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• W2057209737 abstract "In this study we show that expression of active Cdc42Hs and Rac1 GTPases, two Rho family members, leads to the reorganization of the vimentin intermediate filament (IF) network, showing a perinuclear collapse. Cdc42Hs displays a stronger effect than Rac1 as 90% versus 75% of GTPase-expressing cells show vimentin collapse. Similar vimentin IF modifications were observed when endogenous Cdc42Hs was activated by bradykinin treatment, endogenous Rac1 by platelet-derived growth factor/epidermal growth factor, or both endogenous proteins upon expression of active RhoG. This reorganization of the vimentin IF network is not associated with any significant increase in soluble vimentin. Using effector loop mutants of Cdc42Hs and Rac1, we show that the vimentin collapse is mostly independent of CRIB (Cdc42Hs or Rac-interacting binding)-mediated pathways such as JNK or PAK activation but is associated with actin reorganization. This does not result from F-actin depolymerization, because cytochalasin D treatment or Scar-WA expression have merely no effect on vimentin organization. Finally, we show that genistein treatment of Cdc42 and Rac1-expressing cells strongly reduces vimentin collapse, whereas staurosporin, wortmannin, LY-294002,R p-cAMP, or RII, the regulatory subunit of protein kinase A, remain ineffective. Moreover, we detected an increase in cellular tyrosine phosphorylation content after Cdc42Hs and Rac1 expression without modification of the vimentin phosphorylation status. These data indicate that Cdc42Hs and Rac1 GTPases control vimentin IF organization involving tyrosine phosphorylation events. In this study we show that expression of active Cdc42Hs and Rac1 GTPases, two Rho family members, leads to the reorganization of the vimentin intermediate filament (IF) network, showing a perinuclear collapse. Cdc42Hs displays a stronger effect than Rac1 as 90% versus 75% of GTPase-expressing cells show vimentin collapse. Similar vimentin IF modifications were observed when endogenous Cdc42Hs was activated by bradykinin treatment, endogenous Rac1 by platelet-derived growth factor/epidermal growth factor, or both endogenous proteins upon expression of active RhoG. This reorganization of the vimentin IF network is not associated with any significant increase in soluble vimentin. Using effector loop mutants of Cdc42Hs and Rac1, we show that the vimentin collapse is mostly independent of CRIB (Cdc42Hs or Rac-interacting binding)-mediated pathways such as JNK or PAK activation but is associated with actin reorganization. This does not result from F-actin depolymerization, because cytochalasin D treatment or Scar-WA expression have merely no effect on vimentin organization. Finally, we show that genistein treatment of Cdc42 and Rac1-expressing cells strongly reduces vimentin collapse, whereas staurosporin, wortmannin, LY-294002,R p-cAMP, or RII, the regulatory subunit of protein kinase A, remain ineffective. Moreover, we detected an increase in cellular tyrosine phosphorylation content after Cdc42Hs and Rac1 expression without modification of the vimentin phosphorylation status. These data indicate that Cdc42Hs and Rac1 GTPases control vimentin IF organization involving tyrosine phosphorylation events. epidermal growth factor platelet-derived growth factor bradykinin intermediate filament green fluorescence protein phosphate-buffered saline 4-morpholineethanesulfonic acid Cdc42Hs or Rac-interacting binding phosphatidylinositol 3-kinase c-Jun NH2-terminal kinase p21-activated kinase Cdc42-associated tyrosine kinase The Rho family of Ras-like GTPases are clustered in two distinct subgroups: the Rac/Cdc42 subgroup, including Rac1, -2, and -3, RhoG, Cdc42Hs, TC10, chp (Cdc42 homologousprotein), and RhoH, and the Rho subgroup, in which RhoA, -B, and -C, RhoD, RhoL, and Rnd1, -2, and -3 are found. Many cell functions, including maintenance of morphology (1Tapon N. Hall A. Curr. Opin. Cell Biol. 1997; 9: 86-92Crossref PubMed Scopus (692) Google Scholar), motility (2Aepfelbacher M. Vauti F. Weber P.C. Glomset J.