Brief Communication 1387

The novel Rho-family GTPase Rif regulates coordinatedactin-based membrane rearrangementsSara Ellis and Harry Mellor

Small GTPases of the Rho family have a critical role incontrolling cell morphology, motility and adhesionthrough dynamic regulation of the actin cytoskeleton[1,2]. Individual Rho GTPases have been shown toregulate distinct components of the cytoskeletalarchitecture; RhoA stimulates the bundling of actinfilaments into stress fibres [3], Rac reorganises actin toproduce membrane sheets or lamellipodia [4] andCdc42 causes the formation of thin, actin-rich surfaceprojections called filopodia [5]. We have isolated a newRho-family GTPase, Rif (Rho in filopodia), and shownthat it represents an alternative signalling route to thegeneration of filopodial structures. Coordinatedregulation of Rho-family GTPases can be used togenerate more complicated actin rearrangements, suchas those underlying cell migration [6]. In addition toinducing filopodia, Rif functions cooperatively withCdc42 and Rac to generate additional structures,increasing the diversity of actin-based morphology.

Address: Department of Biochemistry, School of Medical Sciences,University of Bristol, Bristol BS8 1TD, UK.

Correspondence: Harry MellorE-mail: h.mellor@bristol.ac.uk

Received: 27 June 2000Revised: 15 August 2000Accepted: 8 September 2000

Published: 20 October 2000

Current Biology 2000, 10:1387–1390

0960-9822/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.

Results and discussionThe Rif GTPase was identified from partial cDNAsequences in the human Expressed Sequence Tag (EST)database, in a search for novel Rho-family GTPases. Thefull-length (211 amino acid) protein contains the conservedα3′ helix insert region, unique to the Rho GTPases(Figure 1), but is generally quite distantly related, showingbetween 32–49% identity to other family members. Thehighest similarities were with Rac2 (49%), RhoD (48%) andRhoA (47%). Rif showed only 43% homology to Cdc42. Rifis widely expressed in human tissues, with the highest levelsof mRNA in colon, stomach and spleen (see Supplementarymaterial). This was also reflected in the sources of theEST clones, which were largely from colon cDNA libraries.

Rif was also highly expressed in HeLa cells (see Supple-mentary material) and so we overexpressed Rif with an

amino-terminal Myc-epitope tag in this cell line toexamine its effects on cell morphology. Expression of aconstitutively active Rif-QL mutant caused the formationof bulbous peripheral protrusions in 50% of cells (75 cells,n = 3; Figure 2a). Wild-type Rif also caused formation ofthese structures, but to a lesser extent (25% of 75 cells,n = 3; data not shown). The protrusions also containedF-actin (data not shown) suggesting an effect of Rif on theactin cytoskeleton. The full Rif phenotype was, however,seen only on removal of the epitope tag. Untagged wild-type Rif (Figure 2d) or the constitutively active Rif-QLmutant (Figure 2b) were entirely localised to the plasmamembrane and their expression caused cells to present a‘hairy’ appearance due to the formation of numerous long,actin-rich (Figure 2c) filopodial structures. These extendedfrom the cell perimeter, but also covered the apical cellsurface. The apical filopodia collapse to some extent onfixation and lie over the body of the cells in these pro-jected confocal images. Expression of wild-type Rif or Rif-QL also caused a modest increase in actin stress fibreformation within the cell (Figure 2c, the intracellular actinis partially obscured by the Rif-induced filopodial struc-tures at the top of the cell). A similar observation has beenmade after expression of activated Cdc42 in some celltypes [5]. We speculate that the bulbous protrusions seenwith epitope-tagged Rif represent thwarted attempts atfilopodia, and that the amino terminus of Rif is importantfor this function.

We examined the Rif-induced filopodial structures furtherby time-lapse microscopy of live cells, using actin taggedwith green fluorescent protein (GFP–actin) to mark thesestructures. Filopodia induced by Rif-QL were highlydynamic and filopodial extension could be seen even overa short time frame (see Supplementary material). Cellsexpressing an inactive, constitutively GDP-bound Rif-TNmutant showed only the sparse, short membrane protru-sions seen in untransfected cells (Figure 2e,f and Supple-mentary material), suggesting that Rif activity is requiredfor its function. Rif-TN had a punctate, perinuclear stain-ing pattern suggesting association with intracellular vesi-cles (Figure 2e). This is reminiscent of the Arf6 GTPase,which redistributes from endosomes to the plasma mem-brane on activation [7], and indeed Rif-TN colocalisedwith Arf6-positive endosomes (data not shown). The for-mation of Rif filopodial structures was dependent onF-actin, as treatment with actin-depolymerising agentscytochalasin D (Figure 2g,h) or latrunculin B (data notshown) caused their collapse into cloverleaf-shaped mem-brane protrusions centred on actin foci.

