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1.  FilGAP, a Rho/Rho-associated protein kinase–regulated GTPase-activating protein for Rac, controls tumor cell migration 
Molecular Biology of the Cell  2012;23(24):4739-4750.
In three-dimensional matrices, tumor cells can move with either an elongated/mesenchymal or a round/amoeboid morphology. This study identifies FilGAP, a Rac GTPase-activating protein (GAP), as a mediator of Rho/Rho-associated protein kinase–dependent amoeboid movement of carcinoma cells. FilGAP may contribute to tumor invasion and metastasis by controlling the amoeboid mode of movement.
Tumor cells exhibit two interconvertible modes of cell motility referred to as mesenchymal and amoeboid migration. Mesenchymal mode is characterized by elongated morphology that requires high GTPase Rac activation, whereas amoeboid mode is dependent on actomyosin contractility induced by Rho/Rho-associated protein kinase (ROCK) signaling. While elongated morphology is driven by Rac-induced protrusion at the leading edge, how Rho/ROCK signaling controls amoeboid movement is not well understood. We identified FilGAP, a Rac GTPase-activating protein (GAP), as a mediator of Rho/ROCK-dependent amoeboid movement of carcinoma cells. We show that depletion of endogenous FilGAP in carcinoma cells induced highly elongated mesenchymal morphology. Conversely, forced expression of FilGAP induced a round/amoeboid morphology that requires Rho/ROCK-dependent phosphorylation of FilGAP. Moreover, depletion of FilGAP impaired breast cancer cell invasion through extracellular matrices and reduced tumor cell extravasation in vivo. Thus phosphorylation of FilGAP by ROCK appears to promote amoeboid morphology of carcinoma cells, and FilGAP contributes to tumor invasion.
doi:10.1091/mbc.E12-04-0310
PMCID: PMC3521682  PMID: 23097497
2.  Rac and Rho GTPases in cancer cell motility control 
Rho GTPases represent a family of small GTP-binding proteins involved in cell cytoskeleton organization, migration, transcription, and proliferation. A common theme of these processes is a dynamic reorganization of actin cytoskeleton which has now emerged as a major switch control mainly carried out by Rho and Rac GTPase subfamilies, playing an acknowledged role in adaptation of cell motility to the microenvironment. Cells exhibit three distinct modes of migration when invading the 3 D environment. Collective motility leads to movement of cohorts of cells which maintain the adherens junctions and move by photolytic degradation of matrix barriers. Single cell mesenchymal-type movement is characterized by an elongated cellular shape and again requires extracellular proteolysis and integrin engagement. In addition it depends on Rac1-mediated cell polarization and lamellipodia formation. Conversely, in amoeboid movement cells have a rounded morphology, the movement is independent from proteases but requires high Rho GTPase to drive elevated levels of actomyosin contractility. These two modes of cell movement are interconvertible and several moving cells, including tumor cells, show an high degree of plasticity in motility styles shifting ad hoc between mesenchymal or amoeboid movements. This review will focus on the role of Rac and Rho small GTPases in cell motility and in the complex relationship driving the reciprocal control between Rac and Rho granting for the opportunistic motile behaviour of aggressive cancer cells. In addition we analyse the role of these GTPases in cancer progression and metastatic dissemination.
doi:10.1186/1478-811X-8-23
PMCID: PMC2941746  PMID: 20822528
3.  Shear stress–induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases 
The Journal of Cell Biology  2003;161(2):429-439.
Shear stress induces endothelial polarization and migration in the direction of flow accompanied by extensive remodeling of the actin cytoskeleton. The GTPases RhoA, Rac1, and Cdc42 are known to regulate cell shape changes through effects on the cytoskeleton and cell adhesion. We show here that all three GTPases become rapidly activated by shear stress, and that each is important for different aspects of the endothelial response. RhoA was activated within 5 min after stimulation with shear stress and led to cell rounding via Rho-kinase. Subsequently, the cells respread and elongated within the direction of shear stress as RhoA activity returned to baseline and Rac1 and Cdc42 reached peak activation. Cell elongation required Rac1 and Cdc42 but not phosphatidylinositide 3-kinases. Cdc42 and PI3Ks were not required to establish shear stress–induced polarity although they contributed to optimal migration speed. Instead, Rho and Rac1 regulated directionality of cell movement. Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement. Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow.
doi:10.1083/jcb.200210135
PMCID: PMC2172912  PMID: 12719476
Rho GTPases; actin cytoskeleton; Rho-kinase; migration; polarity
4.  Rho GTPases Control Polarity, Protrusion, and Adhesion during Cell Movement  
The Journal of Cell Biology  1999;144(6):1235-1244.
