Rho, Rac and Cdc42 regulate actin organization and cell ... · Rho, Rac and Cdc42 are small GTP-binding proteins of the Rho family, a subset of the Ras superfamily (Hall, 1994). The

707Journal of Cell Science 110, 707-720 (1997)Printed in Great Britain © The Company of Biologists Limited 1997JCS4373

Rho, Rac and Cdc42 regulate actin organization and cell adhesion in

macrophages

William E. Allen1,2, Gareth E. Jones2, Jeffrey W. Pollard3 and Anne J. Ridley1,4,*1The Ludwig Institute for Cancer Research, 91 Riding House Street, London W1P 8BT, UK 2The Randall Institute, Drury Lane, King’s College London, UK 3Albert Einstein College of Medicine, New York, USA4Department of Biochemistry and Molecular Biology, Gower Street, London WC1E 6BT, UK

*Author for correspondence

Rho family proteins are known to regulate actin organiz-ation in fibroblasts, but their functions in cells ofhaematopoietic origin have not been studied in detail.Bac1.2F5 cells are a colony-stimulating factor-1 (CSF-1)-dependent murine macrophage cell line; CSF-1 stimulatestheir proliferation and motility, and acts as a chemoattrac-tant. CSF-1 rapidly induced actin reorganization in Bac1cells: it stimulated the formation of filopodia, lamellipodiaand membrane ruffles at the plasma membrane, as well asthe appearance of fine actin cables within the cell interior.Microinjection of constitutively activated (V12)Rac1 stim-ulated lamellipodium formation and membrane ruffling.The dominant inhibitory Rac mutant, N17Rac1, inhibitedCSF-1-induced lamellipodium formation, and also inducedcell rounding. V12Cdc42 induced the formation of longfilopodia, while the dominant inhibitory mutant N17Cdc42prevented CSF-1-induced formation of filopodia but not

lamellipodia. V14RhoA stimulated actin cable assemblyand cell contraction, while the Rho inhibitor, C3 trans-ferase, induced the loss of actin cables. Bac1 cells had cell-to-substratum adhesion sites containing ββ1 integrin,pp125FAK, paxillin, vinculin, and tyrosine phosphorylatedproteins. These ‘focal complexes’ were present in growingand CSF-1-starved cells, but were disassembled in cellsinjected with N17Cdc42 or N17Rac1. Interestingly, ββ1integrin did not disperse until long after focal phosphoty-rosine and vinculin staining had disappeared. We concludethat in Bac1 macrophages Cdc42, Rac and Rho regulate theformation of distinct actin filament-based structures, andthat Cdc42 and Rac are also required for the assembly ofadhesion sites to the extracellular matrix.

Key words: Rho family protein, Macrophage, Actin cytoskeleton,Cell adhesion, CSF-1

SUMMARY

INTRODUCTION

Rho, Rac and Cdc42 are small GTP-binding proteins of theRho family, a subset of the Ras superfamily (Hall, 1994). TheRho family has 11 mammalian members, in addition to manyhomologues in other species, from yeast to Drosophila andDictyostelium (Ridley, 1996). They act as molecular switches,cycling between an active GTP-bound form, and an inactiveGDP-bound form. Their activity in cells is presumed to beregulated by a combination of GTPase activating proteins(GAPs), guanine nucleotide exchange factors and guaninenucleotide dissociation inhibitors (GDIs) (Hall, 1994; Ridley,1995a).

Rho, Rac and Cdc42 have been shown to regulate actin reor-ganization induced by a wide range of extracellular factors inseveral adherent cell types (Hall, 1994; Machesky and Hall,1996). In addition, Rac is a key regulator of the superoxide-generating NADPH oxidase in phagocytes (Ridley, 1995b).More recently, many other functions have been ascribed tothese proteins, from transcription regulation to growth control,and from endocytosis to secretion. At the same time, severalpotential ‘target’ proteins for Rho, Rac and Cdc42 have been

identified, which can interact directly with the GTP-boundform of each protein in vitro. These include serine/threoninekinases and ‘adaptor’ proteins with no enzymatic activity(Ridley, 1996).

Rho itself was the first Rho family protein to be implicatedin the regulation of the actin cytoskeleton. In fibroblasts, itmediates the formation of stress fibres and focal adhesionsinduced by a variety of growth factors and by adhesion tofibronectin (Hall, 1994; Barry et al., 1996). Rho is also requiredfor cell contractility: in fibroblasts and neuronal cells,lysophosphatidic acid (LPA)-induced contractility is preventedby the Rho inhibitor C3 transferase (Jalink et al., 1994;Chrzanowska-Wodnicka and Burridge, 1996). Recent evidencesupports a model where Rho induces the interaction of myosinwith actin filaments by stimulating the phosphorylation ofmyosin light chain (reviewed by Ridley, 1996). It appears thata protein kinase which interacts with Rho, ROKα/Rho-kinase,is a key signalling molecule in this pathway, as overexpressionof ROKα/Rho-kinase can stimulate stress fibre formation evenwhen Rho activity is inhibited (Leung et al., 1996).

Rac and Cdc42 regulate the formation of distinct actinfilament-containing structures in fibroblasts: Rac regulates

708 W. E. Allen and others

lamellipodium formation and membrane ruffling, while Cdc42induces the formation of filopodia (Machesky and Hall, 1996).Rac and Cdc42 also mediate the formation of small focal‘complexes’ in fibroblasts, which are distinct from Rho-induced focal adhesions. These complexes are associated withlamellipodia and filopodia, and have been shown to containvinculin, paxillin, pp125FAK (focal adhesion kinase) and β3integrin (Nobes and Hall, 1995; Hotchin and Hall, 1995). Lessis known about the molecular mechanisms underlying theresponses to Rac and Cdc42 than is known about Rho sig-nalling, although two proteins, POR1 and the Wiskott-AldrichSydrome protein (WASp), have recently been identified aspotential mediators of Rac- and Cdc42-induced responsesrespectively (Van Aelst et al., 1996; Symons et al., 1996). Inaddition, studies in platelets have suggested that production ofphosphatidylinositol(4,5)bisphosphate is important for Rac-induced actin polymerization (Hartwig et al., 1995).