Cdc42-induced filopodia have been shown to contain vin-culin-rich focal complexes at their tips [5]. The Rif-induced structures differed in this respect; unlike Cdc42,Rif-QL expression had no discernible effect on the distri-bution of focal complexes/adhesions, which were presentat the base of the Rif-induced protrusions, between adja-cent structures (Figure 2i,j). Rif also differs from Cdc42 in

its binding specificity. The cellular functions of Cdc42 aremediated by interaction of the activated small GTPasewith CRIB (Cdc42/Rac interactive binding) domains ofdownstream effectors [8]. The Wiskott–Aldrich syndromeprotein (WASP) and N-WASP are two such proteins, andhave been shown to regulate Cdc42-mediated actinrearrangements by recruiting the Arp2/3 complex to sites

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Figure 1

An alignment of Rif with a selection of otherRho family members. Multiple alignments ofhuman sequences were performed using theClustal V algorithm in MegAlign 4.05(DNASTAR Inc.). Regions of specific interestare shown. TC10 is related to Cdc42 (66%identical), interacts with a similar subset ofeffectors, and induces filopodia [15]. Thesequence of the Cdc42 splice variant G25Kis identical to Cdc42 over the regions shown.Elements of secondary structure are identifiedbelow the alignment (H1 and H2 are 310helices), including the α3′ helix, an insertunique to the Rho family GTPases and

present in Rif. Conserved residues involved inGTP hydrolysis are marked (asterisk). Regionsof contact between Cdc42 and the WASPCRIB domain are highlighted with red

triangles. Blue triangles indicate four residuesthat have been shown to be involved in CRIBdomain binding by Cdc42, and which are notconserved in Rif.

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Phosphatebinding Switch 1 Switch 2

25 923 911

H1 H2

**

*138

Insert region182

VGDGGCGKTSLLMVYSQGSFPEHYAPSVFEKYTASVTVGSKEVTLNLYDTAGQEDYDRLRPLSYQN..DKEQLRKLRAAQL..EDVFREAAKVAL RifVGDGAVGKTCLLISYTTNKFPSEYVPTVFDNYAVTVMIGGEPYTLGLFDTAGQEDYDRLRPLSYPQ..DPSTIEKLAKNKQ..KNVFDEAILAAL Cdc42VGDGAVGKTCLLMSYANDAFPEEYVPTVFDHYAVSVTVGGKQYLLGLYDTAGQEDYDRLRPLSYPM..DPKTLARLNDMKE..KTVFDEAIIAIL TC10VGDGAVGKTCLLISYTTNAFPGEYIPTVFDNYSANVMVDSKPVNLGLWDTAGQEDYDRLRPLSYPQ..DKDTIEKLKEKKL..KTVFDEAIRAVL Rac2VGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVADIEVDGKQVELALWDTAGQEDYDRLRPLSYPD..DEHTRRELAKMKQ..REVFEMATRAAL RhoA

α1 β2 β3 α3′ α5

Figure 2

Rif induces the formation of actin-dependentfilopodial structures. (a) HeLa cells weretransfected with Myc-tagged Rif-QL andstained with the 9E10 antibody. All otherpanels show untagged Rif constructs.(b,c) Cells transfected with constitutivelyactivated Rif-QL, stained with (b) polyclonalanti-Rif, and (c) co-stained with TRX-P todetect F-actin. (d) Cells transfected withwild-type Rif, stained with polyclonal anti-Rif.(e,f ) Cells transfected with constitutivelyinactive Rif-TN, stained with (e) polyclonalanti-Rif, and (f) co-stained with TRX-P todetect F-actin. (g,h) Cells transfected withRif-QL and treated with 2 µm cytochalasin Dfor 20 min before fixation, stained with(g) polyclonal anti-Rif, and (h) co-stainedwith TRX-P to detect F-actin. (i) Cellstransfected with Rif-QL and stained withpolyclonal anti-Rif (green) and monoclonalanti-vinculin, for focal adhesions (red);(j) shows the vinculin staining alone.The scale bar represents 10 µm.

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(a) (b) (g)

(e) (c) (i)

(f) (j)

(d) (h)

of Cdc42 activation [9]. We tested the ability of Rif tointeract with WASP in vivo, using an immunoprecipita-tion-based assay. Whereas activated Cdc42 bound stronglyto WASP, Rif showed no interaction (data not shown).

