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International audience; Epithelial tubes perform functions that are essential for the survival of multicellular organisms. Understanding how their polarised features are maintained is therefore crucial. By analysing the function of the clathrin adaptor AP-1 in the C. elegans intestine, we found that AP-1 is required for epithelial polarity maintenance. Depletion of AP-1 subunits does not affect epithelial polarity establishment or the formation of the intestinal lumen. However, the loss of AP-1 affects the polarised distribution of both apical and basolateral transmembrane proteins. Moreover, it triggers de novo formation of ectopic apical lumens between intestinal cells along the lateral membranes later during embryogenesis. We also found that AP-1 is specifically required for the apical localisation of the small GTPase CDC-42 and the polarity determinant PAR-6. Our results demonstrate that AP-1 controls an apical trafficking pathway required for the maintenance of epithelial polarity in vivo in a tubular epithelium.

Breaking the Barrier Being able to deliver drugs into the brain to treat degenerative diseases such as Alzheimer's or Parkinson's requires the ability to traverse the blood-brain barrier (BBB). Understanding the formation of the very specific adherent junctions (AJ) and tight junctions present at the BBB cell junctions is a prerequisite to the design of such therapeutics. However, diminishing the expression of any one component involved in the formation of these intercellular junctions destroys them. Coureuil et al. (p. 83 , published online 11 June) exploited the specific recruitment of AJ proteins by Neisseria meningitidis to dissect this process. Adhesion of the bacteria to human brain endothelial cells recruited the polarity complex Par3/Par6/PKCζ required for the establishment of eukaryotic cell polarity and the formation of intercellular junctions. The bacterial recruitment of the polarity complex depleted junctional proteins at the cell-cell interface opening the intercellular junctions at the brainendothelial interface.

In the germinative zone of the adult rodent brain, neural progenitors migrate into niches delimited by astrocyte processes and differentiate into neuronal precursors. In the present study, we report a modulating role for the scaffolding protein IQGAP1 in neural progenitor migration. We have identified IQGAP1 as a new marker of amplifying neural progenitor and neuronal precursor cells of the subventricular zone (SVZ) and the rostral migratory stream (RMS) in the adult mouse brain. To determine functions for IQGAP1 in neural progenitors, we compared the properties of neural progenitor cells from wild-type andIqgap1-null mutant micein vivoandin vitro. Thein vivostudies reveal a delay in the transition ofde novoneural progenitors into neuronal precursor cells inIqgap1-null mice.In vitro, we demonstrated that IQGAP1 acts as a downstream effector in the vascular endothelial growth factor (VEGF)-dependent migratory response of neural progenitors that also impacts on their neuronal differentiation. The Rho-family GTPases cdc42/Rac1 and Lis1 are major partners of IQGAP1 in this migratory process. Finally, astrocytes of the neurogenic SVZ and RMS are shown to express VEGF. We propose that VEGF synthesized by astrocytes could be involved in the guidance of neural progenitors to neurogenic niches and that IQGAP1 is an effector of the VEGF-dependent migratory signal.

International audience; Most macrophages remain uninfected in HIV-1-infected patients. Nevertheless, the phagocytic capacity of phagocytes from these patients is impaired, favouring the multiplication of opportunistic pathogens. The basis for this phagocytic defect is not known. HIV-1 Tat protein is efficiently secreted by infected cells. Secreted Tat can enter uninfected cells and reach their cytosol. Here we found that extracellular Tat, at the subnanomolar concentration present in the sera of HIV-1-infected patients, inhibits the phagocytosis of Mycobacterium avium or opsonized Toxoplasma gondii by human primary macrophages. This inhibition results from a defect in mannose- and Fcγ-receptor-mediated phagocytosis, respectively. Inhibition relies on the interaction of Tat with phosphatidylinositol (4,5)bisphosphate that interferes with the recruitment of Cdc42 to the phagocytic cup, thereby preventing Cdc42 activation and pseudopod elongation. Tat also inhibits FcγR-mediated phagocytosis in neutrophils and monocytes. This study provides a molecular basis for the phagocytic defects observed in uninfected phagocytes following HIV-1 infection.

