The Rho GTPases are related to the Ras proto-oncogenes and consist of 22 family members. These proteins have important roles in regulating the organization of the actin filament system, and thereby the morphogenesis of vertebrate cells as well as their ability to migrate. In an effort to compare the effects of all members of the Rho GTPase family, active Rho GTPases were transfected into porcine aortic endothelial cells and the effects on the actin filament system were monitored. Cdc42, TCL (TC10-like), Rac1–Rac3 and RhoG induced the formation of lamellipodia, whereas Cdc42, Rac1 and Rac2 also induced the formation of thick bundles of actin filaments. In contrast, transfection with TC10 or Chp resulted in the formation of focal adhesion-like structures, whereas Wrch-1 induced long and thin filopodia. Transfection with RhoA, RhoB or RhoC induced the assembly of stress fibres, whereas Rnd1–Rnd3 resulted in the loss of stress fibres, but this effect was associated with the formation of actin- and ezrin-containing dorsal microvilli. Cells expressing RhoD and Rif had extremely long and flexible filopodia. None of the RhoBTB or Miro GTPases had any major influence on the organization of the actin filament system; instead, RhoBTB1 and RhoBTB2 were present in vesicular structures, and Miro-1 and Miro-2 were present in mitochondria. Collectively, the data obtained in this study to some extent confirm earlier observations, but also allow the identification of previously undetected roles of the different members of the Rho GTPases.
Abbreviations used: CRIB domain, Cdc42 Rac interactive binding domain; FBS, foetal bovine serum; GAL4-DB, GAL4 DNA-binding domain; GAP, GTPase-activating protein; GDI, guanine nucleotide dissociation inhibitor; GEF, guanine nucleotide exchange factor; GST, glutathione S-transferase; HA, haemagglutinin; PAE, porcine aortic endothelial; PAK, p21-activated kinase; PDGF, platelet-derived growth factor; SH3, Src homology 3; TCL, TC10-like; TC10L75 (etc.), TC10 in which residue 75 has been replaced by leucine (etc.); TRITC, tetramethyl rhodamine isothiocyanate; WASP, Wiskott–Aldrich syndrome protein; N-WASP, neuronal WASP.
Present address: Department of Medical Sciences, University Hospital, S-751 85 Uppsala, Sweden.