The basis for this becomes clear on examination of the Rifpeptide sequence. Cdc42 interacts with CRIB domains atthree contact points; however, only the Cdc42 Switch1region and α5 helix are thought to confer specificity ofbinding [10]. Rif shows only weak homology to Cdc42 inthese regions (Figure 1). Owen and co-workers havecarried out detailed analysis of the residues in Cdc42 thatspecify binding to WASP, and two other Cdc42 effectors;ACK and PAK [11]. Asp38 in Cdc42 makes a hydrogenbond with one of the conserved histidine residues in theISXPX…HXXH CRIB consensus. Mutation of this residueto glutamic acid causes a 200-fold reduction in the affinityof Cdc42 for WASP (also an ~30-fold reduction in PAK orACK affinity). Similarly, Cdc42 mutation T35S causes asignificant loss of affinity of Cdc42 for ACK (43-fold),PAK (26-fold) or WASP (35-fold), as does the V42A muta-tion (ACK 17-fold, PAK 2-fold, WASP 3-fold). All threesubstitutions are present naturally in Rif (Figure 1).Leu174 in Cdc42 forms part of a pocket for the conservedisoleucine in the CRIB consensus. Mutation to an alaninecauses a 30-fold reduction in WASP binding (30-fold ACK,

2.5-fold PAK). In Rif this residue is a lysine. Takentogether, it would seem that Rif is highly unlikely to inter-act with Cdc42-binding CRIB domains.

RhoG is a Rho-family GTPase that regulates the actincytoskeleton by activating Cdc42 and Rac [12]. This raisesthe possibility that Rif might be inducing filopodia indi-rectly, through activation of Cdc42. However, Rif-inducedfilopodial structures were not affected by coexpression ofa dominant-negative mutant of Cdc42 (Figure 3a,b), or byexpression of the Cdc42-binding domain of the WASPprotein (data not shown), which blocks Cdc42 actionin vivo [13]. Similarly, Cdc42-induced filopodia were notblocked by expression of the Rif-TN mutant (data notshown). Taken together, these data suggest that Rif andCdc42 regulate filopodia through distinct pathways.

Surprisingly, coexpression of an activated mutant ofCdc42 with Rif-QL dramatically modulated the Rif phe-notype. Peripheral filopodia were retained (Figure 3c,e),but apical structures developed into swollen, finger-likeprojections (Figure 3c,f). These much larger structuresstained heavily with Rif and actin (Figure 3c), whereasCdc42 staining was diffuse (Figure 3d). These structureswere not seen with either Rif-QL or activated Cdc42alone (Figure 3g,h), suggesting that the two small GTPases

Brief Communication 1389

Figure 3

Rif cooperates with Cdc42 and Rac togenerate diversity in actin-based morphology.(a,b) HeLa cells were co-transfected withconstitutively active Rif-QL and the dominant-negative Cdc42N17 mutant and stained with(a) the 9E10 antibody for Cdc42 and(b) polyclonal anti-Rif. (c,d) Cells wereco-transfected with Rif-QL and theconstitutively active Cdc42V12 mutant andstained with (c) polyclonal anti-Rif (green),(c) TRX-P for F-actin (red) and (d) 9E10 todetect the Cdc42. (e,f) Confocal sectionsthrough two cells coexpressing Rif-QL andCdc42V12, stained with polyclonal anti-Rif;(e) is a section through the middle of the cellsshowing retention of peripheral filopodia, (f) isa section from the top of the cells, through theswollen apical projections. (g,h) Cellstransfected with Cdc42V12 and (g) stainedfor Cdc42 with the 9E10 antibody and(h) stained with TRX-P for F-actin. (i) Cellstransfected with the constitutively activatedRacV12 mutant, stained with 9E10 antibodyto detect Rac (green) and TRX-P to detectF-actin (red); colocalisation appears yellow.(j–l) Cells co-transfected with Rif-QL andRacV12, stained with (j) polyclonal anti-Rifand (k) 9E10 antibody for Rac (compare(k) with (i)). Cells in (j,k) were also stainedwith TRX-P for F-actin and (l) shows amagnified portion of the cells with the merged

images of Rif-QL (green), RacV12 (blue)and F-actin (red); colocalisation of thethree signals appears white (neither Rac or

Rif colocalised with actin stress fibreswhich are therefore red). The scale barrepresents 10 µm.