International audience; Rho GTPases regulate the morphology of cells stimulated by extracellular ligands. Their activation is controlled by guanine exchange factors (GEF) that catalyze their binding to GTP. The multidomain Trio protein represents an emerging class of &Rgr; regulators that contain two GEF domains of distinct specificities. We report here the characterization of Rho signaling pathways activated by the N-terminal GEF domain of Trio (TrioD1). In fibroblasts, TrioD1 triggers the formation of particular cell structures, similar to those elicited by RhoG, a GTPase known to activate both Rac1 and Cdc42Hs. In addition, the activity of TrioD1 requires the microtubule network and relocalizes RhoG at the active sites of the plasma membrane. Using a classical in vitro exchange assay, TrioD1 displays a higher GEF activity on RhoG than on Rac1. In fibroblasts, expression of dominant negative RhoG mutants totally abolished TrioD1 signaling, whereas dominant negative Rac1 and Cdc42Hs only led to partial and complementary inhibitions. Finally, expression of a Rho Binding Domain that specifically binds RhoG(GTP) led to the complete abolition of TrioD1 signaling, which strongly supports Rac1 not being activated by TrioD1 in vivo. These data demonstrate that Trio controls a signaling cascade that activates RhoG, which in turn activates Rac1 and Cdc42Hs.

International audience; 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.

International audience; Fibronectin (FN) fibrillogenesis is an essential biological process mediated by alpha5beta1 integrin and cellular contractile forces. Assembly of a FN matrix by activated endothelial cells occurs during angiogenic blood vessel remodeling and signaling components that control this event represent attractive therapeutic targets. Here we examined the role of individual Rho GTPases in FN matrix remodeling by selectively attenuating their expression in cultured endothelial cells. Whereas pharmacological ablation of myosin-regulated contractility abrogated matrix assembly, no significant decrease was detected in the amount of FN deposited by RhoA, RhoB-, RhoC-, Rac1-, or Cdc42-depleted cells. Rather, distinct differences in fiber arrangement were observed. Most strikingly, RhoA silenced cells assembled a fine FN meshwork beneath alpha5beta1 integrin-based fibrillar adhesions, in the absence of classical focal adhesions and actin stress fibers, indicating that alpha5beta1 integrin translocation and FN fibril elongation can occur in low tension states such as those encountered by newly-forming vessels in tissue. In contrast, highly contractile Cdc42-deficient cells deposited FN globules and Rac-deficient cells assembled long arrays, reflecting their increased motility. We propose that regulation of FN scaffolds by Rho GTPase signaling impacts bidirectional communications and mechanical interactions between endothelial cells and their extracellular matrix during vascular morphogenesis.

International audience; Pathogen recognition and engulfment by phagocytic cells of the blood cell lineage constitute the first line of defense against invading pathogens. This cellular immune response is conserved throughout evolution and depends strictly on cytoskeletal changes regulated by the RhoGTPases family. Many pathogens have developed toxins modifying RhoGTPases activity to their own benefit. In particular, the Exoenzyme S (ExoS) toxin of the Gram-negative bacteria Pseudomonas aeruginosa is directly injected into the host cell cytoplasm and contains a GAP domain (ExoSGAP) targeting RhoGTPases. Searching for the contribution of each RhoGTPases, Rho1, Rac1, Rac2, Mtl (Mig2-like) and Cdc42 to fly resistance to P. aeruginosa infections, we found that Rac2 is required to resist to P. aeruginosa and to other Gram-negative or Gram-positive bacteria. The Rac2 immune-deficient phenotype is attributable to defective engulfment of pathogens since Rac2-mutant macrophages exhibited strong reduction in the phagocytosis level of both Gram-negative and Gram-positive bacterial particles whereas systemic immune signaling pathways, including Toll, Immune deficiency and Jun kinases, were not affected. Co-expression of Rac2 and ExoSGAP rescued the increased sensitivity to P. aeruginosa observed in ExoSGAP-expressing flies suggesting that Rac2 is the main host factor whose function is inhibited by the GAP domain of the ExoS toxin.