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4263-4267Crossref PubMed Scopus (44) Google Scholar), adhesion (3Braga V.M. Machesky L.M. Hall A. Hotchin N.A. J. Cell Biol. 1997; 137: 1421-1431Crossref PubMed Scopus (653) Google Scholar, 4Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3714) Google Scholar), cell division (5Dutartre H. Davoust J. Gorvel J.P. Chavrier P. J. Cell Sci. 1996; 109: 367-377PubMed Google Scholar) and proliferation (6Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1056) Google Scholar), smooth muscle contraction (7Hirata K. Kikuchi A. Sasaki T. Kuroda S. Kaibuchi K. Matsuura Y. Seki H. Saida K. Takai Y. J. Biol. Chem. 1992; 267: 8719-8722Abstract Full Text PDF PubMed Google Scholar), and vesicular transport (8Murphy C. Saffrich R. Grummt M. Gournier H. Rybin V. Rubino M. Auvinen P. Lutcke A. Parton R.G. Zerial M. Nature. 1996; 384: 427-432Crossref PubMed Scopus (192) Google Scholar, 9Lamaze C. Chuang T.H. Terlecky L.J. Bokoch G.M. Schmid S.L. Nature. 1996; 382: 177-179Crossref PubMed Scopus (331) Google Scholar) are regulated by these small GTP-binding proteins of the Rho family. Many reports have shown that three of these Rho GTPases, RhoA, Rac1, and Cdc42Hs, tightly regulate the actin filaments organization. In fibroblasts, lysophosphatidic acid-stimulated stress fiber formation requires RhoA (10Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3066) Google Scholar, 11Ridley A.J. Hall A. EMBO J. 1994; 13: 2600-2610Crossref PubMed Scopus (440) Google Scholar). Epidermal growth factor (EGF)1-, platelet-derived growth factor (PDGF)-, and insulin-dependent cortical actin polymerization such as ruffles and lamellipodia requires Rac1 (4Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3714) Google Scholar, 10Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3066) Google Scholar), whereas bradykinin (Bdk)-stimulated filopodia formation requires Cdc42Hs (4Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3714) Google Scholar, 12Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (881) Google Scholar). Activation hierarchies exist among Rho GTPases. RhoG acts upstream of Rac1 and Cdc42Hs (13Gauthier R.C. Vignal E. Meriane M. Roux P. Montcourier P. Fort P. Mol. Biol. Cell. 1998; 9: 1379-1394Crossref PubMed Scopus (135) Google Scholar), and Cdc42 activation leads to subsequent activation of Rac1 (4Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3714) Google Scholar). Evidence also suggests the existence of an antagonism between RhoA and Rac1/Cdc42Hs (14Kozma R. Sarner S. Ahmed S. Lim L. Mol. Cell. Biol. 1997; 17: 1201-1211Crossref PubMed Scopus (535) Google Scholar, 15Moorman J.P. Luu D. Wickham J. Bobak D.A. Hahn C.S. Oncogene. 1999; 18: 47-57Crossref PubMed Scopus (91) Google Scholar, 16Rottner K. Hall A. Small J.V. Curr. Biol. 1999; 9: 640-648Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar). Once in a GTP-bound and -activated conformation, each of these GTPases appear to interact with specific downstream effector proteins. More than 20 candidate targets have been identified so far, such as protein kinases PAKs, ACKs, PKN-related kinases (PRKs), and Rho kinases (ROCKs); lipid kinases phosphatidylinositol 3-kinase, PIP5K, and PLD and non-kinase proteins such as Wiskott-Aldrich