Cell movement is essential during embryogenesis to establish tissue patterns and to drive morphogenetic pathways and in the adult for tissue repair and to direct cells to sites of infection. Animal cells move by crawling and the driving force is derived primarily from the coordinated assembly and disassembly of actin filaments. The small GTPases, Rho, Rac, and Cdc42, regulate the organization of actin filaments and we have analyzed their contributions to the movement of primary embryo fibroblasts in an in vitro wound healing assay. Rac is essential for the protrusion of lamellipodia and for forward movement. Cdc42 is required to maintain cell polarity, which includes the localization of lamellipodial activity to the leading edge and the reorientation of the Golgi apparatus in the direction of movement. Rho is required to maintain cell adhesion during movement, but stress fibers and focal adhesions are not required. Finally, Ras regulates focal adhesion and stress fiber turnover and this is essential for cell movement. We conclude that the signal transduction pathways controlled by the four small GTPases, Rho, Rac, Cdc42, and Ras, cooperate to promote cell movement.
PMCID: PMC2150589  PMID: 10087266
Rho GTPases; Ras; polarity; focal adhesion; wound healing
5.  Cdc42 and Rac Family GTPases Regulate Mode and Speed but Not Direction of Primary Fibroblast Migration during Platelet-Derived Growth Factor-Dependent Chemotaxis ▿ †  
Molecular and Cellular Biology  2009;29(10):2730-2747.
Cdc42 and Rac family GTPases are important regulators of morphology, motility, and polarity in a variety of mammalian cell types. However, comprehensive analysis of their roles in the morphological and behavioral aspects of chemotaxis within a single experimental system is still lacking. Here we demonstrate using a direct viewing chemotaxis assay that of all of the Cdc42/Rac1-related GTPases expressed in primary fibroblasts, Cdc42, Rac1, and RhoG are required for efficient migration towards platelet-derived growth factor (PDGF). During migration, Cdc42-, Rac1-, and RhoG-deficient cells show aberrant morphology characterized as cell elongation and cell body rounding, loss of lamellipodia, and formation of thick membrane extensions, respectively. Analysis of individual cell trajectories reveals that cell speed is significantly reduced, as well as persistence, but to a smaller degree, while the directional response to the gradient of PDGF is not affected. Combined knockdown of Cdc42, Rac1, and RhoG results in greater inhibition of cell speed than when each protein is knocked down alone, but the cells are still capable of migrating toward PDGF. We conclude that, Cdc42, Rac1, and RhoG function cooperatively during cell migration and that, while each GTPase is implicated in the control of morphology and cell speed, these and other Cdc42/Rac-related GTPases are not essential for the directional response toward PDGF.
doi:10.1128/MCB.01285-08
PMCID: PMC2682035  PMID: 19273601
6.  Paxillin kinase linker (PKL) regulates Vav2 signaling during cell spreading and migration 
Molecular Biology of the Cell  2013;24(12):1882-1894.
Rho GTPases play an important role in cell migration. Here the activity of the Rho GTPase GEF Vav2 is shown to be regulated by a phosphorylation-dependent interaction with PKL (GIT2). PKL is required for Vav2 activation, and, in turn, Vav2 regulates the localization of PKL and β-PIX to focal adhesions and to the leading edge of migrating cells.
The Rho family of GTPases plays an important role in coordinating dynamic changes in the cell migration machinery after integrin engagement with the extracellular matrix. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs) and negatively regulated by GTPase-activating proteins (GAPs). However, the mechanisms by which GEFs and GAPs are spatially and temporally regulated are poorly understood. Here the activity of the proto-oncogene Vav2, a GEF for Rac1, RhoA, and Cdc42, is shown to be regulated by a phosphorylation-dependent interaction with the ArfGAP PKL (GIT2). PKL is required for Vav2 activation downstream of integrin engagement and epidermal growth factor (EGF) stimulation. In turn, Vav2 regulates the subsequent redistribution of PKL and the Rac1 GEF β-PIX to focal adhesions after EGF stimulation, suggesting a feedforward signaling loop that coordinates PKL-dependent Vav2 activation and PKL localization. Of interest, Vav2 is required for the efficient localization of PKL and β-PIX to the leading edge of migrating cells, and knockdown of Vav2 results in a decrease in directional persistence and polarization in migrating cells, suggesting a coordination between PKL/Vav2 signaling and PKL/β-PIX signaling during cell migration.
doi:10.1091/mbc.E12-09-0654
PMCID: PMC3681694  PMID: 23615439
7.  Rho GDP Dissociation Inhibitor-Mediated Disruption of Rho GTPase Activity Impairs Lens Fiber Cell Migration, Elongation and Survival 
Developmental biology  2008;315(1):217-231.