Our aim was to shed light on different steps leading from metabotropic receptor activation to changes in cell shape, such as those that characterize the morphological plasticity of neurohypophysial astrocytes (pituicytes). Using explant cultures of adult rat pituicytes, we have previously established that adenosine A1 receptor activation induces stellation via inhibition of RhoA monomeric GTPase and subsequent disruption of actin stress fibers. Here, we rule out RhoA phosphorylation as a mechanism for that inhibition. Rather, our results are more consistent with involvement of a GTPase-activating protein (GAP). siRNA and pull-down experiments suggest that a step downstream of RhoA might involve Cdc42, another GTPase of the Rho family. However, RhoA activation, e.g., in the presence of serum, induces stress fibers, whereas direct Cdc42 activation appears to confine actin within a submembrane - i.e., cortical - network, which also prevents stellation. Therefore, we propose that RhoA may activate Cdc42 in parallel with an effector, such as p160Rho-kinase, that induces and maintains actin stress fibers in a dominant fashion. Rac1 is not involved in the stellation process per se but appears to induce a dendritogenic effect. Ultimately, it may be stated that pituicyte stellation is inducible upon mere actin depolymerization, and preventable upon actin organization, be it in the form of stress fibers or in a cortical configuration.

International audience; Cytoskeletal rearrangements are central to endothelial cell physiology and are controlled by soluble factors, matrix proteins, cell-cell interactions, and mechanical forces. We previously reported that aortic endothelial cells can rearrange their cytoskeletons into complex actin-based structures called podosomes when a constitutively active mutant of Cdc42 is expressed. We now report that transforming growth factor beta (TGF-beta) promotes podosome formation in primary aortic endothelial cells. TGF-beta-induced podosomes assembled together into large ring- or crescent-shaped structures. Their formation was dependent on protein synthesis and required functional Src, phosphatidylinositide 3-kinase, Cdc42, RhoA, and Smad signaling. MT1-MMP and metalloprotease 9 (MMP9), both upregulated by TGF-beta, were detected at sites of podosome formation, and MT1-MMP was found to be involved in the local degradation of extracellular matrix proteins beneath the podosomes and required for the invasion of collagen gels by endothelial cells. We propose that TGF-beta plays an important role in endothelial cell physiology by inducing the formation of podosomal structures endowed with metalloprotease activity that may contribute to arterial remodeling.

We investigated the role of the highly conserved G protein Rac1 in the opportunistic pathogen Candida albicans . We identified and disrupted RAC1 and show here that, in contrast to CDC42 , it is not necessary for viability or serum-induced hyphal growth but is essential for filamentous growth when cells are embedded in a matrix. Rac1 is localized to the plasma membrane, yet its distribution is more homogenous than that of Cdc42, with no enrichment at the tips of either buds or hyphae. In addition, fluorescence recovery after photobleaching results indicate that Rac1 and Cdc42 have different dynamics at the membrane. Furthermore, overexpression of Rac1 does not complement Cdc42 function, and conversely, overexpression of Cdc42 does not complement Rac1 function. Thus, Rac1 and Cdc42, although highly similar to one another, have different roles in C. albicans development.

The CNF1 toxin is produced by uropathogenic and meningitis-causing Escherichia coli. CNF1 catalyzes the constitutive activation of Rho proteins by deamidation. The threshold of activation of Rho proteins by CNF1 is, however, attenuated because of a concomitant decrease of their cellular levels. Depletion of activated-Rac1 is catalyzed by ubiquitin-mediated proteasomal degradation. Consequently, we show by effector-binding pull-down that co-treatment of intoxicated cells with the MG132 proteasome-inhibitor results in a higher level of activation of Rac, as well as RhoA and Cdc42. We show that CNF1 induces the transient recruitment of Rho proteins to cellular membranes. Interestingly, at the difference of Rac and Cdc42, the inhibition of the proteasome during CNF1 treatment does not result in a significant accumulation of RhoA to cellular membranes. Using an in vivo ubiquitylation assay, we evidence that mutation of the geranylgeranyl acceptor cysteine of Rac1 (Rac1C189G) abolished the sensitivity of permanently activated-Rac1 to ubiquitylation, whereas Rac1C189G remained able to bind to the effector-binding domain of p21-PAK. Collectively, these results indicate that association with the cellular membranes is a necessary step for activated-Rac1 ubiquitylation.