syndrome protein (WASP), partner of Rac1 (POR1), plenty of SH35 (POSH) p67PHOX, IQGAPs, Rhotekin, and others (for a review see Aspenstrom (17Aspenstrom P. Curr. Opin. Cell Biol. 1999; 11: 95-102Crossref PubMed Scopus (285) Google Scholar)). Although the role of Rho GTPases on actin cytoskeleton organization has been extensively studied, little is known on their effects on one of the other major component of the cytoskeleton of eucaryotic cells, the intermediate filaments (IFs). IFs consist of a heterogeneous tissue-specific family of proteins, which are prevalent in the perinuclear region and extend radially through the cytoplasm, eventually forming close associations with the cell surface, concentrated in regions containing desmosomes (cadherin-mediated cell-to-cell junctions), hemidesmosomes (integrin-mediated adhesive junctions), and other types of adhesion sites (18Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1274) Google Scholar, 19Kowalczyk A.P. Bornslaeger E.A. Norvell S.M. Palka H.L. Green K.J. Int. Rev. Cytol. 1999; 185: 237-302Crossref PubMed Google Scholar). Cytoskeletal IF also interact with other cytoskeletal elements such as microtubules and microfilaments (20Draberova E. Draber P. J. Cell Sci. 1993; 106: 1263-1273PubMed Google Scholar, 21Prahlad V. Yoon M. Moir R.D. Vale R.D. Goldman R.D. J. Cell Biol. 1998; 143: 159-170Crossref PubMed Scopus (271) Google Scholar, 22Gyoeva F.K. Gelfand V.I. Nature. 1991; 353: 445-448Crossref PubMed Scopus (178) Google Scholar, 23Chou Y.H. Skalli O. Goldman R.D. Curr. Opin. Cell Biol. 1997; 9: 49-53Crossref PubMed Scopus (69) Google Scholar). Interaction with plasma membrane and other cytoskeletal elements involve a number of IF-associated proteins that are essential for maintaining the integrity of IF network (24Djabali K. Histol. Histopathol. 1999; 14: 501-509PubMed Google Scholar). Recently, it has been shown that, at least in actively growing cells, IF are dynamic structures. IF phosphorylation appears to be one of the most predominant biochemical events in coordinating intracellular organization of the IF network (25Inagaki M. Matsuoka Y. Tsujimura K. Ando S. Tokui T. Takahashi T. Inagaki N. Bioessays. 1996; 18: 481-487Crossref Scopus (160) Google Scholar). Cytoplasmic IF disassembled when phosphorylated by protein kinase A, protein kinase C, calcium calmodulin kinase II (CaMKII), and Cdc2 kinases. Interestingly, protein kinase N (PKN), a protein kinase activated by Rho, associates and phosphorylates a subunit of neuron-specific intermediate filament, NFL (26Mukai H. Toshimori M. Shibata H. Kitagawa M. Shimakawa M. Miyahara M. Sunakawa H. Ono Y. J. Biol. Chem. 1996; 271: 9816-9822Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) and ROKα (RhoA-binding kinase α) phosphorylates glial fibrillary acidic protein (GFAP) (27Kosako H. Amano M. Yanagida M. Tanabe K. Nishi Y. Kaibuchi K. Inagaki M. J. Biol. Chem. 1997; 272: 10333-10336Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) and vimentin (28Sin W.C. Chen X.Q. Leung T. Lim L. Mol. Cell. Biol. 1998; 18: 6325-6339Crossref PubMed Google Scholar). In the present study we analyzed the possible role of two Rho GTPases, Rac1 and Cdc42Hs, in regulating the vimentin IF organization. We show that expression of active Rac1 and Cdc42Hs (V12 or L61 mutants) modified vimentin IF distribution: the normally well-spread distribution of IF became dramatically reorganized around the nucleus. Furthermore, Cdc42Hs and Rac1-dependent vimentin collapse was both different and more pronounced than RhoA-dependent vimentin reorganization. This vimentin collapse was observed following activation of endogenous Cdc42Hs by bradykinin and Rac1 by PDGF/EGF or after