To explore the role of the Rho GTPases in lens morphogenesis, we overexpressed bovine Rho GDP dissociation inhibitor (RhoGDIα), which serves as a negative regulator of Rho, Rac and Cdc42 GTPase activity, in a lens-specific manner in transgenic mice. This was achieved using a chimeric promoter of δ-crystallin enhancer and αA-crystallin, which is active at embryonic day 12. Several individual transgenic (Tg) lines were obtained, and exhibited ocular specific phenotype comprised of microphthalmic eyes with lens opacity. The overexpression of bovine RhoGDIα disrupted membrane translocation of Rho, Rac and Cdc42 GTPases in Tg lenses. Transgenic lenses also revealed abnormalities in the migration pattern, elongation and organization of lens fibers. These changes appeared to be associated with impaired organization of the actin cytoskeleton and cell-cell adhesions. At E14.5, the size of the RhoGDIα Tg lenses was larger compared to wild type (WT) and the central lens epithelium and differentiating fibers exhibited an abnormal increase of bromo-deoxy-uridine incorporation. Postnatal Tg eyes, however, were much smaller in size compared to WT eyes, revealing increased apoptosis in the disrupted lens fibers. Taken together, these data demonstrate a critical role for Rho GTPase dependent signaling pathways in processes underlying morphogenesis, fiber cell migration, elongation and survival in the developing lens.
doi:10.1016/j.ydbio.2007.12.039
PMCID: PMC2364637  PMID: 18234179
Rho GTPases; RhoGDIα; Lens; Cytoskeleton; Elongation; Differentiation; Migration and Transgenic Mouse
8.  Constitutive p21-activated Kinase (PAK) Activation in Breast Cancer Cells as a Result of Mislocalization of PAK to Focal AdhesionsD⃞ 
Molecular Biology of the Cell  2004;15(6):2965-2977.
Cytoskeletal remodeling is critical for cell adhesion, spreading, and motility. p21-activated kinase (PAK), an effector molecule of the Rho GTPases Rac and Cdc42, has been implicated in cytoskeletal remodeling and cell motility. PAK kinase activity and subcellular distribution are tightly regulated by rapid and transient localized Rac and Cdc42 activation, and by interactions mediated by adapter proteins. Here, we show that endogenous PAK is constitutively activated in certain breast cancer cell lines and that this active PAK is mislocalized to atypical focal adhesions in the absence of high levels of activated Rho GTPases. PAK localization to focal adhesions in these cells is independent of PAK kinase activity, NCK binding, or GTPase binding, but requires the association of PAK with PIX. Disruption of the PAK–PIX interaction with competitive peptides displaces PAK from focal adhesions and results in a substantial reduction in PAK hyperactivity. Moreover, disruption of the PAK–PIX interaction is associated with a dramatic decrease of PIX and paxillin in focal adhesions, indicating that PAK localization to these structures via PIX is required for the maintenance of paxillin- and PIX-containing focal adhesions. Abnormal regulation of PAK localization and activity may contribute to the tumorigenic properties of certain breast cancer cells.
doi:10.1091/mbc.E03-08-0604
PMCID: PMC420118  PMID: 15047871
9.  Individual Rac GTPases Mediate Aspects of Prostate Cancer Cell and Bone Marrow Endothelial Cell Interactions 
Journal of Signal Transduction  2011;2011:541851.
The Rho GTPases organize the actin cytoskeleton and are involved in cancer metastasis. Previously, we demonstrated that RhoC GTPase was required for PC-3 prostate cancer cell invasion. Targeted down-regulation of RhoC led to sustained activation of Rac1 GTPase and morphological, molecular and phenotypic changes reminiscent of epithelial to mesenchymal transition. We also reported that Rac1 is required for PC-3 cell diapedesis across a bone marrow endothelial cell layer. In the current study, we queried whether Rac3 and RhoG GTPases also have a role in prostate tumor cell diapedesis. Using specific siRNAs we demonstrate roles for each protein in PC-3 and C4-2 cell adhesion and diapedesis. We have shown that the chemokine CCL2 induces tumor cell diapedesis via Rac1 activation. Here we find that RhoG partially contributes to CCL2-induced tumor cell diapedesis. We also find that Rac1 GTPase mediates tight binding of prostate cancer cells to bone marrow endothelial cells and promotes retraction of endothelial cells required for tumor cell diapedesis. Finally, Rac1 leads to β1 integrin activation, suggesting a mechanism that Rac1 can mediate tight binding with endothelial cells. Together, our data suggest that Rac1 GTPase is key mediator of prostate cancer cell-bone marrow endothelial cell interactions.
doi:10.1155/2011/541851
PMCID: PMC3135208  PMID: 21776386
10.  Control of the cytokinetic apparatus by flux of the Rho GTPases 
Biochemical Society transactions  2008;36(Pt 3):378-380.
