L., Dielmann, D. and Hall, A. (
1992
). The small GTP-binding protein Rac regulates growth factor-induced membrane ruffling.
Cell
70
,
401
-410.
Rohatgi, R., Ho, H. H. and Kirschner, M. W.(
2000
). Mechanism of N-WASP activation by Cdc42 and phosphatidylinositol 4,5-bisphosphate.
J. Cell Biol.
150
,
1299
-1309.
Snapper, S. B., Takeshima, F., Anton, I., Liu, C. H., Thomas, S. M., Nguyen, D., Dudley, D., Fraser, H., Purich, D., Lopez-Ilasaca, M. et al. (
2001
). N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility.
Nat. Cell Biol.
3
,
897
-904.
Svitkina, T. M. and Borisy, G. G. (
1999
). Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia.
J. Cell Biol.
145
,
1009
-1026.
Symons, M., Derry, J. M. J., Kariak, B., Jiang, S., Lemahieu,V., McCormick, F., Francke, U. and Abo, A. (
1996
). Wiskott-Aldrich syndrome protein, a novel effector for the GTPase Cdc42Hs, is implicated in actin polymerization.
Cell
84
,
723
-734.
Thelen, M., Wymann, M. P. and Langen, H.(
1994
). Wortmannin binds specifically to 1-phosphatidylinositol 3-kinase while inhibiting guanine nucleotide-binding protein-coupled receptor signaling in neutrophil leukocytes.
Proc. Natl. Acad. Sci. USA
91
,
4960
-4964.
Veithen, A., Cupers, P., Baudhuin, P. and Courtoy, P. J.(
1996
). v-Src induces constitutive macropinocytosis in rat fibroblasts.
J. Cell Sci.
109
,
2005
-2012.
Vetterkind, S., Miki, H., Takenawa, T., Klawitz, I.,Scheidtmann, K. H. and Preuss, U. (
2002
). The rat homologue of Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP)associates with actin filaments, recruits N-WASP from the nucleus, and mediates mobilization of actin from stress fibers in favor of filopodia formation.
J. Biol. Chem.
277
,
87
-95.
Warn, R., Brown, D., Dowrick, P., Prescott, A. and Warn, A.(
1993
). Cytoskeletal changes associated with cell motility. In
Cell Behaviour: Adhesion and Motility
, Vol.
47
(ed. G. Jones, C. Wigley and R. Warn), pp.
325
-338. Cambridge, UK: The Company of Biologists.
Wennstrom, S., Hawkins, P., Cooke, F., Hara, K., Yonezawa, K.,Kasuga, M., Jackson, T., Claesson-Welsh, L. and Stephens, L.(
1994a
). Activation of phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling.
Curr. Biol.
4
,
385
-393.
Wennstrom, S., Siegbahn, A., Yokote, K., Arvidsson, A. K.,Heldin, C. H., Mori, S. and Claesson-Welsh, L. (
1994b
). Membrane ruffling and chemotaxis transduced by the PDGF beta-receptor require the binding site for phosphatidylinositol 3′ kinase.
Oncogene
9
,
651
-660.
Westermark, B., Siegbahn, A., Heldin, C. H. and Claesson-Welsh,L. (
1990
). B-type receptor for platelet-derived growth factor mediates a chemotactic response by means of ligand-induced activation of the receptor protein-tyrosine kinase.
Proc. Natl. Acad. Sci. USA
87
,
128
-132.
Westphal, R. S., Soderling, S. H., Alto, N. M., Langeberg, L. K. and Scott, J. D. (
2000
). Scar/WAVE-1, a Wiskott-Aldrich syndrome protein, assembles an actin-associated multi-kinase scaffold.
EMBO J.
19
,
4589
-4600.

Supplementary information