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Brief Communication 1387

The novel Rho-family GTPase Rif regulates coordinated -based membrane rearrangements Sara Ellis and Harry Mellor

Small GTPases of the Rho family have a critical role in amino-terminal Myc-epitope tag in this cell line to controlling cell morphology, motility and adhesion examine its effects on cell morphology. Expression of a through dynamic regulation of the actin constitutively active Rif-QL mutant caused the formation [1,2]. Individual Rho GTPases have been shown to of bulbous peripheral protrusions in 50% of cells (75 cells, regulate distinct components of the cytoskeletal n = 3; Figure 2a). Wild-type Rif also caused formation of architecture; RhoA stimulates the bundling of actin these structures, but to a lesser extent (25% of 75 cells, filaments into stress fibres [3], Rac reorganises actin to n = 3; data not shown). The protrusions also contained produce membrane sheets or lamellipodia [4] and F-actin (data not shown) suggesting an effect of Rif on the Cdc42 causes the formation of thin, actin-rich surface actin cytoskeleton. The full Rif phenotype was, however, projections called [5]. We have isolated a new seen only on removal of the epitope tag. Untagged wild- Rho-family GTPase, Rif (Rho in filopodia), and shown type Rif (Figure 2d) or the constitutively active Rif-QL that it represents an alternative signalling route to the mutant (Figure 2b) were entirely localised to the plasma generation of filopodial structures. Coordinated membrane and their expression caused cells to present a regulation of Rho-family GTPases can be used to ‘hairy’ appearance due to the formation of numerous long, generate more complicated actin rearrangements, such actin-rich (Figure 2c) filopodial structures. These extended as those underlying cell migration [6]. In addition to from the cell perimeter, but also covered the apical cell inducing filopodia, Rif functions cooperatively with surface. The apical filopodia collapse to some extent on Cdc42 and Rac to generate additional structures, fixation and lie over the body of the cells in these pro- increasing the diversity of actin-based morphology. jected confocal images. Expression of wild-type Rif or Rif- QL also caused a modest increase in actin stress fibre Address: Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK. formation within the cell (Figure 2c, the intracellular actin is partially obscured by the Rif-induced filopodial struc- Correspondence: Harry Mellor tures at the top of the cell). A similar observation has been E-mail: [email protected] made after expression of activated Cdc42 in some cell Received: 27 June 2000 types [5]. We speculate that the bulbous protrusions seen Revised: 15 August 2000 with epitope-tagged Rif represent thwarted attempts at Accepted: 8 September 2000 filopodia, and that the amino terminus of Rif is important Published: 20 October 2000 for this function.

Current Biology 2000, 10:1387–1390 We examined the Rif-induced filopodial structures further by time-lapse microscopy of live cells, using actin tagged 0960-9822/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. with green fluorescent protein (GFP–actin) to mark these structures. Filopodia induced by Rif-QL were highly Results and discussion dynamic and filopodial extension could be seen even over The Rif GTPase was identified from partial cDNA a short time frame (see Supplementary material). Cells sequences in the human Expressed Sequence Tag (EST) expressing an inactive, constitutively GDP-bound Rif-TN database, in a search for novel Rho-family GTPases. The mutant showed only the sparse, short membrane protru- full-length (211 amino acid) protein contains the conserved sions seen in untransfected cells (Figure 2e,f and Supple- α3′ helix insert region, unique to the Rho GTPases mentary material), suggesting that Rif activity is required (Figure 1), but is generally quite distantly related, showing for its function. Rif-TN had a punctate, perinuclear stain- between 32–49% identity to other family members. The ing pattern suggesting association with intracellular vesi- highest similarities were with Rac2 (49%), RhoD (48%) and cles (Figure 2e). This is reminiscent of the Arf6 GTPase, RhoA (47%). Rif showed only 43% homology to Cdc42. Rif which redistributes from endosomes to the plasma mem- is widely expressed in human tissues, with the highest levels brane on activation [7], and indeed Rif-TN colocalised of mRNA in colon, stomach and spleen (see Supplementary with Arf6-positive endosomes (data not shown). The for- material). This was also reflected in the sources of the mation of Rif filopodial structures was dependent on EST clones, which were largely from colon cDNA libraries. F-actin, as treatment with actin-depolymerising agents cytochalasin D (Figure 2g,h) or latrunculin B (data not Rif was also highly expressed in HeLa cells (see Supple- shown) caused their collapse into cloverleaf-shaped mem- mentary material) and so we overexpressed Rif with an brane protrusions centred on actin foci. 1388 Current Biology Vol 10 No 21