Rho, Rac and Cdc42-mediated responses have been studiedin most detail in Swiss 3T3 fibroblasts, but distinct, oftendifferent, effects of these proteins on actin organization havebeen observed in other cell types. In MDCK epithelial cells,for example, Rac is required for the formation of lamellipodiabut, unlike in fibroblasts, microinjection of activated Rac is notsufficient by itself to induce lamellipodium formation. In KBcells, which are derived from a human epithelial carcinoma,Rac and Rho each regulate a phenotypically distinct type ofruffling response (Nishiyama et al., 1994). Both Rho andCdc42 have been implicated in the regulation of podosome-likeactin-containing structures, Rho in osteoclasts (which are ofthe same lineage as macrophages) (Zhang et al., 1995) andCdc42 in human HeLa cells (Dutartre et al., 1996). In neuronalcells, which do not have stress fibres, several extracellularfactors stimulate Rho-dependent rounding up and retractionof neurites (Jalink et al., 1994; Postma et al., 1996). Rho hasalso been implicated in cell-cell adhesion: it regulates plateletaggregation and integrin-mediated lymphocyte interactions(Morii et al., 1992; Tominaga et al., 1993). All of these studiessupport the hypothesis that Rho family proteins mediate actinreorganization, but they also show that the precise responseregulated by each protein is highly cell-type dependent.

To date no study has looked in detail at the roles of Rhofamily proteins in regulating cytoskeletal organization inhaemopoietic cells, which lack the stress fibres and associatedfocal adhesions found in many cultured cells. Cells of themonocyte/macrophage lineage are particularly interestingbecause they are highly locomotory, and their ability to migratetowards a source of chemoattractant is essential for theirrecruitment to sites of inflammation (Downey, 1994). Cellmotility is a highly regulated process dependent on the coor-dinated formation of actin-containing structures (Mitchisonand Cramer, 1996; Lauffenburger and Horwitz, 1996). Toinvestigate Rho, Rac and Cdc42 function in macrophages, wehave chosen to use Bac1.2F5 cells as a model system. Bac1cells are a cloned, SV40-immortalized murine macrophage cellline, which resemble primary macrophages in their dependenceon colony-stimulating factor-1 (CSF-1) for both viability andproliferation even in the presence of foetal calf serum (FCS)(Morgan et al., 1987; Boocock et al., 1989). In the absence ofCSF-1, Bac1 cells become rounded, and on re-addition of CSF-1, the cells rapidly spread, producing membrane ruffles andlamellipodia (Boocock et al., 1989). Migration studies have

shown that CSF-1-starved Bac1 cells become immobile, butregain their locomotory behaviour after addition of CSF-1, andare chemotactic to CSF-1 (Webb et al., 1996).

We have analysed the organisation of the actin cytoskeletonand cell-to-substratum adhesions in growing, CSF-1-starvedand CSF-1-stimulated Bac1 macrophages. Subsequently, theroles of Rho, Rac and Cdc42 in regulating the formation ofactin cytoskeletal structures and cell-to-substratum adhesionswas investigated.

MATERIALS AND METHODS

Cell cultureCells used in these experiments were a subclone of the cloned mousecell line Bac1.2F5 (Morgan et al., 1987). Unless otherwise stated allmedia and supplements were supplied by ICN Flow. These cells weremaintained in growth medium consisting of DMEM supplementedwith 10% (v/v) foetal calf serum (FCS, Biopharm UK), 1.32 nM CSF-1 (Chiron Corp.), 2 mM L-glutamine, 0.15 mM L-asparagine, 15 nMβ-mercaptoethanol, 77.5 units ml−1 streptomycin and 25 units ml−1

penicillin. Cells were grown at 37°C in T25 tissue culture flasks(Falcon), maintained in a humid atmosphere of 10% CO2. Cells weresubcultured twice weekly by dissociation with 0.04% EDTA inphosphate buffered saline (PBS), and re-suspended in growth mediumat 2×105 cells ml−1. To obtain quiescent cultures, cells were main-tained for 24 hours in medium as above, but lacking CSF-1. QuiescentBac1 macrophages were re-stimulated by the addition of 1.32 nMCSF-1 after 24 hours in the absence of CSF-1.

Expression and purification of recombinant proteinsV14RhoA, V14A37RhoA, V12Rac1, V12A35Rac1, N17Rac1,

V12Cdc42, N17Cdc42 and C3 transferase were expressed inEscherichia coli as glutathione-S-transferase (GST)-fusion proteinsand purified essentially as described by Ridley and Hall (1992). Activeprotein concentrations for GTP-binding proteins were determined bya filter binding assay using [3H]GDP (Hall and Self, 1986). As pre-viously reported, N17Cdc42 and N17Rac1 were found to have a lowaffinity for [3H]GDP (Self and Hall, 1995), compared to their con-centrations as determined by gel electrophoresis. The protein con-centrations for N17Rac1 and N17Cdc42 were therefore estimatedusing a protein assay kit (Bio-Rad). The protein concentration of C3transferase was also determined by this method.

MicroinjectionTo obtain cells for microinjection, 5×104 cells per well were seededin 15 mm diameter wells containing 13 mm circular glass coverslips.For microinjection, coverslips were marked with a cross using a glasspen, to facilitate the localisation of microinjected cells. For analysisof cells growing in CSF-1, cells were microinjected 48 hours afterseeding. To obtain quiescent, cytokine-starved cells for microinjec-tion, cells were seeded as above and maintained in growth mediumfor 24 hours, followed by 24 hours in medium without CSF-1.V12Rac1, V12Cdc42, V14RhoA, V12A35Rac1 and V14A37RhoAproteins were injected at betwen 200 and 300 µg/ml (determined by[3H]GDP binding) unless otherwise indicated, C3 transferase wasinjected at approximately 2 µg/ml, and N17Rac1 and N17Cdc42 wereinjected at approximately 0.75 mg/ml (total protein concentration,determined by Bradford assay). To identify microinjected cells, ratimmunoglobulin G (IgG) (Pierce) or rabbit IgG (Sigma) at 0.5 mg/mlwas microinjected together with the recombinant proteins.