Cytokinesis in animal cells is powered by the cytokinetic apparatus, a ring of filamentous actin and myosin-2 that underlies the plasma membrane and closes between the separating chromosomes. Formation of the cytokinetic apparatus is at least partially dependent on the small GTPase, Rho. Similar to other small GTPases, Rho cycles between the active (GTP-bound) and inactive (GDP-bound) states. Because of this switch-like behaviour, Rho and other members of the Rho GTPase family, such as Rac and Cdc42, have long been thought to work in a manner such that their activation and inactivation are not tightly coupled. That is, a given Rho-dependent event, such as cytokinesis, has been thought to be initiated by activation of Rho, and then, many minutes later, terminated by inactivation of Rho. Here we discuss evidence suggesting that in fact Rho undergoes rapid movement through the GTPase cycle throughout the entire process of cytokinesis, and that this cycling is necessary for proper cytokinetic apparatus function.
doi:10.1042/BST0360378
PMCID: PMC3570265  PMID: 18481962
cell division; cytokinesis; filamentous actin (F-actin); myosin-2; Rho; signalling
11.  Antagonistic Regulation of Actin Dynamics and Cell Motility by TRPC5 and TRPC6 Channels 
Science signaling  2010;3(145):ra77.
The Rho family of small guanosine triphosphatases (Rho GTPases: RhoA, Cdc42, and Rac1) regulates many aspects of cell behavior, including actin dynamics and cell migration. The generation of calcium ion (Ca2+) microdomains is critical in promoting cell migration because they control the localized activity of Rho GTPases. We identified receptor-activated TRPC5 and TRPC6 (transient receptor potential canonical type 5 and 6) channels as antagonistic regulators of actin remodeling and cell motility in fibroblasts and kidney podocytes. We show that TRPC5 is in a molecular complex with Rac1, whereas TRPC6 is in a molecular complex with RhoA. TRPC5-mediated Ca2+ influx induces Rac1 activation, thereby promoting cell migration, whereas TRPC6-mediated Ca2+ influx increases RhoA activity, thereby inhibiting cell migration. Our data unveil antagonistic Ca2+ influx pathways as a conserved signaling mechanism for the integrated regulation of cell migration.
doi:10.1126/scisignal.2001200
PMCID: PMC3071756  PMID: 20978238
12.  Phosphorylation of RhoGDI by Src Regulates Rho GTPase Binding and Cytosol-Membrane Cycling 
Molecular Biology of the Cell  2006;17(11):4760-4768.
Rho GTPases (Rac, Rho, and Cdc42) play important roles in regulating cell function through their ability to coordinate the actin cytoskeleton, modulate the formation of signaling reactive oxidant species, and control gene transcription. Activation of Rho GTPase signaling pathways requires the regulated release of Rho GTPases from RhoGDI complexes, followed by their reuptake after membrane cycling. We show here that Src kinase binds and phosphorylates RhoGDI both in vitro and in vivo at Tyr156. Analysis of Rho GTPase–RhoGDI complexes using in vitro assays of complexation and in vivo by coimmunoprecipitation analysis indicates that Src-mediated phosphorylation of Tyr156 causes a dramatic decrease in the ability of RhoGDI to form a complex with RhoA, Rac1, or Cdc42. Phosphomimetic mutation of Tyr156→Glu results in the constitutive association of RhoGDIY156E with the plasma membrane and/or associated cortical actin. Substantial cortical localization of tyrosine-phosphorylated RhoGDI is also observed in fibroblasts expressing active Src, where it is most evident in podosomes and regions of membrane ruffling. Expression of membrane-localized RhoGDIY156E mutant is associated with enhanced cell spreading and membrane ruffling. These results suggest that Src-mediated RhoGDI phosphorylation is a novel physiological mechanism for regulating Rho GTPase cytosol membrane–cycling and activity.
doi:10.1091/mbc.E06-06-0533
PMCID: PMC1635405  PMID: 16943322
13.  Cellular Cytoskeleton Dynamics Modulates Non-Viral Gene Delivery through RhoGTPases 
PLoS ONE  2012;7(4):e35046.
Although it is well accepted that the constituents of the cellular microenvironment modulate a myriad of cellular processes, including cell morphology, cytoskeletal dynamics and uptake pathways, the underlying mechanism of how these pathways influence non-viral gene transfer have not been studied. Transgene expression is increased on fibronectin (Fn) coated surfaces as a consequence of increased proliferation, cell spreading and active engagement of clathrin endocytosis pathway. RhoGTPases mediate the crosstalk between the cell and Fn, and regulate cellular processes involving filamentous actin, in-response to cellular interaction with Fn. Here the role of RhoGTPases specifically Rho, Rac and Cdc42 in modulation of non-viral gene transfer in mouse mesenchymal stem (mMSCs) plated in a fibronectin microenvironment was studied. More than 90% decrease in transgene expression was observed after inactivation of RhoGTPases using difficile toxin B (TcdB) and C3 transferase. Expression of dominant negative RhoA (RhoAT19N), Rac1(Rac1T17N) and Cdc42 (Cdc42T17N) also significantly reduced polyplex uptake and transgene expression. Interactions of cells with Fn lead to activation of RhoGTPases. However, further activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes (RhoAQ63L, Rac1Q61L and Cdc42Q61L) did not further enhance transgene expression in mMSCs, when plated on Fn. In contrast, activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes for cells plated on collagen I, which by itself did not increase RhoGTPase activation, resulted in enhanced transgene expression. Our study shows that RhoGTPases regulate internalization and effective intracellular processing of polyplexes that results in efficient gene transfer.
