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The Novel Rho-Family Gtpase Rif Regulates Coordinated Actin-Based Membrane Rearrangements Sara Ellis and Harry Mellor

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The Novel Rho-Family Gtpase Rif Regulates Coordinated Actin-Based Membrane Rearrangements Sara Ellis and Harry Mellor View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Brief Communication 1387 The novel Rho-family GTPase Rif regulates coordinated actin-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 cytoskeleton 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 filopodia [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).
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