active RhoG expression. Because the IF network was closely associated with the actin microfilaments, the dynamics of which was highly regulated by Rho GTPases, we expressed two effector loop mutants of Rac1 and Cdc42Hs that had a differential effect on F-actin organization. Interestingly, the Y40C mutants of Cdc42Hs and Rac1, which still induced F-actin rearrangements, promoted the collapse of IF as efficiently as did the V12 or L61 mutants. The F37A mutants of Cdc42Hs and Rac1, which had lost their ability to induce filopodia and membrane ruffling, respectively, no longer induced the collapse of IF. In addition, by using various drugs known to have kinase inhibitory activity, we show that vimentin IF reorganization involved tyrosine phosphorylation events. Rat embryo (REF-52) or human (Hs-68) fibroblasts were cultured at 37 °C in the presence of 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were plated on 18-mm diameter glass coverslips 16–24 h before transfection. Cells were treated with 100 nm cytochalasin D (Sigma, France) for 5–60 min. PDGF/EGF (5 ng ml−1; Sigma, France) and bradykinin (100 ng ml−1; Sigma) were added for 15–60 min.R p-cAMP (Sigma) was used at 3 mm, staurosporin (Sigma) at 50 nm, genistein (Sigma) at 100 μm, LY-294002 (Sigma) at 10 μm, and wortmannin (Sigma) at 100 nm. These drugs were added for 15–120 min. The regulatory subunit of protein kinase A, RII (40 units/μl in the needle; Promega), was microinjected in GFP-expressing cells. Cells were transfected with plasmids encoding GFP-tagged RhoGV12, Rac1V12, Cdc42HsV12, RhoAV14, Rac1L61, Rac1L61F37A, Rac1L61Y40C, Cdc42HsL61, Cdc42HsL61F37A, Cdc42HsL61Y40C, and Myc-tagged Scar-WA using the LipofectAMINE method (Life Technologies, Gaithersburg, MD) as described previously ((13Gauthier R.C. Vignal E. Meriane M. Roux P. Montcourier P. Fort P. Mol. Biol. Cell. 1998; 9: 1379-1394Crossref PubMed Scopus (135) Google Scholar)). As a control, cells were transfected with empty vector (pEGFPC1). 4 h after transfection, Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum was added and cells were fixed at different times thereafter. 18 h after transfection, cells were fixed for 5 min in 3.7% formalin (in PBS) followed by a 2-min permeabilization in 0.1% Triton X-100 (in PBS) and incubation in PBS containing 0.1% bovine serum albumin. Expression of GFP-tagged proteins was visualized directly. Cells were stained for vimentin distribution using a mouse monoclonal anti-vimentin (Sigma, France) (1:200 dilution), followed by incubation with affinity-purified tetramethyl-rhodamin-5 (and 6) isothiocyanate-conjugated goat anti-mouse antibody (Cappel-ICN) (1:40 dilution). Cells were stained for F-actin using coumarin phenyl isothiocyanate-conjugated phalloidin (Sigma, France) and for phosphotyrosine epitopes using the 4G10 monoclonal antibody. Expression of Myc epitope-tagged proteins was visualized after a 60-min incubation with 9E10 anti-Myc monoclonal antibody (gift from D. Mathieu, Montpellier, France) (one-half dilution in PBS/bovine serum albumin), followed by incubation with affinity-purified fluorescein isothiocyanate-conjugated goat anti-mouse antibody (Cappel, ICN). Cells were washed in PBS, mounted in Mowviol (Aldrich, Milwaukee, WI), and observed using a DMR B Leica microscope using a 40× (NA 1.00) or 63× (NA 1.32) planapochromatic lens. Images obtained were captured with a MicroMax 1300 Y/HS (B/W) cooled (−10 °C) charge-coupled device camera as 16-bit images, and using a MetaMorph (v.4.11) control program (RS-Princeton Instruments) run by a PC-compatible microcomputer. Images were saved in TIFF format (16 bit) and subsequently adapted as TIFF 8-bit format after they were opened with