The Rho G protein Cdc42 and its exchange factor Cdc24 are required for hyphal growth of the human fungal pathogen Candida albicans . Previously, we reported that strains ectopically expressing Cdc24 or Cdc42 are unable to form hyphae in response to serum. Here we investigated the role of these two proteins in hyphal growth, using quantitative real-time PCR to measure induction of hypha-specific genes together with time lapse microscopy. Expression of the hypha-specific genes examined depends on the cyclic AMP-dependent protein kinase A pathway culminating in the Efg1 and Tec1 transcription factors. We show that strains with reduced levels of CDC24 or CDC42 transcripts induce hypha-specific genes yet cannot maintain their expression in response to serum. Furthermore, in serum these mutants form elongated buds compared to the wild type and mutant budding cells, as observed by time lapse microscopy. Using Cdc24 fused to green fluorescent protein, we also show that Cdc24 is recruited to and persists at the germ tube tip during hyphal growth. Altogether these data demonstrate that the Cdc24/Cdc42 GTPase module is required for maintenance of hyphal growth. In addition, overexpression studies indicate that specific levels of Cdc24 and Cdc42 are important for invasive hyphal growth. In response to serum, CDC24 transcript levels increase transiently in a Tec1-dependent fashion, as do the G-protein RHO3 and the Rho1 GTPase activating protein BEM2 transcript levels. These results suggest that a positive feedback loop between Cdc24 and Tec1 contributes to an increase in active Cdc42 at the tip of the germ tube which is important for hypha formation.

Haematopoietic cell kinase (Hck) is a protein tyrosine kinase of the Src family specifically expressed in phagocytes as two isoforms, p59Hck and p61Hck, present at the plasma membrane and lysosomes, respectively. We report that ectopic expression of a constitutively active mutant of p61Hck (p61Hck(ca)) triggered the de novo formation of actin-rich rings at the ventral face of the cells that we characterized as bona fide podosome rosettes, structures involved in cell migration. Their formation required the adaptor domains and the kinase activity of p61Hck, the integrity of microfilament and microtubule networks and concerted action of Cdc42, Rac and Rho. Podosome rosette formation was either abolished when p61Hck(ca) was readdressed from lysosomes to the cytosol or triggered when p59Hck(ca) was relocalized to lysosomes. Lysosomal markers were present at podosome rosettes. By stimulating exocytosis of p61Hck(ca) lysosomes with a calcium ionophore, the formation of podosome rosettes was enhanced. Interestingly, we confirm that, in human macrophages, Hck and lysosomal markers were present at podosomes which were spatially reorganized as clusters, a foregoing step to form rosettes, upon expression of p61Hck(ca). We propose that lysosomes, under the control of p61Hck, are involved in the biogenesis of podosomes, a key phenomenon in the migration of phagocytes.

During Saccharomyces cerevisiae mating, chemotropic growth and cell fusion are critical for zygote formation. Cdc24p, the guanine nucleotide exchange factor for the Cdc42 G protein, is necessary for oriented growth along a pheromone gradient during mating. To understand the functions of this critical Cdc42p activator, we identified additional cdc24 mating mutants. Two mating-specific mutants, the cdc24-m5 and cdc24-m6 mutants, each were isolated with a mutated residue in the conserved catalytic domain. The cdc24-m6 mutant responds normally to pheromone and orients its growth towards a mating partner yet accumulates prezygotes during mating. cdc24-m6 prezygotes have two apposed intact cell walls and do not correctly localize proteins required for cell fusion, despite normal exocytosis. Our results indicate that the exchange factor Cdc24p is necessary for maintaining or restricting specific proteins required for cell fusion to the cell contact region during mating.

In epithelial cells, endocytic activity is mostly dedicated to nutrient and macromolecule uptake. To invade these cells, Chlamydiaceae, like other pathogens, have evolved strategies that utilise the existing endocytic machineries and signalling pathways, but little is known about the host cell molecules involved. In this report, we show that within five minutes of infection of HeLa cells by Chlamydia caviae GPIC strain several events take place in the immediate vicinity of invasive bacteria: GM1-containing microdomains cluster, tyrosine-phosphorylated proteins accumulate, and intense actin polymerization occurs. We show that actin polymerization is controlled by the small GTPases Cdc42 and Rac, which become activated upon infection. Expression of dominant negative forms of these GTPases inhibits C. caviae entry and leads to abnormal actin polymerization. In contrast, the small GTPase Rho does not seem essential for bacterial entry. Finally, phosphatidylinositol 3-kinase activity is also required for internalization of C. caviae, probably downstream of the other molecular events reported here. We present the first scheme of the events occurring at the sites of invasion of epithelial cells by a member of the Chlamydiaceae family.