doi:10.1371/journal.pone.0035046
PMCID: PMC3324413  PMID: 22509380
14.  Targeting and activation of Rac1 are mediated by the exchange factor β-Pix 
The Journal of Cell Biology  2006;172(5):759-769.
Rho guanosine triphosphatases (GTPases) are critical regulators of cytoskeletal dynamics and control complex functions such as cell adhesion, spreading, migration, and cell division. It is generally accepted that localized GTPase activation is required for the proper initiation of downstream signaling events, although the molecular mechanisms that control targeting of Rho GTPases are unknown. In this study, we show that the Rho GTPase Rac1, via a proline stretch in its COOH terminus, binds directly to the SH3 domain of the Cdc42/Rac activator β-Pix (p21-activated kinase [Pak]–interacting exchange factor). The interaction with β-Pix is nucleotide independent and is necessary and sufficient for Rac1 recruitment to membrane ruffles and to focal adhesions. In addition, the Rac1–β-Pix interaction is required for Rac1 activation by β-Pix as well as for Rac1-mediated spreading. Finally, using cells deficient for the β-Pix–binding kinase Pak1, we show that Pak1 regulates the Rac1–β-Pix interaction and controls cell spreading and adhesion-induced Rac1 activation. These data provide a model for the intracellular targeting and localized activation of Rac1 through its exchange factor β-Pix.
doi:10.1083/jcb.200509096
PMCID: PMC2063707  PMID: 16492808
15.  Atypical RhoV and RhoU GTPases control development of the neural crest 
Small GTPases  2011;2(6):310-313.
This review addresses the developmental roles of two GTPases of the Rho family, RhoV/Chp and RhoU/Wrch. These two GTPases form a distinct subfamily related to Rac and Cdc42 proteins and were detected in a screen for Rho members that are particularly expressed in the neural crest, an embryonic tissue peculiar to vertebrates. The neural crest represents a physiological model of normal epithelial to mesenchymal transition (EMT), in which epithelial cells at the border of neural and non-neural ectoderm differentiate, lose their intercellular connections and migrate throughout the embryo. We showed that RhoV, transiently induced by the canonical Wnt pathway, is required for the full differentiation of neural crest cells, while RhoU, induced later by the non-canonical Wnt pathway, is necessary for the migration process. These two GTPases, which are highly conserved across vertebrates, are thus tightly functionally linked to Wnt signaling, whose implication in embryonic development and cancer progression is well established. In the light of the recent literature, we discuss how RhoV and RhoU may achieve their physiological functions.
doi:10.4161/sgtp.18086
PMCID: PMC3337159  PMID: 22545228
atypical Rho GTPases; development; neural crest; RhoU; RhoV; Wnt signaling; Xenopus
16.  Activation of Cdc42, Rac, PAK, and Rho-Kinase in Response to Hepatocyte Growth Factor Differentially Regulates Epithelial Cell Colony Spreading and Dissociation 
Molecular Biology of the Cell  2000;11(5):1709-1725.
Hepatocyte growth factor (HGF), the ligand for the Met receptor tyrosine kinase, is a potent modulator of epithelial–mesenchymal transition and dispersal of epithelial cells, processes that play crucial roles in tumor development, invasion, and metastasis. Little is known about the Met-dependent proximal signals that regulate these events. We show that HGF stimulation of epithelial cells leads to activation of the Rho GTPases, Cdc42 and Rac, concomitant with the formation of filopodia and lamellipodia. Notably, HGF-dependent activation of Rac but not Cdc42 is dependent on phosphatidylinositol 3-kinase. Moreover, HGF-induced lamellipodia formation and cell spreading require phosphatidylinositol 3-kinase and are inhibited by dominant negative Cdc42 or Rac. HGF induces activation of the Cdc42/Rac-regulated p21-activated kinase (PAK) and c-Jun N-terminal kinase, and translocation of Rac, PAK, and Rho-dependent Rho-kinase to membrane ruffles. Use of dominant negative and activated mutants reveals an essential role for PAK but not Rho-kinase in HGF-induced epithelial cell spreading, whereas Rho-kinase activity is required for the formation of focal adhesions and stress fibers in response to HGF. We conclude that PAK and Rho-kinase play opposing roles in epithelial–mesenchymal transition induced by HGF, and provide new insight regarding the role of Cdc42 in these events.
