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An important consequence of cell swelling is the reorganization of the F-actin cytoskeleton in different cell types. We demonstrate in this study by means of rhodaminephalloidin labeling and fluorescence microscopy that a drastic reorganization of F-actin occurs in swollen Rat-1 fibroblasts: stress fibers disappear and F-actin patches are formed in peripheral extensions at the cell border. Moreover, we demonstrate that activation of both Rac and Cdc42, members of the family of small Rho GTPases, forms the link between the hypotonic stimulation and F-actin reorganization. Indeed, inhibition of the small GTPases RhoA, Rac, and Cdc42 (by Clostridium difficile toxin B) prevents the hypotonicity-induced reorganization of the actin cytoskeleton, whereas inhibition of RhoA alone (by C. limosum C3 exoenzyme) does not preclude this rearrangement. Second, a direct activation and translocation toward the actin patches underneath the plasma membrane is observed for endogenous Rac and Cdc42 (but not for RhoA) during cell swelling. Finally, transfection of Rat-1 fibroblasts with constitutively active RhoA, dominant negative Rac, or dominant negative Cdc42 abolishes the swelling-induced actin reorganization. Interestingly, application of cRGD, a competitor peptide for fibronectin-integrin association, induces identical membrane protrusions and changes in the F-actin cytoskeleton that are also inhibited by C. difficile toxin B and dominant negative Rac or Cdc42. Moreover, cRGD also induces a redistribution of endogenous Rac and Cdc42 to the newly formed submembranous F-actin patches. We therefore conclude that hypotonicity and cRGD remodel the F-actin cytoskeleton in Rat-1 fibroblasts in a Rac/Cdc42-dependent way. Rho; actin; swelling

RhoA exerts a permissive effect on volumeregulated anion channels in vascular endothelial cells. Am J Physiol Cell Physiol 283: Cl15-C125, 2002. First published February 20, 2002; 10.1152/ajpcell.00038.2001.--Cell swelling triggers in most cell types an outwardly rectifying anion current, [I.sub.Cl.swell], via volume-regulated anion channels (VRACs). We have previously demonstrated in calf pulmonary artery endothelial (CPAE) cells that inhibition of the Rho/Rho kinase/myosin light chain phosphorylation pathway reduces the swelling-dependent activation of [I.sub.Cl.swell]. However, these experiments did not allow us to discriminate between a direct activator role or a permissive effect. We now show that the Rho pathway did not affect VRAC activity if this pathway was activated by transfecting CPAE cells with constitutively active isoforms of G[alpha] (a Rho activating heterotrimeric protein subunit), Rho, or Rho kinase. Furthermore, biochemical and morphological analysis failed to demonstrate activation of the Rho pathway during hypotonic cell swelling. Finally, manipulating the Rho pathway with either guanosine 5'-O-(3-thiotriphosphate) or C3 exoenzyme had no effect on VRACs in caveolin-l-expressing Caco-2 cells. We conclude that the Rho pathway exerts a permissive effect on VRACs in CPAE cells, i.e., swelling-induced opening of VRACs requires a functional Rho pathway, but not an activation of the Rho pathway. cytoskeleton; actin; myosin phosphorylation; RhoA pathway

Inhibition of VRAC by c-Src tyrosine kinase targeted to caveolae is mediated by the Src homology domains. Am J Physiol Cell Physiol 281: C248-C256, 2001.--We used the whole cell patch-clamp technique in calf pulmonary endothelial (CPAE) cells to investigate the effect of wild-type and mutant c-Src tyrosine kinase on [I.sub.Cl,swell], the swelling-induced [Cl.sup.-] current through volume-regulated anion channels (VRAC). Transient transfection of wild-type c-Src in CPAE cells did not significantly affect [I.sub.Cl,swell]. However, transfection of c-Src with a [Ser.sup.3]Cys mutation that introduces a dual acylation signal and targets c-Src to lipid rafts and caveolae strongly repressed hypotenicity-induced [I.sub.Cl,swell] in CPAE cells. Kinase activity was dispensable for the inhibition of [I.sub.Cl.swell], since kinase-deficient c-Src [Ser.sup.3]Cys either with an inactivating point mutation in the kinase domain or with the entire kinase domain deleted still suppressed VRAC activity. Again, the [Ser.sup.3]Cys mutation was required to obtain maximal inhibition by the kinase-deleted c-Src. In contrast, the inhibitory effect was completely lost when the Src homology domains 2 and 3 were deleted in c-Src. We therefore conclude that c-Src-mediated inhibition of VRAC requires compartmentalization of c-Src to caveolae and that the Src homology domains 2 and/or 3 are necessary and sufficient for inhibition. anion channel; caveola; tyrosine kinase; cell volume; volume-regulated anion channel