Figure 1

An alignment of Rif with a selection of other Phosphate Rho family members. Multiple alignments of binding Switch 1 Switch 2 Insert region human sequences were performed using the * 138 182 * * Clustal V algorithm in MegAlign 4.05 25 VGDGGCGKTSLLMVYSQGSFPEHYAPSVFEKYTASVTVGSKEVTLNLYDTAGQEDYDRLRPLSYQN..DKEQLRKLRAAQL..EDVFREAAKVAL Rif 9 VGDGAVGKTCLLISYTTNKFPSEYVPTVFDNYAVTVMIGGEPYTLGLFDTAGQEDYDRLRPLSYPQ..DPSTIEKLAKNKQ..KNVFDEAILAAL Cdc42 (DNASTAR Inc.). Regions of specific interest 23 VGDGAVGKTCLLMSYANDAFPEEYVPTVFDHYAVSVTVGGKQYLLGLYDTAGQEDYDRLRPLSYPM..DPKTLARLNDMKE..KTVFDEAIIAIL TC10 9 VGDGAVGKTCLLISYTTNAFPGEYIPTVFDNYSANVMVDSKPVNLGLWDTAGQEDYDRLRPLSYPQ..DKDTIEKLKEKKL..KTVFDEAIRAVL Rac2 are shown. TC10 is related to Cdc42 (66% 11 VGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVADIEVDGKQVELALWDTAGQEDYDRLRPLSYPD..DEHTRRELAKMKQ..REVFEMATRAAL RhoA identical), interacts with a similar subset of α β β α ′α effectors, and induces filopodia [15]. The 1 2 3 H1 H2 3 5 sequence of the Cdc42 splice variant G25K Current Biology is identical to Cdc42 over the regions shown. Elements of secondary structure are identified present in Rif. Conserved residues involved in triangles. Blue triangles indicate four residues below the alignment (H1 and H2 are 310 GTP hydrolysis are marked (asterisk). Regions that have been shown to be involved in CRIB helices), including the α3′ helix, an insert of contact between Cdc42 and the WASP domain binding by Cdc42, and which are not unique to the Rho family GTPases and CRIB domain are highlighted with red conserved in Rif.

Cdc42-induced filopodia have been shown to contain vin- its binding specificity. The cellular functions of Cdc42 are culin-rich focal complexes at their tips [5]. The Rif- mediated by interaction of the activated small GTPase induced structures differed in this respect; unlike Cdc42, with CRIB (Cdc42/Rac interactive binding) domains of Rif-QL expression had no discernible effect on the distri- downstream effectors [8]. The Wiskott–Aldrich syndrome bution of focal complexes/adhesions, which were present protein (WASP) and N-WASP are two such proteins, and at the base of the Rif-induced protrusions, between adja- have been shown to regulate Cdc42-mediated actin cent structures (Figure 2i,j). Rif also differs from Cdc42 in rearrangements by recruiting the Arp2/3 complex to sites

Figure 2

Rif induces the formation of actin-dependent (a) (b) (g) filopodial structures. (a) HeLa cells were transfected with Myc-tagged Rif-QL and stained with the 9E10 antibody. All other panels show untagged Rif constructs. (b,c) Cells transfected with constitutively activated Rif-QL, stained with (b) polyclonal anti-Rif, and (c) co-stained with TRX-P to detect F-actin. (d) Cells transfected with (d) (h) wild-type Rif, stained with polyclonal anti-Rif. (e,f) Cells transfected with constitutively inactive Rif-TN, stained with (e) polyclonal anti-Rif, and (f) co-stained with TRX-P to detect F-actin. (g,h) Cells transfected with Rif-QL and treated with 2 µm cytochalasin D for 20 min before fixation, stained with (g) polyclonal anti-Rif, and (h) co-stained with TRX-P to detect F-actin. (i) Cells (e) (c) (i) transfected with Rif-QL and stained with polyclonal anti-Rif (green) and monoclonal anti-vinculin, for focal adhesions (red); (j) shows the vinculin staining alone. The scale bar represents 10 µm.