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(a) (c)

(d)

(b)

(i) (j) (k) (l)

(e) (g)

(f) (h)

cooperate in their formation. The structures were also seenon coexpression of wild-type Rif with activated Cdc42,but to a lesser extent than with constitutively activatedRif-QL (data not shown). Modulation of the Rif pheno-type was also seen with the Rac GTPase. Coexpression ofRif-QL with an activated Rac mutant led to shortening ofthe Rif filopodial structures into numerous bristles(Figure 3j,k). These covered the cell surface, but wereparticularly localised to regions of cell–cell contact. Unlikethe interaction with Cdc42, activated Rac colocalisedextensively with the Rif structures, which also stainedheavily for actin (Figure 3l). As with Cdc42, a dominant-negative mutant of Rac was without effect on the Rif-induced filopodial structures (data not shown). Rif-inducedactin rearrangements were seen in the other cell linestested (NIH 3T3, CHO, MDCK, Hep2, Caco, data notshown), but varied in magnitude compared to HeLa cells.We speculate that the high endogenous level of Rif in thiscell line may be matched by equivalent expression of therelevant downstream signalling machinery. We are currentlyinvestigating what this machinery might be. The effects ofthe epitope tag on the Rif phenotype show that the freeamino terminus of the protein is required for proper locali-sation and/or function. Notably, the first 19 amino acids ofRif show no homology to other Rho family members andmay therefore mediate a unique protein–protein interaction.

Recent evidence has suggested that the basic buildingblocks of actin architecture, controlled by single Rhofamily GTPases, can be used combinatorially by cells tocreate diverse cellular structures and complicated mechan-ical movements. Identification of the Rif GTPase adds tothis diversity, as well as to the complexity of the signallingpathways behind it.

Materials and methodsCloning of RifThe NCBI human EST database was searched for novel Rho-relatedGTPases using the tblastn algorithm. Overlapping ESTs encoding Rifwere obtained from the UK HGMP Resource Centre (Hinxton, UK).Mammalian expression vector constructs were generated in pcDNA3(Invitrogen) using standard molecular biology techniques. A GTPase-deficient, activated construct, Rif-QL, and a constitutively GDP-boundinactive mutant, Rif-TN, were engineered by using the QuikChangesite-directed mutagenesis system (Stratagene) to change Gln75 toleucine and Thr33 to asparagine, respectively. Corresponding Rif con-structs with an amino-terminal Myc epitope tag were also produced.Mammalian expression vector constructs encoding Rac, Cdc42 andWASP cDNAs were a generous gift from Kate Nobes and Alan Hall.We thank Laura Machesky for a mammalian expression vector encod-ing GFP fused to chicken β-actin. The cDNA sequence of Rif has beendeposited in the GenBank database (accession number AF239923).

AntibodiesA synthetic peptide, corresponding to the first 20 amino acids of Rif,was used to generate rabbit polyclonal antibodies. These were affinity-purified using the peptide coupled to SulfoLink gel (Pierce). The mono-clonal anti-Myc epitope antibody 9E10, anti-Cdc42 and anti-vinculinantibodies were from Santa Cruz, Transduction Labs and Sigma,respectively. Cy2-, Cy3- and Cy5-conjugated secondary antibodies werefrom Jackson Laboratories.

Cell culture, transfection and immunofluorescence microscopyHeLa cells were cultured on glass coverslips in DMEM supplementedwith 10% FBS. Cells were transfected with mammalian expressionvectors using Transfast lipid (Promega). After exposure to the lipid/DNAmix for 2 h, the cells were washed into serum-free DMEM containing 0.1%fatty acid-free BSA and left for approximately 16 h prior to experimenta-tion. Cells were processed for immunofluorescence microscopy asdescribed previously [14]. Where applicable, F-actin was stained withTexas Red-X phalloidin (TRX-P, Molecular Probes). Cells were viewedusing a Leica DM RBE confocal microscope under a Plan Apo x63/1.32oil immersion objective. Cy2, Cy3 and Cy5 were excited using the488 nm, 568 nm and 647 nm lines of a Kr-Ar laser, respectively. Series ofimages were taken at 0.5 µm intervals through the Z-plane of the cell and,unless indicated, were processed to form a projected image of the cell.

Supplementary materialSupplementary material including analysis of Rif mRNA expression andmovies of Rif-induced filopodial structures is available at http://current-biology.com/supmat/supmatin.htm.

AcknowledgementsH.M. is a recipient of a Career Development Award Fellowship from theWellcome Trust. S.E. is supported by an MRC studentship. This work wassupported by grants from the Wellcome Trust and the Royal Society to HMand by MRC Infrasructure Award G4500006 to the School of Medical Sci-ences Imaging Facility. We thank George Banting, Alan Hall and KateNobes for useful comments.

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