PMCID: PMC14878  PMID: 10793146
17.  Cell type-specific roles for Cdc42, Rac, and RhoL in Drosophila oogenesis 
The Journal of Cell Biology  1996;133(3):617-630.
The Rho subfamily of GTPases has been shown to regulate cellular morphology. We report the discovery of a new member of the Rho family, named RhoL, which is equally similar to Rac, Rho, and Cdc42. Expression of a dominant-negative RhoL transgene in the Drosophila ovary caused nurse cells to collapse and fuse together. Mutant forms of Cdc42 mimicked this effect. Expression of constitutively active RhoL led to nurse cell subcortical actin breakdown and disruption of nurse cell- follicle cell contacts, followed by germ cell apoptosis. In contrast, Rac activity was specifically required for migration of a subset of follicle cells called border cells. All three activities were necessary for normal transfer of nurse cell cytoplasm to the oocyte. These results suggest that Rho protein activities have cell type-specific effects on morphogenesis.
PMCID: PMC2120826  PMID: 8636236
18.  Inactivation of small Rho GTPases by the multifunctional RTX toxin from Vibrio cholerae 
Cellular microbiology  2007;9(5):1324-1335.
Many bacterial toxins target small Rho GTPases in order to manipulate the actin cytoskeleton. The depolymerization of the actin cytoskeleton by the Vibrio cholerae RTX toxin was previously identified to be due to the unique mechanism of covalent actin cross-linking. However, identification and subsequent deletion of the actin cross-linking domain within the RTX toxin revealed that this toxin has an additional cell rounding activity. In this study, we identified that the multifunctional RTX toxin also disrupts the actin cytoskeleton by causing the inactivation of small Rho GTPases, Rho, Rac and Cdc42. Inactivation of Rho by RTX was reversible in the presence of cycloheximide and by treatment of cells with CNF1 to constitutively activate Rho. These data suggest that RTX targets Rho GTPase regulation rather than affecting Rho GTPase directly. A novel 548 amino acid region of RTX was identified to be responsible for the toxin-induced inactivation of the Rho GTPases. This domain did not carry GAP or phosphatase activities. Overall, these data show that the RTX toxin reversibly inactivates Rho GTPases by a mechanism distinct from other Rho modifying bacterial toxins.
doi:10.1111/j.1462-5822.2006.00876.x
PMCID: PMC2258554  PMID: 17474905
Rho GTPase; Vibrio cholerae RTX toxin; actin; Lethal Factor; Cytotoxic Necrotizing Factor
19.  Binding of Rac1, Rnd1, and RhoD to a Novel Rho GTPase Interaction Motif Destabilizes Dimerization of the Plexin-B1 Effector Domain*S 
The Journal of biological chemistry  2007;282(51):37215-37224.
Plexins are the first known transmembrane receptors that interact directly with small GTPases. On binding to certain Rho family GTPases, the receptor regulates the remodeling of the actin cytoskeleton and alters cell movement in response to semaphorin guidance cues. In a joint solution NMR spectroscopy and x-ray crystallographic study, we characterize a 120-residue cytoplasmic independent folding domain of plexin-B1 that directly binds three Rho family GTPases, Rac1, Rnd1, and RhoD. The NMR data show that, surprisingly, the Cdc42/Rac interactive binding-like motif of plexin-B1 is not involved in this interaction. Instead, all three GTPases interact with the same region, β-strands 3 and 4 and a short α-helical segment of the plexin domain. The 2.0 Å resolution x-ray structure shows that these segments are brought together by the tertiary structure of the ubiquitin-like fold. In the crystal, the protein is dimerized with C2 symmetry through a four-stranded antiparallel β-sheet that is formed outside the fold by a long loop between the monomers. This region is adjacent to the GTPase binding motifs identified by NMR. Destabilization of the dimer in solution by binding of any one of the three GTPases suggests a model for receptor regulation that involves bidirectional signaling. The model implies a multifunctional role for the GTPase-plexin interaction that includes conformational change and a localization of active receptors in the signaling mechanism.
doi:10.1074/jbc.M703800200
PMCID: PMC2655321  PMID: 17916560
20.  Involvement of Rho Family GTPases in p19Arf- and p53-Mediated Proliferation of Primary Mouse Embryonic Fibroblasts 
Molecular and Cellular Biology  2004;24(3):1426-1438.
