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.
Highlights
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 homologous protein), and RhoH, and the Rho subgroup, in which RhoA, -B, and -C, RhoD, RhoL, and Rnd1, -2, and -3 are found
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)
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
Summary
Cells Culture, Treatment, and Microinjection—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 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). 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). Detection of Tyrosine-phosphorylated Vimentin—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 mM sodium pyrophosphate, 0.1% SDS, 1% deoxycholate, 10% glycerol, 20 mM TrisHCl, pH 7.4, 150 mM NaCl, 2.5 mM EDTA) supplemented with 20 mM -glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 100 M Na3VO4. Membranes were probed with the 4G10 anti-phosphotyrosine antibody as described above
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