ImmunofluorescenceFor immunofluorescence studies, cells were fixed in 4% formaldehydein PBS for 20 minutes at room temperature. In some experiments, 3%

709Rho, Rac and Cdc42 in macrophages

Fig. 1. CSF-1-induced actin reorganization in Bac1 cells. Bac1 cells were stained with TRITC-labelled phalloidin to reveal actin filaments.Confocal micrographs are shown of growing cells (A,B), CSF-1-starved cells (C), and cells stimulated with CSF-1 for 5 minutes (D), 15minutes (E), or 30 minutes (F). Arrows in A and F indicate actin cables; the arrow in C indicates a retraction fibre; arrows in E indicatefilopodia and lamellipodia. Bars: 10 µm (A and F); 25 µm (B); A,C,D,E are at the same magnification.

sucrose (w/v) was included during fixation. Cells were permeabilisedwith 0.5% Triton X-100 in PBS for 5 minutes. For localization of actinfilaments, cells were incubated with 0.1 µg/ml TRITC-labelled phal-loidin (Sigma Chemical Co.) for 45 minutes. For localisation of phos-photyrosine, paxillin, pp125FAK and vinculin, cells were incubated for60 minutes at room temperature with a 1:500 dilution of mouse anti-phosphotyrosine antibody (Sigma Chemical Co.), a 1:200 dilution ofmouse anti-paxillin antibody (Transduction Laboratories), a 1:200dilution of mouse anti-pp125FAK antibody (Transduction Laboratories)or a 1:20 dilution of mouse anti-vinculin antibody (F9, a gift from DavidCritchley). This was followed by a 45 minute incubation with 1:200

TRITC-conjugated anti-mouse IgG (Jackson). For localisation of β1integrin, cells were fixed at 4°C in 4% formaldehyde/3% sucrose andpermeabilised as above, blocked for 15 minutes at room temperaturewith filtered pre-immune goat serum, incubated for 40 minutes at roomtemperature with a rat anti-mouse β1 integrin antibody (9EG7, a giftfrom Dietmar Westweber), rinsed three times in PBS and then incubatedfor 40 minutes at room temperature with FITC-goat anti-rat IgG (1:50,Sigma) or TRITC-goat anti-rat IgG (1:50, Cappell). Injected rat IgG orrabbit IgG was localised with FITC-conjugated goat anti-rat IgG (1:400,Sigma), TRITC-conjugated goat anti-rabbit IgG (1:50, Sigma) orAMCA-conjugated donkey anti-rabbit IgG (1:50, Dako) as appropriate.



Fig. 2. Characterization of focal complexes in Bac1 cells. GrowingBac1 cells were co-stained with an anti-β1 integrin antibody (A) andan anti-phosphotyrosine antibody (B). A lower magnification imageof growing cells stained with the anti-phosphotyrosine antibody (C)is also shown. Co-staining of growing cells with anti-phosphotyrosine antibody and with TRITC-phalloidin to reveal actinfilaments (D) shows the localization of oblong-shaped focal contactsin lamellipodia and at the bases of filopodia. Growing cells were alsostained with an anti-vinculin antibody (E), or with an anti-pp125FAK

antibody (F). CSF-1-starved cells (G) and cells restimulated withCSF-1 (H) were stained with an anti-phosphotyrosine antibody. Bars:10 µm (A,D,F); 25 µm (C). A,B,E-H are at the same magnification.

Images of cells were obtained using a Bio-Rad MRC-500 confocallaser scanning microscope, with the exception of the image in Fig.3B. This was visualised using a Leica TCS NT confocal laserscanning head attached to a Leica DM RXA optical microscope (×100oil objective). The software was Leica Power Scan. The Z-seriesconsisted of 32 optical sections which were built into a 3-D recon-struction using the Power Scan software. We are grateful to MatthewIsoukis for assistance in generating this image.

RESULTS

Effects of CSF-1 withdrawal and restimulation onactin organization in Bac1 cellsThe organization of the macrophage actin cytoskeleton wasinvestigated in growing Bac1 cells and in cells starved and re-stimulated with CSF-1. Bac1 macrophages maintained in thepresence of CSF-1 exhibited a variety of morphologies con-sistent with their motile status (Fig. 1A and B). They possessedlamellipodia and filopodia containing a fine network of actinfilaments at the level of the substratum (Fig. 1A and B), andsome membrane ruffles on the dorsal surface (above the levelof the section in Fig. 1A). In addition, they possessed actincables within the body of the cells (e.g. arrow in Fig. 1A).These structures were much finer than the actin stress fibresfound in cultured fibroblasts and many other adherent celltypes. The cables ran parallel to each other through thecytoplasm, around the nucleus, but were not observed beneaththe nucleus, and rarely above the nucleus.

As previously observed (Boocock et al., 1989), cells main-tained in the absence of CSF-1 for a period of 24-48 hours hada rounded contracted morphology and retraction fibres, butremained attached to the substratum. What little polymerisedactin they contained showed a punctate distribution (Fig. 1C).Only very rarely were fine actin cables observed, and few cellshad lamellipodia or filopodia. Stimulation of these starved cellswith CSF-1 rapidly induced actin reorganisation. Within 5minutes, new membrane ruffles, lamellipodia and filopodiawere apparent (Fig. 1D). Maximum membrane ruffling andlamellipodial activity was observed between 5 and 10 minutesafter CSF-1 addition (as previously observed, Boocock et al.,1989), whereas cells possessed many long filopodia at all time-points between 5 and 30 minutes (Fig. 1D-F). New actincables, similar to those observed in the body of growing cells,were first evident at 15 minutes, and were clearly observed by30 minutes after CSF-1 addition (e.g. arrow in Fig. 1F). At 30minutes, the actin organisation of re-stimulated macrophagesresembled that seen in growing cells (compare Fig. 1A and F).

