No abstract

GTPases of the Rho family are molecular switches that play important roles in converting and amplifying external signals into cellular effects. Originally demonstrated to control the dynamics of the F-actin cytoskeleton, Rho GTPases have been implicated in many basic cellular processes that influence cell proliferation, differentiation, motility, adhesion, survival, or secretion. To elucidate the evolutionary history of the Rho family, we have analyzed over 20 species covering major eukaryotic clades from unicellular organisms to mammals, including platypus and opossum, and have reconstructed the ontogeny and the chronology of emergence of the different subfamilies. Our data establish that the 20 mammalian Rho members are structured into 8 subfamilies, among which Rac is the founder of the whole family. Rho, Cdc42, RhoUV, and RhoBTB subfamilies appeared before Coelomates and RhoJQ, Cdc42 isoforms, RhoDF, and Rnd emerged in chordates. In vertebrates, gene duplications and retrotranspositions increased the size of each chordate Rho subfamily, whereas RhoH, the last subfamily, arose probably by horizontal gene transfer. Rac1b, a Rac1 isoform generated by alternative splicing, emerged in amniotes, and RhoD, only in therians. Analysis of Rho mRNA expression patterns in mouse tissues shows that recent subfamilies have tissue-specific and low-level expression that supports their implication only in narrow time windows or in differentiated metabolic functions. These findings give a comprehensive view of the evolutionary canvas of the Rho family and provide guides for future structure and evolution studies of other components of Rho signaling pathways, in particular regulators of the RhoGEF family.

Phorbol ester induces actin cytoskeleton rearrangements in cultured vascular smooth muscle cells. Calponin and SM22 alpha are major components of differentiated smooth muscle and potential regulators of actin cytoskeleton interactions. Here we show that actin fibers decorated with h1 CaP remain stable, whereas SM22 alpha-decorated actin bundles undergo rapid reorganization into podosomes within 30 min of PDBu exposure. Ectopic expression of GFP alpha-actinin had no effect on the stability of the actin cytoskeleton and alpha-actinin was transported rapidly into PDBu-induced podosomes. Our results demonstrate the involvement of CaP and SM22 alpha in coordinating the balance between stabilization and dynamics of the actin cytoskeleton in mammalian smooth muscle. We provide evidence for the existence of two functionally distinct actin filament populations and introduce a molecular mechanism for the stabilization of the actin cytoskeleton by the unique actin-binding interface formed by calponin family-specific CLIK23 repeats.

RhoG is a member of the Rho family of GTPases that shares 72% and 62% sequence identity with Rac1 and Cdc42Hs, respectively. We have expressed mutant RhoG proteins fused to the green fluorescent protein and analyzed subsequent changes in cell surface morphology and modifications of cytoskeletal structures. In rat and mouse fibroblasts, green fluorescent protein chimera and endogenous RhoG proteins colocalize according to a tubular cytoplasmic pattern, with perinuclear accumulation and local concentration at the plasma membrane. Constitutively active RhoG proteins produce morphological and cytoskeletal changes similar to those elicited by a simultaneous activation of Rac1 and Cdc42Hs, i.e., the formation of ruffles, lamellipodia, filopodia, and partial loss of stress fibers. In addition, RhoG and Cdc42Hs promote the formation of microvilli at the cell apical membrane. RhoG-dependent events are not mediated through a direct interaction with Rac1 and Cdc42Hs targets such as PAK-1, POR1, or WASP proteins but require endogenous Rac1 and Cdc42Hs activities: coexpression of a dominant negative Rac1 impairs membrane ruffling and lamellipodia but not filopodia or microvilli formation. Conversely, coexpression of a dominant negative Cdc42Hs only blocks microvilli and filopodia, but not membrane ruffling and lamellipodia. Microtubule depolymerization upon nocodazole treatment leads to a loss of RhoG protein from the cell periphery associated with a reversal of the RhoG phenotype, whereas PDGF or bradykinin stimulation of nocodazole-treated cells could still promote Rac1- and Cdc42Hs-dependent cytoskeletal reorganization. Therefore, our data demonstrate that RhoG controls a pathway that requires the microtubule network and activates Rac1 and Cdc42Hs independently of their growth factor signaling pathways.