Contains fulltext : 58052.pdf (Publisher’s version ) (Closed access) Tissue invasion by tumor cells involves their migration across basement membranes through activation of extracellular matrix degradation and cell motility mechanisms. Chemokines binding to their receptors provide chemotactic cues guiding cells to specific tissues and organs; they therefore could potentially participate in tumor cell dissemination. Melanoma cells express CXCR4, the receptor for the chemokine stromal cell-derived factor-1alpha (SDF-1alpha). Using Matrigel as a model, we show that SDF-1alpha promotes invasion of melanoma cells across basement membranes. Stimulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) activity by SDF-1alpha was necessary for invasion, involving at least up-regulation in the expression of this metalloproteinase, as detected in the highly metastatic BLM melanoma cell line. Moreover, SDF-1alpha triggered the activation of the GTPases RhoA, Rac1, and Cdc42 on BLM cells, and expression of dominant-negative forms of RhoA and Rac1, but not Cdc42, substantially impaired the invasion of transfectants in response to SDF-1alpha, as well as the increase in MT1-MMP expression. Furthermore, CXCR4 expression on melanoma cells was notably augmented by transforming growth factor-beta1, a Matrigel component, whereas anti-transforming growth factor-beta antibodies inhibited increases in CXCR4 expression and melanoma cell invasion toward SDF-1alpha. The identification of SDF-1alpha as a potential stimulatory molecule for MT1-MMP as well as for RhoA and Rac1 activities during melanoma cell invasion, associated with an up-regulation in CXCR4 expression by interaction with basement membrane factors, could contribute to better knowledge of mechanisms stimulating melanoma cell dissemination.

International audience; In human neutrophils, beta2 integrin engagement mediated a decrease in GTP-bound Rac1 and Rac2. Pretreatment of neutrophils with LY294002 or PP1 (inhibiting phosphatidylinositol 3-kinase (PI 3-kinase) and Src kinases, respectively) partly reversed the beta2 integrin-induced down-regulation of Rac activities. In contrast, beta2 integrins induced stimulation of Cdc42 that was independent of Src family members. The PI 3-kinase dependence of the beta2 integrin-mediated decrease in GTP-bound Rac could be explained by an enhanced Rac-GAP activity, since this activity was blocked by LY204002, whereas PP1 only had a minor effect. The fact that only Rac1 but not Rac2 (the dominating Rac) redistributed to the detergent-insoluble fraction and that it was independent of GTP loading excludes the possibility that down-regulation of Rac activities was due to depletion of GTP-bound Rac from the detergent-soluble fraction. The beta2 integrin-triggered relocalization of Rac1 to the cytoskeleton was enabled by a PI 3-kinase-induced dissociation of Rac1 from LyGDI. The dissociations of Rac1 and Rac2 from LyGDI also explained the PI 3-kinase-dependent translocations of Rac GTPases to the plasma membrane. However, these accumulations of Rac in the membrane, as well as that of p47phox and p67phox, were also regulated by Src tyrosine kinases. Inasmuch as Rac GTPases are part of the NADPH oxidase and the respiratory burst is elicited in neutrophils adherent by beta2 integrins, our results indicate that activation of the NADPH oxidase does not depend on the levels of Rac-GTP but instead requires a beta2 integrin-induced targeting of the Rac GTPases as well as p47phox and p67phox to the plasma membrane.