Bac1 macrophages have focal complexes containingββ1 integrinPrevious studies using inteference reflection microscopyshowed that Bac1 macrophages did not possess fibroblast-likefocal adhesions to the substratum (Boocock et al., 1989). Apunctate distribution of several proteins known to be associ-ated with fibroblast focal adhesions has, however, beenobserved in macrophages (Rosen et al., 1990; de Nichilo andBurns, 1993; Wojciak-Stothard et al., 1996). To examine theinteractions of Bac1 cells with their substratum in more detail,the localisation of a number of focal adhesion proteins wasstudied. In growing Bac1 cells, β1 integrin showed a punctatedistribution (Fig. 2A), which co-localized with clusters ofphosphotyrosine staining (Fig. 2B). Small punctate structuresstaining for phosphotyrosine were distributed over the lowersurface of the cell, while larger oblong structures localized inlamellipodia and at the bases of filopodia (Fig. 2C and D).These structures were considerably smaller than Swiss 3T3fibroblast focal adhesions (data not shown), and were notclearly associated with bundles of actin filaments. They willtherefore be referred to as ‘focal complexes’ (see also Nobesand Hall, 1995).

In addition to β1 integrin, the Bac1 focal complexescontained vinculin (Fig. 2E), pp125FAK (Fig. 2F) and paxillin(data not shown). The nuclear staining observed in these andin some subsequent photographs is due to non-specific bindingof the secondary antibodies (data not shown). Western blottingof cell lysates with anti-pp125FAK (H. Flinn and A. Ridley,unpublished data) and anti-vinculin (data not shown) anti-bodies confirmed that pp125FAK and vinculin were expressedin Bac1 cells.

The focal complexes were retained in cells which had beenCSF-1-starved for 24 or 48 hours, although their distributionwas altered, reflecting the change in cell morphology (Fig. 2Gand data not shown). Re-stimulation with CSF-1 led to a rapidredistribution of focal complexes and the appearance of largeroblong complexes, reflecting the formation of lamellipodia andfilopodia (Fig. 2H).

Rac regulates the formation of lamellipodia andmembrane rufflesRac has previously been shown to regulate growth factor-induced formation of lamellipodia and membrane ruffles (Hall,1994). The role of Rac in regulating actin organisation in Bac1cells was investigated first by microinjection of the constitu-tively active mutant V12Rac1 (Ridley et al., 1992). In bothgrowing Bac1 cells and CSF-1-starved cells, large lamellipo-dia formed within 10 minutes of injection (Fig. 3A and datanot shown), and membrane ruffles were observed on the dorsalsurface of cells (Fig. 3B). The lamellipodia and membraneruffles formed resembled those observed on CSF-1-stimulatedcells (Fig. 1C; see also Boocock et al., 1989), but were moreextensive. Often, one or two large lamellipodia encompassedthe entire circumference of the cell. This V12Rac1-inducedresponse persisted for up to 6 hours, but by 24 hours allmicroinjected cells had reverted to the morphology of unin-jected cells.

In addition to stimulating lamellipodium formation,V12Rac1 also stimulated the formation of actin cables wheninjected into CSF-1-starved cells, similar in morphology tothose observed in growing or CSF-1-stimulated cells. As with



Fig. 3. Rac regulates the formation of lamellipodia and focalcomplexes in Bac1 cells. Actin filaments are shown in a CSF-1-starved cell microinjected with V12Rac1 and fixed after 60 minutes(A,B). The image in A shows a single confocal section through aninjected cell at the level of the substratum. The large arrow indicatesthe injected cell, arrowheads indicate lamellipodia, small arrowsshow actin cables. The image in B is a z section 3-D reconstructionof the same cell (see Materials and Methods) to show bothlamellipodia (arrowhead) and membrane ruffles on the dorsal surface(small arrows). (C) The localization of focal complexes is shown in agrowing V12Rac1-injected cell (arrow) fixed 15 minutes afterinjection and stained with an anti-phosphotyrosine antibody. In D,CSF-1-starved cells were injected with N17Rac1 then cells werestimulated with CSF-1 for 5 minutes before fixing and staining withTRITC-phalloidin. The arrow indicates an injected cell, thearrowhead indicates retraction fibres. In E and F, cells injected withN17Rac1 were fixed after 30 minutes and co-stained with FITC-labelled anti-rat IgG to localize injected cells (E) and with an anti-phosphotyrosine antibody to reveal focal complexes (F). In G,growing cells were injected with N17Rac1, fixed after 60 minutesand stained with an anti-β1 integrin antibody. Both cells in the imagewere microinjected (arrows). Bars: 10 µm (A,B,C,G). C-F are at thesame magnification.

CSF-1 stimulation, the timecourse of actin cable formation wasslower than that of lamellipodium formation: actin cables werefirst observed in cells 30 minutes after injection of V12Rac1,and were clearly present at 60 minutes (Fig. 3A). As a control,the inactive V12A35Rac1 protein (Ridley et al., 1992) wasinjected. This induced no morphological changes or alterationsin actin organization at 15 or 30 minutes after injection. Asdescribed above, CSF-1-starved cells retain focal complexes(Fig. 2G). Injection of V12Rac1 induced a similar reorganiza-tion of focal complexes as CSF-1 stimulation, with distinctoblong complexes concentrated along the inner edges of thelamellipodia (Fig. 3C, compare with Fig. 2H).