The Rho family GTPases Rac1, RhoA, and Cdc42 function as molecular switches that transduce intracellular signals regulating gene expression and cell proliferation as well as cell migration. p19Arf and p53, on the other hand, are tumor suppressors that act both independently and sequentially to regulate cell proliferation. To investigate the functional interaction and cooperativeness of Rho GTPases with the p19Arf-p53 pathway, we examined the contribution of Rho GTPases to the gene transcription and cell proliferation unleashed by deletion of p19Arf or p53 in primary mouse embryo fibroblasts. We found that (i) p19Arf or p53 deficiency led to a significant increase in PI 3-kinase activity, which in turn upregulated RhoA and Rac1 activities; (ii) deletion of p19Arf or p53 led to an increase in cell growth rate that was in part dependent on RhoA, Rac1, and Cdc42 activities; (iii) p19Arf or p53 deficiency caused an enhancement of the growth-related transcription factor NF-κB and cyclin D1 activities that are partly dependent on RhoA or Cdc42 but not on Rac1; (iv) forced expression of the activating mutants of Rac1, RhoA, or Cdc42 caused a hyperproliferative phenotype of the p19Arf−/− and p53−/− cells and promoted transformation of both cells; (v) RhoA appeared to contribute to p53-regulated cell proliferation by modulating cell cycle machinery, while hyperactivation of RhoA further suppressed a p53-independent apoptotic signal; and (vi) multiple pathways regulated by RhoA, including that of Rho-kinase, were required for RhoA to fully promote the transformation of p53−/− cells. Taken together, these results provide strong evidence indicating that signals through the Rho family GTPases can both contribute to cell growth regulation by p19Arf and p53 and cooperate with p19Arf or p53 deficiency to promote primary cell transformation.
doi:10.1128/MCB.24.3.1426-1438.2004
PMCID: PMC321455  PMID: 14729984
21.  Multiple roles for RhoA during T cell transendothelial migration 
Small GTPases  2010;1(3):174-179.
T cells need to cross endothelial barriers during immune surveillance and inflammation. This involves T-cell adhesion to the endothelium followed by polarization and crawling with a lamellipodium at the front and contractile uropod at the back. T cells subsequently extend lamellipodia and filopodia under the endothelium in order to transmigrate. Rho GTPases play key roles in cell migration by regulating cytoskeletal dynamics and cell adhesion. We have found that the Rho GTPase RhoA is required for efficient T-cell polarization and migration on endothelial cells as well as transendothelial migration. RhoA-depleted cells lack both lamellipodia and uropods, and instead have narrow protrusions extending from a rounded cell body. Using a RhoA activity biosensor, we have shown that RhoA is active at the leading edge in lamellipodia and filopodia of crawling and transmigrating T cells, as well as in the uropod. In lamellipodia, its activity correlates with both protrusion and retraction. We predict that RhoA signals via the formin mDIA 1 during lamellipodial protrusion whereas it induces lamellipodial retraction via the kinase ROCK and actomyosin contractility. We propose that different guanine-nucleotide exchange factors (GEFs) are responsible for coordinating RhoA activation and signaling in different regions of transmigrating T cells.
doi:10.4161/sgtp.1.3.14724
PMCID: PMC3116607  PMID: 21686273
Rho GTPases; T cells; diapedesis; endothelial cells; actin cytoskeleton; cell migration; uropod; actomyosin
22.  miR-124-regulated RhoG 
Small GTPases  2013;4(1):42-46.
RhoG is a member of the Rho family of small GTPases sharing highest sequence similarity with Rac and Cdc42. Mig-2 and Mtl represent the functional equivalents of RhoG in Caenorhabditis elegans and Drosophila, respectively. RhoG has attracted great interest because it plays a central role in the regulation of cytoskeletal reorganization in various physiological and pathophysiological situations. For example, it is fundamental to phagocytotic processes, is able to regulate gene expression, cell survival and proliferation, and is involved in cell migration and in the invasion of pathogenic bacteria. The activation of Rac1 via an ELMO/Dock180 module has been elaborated to be important for RhoG signaling. Although a stimulatory role for neurite outgrowth in the pheochromocytoma PC12 cell line has been assigned to RhoG, the exact function of this GTPase for the development of the processes of primary neurons remains to be clarified. In this view, we discuss the impact of RhoG on axonal and dendritic differentiation, its role as a conductor of Rac1 and Cdc42 activity and the functional regulation of RhoG expression by the microRNA miR-124.
doi:10.4161/sgtp.22922
PMCID: PMC3620101  PMID: 23303397
RhoG; Rac1; Cdc42; microRNA miR-124; ELMO/Dock180; GEF; axonal branching; dendritic branching; cytoskeletal reorganization
23.  RHOB LINKS PDGF SIGNALING TO CELL MIGRATION BY COORDINATING ACTIVATION AND LOCALIZATION OF CDC42 AND RAC 
Journal of cellular biochemistry  2011;112(6):1572-1584.