International audience; Interactions between the Eph receptor tyrosine kinase and ephrin ligands transduce short-range signals regulating axon pathfinding, development of the cardiovascular system, as well as migration and spreading of neuronal and non-neuronal cells. Some of these effects are believed to be mediated by alterations in actin dynamics. The members of the small Rho GTPase family elicit various effects on actin structures and are probably involved in Eph receptor-induced actin modulation. EphrinA1 is proposed to contribute to angiogenesis as it is strongly expressed at sites of neovascularization. Moreover, angiogenic factors induce the expression of ephrinA1 in endothelial cells. In this study, using rat vascular smooth muscle cells (VSMCs), we investigated the contribution of the small Rho GTPases in ephrinA1-induced integrin inactivation. EphrinA1 did not significantly affect early adhesion of VSMCs on purified laminin or fibronectin, but strongly impaired cell spreading. The Rho kinase inhibitor Y-27632 partly reversed the ephrinA1 effect, suggesting involvement of Rho in this model. However, inhibition of RhoA synthesis with short interfering (si)RNA had a modest effect, suggesting that RhoA plays a limited role in ephrinA1-mediated inhibition of spreading in VSMCs. The ephrinA1-mediated morphological alterations correlated with inhibition of Rac1 and p21-activated kinase 1 (PAK1) activity, and were antagonized by the expression of a constitutively active Rac mutant. Moreover, repression of Rac1 synthesis with siRNA amplifies the ephrinA1-induced inhibition of spreading. Finally, sphingosine-1-phosphate (S1P), a lipid mediator known to inhibit Rac activation in VSMCs amplifies the ephrinA1 effect. In conclusion, our results emphasize the role of the Rac/PAK pathway in ephrinA1-mediated inhibition of spreading. In this way, ephrinA1, alone or in synergy with S1P, can participate in blood vessel destabilization, a prerequisite for angiogenesis.

International audience; Entamoeba histolytica migration is essential for the development of amoebiasis, a human disease characterised by invasion and destruction of tissues. Amoebic motility requires both polarisation of the cell and formation of a predominant pseudopod. As p21-activated kinases PAKs are known to regulate eukaryotic cell motility and morphology, we investigated the role of PAK in E. histolytica. We showed that the C-terminal domain of EhPAK comprised a constitutive kinase activity in vitro and that overproduction of this fragment, in E. histolytica, caused a significant reduction in amoeboid migration, as measured by dynamic image analysis, indicating an involvement of EhPAK in this process. A dramatic loss of polarity, as indicated by the increased number of membrane extensions all around E. histolytica, was also observed, suggesting that the N-terminal domain of EhPAK was necessary for maintenance of cell polarity. To support this view, we showed that despite the absence of the consensus motif to bind to Rac and Cdc42, the N-terminal domain of EhPAK bound to Rac1, suggesting that the N-terminal region was a regulatory domain. In addition, we also found an increased rate of human red blood cell phagocytosis, suggesting for the first time an active role for a PAK protein in this process. Taking together, the results suggest strongly that EhPAK is a key regulatory element in polarity, motility and phagocytosis of E. histolytica.

International audience; Signals from the extracellular matrix are essential for the survival of many cell types. Dominant-negative mutants of two members of Rho family GTPases, Rac1 and Cdc42, mimic the loss of anchorage in primary mouse fibroblasts and are potent inducers of apoptosis. This pathway of cell death requires the activation of both the p53 tumor suppressor and the extracellular signal-regulated mitogen-activated protein kinases (Erks). Here we characterize the proapoptotic Erk signal and show that it differs from the classically observed survival-promoting one by the intensity of the kinase activation. The disappearance of the GTP-bound forms of Rac1 and Cdc42 gives rise to proapoptotic, moderate activation of the Raf-MEK-Erk cascade via a signaling pathway involving the kinases phosphatidlyinositol 3-kinase and Akt. Moreover, concomitant activation of p53 and inhibition of Akt are both necessary and sufficient to signal anoikis in primary fibroblasts. Our data demonstrate that the GTPases of the Rho family control three major components of cellular signal transduction, namely, p53, Akt, and Erks, which collaborate in the induction of apoptosis due to the loss of anchorage.

International audience; The Rho family of GTP-binding proteins plays a critical role in a variety of cellular processes, including cytoskeletal reorganization and activation of kinases such as p38 and C-jun N-terminal kinase (JNK) MAPKs. We report here that dominant negative forms of Rac1 and Cdc42Hs inhibit the expression of the muscle-specific genes myogenin, troponin T, and myosin heavy chain in L6 and C2 myoblasts. Such inhibition correlates with decreased p38 activity. Active RhoA, RhoG, Rac1, and Cdc42Hs also prevent myoblast-to-myotube transition but affect distinct stages: RhoG, Rac1, and Cdc42Hs inhibit the expression of all muscle-specific genes analyzed, whereas active RhoA potentiates their expression but prevents the myoblast fusion process. We further show by two different approaches that the inhibitory effects of active Rac1 and Cdc42Hs are independent of their morphogenic activities. Rather, myogenesis inhibition is mediated by the JNK pathway, which also leads to a cytoplasmic redistribution of Myf5. We propose that although Rho proteins are required for the commitment of myogenesis, they differentially influence this process, positively for RhoA and Rac1/Cdc42Hs through the activation of the SRF and p38 pathways, respectively, and negatively for Rac1/Cdc42Hs through the activation of the JNK pathway.