To determine the role of Rac in CSF-1-induced actin reor-ganization, the dominant negative inhibitor N17Rac1 (Ridleyet al. 1992) was microinjected into growing and CSF-1-starvedcells. This allowed the activity of endogenous Rac proteins tobe inhibited. Injection of N17Rac1 into CSF-1-starved cellsinhibited the formation of lamellipodia and membrane rufflesfollowing CSF-1 re-stimulation (Fig. 3D). These cells alsoexhibited many retraction fibres; time-lapse videomicroscopyof injected cells confirmed that these fibres did not rapidlyprotrude and retract and were therefore not filopodia (data notshown).. The injected cells remained rounded for at least 3hours following CSF-1 addition. In the absence of CSF-1,retraction fibres were not observed, presumably because thecells were rounded up. Injection of N17Rac1 into growing cellsled to a loss of lamellipodia and membrane ruffles; cells alsorounded up and this was accompanied by the appearance ofmany long retraction fibres (data not shown).

Cdc42 regulates filopodium formationCdc42 has been reported to induce filopodium formation infibroblasts (Nobes and Hall, 1995; Kozma et al., 1995). CSF-1-starved Bac1 cells injected with the constitutively activeV12Cdc42 protein formed numerous long filopodia within 15minutes. Time-lapse videomicroscopy confirmed that these wereactively protruding and retracting filopodia (data not shown). Inaddition it was observed that the actin filaments within the cell

formed a fine meshwork unlike the actin cables observed ingrowing cells (Fig. 4A). These morphology changes persistedfor 4 to 6 hours after injection. When V12Cdc42 was injectedat a concentration of 200 µg/ml, membrane ruffles or lamel-lipodia were only rarely observed in injected cells. At a lowerconcentration of V12Cdc42 (25 µg/ml), however, lamellipodiawere more consistently observed in cells between 15 and 30minutes after injection (data not shown). This suggests that Racmay be activated downstream of Cdc42, but that lamellipodiumformation is inhibited by high levels of Cdc42 activity.

Growing Bac1 cells possess many filopodia (Fig. 1A), andsimilarly CSF-1 stimulates the formation of filopodia (Fig. 1D-F). To determine whether Cdc42 regulates the formation offilopodia, the dominant negative inhibitor N17Cdc42 wasmicroinjected into CSF-1-starved cells, which were then stim-ulated with CSF-1. N17Cdc42 initially induced cell roundingand retraction (Fig. 4C), similar to the effect of N17Rac1, butthis effect was transient. By 15 minutes after stimulation withCSF-1, N17Cdc42-injected cells clearly exhibited lamellipodiabut not filopodia (Fig. 4D). These results suggest that CSF-1is able to induce Rac-mediated lamellipodium formation in theabsence of functional Cdc42.

Cdc42 controls focal complex formation via RacThe rapid retraction of Bac1 cells injected with N17Rac1suggested that their cell to substratum adhesions were beingaltered. Indeed, cells injected with N17Rac1 rapidly lost theirfocal complexes within 5 minutes of injection, as imaged byphosphotyrosine (Fig. 3E and F) and vinculin staining (data notshown). Interestingly the β1 integrin staining pattern remainedpunctate at 15 minutes after injection, despite the loss of cyto-plasmic components of the focal complexes. At 60 minutes, themajority of cells had little or no detectable punctate staining ofof β1 integrin, although punctate localization was observed insome cells (Fig. 3G, compare to Fig. 2A). By 90 minutes, β1integrin was not detectably clustered in any injected cells (datanot shown). As a control the inactive mutant proteinV12A35Rac1 was injected. This protein did not induce anychanges in focal complex organisation (data not shown). Theseresults suggest that Rac regulates both the formation of lamel-lipodia and focal complexes, and that in the absence of focalcomplexes the cells round up, although they did not detachfrom the substratum.

In V12Cdc42-injected cells, phosphotyrosine-containingfocal complexes were reorganized into distinct oblong struc-tures often localised at the proximal end of filopodia (Fig. 4B).As with N17Rac1, N17Cdc42-injected cells lost their focalcomplexes within 5 minutes of injection, as imaged by phos-photyrosine staining (Fig. 4E and F) and vinculin staining (datanot shown), while β1 integrin staining remained as for controlcells at 15 minutes, was diminished at 60 minutes and notdetectably clustered at 90 minutes (data not shown and Fig.4G).

The above results show that both Rac1 and Cdc42 regulatethe formation and distribution of focal complexes. To investi-gate if Cdc42 and Rac control focal complexes via parallelpathways, or by one activating the other, the constitutivelyactive form of Cdc42 (V12Cdc42) and the dominant negativeRac inhibitor (N17Rac1) were microinjected in combinationinto Bac1 cells. Similarly, activated Rac (V12Rac1) and theCdc42 inhibitor (N17Cdc42) were also injected in combina-


Fig. 4. Cdc42 regulates the formation of filopodia and focal complexesin Bac1 cells. CSF-1-starved Bac1 cells were microinjected withV12Cdc42, fixed after 30 minutes, and stained with TRITC-phalloidinto reveal actin filaments (A) or with an anti-phosphotyrosine antibodyto localize focal complexes (B). Microinjected cells are indicated withlong arrows; the arrowhead in A indicates filopodia. (C and D) Actinfilaments are shown in CSF-1-starved cells injected with N17Cdc42,then stimulated with CSF-1 for 5 minutes (C) or 15 minutes (D).Microinjected cells are indicated with arrows; the arrowhead (C)indicates retraction fibres. (E-G) Growing cells were injected withN17Cdc42 and rat IgG, fixed after 30 minutes (E,F) or 60 minutes (G),and stained with FITC-labelled anti-rat IgG to identify injected cells(E), with a mouse anti-phosphotyrosine antibody followed by TRITC-labelled anti-mouse IgG (F) or with a rat anti-β1 integrin antibodyfollowed by TRITC-labelled anti-rat IgG (G). Arrows indicate injectedcells. Bars: 10 µm (A,C,G); A,B,D-F are at the same magnification.


