Emmanuel Vignal

Characterization of TCL, a New GTPase of the Rho Family related to TC10 and Cdc42

GTPases of the Rho family control a wide variety of cellular processes such as cell morphology, motility, proliferation, differentiation, and apoptosis. We report here the characterization of a new Rho member, which shares 85% and 78% amino acid similarity to TC10 and Cdc42, respectively. This GTPase, termed as TC10-like (TCL) is encoded by an unexpectedly large locus, made of five exons spanning over 85 kilobases on human chromosome 14. TCL mRNA is 2.5 kilobases long and is mainly expressed in heart. In vitro, TCL shows rapid GDP/GTP exchange and displays higher GTP dissociation and hydolysis rates than TC10. Using the yeast two-hybrid system and GST pull-down assays, we show that GTP-bound but not GDP-bound TCL protein directly interacts with Cdc42/Rac interacting binding domains, such as those found in PAK and WASP. Despite its overall similarity to TC10 and Cdc42, the constitutively active TCL mutant displays distinct morphogenic activity in REF-52 fibroblasts, producing large and dynamic F-actin-rich ruffles on the dorsal cell membrane. Interestingly, TCL morphogenic activity is blocked by dominant negative Rac1 and Cdc42 mutants, suggesting a cross-talk between these three Rho GTPases.

A Cell Active Chemical GEF Inhibitor Selectively Targets the Trio/RhoG/Rac1 Signaling Pathway

RhoGEFs (guanine nucleotide exchange factors of the Rho GTPase family) are upstream regulators of cell adhesion and migration pathways, thus representing attractive yet relatively unexplored targets for the development of anti-invasive drugs. We screened for chemical inhibitors of TrioN, the N-terminal GEF domain of the multidomain Trio protein, and identified ITX3 as a nontoxic inhibitor. In transfected mammalian cells, ITX3 blocked TrioN-mediated dorsal membrane ruffling and Rac1 activation while having no effect on GEF337-, Tiam1-, or Vav2-mediated RhoA or Rac1 activation. ITX3 specifically inhibited endogenous TrioN activity, as evidenced by its ability to inhibit neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells or C2C12 differentiation into myotubes. This study introduces a selective cell active inhibitor of the Trio/RhoG/Rac1 pathway and validates RhoGEFs as druggable targets.

MBNL1 and RBFOX2 cooperate to establish a splicing programme involved in pluripotent stem cell differentiation

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) has provided huge insight into the pathways, mechanisms and transcription factors that control differentiation. Here we use high-throughput RT-PCR technology to take a snapshot of splicing changes in the full spectrum of high- and low-expressed genes during induction of fibroblasts, from several donors, into iPSCs and their subsequent redifferentiation. We uncover a programme of concerted alternative splicing changes involved in late mesoderm differentiation and controlled by key splicing regulators MBNL1 and RBFOX2. These critical splicing adjustments arise early in vertebrate evolution and remain fixed in at least 10 genes (including PLOD2, CLSTN1, ATP2A1, PALM, ITGA6, KIF13A, FMNL3, PPIP5K1, MARK2 and FNIP1), implying that vertebrates require alternative splicing to fully implement the instructions of transcriptional control networks.

Cdc42Hs and Rac1 GTPases induce the collapse of the vimentin intermediate filament network.

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.

Activity of the RhoU/Wrch1 GTPase is critical for cranial neural crest cell migration.

The neural crest (NC) is a stem cell-like population that arises at the border of neural and non-neural ectoderm. During development, NC undergoes an epithelio-mesenchymal transition (EMT), i.e. loss of epithelial junctions and acquisition of pro-migratory properties, invades the entire embryo and differentiates into a wide diversity of terminal tissues. We have studied the implication of Rho pathways in NC development and previously showed that RhoV is required for cranial neural crest (CNC) cell specification. We show here that the non-canonical Wnt response rhoU/wrch1 gene, closely related to rhoV, is also expressed in CNC cells but at later stages. Using both gain- and loss-of-function experiments, we demonstrate that the level of RhoU expression is critical for CNC cell migration and subsequent differentiation into craniofacial cartilages. In in vitro cultures, RhoU activates pathways that cooperate with PAK1 and Rac1 in epithelial adhesion, cell spreading and directional cell migration. These data support the conclusion that RhoU is an essential regulator of CNC cell migration.

Signalling pathways controlled by the GTPase RhoG

The small Rho-related GTP binding proteins of the Ras superfamily are clustered in two distinct subgoups: The Rac/Cdc42 subgroup (Rat (l-3), RhoG, Cdc42, TClO, chp, and TTF/RhoH) and the Rho subgroup(Rho(A-C), RhoD, RhoL, and Rnd(l-3)). Rho proteins control multiple biochemical pathways leading to cytoskeletal rearrangements, kinase activation and gene transcription, implicated in physiological processes such as cell proliferation, motility or endo/exocytosis. Like other Ras-like proteins, most Rho GTPases are bound to GDP and inactive in resting cells. Cell stimulation with extracellular ligands triggers the exchange of GDP for GTP which makes the GTPase competent to bind its downstream effecters. Inactivation of the GTPase is achieved by GTP hydrolysis into GDP. The oscillation between active and inactive states is controlled by GTPase-activating proteins (GAPS), which enhance the intrinsic GTF’ase activity, and by guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTE’. Rhospecific GEFs form a growing family of more than 30 related proteins, which share a region of about 200 residues termed Dbl-Homology (DH) domain, responsible for their nucleotide exchange activity in vitro. A particular class of these Rho regulators is represented by the multidomain Trio protein, which contains two GEF domains of distinct specificities. GEF proteins are key regulators of cell growth control since deregulated expression of many Dblrelated proteins has been reported to efficiently promote cell transformation and invasion. Despite their biological role, the specificity of RhoGEF activity with respect to Rho proteins remains largely unknown. Most studies on Rho proteins have focused on the role of RhoA, Racl and Cdc42Hs in the control of the cytoskeleton, and have established that these GTPases regulate the formation of polymerized F-actin containing structures: membrane ruffles and lamellipodia, controlled by Rat, membrane microvilli and 551