International audience; BACKGROUND: Ras-mediated transformation of mammalian cells has been shown to activate multiple signalling pathways, including those involving mitogen-activated protein kinases and the small GTPase Rho. Members of the Rho family affect cell morphology by controlling the formation of actin-dependent structures: specifically, filopodia are induced by Cdc42Hs, lamellipodia and ruffles by Rac, and stress fibers by RhoA. In addition, Rho GTPases are involved in progression through the G1 phase of the cell cycle, and Rac1 and RhoA have recently been directly implicated in the morphogenic and mitogenic responses to transformation by oncogenic Ras. In order to examine the cross-talk between Ras and Rho proteins, we investigated the effects on focus-forming activity and cell growth of the Rho-family members Cdc42Hs, Rac1 and RhoG by expressing constitutively active or dominant-negative forms in NIH3T3 cells. RESULTS: Expression of Rac1 or RhoG modulated the saturation density to which the cells grew, probably by affecting the level of contact inhibition. Although all three GTPases were required for cell transformation mediated by Ras but not by constitutively active Raf, the selective activation of each GTPase was not sufficient to induce the formation of foci. The coordinated activation of Cdc42Hs, RhoG and Rac1, however, elicited a high focus-forming activity, independent of the mitogen-activated ERK and JNK protein kinase pathways. CONCLUSIONS: Ras-mediated transformation induces extensive changes in cell morphology which require the activity of members of the Rho family of GTPases. Our data show that the pattern of coordinated Rho family activation that elicits a focus-forming activity in NIH3T3 cells is distinct from the regulatory cascade that has been proposed for the control of actin-dependent structures in Swiss 3T3 cells.

Rho-GTPases control a wide range of physiological processes by regulating actin cytoskeleton dynamics [1]. Numerous studies on neuronal cell lines have established that Rac, Cdc42, and RhoG activate neurite extension, while RhoA mediates neurite retraction [2–5]. Guanine nucleotide exchange factors (GEFs) activate Rho-GTPases by accelerating GDP/GTP exchange [6]. Trio displays two Rho-GEF domains, GEFD1, activating the Rac pathway via RhoG, and GEFD2, acting on RhoA, and contains numerous signaling motifs whose contribution to Trio function has not yet been investigated [7–9]. Genetic analyses in Drosophila and in Caenorhabditis elegans indicate that Trio is involved in axon guidance and cell motility via a GEFD1-dependent process, suggesting that the activity of its Rho-GEFs is strictly regulated [10–14]. Here, we show that human Trio induces neurite outgrowth in PC12 cells in a GEFD1-dependent manner. Interestingly, the spectrin repeats and the SH3-1 domain of Trio are essential for GEFD1-mediated neurite outgrowth, revealing an unexpected role for these motifs in Trio function. Moreover, we demonstrate that Trio-induced neurite outgrowth is mediated by the GEFD1-dependent activation of RhoG, previously shown to be part of the NGF (nerve growth factor) pathway [4]. The expression of different Trio mutants interferes with NGF-induced neurite outgrowth, suggesting that Trio may be an upstream regulator of RhoG in this pathway. In addition, we show that Trio protein accumulates under NGF stimulation. Thus, Trio is the first identified Rho-GEF involved in the NGF-differentiation signaling.

International audience; We have recently reported that two Rho family GTPases, Rac1 and Cdc42, are intimately involved in the control of cell survival of murine fibroblasts linked to adherence to the extracellular matrix. Inhibition of either Rac1 or Cdc42 signaling in adherent cells mimics the loss of anchorage and efficiently induces apoptosis in both immortalized and primary cells. In both cases cell death is dependent on the wild-type p53 tumor suppressor and is accompanied by activation of endogenous p53. Here, we describe that the inhibition of Rac1 or Cdc42 signaling leads to MAPK ERK activation via a pathway involving PI(3)K, Akt, Raf, and MEK, but not Ras. The moderate level of ERK activation that accompanies anoikis is an essential component of proapoptotic signaling; whereas sustained, high-intensity ERK signaling promotes survival in the same experimental system.