(f) (j)

Current Biology Brief Communication 1389

Figure 3

Rif cooperates with Cdc42 and Rac to (a) (c) (e) (g) generate diversity in actin-based morphology. (a,b) HeLa cells were co-transfected with constitutively 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 were co-transfected with Rif-QL and the constitutively active Cdc42V12 mutant and stained with (c) polyclonal anti-Rif (green), (b) (f) (h) (c) TRX-P for F-actin (red) and (d) 9E10 to detect the Cdc42. (e,f) Confocal sections through two cells coexpressing Rif-QL and (d) Cdc42V12, stained with polyclonal anti-Rif; (e) is a section through the middle of the cells showing retention of peripheral filopodia, (f) is a section from the top of the cells, through the swollen apical projections. (g,h) Cells transfected with Cdc42V12 and (g) stained (i) (j) (k) (l) for Cdc42 with the 9E10 antibody and (h) stained with TRX-P for F-actin. (i) Cells transfected with the constitutively activated RacV12 mutant, stained with 9E10 antibody to detect Rac (green) and TRX-P to detect F-actin (red); colocalisation appears yellow. (j–l) Cells co-transfected with Rif-QL and RacV12, stained with (j) polyclonal anti-Rif Current Biology and (k) 9E10 antibody for Rac (compare (k) with (i)). Cells in (j,k) were also stained images of Rif-QL (green), RacV12 (blue) Rif colocalised with actin stress fibres with TRX-P for F-actin and (l) shows a and F-actin (red); colocalisation of the which are therefore red). The scale bar magnified portion of the cells with the merged three signals appears white (neither Rac or represents 10 µm.

of Cdc42 activation [9]. We tested the ability of Rif to 2.5-fold PAK). In Rif this residue is a lysine. Taken interact with WASP in vivo, using an immunoprecipita- together, it would seem that Rif is highly unlikely to inter- tion-based assay. Whereas activated Cdc42 bound strongly act with Cdc42-binding CRIB domains. to WASP, Rif showed no interaction (data not shown). RhoG is a Rho-family GTPase that regulates the actin The basis for this becomes clear on examination of the Rif cytoskeleton by activating Cdc42 and Rac [12]. This raises peptide sequence. Cdc42 interacts with CRIB domains at the possibility that Rif might be inducing filopodia indi- three contact points; however, only the Cdc42 Switch1 rectly, through activation of Cdc42. However, Rif-induced region and α5 helix are thought to confer specificity of filopodial structures were not affected by coexpression of binding [10]. Rif shows only weak homology to Cdc42 in a dominant-negative mutant of Cdc42 (Figure 3a,b), or by these regions (Figure 1). Owen and co-workers have expression of the Cdc42-binding domain of the WASP carried out detailed analysis of the residues in Cdc42 that protein (data not shown), which blocks Cdc42 action specify binding to WASP, and two other Cdc42 effectors; in vivo [13]. Similarly, Cdc42-induced filopodia were not ACK and PAK [11]. Asp38 in Cdc42 makes a hydrogen blocked by expression of the Rif-TN mutant (data not bond with one of the conserved histidine residues in the shown). Taken together, these data suggest that Rif and ISXPX…HXXH CRIB consensus. Mutation of this residue Cdc42 regulate filopodia through distinct pathways. to glutamic acid causes a 200-fold reduction in the affinity of Cdc42 for WASP (also an ~30-fold reduction in PAK or Surprisingly, coexpression of an activated mutant of ACK affinity). Similarly, Cdc42 mutation T35S causes a Cdc42 with Rif-QL dramatically modulated the Rif phe- significant loss of affinity of Cdc42 for ACK (43-fold), notype. Peripheral filopodia were retained (Figure 3c,e), PAK (26-fold) or WASP (35-fold), as does the V42A muta- but apical structures developed into swollen, finger-like tion (ACK 17-fold, PAK 2-fold, WASP 3-fold). All three projections (Figure 3c,f). These much larger structures substitutions are present naturally in Rif (Figure 1). stained heavily with Rif and actin (Figure 3c), whereas Leu174 in Cdc42 forms part of a pocket for the conserved Cdc42 staining was diffuse (Figure 3d). These structures isoleucine in the CRIB consensus. Mutation to an alanine were not seen with either Rif-QL or activated Cdc42 causes a 30-fold reduction in WASP binding (30-fold ACK, alone (Figure 3g,h), suggesting that the two small GTPases 1390 Current Biology Vol 10 No 21