Fig. 5. Cdc42 acts upstream of Rac to regulate focal complex formation. Growing Bac1 cells were microinjected with V12Cdc42 and N17Rac1(and rabbit IgG) (A,B) or with V12Rac1 and N17Cdc42 (and rabbit IgG) (C,D) and fixed approximately 30 minutes after injection. Cells werestained with FITC-labelled anti-rabbit IgG to localize injected cells (A,C) and with a mouse anti-phosphotyrosine antibody followed byTRITC-labelled anti-mouse IgG to localize focal complexes (B,D). Arrows indicate injected cells. Bar, 10 µm.

tion. V12Cdc42 was unable to counteract the dissociation offocal complexes induced by N17Rac1 (Fig. 5A and B), whileV12Rac1 was able to counteract the action of N17Cdc42 (Fig.5C and D). This suggests that Cdc42 acts upstream of Rac toregulate focal complex formation.

RhoA induces Bac1 macrophages to round upUnlike many other cultured adherent cells, macrophages do notpossess stress fibres, and therefore Rho would not be expectedto stimulate stress fibre formation. To investigate whether Rhoinduced changes in actin organization in macrophages,V14RhoA, the constitutively active form, was microinjectedinto growing Bac1 cells. V14RhoA rapidly induced actin redis-tribution, and the cells assumed a rounded contracted mor-phology within 5 minutes, often with processes resemblingretraction fibres (Fig. 6A). Similar results were observed whenV14RhoA was injected into cells starved of CSF-1 for 24hours, although these starved cells lacked retraction fibres (Fig.6B) probably because they were already rounded up by star-vation. The fine actin cables visible in growing uninjected cellswere still apparent when V14RhoA was injected at a concen-tration of 50 µg/ml (Fig. 6A), while at higher protein concen-trations dense whorls of polymerised actin fibres wereobserved (data not shown). This reorganisation of the actincytoskeleton was observed within 10 minutes of injection. The

Rho-induced changes persisted unchanged for 6 hours, but theinjected cells had fully reverted back to their normal actinorganisation after 18 hours. As a control, the inactive mutantprotein V14A37RhoA was injected. This protein did notinduce any changes in the organization of actin at 15 or 30minutes after injection (data not shown).

The Clostridium botulinum exoenzyme C3 transferase,which selectively inhibits the activity of Rho by ADP-ribo-sylating Rho (von Eichel-Streiber et al., 1996), was used toinhibit the function of endogenous Rho within Bac1 cells.Microinjection of C3 transferase into growing cells inducedcell flattening and radial spreading within 15 minutes, accom-panied by the loss of the fine actin cables observed in growingcells (Fig. 6D). These cells still possessed lamellipodia,showing that C3 transferase did not inhibit Rac-regulatedprocesses. In addition, cells co-injected with V12Rac1 and C3transferase had lamellipodia and membrane ruffles, but nodetectable actin cables (data not shown), confirming that Racacts upstream of Rho to induce cable assembly.

At later timepoints, C3 transferase-injected cells assumed ahighly elongated ‘dendritic’ morphology, which was evidentby 2 hours but had become most extreme by 18 hours afterinjection (Fig. 6E). The cells extended long processes, withlamellipodia located at the ends. This response of cells to C3transferase started to revert back to the control state from 24


Fig. 6. Rho regulates the formation of actin cables in Bac1 cells. Growing Bac1 cells (A,C-F) or CSF-1-starved cells (B) were microinjectedwith V14RhoA and fixed after 15 minutes (A-C) or injected with C3 transferase and fixed after 30 minutes (D,F) or 18 hours (E). (A) Cellswere injected with 50 µg/ml V14RhoA; (B-F) cells were injected with V14RhoA at 200 µg/ml. Cells were stained with TRITC-phalloidin toreveal actin filaments (A,B,D,E) or with a mouse anti-phosphotyrosine antibody followed by TRITC-labelled anti-mouse IgG to reveal focalcomplexes (C,F). Arrows indicate injected cells. Arrowhead (A) indicates fine actin cables. Bars, 10 µm; A-C,E,F are at the samemagnification.

hours after injection. These results indicate that formation ofthe fine actin cables is regulated by Rho, and that these cablesmaintain the shape of the cells.

The role of Rho in regulating focal complexes was investi-gated by staining cells injected with V14RhoA or C3 trans-ferase with anti-phosphotyrosine antibodies. V14RhoA-injected cells retained their focal complexes, with a similardistribution of phosphotyrosine staining to that observed in

CSF-1-starved control cells (Fig. 6C). C3 transferase-injectedcells retained their focal complexes as judged by phosphoty-rosine staining at all timepoints examined between 15 minutes(Fig. 6F) and 18 hours after injection, but their distributionbecame altered as the cell morphology became more extreme.In elongated cells no focal complexes were visible along thelong cell extensions, but were abundant in the terminal lamel-lipodia (data not shown). This was consistent with the frequent


observation that the highly elongated cell processes often hadgrown over adjacent cells, indicating that the long extensionsare only attached to the substratum at their ends. From theseresults it can be concluded that Rho does not regulate focalcomplex formation in macrophages.

DISCUSSION

Bac1 macrophages have an actin cytoskeleton which is quitedistinct in organisation from that of fibroblasts. Reflecting themore locomotory function of macrophages, they have fine actincables rather than well defined actin stress fibres. In additionthey have membrane ruffles, lamellipodia and abundantfilopodia. Like Swiss 3T3 fibroblasts, which can be induced tolose their actin stress fibres by culture in the absence of serum,it was shown that Bac1 macrophages lose their actin cables inthe absence of CSF-1. In addition, CSF-1-starved cellsgenerally lack lamellipodia, membrane ruffles and filopodia.CSF-1 rapidly stimulated cell spreading, membrane ruffling,and the formation of lamellipodia and filopodia, and subse-quently the assembly of actin cables. These observationsindicate that Bac1 cells are a suitable model for investigatingthe regulatory mechanisms underlying actin reorganisation, asin the absence of CSF-1 they provide a ‘blank’ starting pointfrom which to observe the effects of growth factors andinjected proteins on actin organisation, a property which hasalso made serum-starved Swiss 3T3 fibroblasts a good model(Ridley et al., 1992; Ridley and Hall, 1992).