Adobe Photoshop for processing and mounting with Adobe Illustrator. Two distinct methods were used to evaluate the amount of soluble vimentin in cell extracts from control REF-52 cells or REF-52 cells transfected with GFP-Cdc42HsV12 or GFP-Rac1V12. After washing with ice-cold PBS, cells were scraped, collected, and centrifuged at 10,000 rpm. Pellets were resuspended in either a lysis buffer containing 1% Nonidet P-40, 10% glycerol, 20 mm Hepes, pH 7.5, 150 mm NaCl, 1 mmphenylmethylsulfonyl fluoride, 20 mm NaF, a protease inhibitor mixture (Sigma), 100 μmNa3VO4 as described previously by Valgeirsdottir et al. (29Valgeirsdottir S. Claesson W.L. Bongcam R.E. Hellman U. Westermark B. Heldin C.H. J. Cell Sci. 1998; 111: 1973-1980PubMed Google Scholar), or 1% Triton, 50 mmMES, 600 mm KCl, 10 mm MgCl2. For the second method, the first soluble pool obtained underwent three successive extractions of 10 min in ice-cold lysis buffer (30Lamb N.J. Fernandez A. Feramisco J.R. Welch W.J. J. Cell Biol. 1989; 108: 2409-2422Crossref PubMed Scopus (109) Google Scholar). The soluble and insoluble fractions obtained with both methods were loaded onto 10% polyacrylamide gels and then transferred onto nitrocellulose. Initial lysates were normalized for protein content (BCA, Sigma). Membranes were saturated in 8% milk in Tris-HCl, pH 7.5, containing 0.1% Tween and subsequently incubated with a mouse monoclonal antibody directed against vimentin (clone V9, Sigma) (1/1000 dilution) followed by peroxidase-conjugated anti-mouse antibody (Amersham Pharmacia Biotech) (1/2000 dilution). After extensive washing, membranes were incubated with chemiluminescence reagent (ECL, PerkinElmer Life Sciences) and analyzed with a PhosphorImager (Molecular Dynamics). Total cell lysates from untransfected or cells transfected with either empty pEGFPN1 vector (MOCK), Cdc42HsV12, or Rac1V12 were obtained by the addition of 1% boiling SDS, 10 mm Tris-HCl, pH 7.4. After scraping, samples (30 μg of protein) were loaded onto a 10% polyacrylamide gel and then transferred onto nitrocellulose. Membranes were treated as described above and incubated with an anti-phosphotyrosine antibody (4G10, 1/200 dilution) followed with peroxidase-conjugated anti-mouse antibody (Amersham Pharmacia Biotech) (dilution 1/2000). After extensive washing, membranes were incubated with chemiluminescence reagent (ECL, PerkinElmer Life Sciences) and analyzed with a PhosphorImager (Molecular Dynamics). Untransfected cells or cells transfected with either empty pEGFPN1 vector (MOCK), Cdc42HsV12, or Rac1V12 were lysed for 20 min in ice-cold modified radioimmune precipitation buffer (1% Triton X-100, 10 mmsodium pyrophosphate, 0.1% SDS, 1% deoxycholate, 10% glycerol, 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 2.5 mm EDTA) supplemented with 20 mmβ-glycerophosphate, 1 mm phenylmethylsulfonyl fluoride, 20 mm NaF, 100 μmNa3VO4. Extracts were immunoprecipitated using a mouse monoclonal anti-vimentin antibody (V9, 1/100 dilution), separated by a 10% polyacrylamide gel, and then transferred onto nitrocellulose. Membranes were probed with the 4G10 anti-phosphotyrosine antibody as described above. To examine the overall effect of two Rho GTPases of the Rac/Cdc subgroup on vimentin IF distribution, mammalian expression plasmids encoding GFP-tagged constitutively active (G12V or Q61L) (for review see Ref. 31Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5200) Google Scholar), Cdc42Hs, or Rac1 were transfected into growing REF-52 cells. In addition, the activated RhoA mutant, RhoAV14, was also expressed. 