cooperate in their formation. The structures were also seen Cell culture, transfection and immunofluorescence microscopy on coexpression of wild-type Rif with activated Cdc42, HeLa cells were cultured on glass coverslips in DMEM supplemented with 10% FBS. Cells were transfected with mammalian expression but to a lesser extent than with constitutively activated vectors using Transfast lipid (Promega). After exposure to the lipid/DNA Rif-QL (data not shown). Modulation of the Rif pheno- mix for 2 h, the cells were washed into serum-free DMEM containing 0.1% type was also seen with the Rac GTPase. Coexpression of fatty acid-free BSA and left for approximately 16 h prior to experimenta- Rif-QL with an activated Rac mutant led to shortening of tion. Cells were processed for immunofluorescence microscopy as the Rif filopodial structures into numerous bristles described previously [14]. Where applicable, F-actin was stained with Texas Red-X phalloidin (TRX-P, Molecular Probes). Cells were viewed (Figure 3j,k). These covered the cell surface, but were using a Leica DM RBE confocal microscope under a Plan Apo x63/1.32 particularly localised to regions of cell–cell contact. Unlike oil immersion objective. Cy2, Cy3 and Cy5 were excited using the the interaction with Cdc42, activated Rac colocalised 488 nm, 568 nm and 647 nm lines of a Kr-Ar laser, respectively. Series of extensively with the Rif structures, which also stained images 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. heavily for actin (Figure 3l). As with Cdc42, a dominant- negative mutant of Rac was without effect on the Rif- Supplementary material induced filopodial structures (data not shown). Rif-induced Supplementary material including analysis of Rif mRNA expression and actin rearrangements were seen in the other cell lines movies of Rif-induced filopodial structures is available at http://current- tested (NIH 3T3, CHO, MDCK, Hep2, Caco, data not biology.com/supmat/supmatin.htm. shown), but varied in magnitude compared to HeLa cells. We speculate that the high endogenous level of Rif in this Acknowledgements H.M. is a recipient of a Career Development Award Fellowship from the cell line may be matched by equivalent expression of the Wellcome Trust. S.E. is supported by an MRC studentship. This work was relevant downstream signalling machinery. We are currently supported by grants from the Wellcome Trust and the Royal Society to HM investigating what this machinery might be. The effects of and by MRC Infrasructure Award G4500006 to the School of Medical Sci- ences Imaging Facility. We thank George Banting, Alan Hall and Kate the epitope tag on the Rif phenotype show that the free Nobes for useful comments. amino terminus of the protein is required for proper locali- sation and/or function. Notably, the first 19 amino acids of References Rif show no homology to other Rho family members and 1. Ridley AJ: Rho. In GTPases. Edited by A Hall. Oxford: Oxford may therefore mediate a unique protein–protein interaction. University Press 2000:89-136. 2. Hall A: Rho GTPases and the actin cytoskeleton. Science 1998, 279:509-514. Recent evidence has suggested that the basic building 3. Ridley AJ, Hall A: The small GTP-binding protein Rho regulates the assembly of focal adhesions and actin stress fibers in response blocks of actin architecture, controlled by single Rho to growth factors. Cell 1992, 70:389-399. family GTPases, can be used combinatorially by cells to 4. Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A: The small create diverse cellular structures and complicated mechan- GTP-binding protein Rac regulates growth factor-induced membrane ruffling. Cell 1992, 70:401-410. ical movements. Identification of the Rif GTPase adds to 5. Nobes CD, Hall A: Rho, Rac, and Cdc42 GTPases regulate the this diversity, as well as to the complexity of the signalling assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 1995, 81:53-62. pathways behind it. 6. Ridley AJ, Allen WE, Peppelenbosch M, Jones GE: Rho family proteins and cell migration. Biochem Soc Symp 1999, 65:111-123. Materials and methods 7. Radhakrishna H, Klausner RD, Donaldson JG: Aluminum fluoride stimulates surface protrusions in cells overexpressing the ARF6 Cloning of Rif GTPase. J Cell Biol 1996, 134:935-947. The NCBI human EST database was searched for novel Rho-related 8. Bishop AL, Hall A: Rho GTPases and their effector proteins. GTPases using the tblastn algorithm. Overlapping ESTs encoding Rif Biochem J 2000, 348:241-255. were obtained from the UK HGMP Resource Centre (Hinxton, UK). 9. Mullins RD: How WASP-family proteins and the Arp2/3 complex Mammalian expression vector constructs were generated in pcDNA3 convert intracellular signals into cytoskeletal structures. Curr Opin (Invitrogen) using standard molecular biology techniques. A GTPase- Cell Biol 2000, 12:91-96. deficient, activated construct, Rif-QL, and a constitutively GDP-bound 10. Abdul-Manan N, Aghazadeh B, Liu GA, Majumdar A, Ouerfelli O, inactive mutant, Rif-TN, were engineered by using the QuikChange Siminovitch KA, et al.: Structure of Cdc42 in complex with the site-directed mutagenesis system (Stratagene) to change Gln75 to GTPase-binding domain of the ‘Wiskott-Aldrich syndrome’ leucine and Thr33 to asparagine, respectively. Corresponding Rif con- protein. Nature 1999, 399:379-383. 11. Owen D, Mott HR, Laue ED, Lowe PN: Residues in Cdc42 that structs with an amino-terminal Myc epitope tag were also produced. specify binding to individual CRIB effector proteins. Biochemistry Mammalian expression vector constructs encoding Rac, Cdc42 and 2000, 39:1243-1250. WASP cDNAs were a generous gift from Kate Nobes and Alan Hall. 12. Gauthier-Rouviere C, Vignal E, Meriane M, Roux P, Montcourier P, We thank Laura Machesky for a mammalian expression vector encod- Fort P: RhoG GTPase controls a pathway that independently ing GFP fused to chicken β-actin. The cDNA sequence of Rif has been activates Rac1 and Cdc42Hs. Mol Biol Cell 1998, 9:1379-1394. deposited in the GenBank database (accession number AF239923). 13. Nobes CD, Hall A: Rho GTPases control polarity, protrusion, and adhesion during cell movement. J Cell Biol 1999, 144:1235-1244. Antibodies 14. Mellor H, Flynn P, Nobes CD, Hall A, Parker PJ: PRK1 is targeted to A synthetic peptide, corresponding to the first 20 amino acids of Rif, endosomes by the small GTPase, RhoB. J Biol Chem 1998, 273:4811-4814. was used to generate rabbit polyclonal antibodies. These were affinity- 15. Neudauer CL, Joberty G, Tatsis N, Macara IG: Distinct cellular purified using the peptide coupled to SulfoLink gel (Pierce). The mono- effects and interactions of the Rho-family GTPase TC10. Curr Biol clonal anti-Myc epitope antibody 9E10, anti-Cdc42 and anti-vinculin 1998, 8:1151-1160. antibodies were from Santa Cruz, Transduction Labs and Sigma, respectively. Cy2-, Cy3- and Cy5-conjugated secondary antibodies were from Jackson Laboratories.