The molecular composition of fibroblast focal adhesions hasbeen well characterised (Burridge et al., 1988; Jockush et al.,1995). In comparison relatively little is known about the cell-to-substratum contacts in monocytes/macrophages, althoughprevious studies have shown a punctate localization of severalproteins, including vinculin, talin, the protein kinase Csubstrate MARCKS, and αvβ5 integrin (Rosen et al., 1990; deNichilo and Burns, 1993; Wojciak-Stothard et al., 1996). Wehave further examined cell-to-substratum contacts in Bac1macrophages, and shown that they have ‘focal complexes’ con-taining several proteins normally associated with fibroblastfocal adhesions, including β1 integrin, paxillin, vinculin andpp125FAK. It is interesting that pp125FAK is expressed in Bac1cells, as the presence of this protein in monocytes/macrophageshas been controversial (Kharbanda et al., 1995; de Nichilo andYamada, 1996). It appears that pp125FAK is not normallydetectably expressed in freshly isolated monocytes ormacrophages (Choi et al., 1993; Lin et al., 1994; de Nichiloand Yamada, 1996) although it has been detected in a humanmonocytic cell line (Lin et al., 1995).

The focal complexes in Bac1 cells were considerablysmaller than fibroblast focal adhesions, and unlike the latter,which are Rho-dependent, macrophage focal complexes wereregulated by Cdc42 and Rac, but not Rho. These complexestherefore resemble the Cdc42/Rac-regulated focal complexesdescribed in Swiss 3T3 cells (Nobes and Hall, 1995). Cdc42acted upstream of Rac to regulate macrophage focalcomplexes, rather than acting in parallel with Rac. It is inter-esting that focal complexes were retained in CSF-1-starvedcells, which generally lack membrane ruffles, lamellipodia oractin cables. This implies that there is a residual level of Cdc42and Rac activity in starved cells that is sufficient to maintain

focal complexes. The molecular mechanism underlying Rac-induced complex assembly remains to be established: unlikeRho, Rac does not appear to stimulate the tyrosine phospho-rylation of pp125FAK and other proteins, at least in fibroblasts(Flinn and Ridley, 1996). A number of proteins interacting withRac have been recently characterized, however (van Aelst etal., 1996; Ridley, 1996, and references therein), and one ofthese may act as a molecular link between Rac and focalcomplex formation.

Interestingly, β1 integrin remained clustered in N17Rac- andN17Cdc42-injected macrophages even when vinculin haddispersed and very few clusters of tyrosine phosphorylatedproteins remained. This implies that integrin/extracellularmatrix interactions in these cells are relatively stable and notstrictly dependent on the colocalization of cytosolic proteins.This stability of integrin/extracellular matrix interactions isalso suggested by the observation that moving cells often leavebehind integrins bound to the extracellular matrix (Hutten-locher et al., 1995). One explanation for the slow dispersal ofintegrin clusters is that once the integrins are in a high-affinitystate, bound to extracellular matrix proteins, they revert onlyslowly to a low-affinity state, when they detach and can diffusewithin the plasma membrane. In fibroblasts, integrins areclearly able to delocalize from focal adhesion sites, as β1integrin distribution became diffuse and focal adhesions disas-sembled within 30 minutes of adding various protein kinaseinhibitors, although in this case earlier timepoints were notexamined (Chrzanowska-Wodnicka and Burridge, 1996). Itwill be interesting to determine whether any focal complexproteins remain clustered with the integrins in N17Rac-injected cells, as studies on bead-induced focal adhesionformation have suggested a hierarchy of focal adhesionproteins associating with integrins, with pp125FAK being mostclosely associated with integrin clustering (Miyamoto et al.,1995).

Microinjection of Rac showed that, as in fibroblasts, itrapidly induced the formation of lamellipodia and membraneruffles. This response closely resembled the early response ofBac1 cells to CSF-1, and indeed Rac was required for CSF-1-induced lamellipodium formation and membrane ruffling. Themembrane ruffling response in CSF-1-stimulated cells istransient and they subsequently polarize (Boocock et al.,1989), whereas V12Rac1-injected cells continue to show adramatic ruffling response for several hours after injection. Inthe CSF-1 response, therefore, it is likely that Rac activation israpidly downregulated and/or localized to discrete points of theplasma membrane, leading to morphological polarization.

Injection of macrophages with activated Cdc42 (V12Cdc42)was sufficient to produce many long and well defined filopodia.This is a much more dramatic response than that observed infibroblasts (Kozma et al., 1995; Nobes and Hall, 1995). Thisreflects the fact that Bac1 cells show considerable filopodialactivity when motile, and also in response to CSF-1 stimula-tion, whereas fibroblasts do not normally have significantnumbers of filopodia. Macrophages are therefore likely to be amuch more sensitive model than fibroblasts for investigatingthe signalling pathways downstream of Cdc42 leading to theformation of filopodia.

Macrophages are known not to form stress fibres, so it wasnot surprising that Rho did not induce stress fibres in Bac1cells, in contrast to its effect in fibroblasts and epithelial cells


Fig. 7. Model for the roles of Cdc42, Rac and Rho in regulating actinorganization and the formation of focal complexes in macrophages.CSF-1 independently activates Cdc42, which induces the formationof filopodia, and Rac, which induces the formation of lamellipodiaand membrane ruffles. Cdc42 also acts upstream of Rac to regulatethe assembly of focal complexes, while Rac activation leads to theRho-mediated assembly of actin cables. High levels of filopodialactivity can inhibit lamellipodial extension.