18 h later, cells were fixed and immunostained with antibodies directed against vimentin and for filamentous actin (F-actin) using rhodamine-conjugated phalloidin (Fig.1 A). Expression of each of these GTPases induced the formation of specific actin-driven structures. Cdc42Hs (Fig. 1 A, panel a) induced filopodia (panel b), Rac1 (panel e) induced lamellipodia/ruffles (panel f), and RhoA (panel i) induced stress fibers (panel j) as previously reported (4Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3714) Google Scholar, 12Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (881) Google Scholar). Interestingly, expression of these GTPases also led to a marked change in the organization of the vimentin network (panels c and g). Indeed, although in control nontransfected cells vimentin showed a well-spread distribution from the perinuclear region to the cell periphery (see control nontransfected cells in panels c, g, andk), in Cdc42Hs- or Rac1-expressing cells, vimentin accumulated at the perinuclear area. Vimentin IF organization was also extensively modified after active RhoA expression (panel k), which is in total agreement with a previous report (32Paterson H.F. Self A.J. Garrett M.D. Just I. Aktories K. Hall A. J. Cell Biol. 1990; 111: 1001-1007Crossref PubMed Scopus (569) Google Scholar). Corresponding Normarski images were shown to precisely localize cell margins (panels d, h, and l). Similar effects of Cdc42Hs, Rac1, and RhoA on vimentin distribution were also observed in Hs-68 human fibroblasts (Fig. 1 B). More than 90% of Cdc42Hs-expressing cells and around 75% of Rac1-expressing cells showed extensive IF reorganization, with the same efficiency in REF-52 and Hs-68 cells. In contrast, less than 50% of RhoA-expressing cells presented a modified vimentin IF network. For both cell types, no significant modification in vimentin IF distribution (less than 5%) was detected upon transfection with pEGFPC1. Bradykinin (Bdk) has been shown to activate Cdc42Hs and induce filopodia formation, whereas platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) are two mitogens that activate Rac1 and produce lamellipodia (4Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3714) Google Scholar, 10Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3066) Google Scholar, 12Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (881) Google Scholar). To activate endogenous Rac1 and Cdc42Hs proteins, we thus stimulated REF-52 cells with Bdk or PDGF/EGF for different periods of time. Cells were then fixed and immunostained for F-actin to control endogenous Cdc42Hs and Rac1 activation and for vimentin IF distribution (Fig.2). In control unstimulated cells, both actin microfilaments and vimentin IF formed a well-spread network from the perinuclear region to the cell periphery (panels a andb). 15 min after Bdk addition, thin F-actin-rich filopodial extensions were detected at the edges of the cells (panel c). Concomitantly, extensive vimentin IF reorganization into perinuclear caps was observed (panel d), similar to the IF redistribution observed in Cdc42HsV12-expressing cells (Fig.1 A, panel g). 15 min after stimulation with PDGF/EGF, F-actin-containing lamellipodia were observed at the cell periphery (panel e) as well as a vimentin IF perinuclear distribution (panel f) comparable to that observed in Rac1V12-expressing cells (panel k). Activation of endogenous Cdc42Hs and Rac1 was also achieved by expressing active RhoG (13Gauthier R.C. Vignal E. Meriane M. Roux P. Montcourier P. Fort P. Mol. Biol. Cell. 1998; 9: 1379-1394Crossref PubMed Scopus (135) Google Scholar, 33Roux P. Gauthier R.C. Doucet B.S. Fort P. Curr. Biol. 1997; 7: 629-637Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In REF-52 cells transfected with GFP-tagged RhoGV12 (panel g), local actin polymerization led to the formation of lamellipodia and filopodia (panel h) as well as vimentin IF perinuclear redistribution (panel i). Because previous work reported that IF reorganization could be associated with changes in vimentin solubility (34Soellner P. Quinlan R.A. Franke W.W. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7929-7933Crossref PubMed Scopus (170) Google Scholar), we analyzed the amount of vimentin extractable using either a mild Nonidet P-40-containing lysis buffer (29Valgeirsdottir S. Claesson W.L. Bongcam R.E. Hellman U. Westermark B. Heldin C.H. J. Cell Sci. 1998; 111: 1973-1980PubMed Google Scholar) or high salt and Triton X-100-containing lysis buffer (30Lamb N.J. Fernandez A. Feramisco J.R. Welch W.J. J. Cell Biol. 1989; 108: 2409-2422Crossref PubMed Scopus (109) Google Scholar) after expression of active Cdc42Hs and Rac1 GTPases. The amount of vimentin present in a soluble form extractable with a mild Nonidet P-40-containing buffer was not modified after expression of these GTPases (Fig. 2 B) as was the case when using the high salt and Triton X-100-containing lysis buffer (data not shown). Taken together, these data show that expression of active Cdc42Hs and Rac1 or activation of endogenous Cdc42Hs and Rac1 all led to vimentin IF perinuclear reorganization without modification of the solubility of vimentin. We next investigated the pathways controlled by Rac1 and Cdc42Hs responsible for vimentin IF collapse. We used effector loop mutants of GTPases previously shown to differentially bind and activate downstream effectors (35Joneson T. McDonough M. Bar-Sagi D. Van Aelst L. Science. 1996; 274: 1374-1376Crossref PubMed Scopus (232) Google Scholar, 36Lamarche N. Tapon N. Stowers L. Burbelo P.D. Aspenstrom P. Bridges T. Chant J. Hall A. Cell. 1996; 87: 519-529Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar, 37Westwick J.K. Lambert Q.T. Clark G.J. Symons M. Van Aelst L. Pestell R.G. Der C.J. Mol. Cell. Biol. 1997; 17: 1324-1335Crossref PubMed Scopus (385) Google Scholar) (Fig.3). The Y40C mutants of Cdc42Hs and Rac1 had lost their ability to interact with CRIB (Cdc42Hs or Rac-interacting binding) motif-containing proteins and did not activate PAK-1 and JNK activity, but they still induced cortical F-actin polymerization, filopodia, and membrane ruffling, respectively. Conversely, the F37A mutants of Cdc42Hs and Rac1 still bound the CRIB motif-containing proteins, activating PAK and JNK, but were less efficient for inducing filopodia or membrane ruffling. Cells expressing the Y40C mutants of Cdc42Hs and Rac1 showed vimentin IF reorganization comparable to the one observed in active Rho GTPases-expressing cells (V12 or L61 mutants) (compare Fig.1 B with Fig. 3). Under the same conditions, expression of F37A mutants of Cdc42Hs and Rac1 did not significantly affect vimentin IF distribution, because only 10–20% of expressing cells showed weak vimentin reorganization. Interaction of IF with microfilaments is thought to regulate IF organization in vivo (23Chou Y.H. Skalli O. Goldman R.D. Curr. Opin. Cell Biol. 1997; 9: 49-53Crossref PubMed Scopus (69) Google Scholar). Rho GTPases of the Rac/Cdc subgroup are well-known key regulators of actin microfilaments (1Tapon N. Hall A. Curr. Opin. Cell Biol. 1997; 9: 86-92Crossref PubMed Scopus (692) Google Scholar), suggesting that the vimentin IF collapse we observed might result from Rac1- or Cdc42-dependent F-actin reorganization. Two subpopulations of actin structures were affected by Rac1 and Cdc42Hs expression: submembranous cortical actin, which was extensively modified to produce ruffles/lamellipodia and microvilli/filopodia, and stress fibers, which were mostly depolymerized. To test the existence of a relationship between stress fibers, depolymerization, and vimentin reorganization, REF-52 cells were treated with cytochalasin D, an F-actin-depolymerizing drug, fixed, and stained for F-actin and vimentin IF distribution (Fig.4 A). 