Increased Cell-Matrix Adhesion upon Constitutive Activation of Rho Proteins by Cytotoxic Necrotizing Factors from E. coli and Y. pseudotuberculosis
Figure 1
Formation of actin filaments upon activation of Rho proteins by CNF toxins. (a) HeLa cells were treated with CNF1 and CNFy for 24 h. The actin cytoskeleton and nuclei of CNF-treated HeLa cells was stained by rhodamine-phalloidin and DAPI, respectively. (b) HeLa cells were treated with CNF1 for 12 h. Reduced electrophoretic mobility of RhoA and the cellular levels of RhoA, Rac1, and Cdc42 were determined by Western blot analysis in time-dependent manner. Beta-actin was used as loading control. A representative Western out of three blots was presented. Signal intensities of Rac1 were quantified, and normalized to the level of beta-actin. (c) HeLa cells were treated with the indicated toxins for 5 h and the cellular levels of active GTP-bound RhoA, Rac1, and Cdc42 were determined by a pull-down assay using either GST-RBD (Rho-binding domain of Rhotekin) for RhoA and GST-PAK-CRIB (p21-binding domain of Pak1) for Rac1 and Cdc42. Total and precipitated Rho proteins were detected by immuno-blot analysis. A sample of lysates from nontreated cells was subjected to nucleotide exchange with the nonhydrolysable GTPγS. Signal intensities () were quantified, and normalized to the level of total Rho proteins. Results displayed are the mean ± SD of three independent experiments. values <0.01 (**) and <0.001 (***) were considered as statistically significant as compared with nontreated cells.