The small GTPase RhoB regulates endocytic trafficking of receptor tyrosine kinases (RTKs) and the non-receptor kinases Src and Akt. While receptor–mediated endocytosis is critical for signaling processes driving cell migration, mechanisms that coordinate endocytosis with the propagation of migratory signals remain relatively poorly understood. In this study, we show that RhoB is essential for activation and trafficking of the key migratory effectors Cdc42 and Rac in mediating the ability of platelet-derived growth factor (PDGF) to stimulate cell movement. Stimulation of the PDGF receptor-β on primary vascular smooth muscle cells (VSMCs) results in RhoB-dependent trafficking of endosome-bound Cdc42 from the perinuclear region to the cell periphery, where the RhoGEF Vav2 and Rac are also recruited to drive formation of circular dorsal and peripheral ruffles necessary for cell migration. Our findings identify a novel RhoB dependent endosomal trafficking pathway that integrates RTK endocytosis with Cdc42/Rac localization and cell movement.
doi:10.1002/jcb.23069
PMCID: PMC3079809  PMID: 21344485
RhoB; Cdc42; Rac; Endosome; platelet-derived growth factor receptor; endocytic trafficking; vascular smooth muscle cells; cell migration
24.  Distinct roles of Rac GTPases and the UNC-73/Trio and PIX-1 Rac GTP exchange factors in neuroblast protrusion and migration in C. elegans 
Small GTPases  2010;1(1):44-61.
The Rac and Cdc42 GTPases as well as the multiple GTP exchange factors that regulate their activity have been implicated in the pathways that drive actin cytoskeleton reorganization, but the individual contributions of these molecules to cell migration remain unknown. Studies shown here examine the roles of CED-10/Rac, MIG-2/RhoG and CDC-42 in the migration of the QL and QR neuroblasts in C. elegans. CED-10/Rac was found to normally limit protrusion and migration, whereas MIG-2/RhoG was required for protrusion and migration. CED-10/Rac and MIG-2/RhoG also had redundant roles in Q protrusion and migration. Surprisingly, CDC-42 was found to have only weak effects on the protrusion and the migration. We found that a mutation of unc-73/Trio, which encodes a GEF for CED-10/Rac and MIG-2/RhoG, caused protrusions that were thin and filopodia-like, suggesting that UNC-73/Trio is required for robust lamellipodia-like protrusion. A screen of the 19 C. elegans Dbl homology Rho GEF genes revealed that PIX-1 was required for proper Q neuroblast protrusion and migration. Genetic analysis indicated that PIX-1 might act in the CED-10/Rac pathway in parallel to MIG-2/RhoG and that PIX-1 has redundant function with UNC-73/Trio in Q neuroblast protrusion and migration. These results indicate that Rho GTPases and GEFs have both unique and overlapping roles in neuronal migration.
doi:10.4161/sgtp.1.1.12991
PMCID: PMC3109480  PMID: 21686119
neuroblast migration; Rac GTPase; C. elegans; UNC-73/Trio; PIX-1; GTP exchange factor
25.  Cdc42 and the Actin-Related Protein/Neural Wiskott-Aldrich Syndrome Protein Network Mediate Cellular Invasion by Cryptosporidium parvum  
Infection and Immunity  2004;72(5):3011-3021.
Cryptosporidium parvum invasion of epithelial cells involves host cell membrane alterations which require a remodeling of the host cell actin cytoskeleton. In addition, an actin plaque, possibly associated with the dense-band region, forms within the host cytoplasm at the host-parasite interface. Here we show that Cdc42 and RhoA, but not Rac1, members of the Rho family of GTPases, are recruited to the host-parasite interface in an in vitro model of human biliary cryptosporidiosis. Interestingly, activation of Cdc42, but not RhoA, was detected in the infected cells. Neural Wiskott-Aldrich syndrome protein (N-WASP) and p34-Arc, actin-regulating downstream effectors of Cdc42, were also recruited to the host-parasite interface. Whereas cellular expression of a constitutively active mutant of Cdc42 promoted C. parvum invasion, overexpression of a dominant negative mutant of Cdc42, or depletion of Cdc42 mRNA by short interfering RNA-mediated gene silencing, inhibited C. parvum invasion. Expression of the WA fragment of N-WASP to block associated actin polymerization also inhibited C. parvum invasion. Moreover, inhibition of host cell Cdc42 activation by dominant negative mutation inhibited C. parvum-associated actin remodeling, membrane protrusion, and dense-band formation. In contrast, treatment of cells with a Rho inhibitor, exoenzyme C3, or cellular overexpression of dominant negative mutants of RhoA and Rac1 had no effect on C. parvum invasion. These data suggest that C. parvum invasion of target epithelia results from the organism's ability to activate a host cell Cdc42 GTPase signaling pathway to induce host cell actin remodeling at the attachment site.
doi:10.1128/IAI.72.5.3011-3021.2004
PMCID: PMC387898  PMID: 15102814

Results 1-25 (813191)