(Ridley and Hall, 1992; Ridley et al., 1995). Rho did, however,regulate both Bac1 cell contractility and the assembly of actincables within the cytoplasm. These results resemble thoseobtained with NIE-115 mouse neuronal cells, where Rhomediates the rapid cell rounding and neurite retraction inducedby a variety of agents (Jalink et al., 1994; Postma et al., 1996).Our results are consistent with the hypothesis that Rho isprimarily involved in maintaining cell shape and tension withinthe actin cytoskeleton. Recently evidence has accumulatedlinking Rho via the protein kinase ROKα/Rho-kinase to inhi-bition of myosin light chain phosphatase, resulting in increasedmyosin light chain phosphorylation (Matsui et al., 1996; Leunget al., 1996; Kimura et al., 1996). Phosphorylation of myosinlight chain favours the formation of myosin filaments and theirinteraction with actin filaments, and is therefore believed tolead to the assembly of contractile actomyosin filaments (Tanet al., 1992; Chrzanowska-Wodnicka and Burridge, 1996).Taken together, these results suggest that the actin cablesobserved in Bac1 cells are the macrophage counterpart tofibroblast stress fibres, and that the ability of Rho to modulatemyosin association with actin filaments underlies Rho-mediated changes in actin organization and cell shape in manyif not all cell types.

CSF-1 stimulation of macrophages appears to activateCdc42 and Rac rapidly, as filopodia and lamellipodia wereobserved immediately after CSF-1 addition. In Swiss 3T3fibroblasts, a link between Cdc42 and Rac has been observed:microinjection of Cdc42 stimulates filopodium formation andsubsequently Rac-mediated lamellipodium formation (Kozmaet al., 1995; Nobes and Hall, 1995). In Bac1 cells stimulatedwith CSF-1, Rac activation is not dependent on Cdc42, aslamellipodia and membrane ruffles were observed inN17Cdc42-injected cells stimulated with CSF-1. Cdc42 can,however, act upstream of Rac, as lamellipodia were observedon cells injected with low concentrations of V12Cdc42. Ourdata on focal complex formation also indicate that Cdc42 actsupstream of Rac. It appears, however, that in macrophages,where the response to injected Cdc42 protein is very dramatic,filopodia formation dominates over lamellipodia formation.When V12Cdc42 is injected at high concentrations, few or nolamellipodia are observed. In addition, Cdc42-injected cellsstimulated with CSF-1 showed few or no lamellipodia ormembrane ruffles compared to uninjected cells. Furthermore,within populations of CSF-1-stimulated cells, those with manyfilopodia generally lacked lamellipodia or membrane ruffles(unpublished observations). In cells with many filopodia, theformation of other actin-based structures such as lamellipodiamay be prevented due to a diversion of limiting amounts ofactin or associated proteins into filopodia. This would maskany activation of Rac downstream of Cdc42.

Following the formation of filopodia and lamellipodia, alater response to CSF-1 was the Rho-mediated formation ofactin cables within the cytoplasm. In this response, Rhoappears to be activated downstream of Rac, as microinjectionof V12Rac1 also stimulated actin cable assembly, and this wasinhibited by C3 transferase. These results are in agreementwith the previously observed link between Rac and Rho inSwiss 3T3 fibroblasts (Ridley et al., 1992), showing that thishierarchical connection is not restricted to fibroblasts. InMDCK epithelial cells, however, no increase in actin stressfibres has been observed in Rac-injected cells (Ridley et al.,

1995), and thus the effect of Rac on Rho presumably dependson the cell type and/or cell conditions.

Our results are consistent with a model for Rho family actionin macrophages (Fig. 7). CSF-1 initially activates Cdc42 andRac in parallel to stimulate the formation of filopodia andlamellipodia/membrane ruffles, although Cdc42 can also actupstream of Rac. Subsequently, Rac acts upstream of Rho toinduce actin cable formation. Formation of large numbers offilopodia, induced by Cdc42, inhibits both the formation oflamellipodia and actin cables. Redistribution of focalcomplexes is also induced by CSF-1, and is regulated by Cdc42acting upstream of Rac.

Our observations on the effects of Cdc42, Rac and Rho in ahighly motile cell type are consistent with an important rolefor these proteins in regulating cell migration. Most theoriessuggest that the first step in cell locomotion is the extension ofcell protrusions at the leading edge (Lauffenburger andHorwitz, 1996; Mitchison and Cramer, 1996). This oftenbegins with the extension of filopodia and subsequently lamel-lipodia, indicating a role for Cdc42 and Rac at this stage. Cellprotrusions then become anchored to the substratum, and againCdc42 and Rac are implicated at this stage, inducing theformation of new integrin-containing focal complexes. Thethird step is the loosening of adhesions between the rear edgeof the cell and the substratum and the contraction of the cellbody to pull after the leading edge protrusion. The results ofthis and other studies indicate that Rho induces the formationof a contractile actomyosin filament network, and couldtherefore be involved in regulating this step of cell motility.Studies of the roles of Rho family proteins in macrophagemigration are now in progress.

We are grateful to Jon Erickson and Rick Cerione for E. coli strainsexpressing recombinant GST-V12Cdc42 and GST-N17Cdc42proteins, and to Ritu Garg for purifying recombinant proteins formicroinjection. We thank Alan Entwistle for assistance with confocallaser scanning microscopy and image generation, and Matthew


Isoukis (Leica UK Limited) for generating the image in Fig. 3B. Weare grateful to David Critchley and Dietmar Westweber for generousgifts of antibodies, and to Maikel Peppelenbosch and other membersof our laboratories for critical discussions of this work. W.E.A. wassupported by the Arthritis and Rheumatism Council (UK).

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(Received 11 November 1996 – Accepted 15 January 1997)

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Rho, Rac and Cdc42 regulate actin organization and cell ... · Rho, Rac and Cdc42 are small GTP-binding proteins of the Rho family, a subset of the Ras superfamily (Hall, 1994). The