30 min after cytochalasin D addition, the level of F-actin staining became barely detectable (panel b), whereas the vimentin IF network remained unaffected (panel c). We next transfected REF-52 cells with Scar-WA, a mutant form of the Arp2/3-interacting protein Scar, which prevents assembly of F-actin structures (38Machesky L.M. Insall R.H. Curr. Biol. 1998; 8: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar). As for cytochalasin-treated cells, expression of Myc-tagged Scar-WA (panel d) led to a decrease of F-actin polymerization (panel e) without any significant vimentin IF modifications (panel e). At variance, as shown in Fig. 4 B, coexpression of Scar-WA (panels b ande) with active Cdc42Hs (panel a) or Rac1 (panel d) as well as cytochalasin D treatment of Cdc42 and Rac1-expressing cells (data not shown) led to inhibition of Cdc42 or Rac1-dependent vimentin reorganization (panels c and f). As shown by the arrow in Fig. 4B (panel d) is a cell expressing only GFP_Rac1V12 showing a collapsed vimentin. These data show that, although an overall F-actin depolymerization did not affect vimentin organization, inhibition of Cdc42Hs- and Rac1-dependent F-actin modification impaired Cdc42Hs and Rac1-induced vimentin redistribution. Phosphorylation has been shown to be a major regulatory pathway coordinating intracellular organization of the IF network, so we used various drugs having known kinase inhibitory activity to address the involvement of phosphorylation events in vimentin IF collapse in Cdc42Hs- or Rac1-expressing cells (TableI). First, we analyzed the effects of inhibiting protein kinase A activity, a protein that induced both the collapse of vimentin IF and F-actin reorganization (30Lamb N.J. Fernandez A. Feramisco J.R. Welch W.J. J. Cell Biol. 1989; 108: 2409-2422Crossref PubMed Scopus (109) Google Scholar) by two ways: microinjection of the RII regulatory subunit of protein kinase A in GFP-Cdc42HsV12- or GFP-Rac1V12-expressing cells or addition of a cAMP antagonist R p-cAMP to GFP-Cdc42HsV12- or GFP-Rac1V12-expressing cells. Second, we treated GFP-Cdc42HsV12- and GFP-Rac1V12-expressing cells with the PI3Kinhibitors LY-294002 and wortmannin (39Yano H. Nakanishi S. Kimura K. Hanai N. Saitoh Y. Fukui Y. Nonomura Y. Matsuda Y. J. Biol. Chem. 1993; 268: 25846-25856Abstract Full Text PDF PubMed Google Scholar). Third, we used staurosporin, an inhibitor of various serine/threonine kinases. In all cases, no modification of the vimentin IF reorganization induced by Cdc42Hs and Rac1 was observed. Finally, we treated GFP-Cdc42HsV12- and GFP-Rac1V12-expressing cells with the tyrosine kinase inhibitor genistein for 15 to 120 min (40Akiyama T. Ishida J. Nakagawa S. Ogawara H. Watanabe S. Itoh N. Shibuya M. Fukami Y. J. Biol. Chem. 1987; 262: 5592-5595Abstract Full Text PDF PubMed Google Scholar). As shown in Fig. 5, cells treated with this compound displayed a less pronounced reorganization of the vimentin IF network upon Cdc42Hs and Rac1 expression (panels a/b and c/e) as compared with Fig. 1. This inhibitory effect on vimentin IF reorganization was not correlated with F-actin modific" @default.
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• W2057209737 date "2000-10-01" @default.
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• W2057209737 title "Cdc42Hs and Rac1 GTPases Induce the Collapse of the Vimentin Intermediate Filament Network" @default.
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