CYK4 inhibits Rac1-dependent PAK1 and ARHGEF7 effector pathways during cytokinesis

In mitosis, animal cells lose their adhesion to the surrounding surfaces and become rounded. During mitotic exit, they reestablish these adhesions and at the same time physically contract and divide. How these competing processes are spatially segregated at the cell cortex remains mysterious. To address this question, we define the specific effector pathways used by RhoA and Rac1 in mitotic cells. We demonstrate that the MKlp1-CYK4 centralspindlin complex is a guanosine triphosphatase-activating protein (GAP) for Rac1 and not RhoA and that CYK4 negatively regulated Rac1 activity at the cell equator in anaphase. Cells expressing a CYK4 GAP mutant had defects in cytokinesis and showed elevated staining for the cell adhesion marker vinculin. These defects could be rescued by depletion of ARHGEF7 and p21-activated kinase, Rac1-specific effector proteins required for cell adhesion. Based on these findings, we propose that CYK4 GAP activity is required during anaphase to inhibit Rac1-dependent effector pathways associated with control of cell spreading and adhesion.     

RhoA and RhoC have distinct roles in migration and invasion by acting through different targets

Several studies suggest that RhoA and RhoC, despite their sequence similarity, have different roles in cell migration and invasion, but the molecular basis for this is not known. Using RNAi, we show that RhoA-depleted cells became elongated and extended multiple Rac1-driven narrow protrusions in 2D and 3D environments, leading to increased invasion. These phenotypes were caused by combined but distinct effects of the Rho-regulated kinases ROCK1 and ROCK2. Depletion of ROCK2 induced multiple delocalized protrusions and reduced migratory polarity, whereas ROCK1 depletion selectively led to cell elongation and defective tail retraction. In contrast, RhoC depletion increased cell spreading and induced Rac1 activation around the periphery in broad lamellipodia, thereby inhibiting directed migration and invasion. These effects of RhoC depletion are mediated by the formin FMNL3, which we identify as a new target of RhoC but not RhoA. We propose that RhoA contributes to migratory cell polarity through ROCK2-mediated suppression of Rac1 activity in lamellipodia, whereas RhoC promotes polarized migration through FMNL3 by restricting lamellipodial broadening.     

Tumor Cell Migration and Invasion Are Enhanced by Depletion of Rap1 GTPase-activating Protein (Rap1GAP)

The functional significance of the widespread down-regulation of Rap1 GTPase-activating protein (Rap1GAP), a negative regulator of Rap activity, in human tumors is unknown. Here we show that human colon cancer cells depleted of Rap1GAP are endowed with more aggressive migratory and invasive properties. Silencing Rap1GAP enhanced the migration of confluent and single cells. In the latter, migration distance, velocity, and directionality were increased. Enhanced migration was a consequence of increased endogenous Rap activity as silencing Rap expression selectively abolished the migration of Rap1GAP-depleted cells. ROCK-mediated cell contractility was suppressed in Rap1GAP-depleted cells, which exhibited a spindle-shaped morphology and abundant membrane protrusions. Tumor cells can switch between Rho/ROCK-mediated contractility-based migration and Rac1-mediated mesenchymal motility. Strikingly, the migration of Rap1GAP-depleted, but not control cells required Rac1 activity, suggesting that loss of Rap1GAP alters migratory mechanisms. Inhibition of Rac1 activity restored membrane blebbing and increased ROCK activity in Rap1GAP-depleted cells, suggesting that Rac1 contributes to the suppression of contractility. Collectively, these findings identify Rap1GAP as a critical regulator of aggressive tumor cell behavior and suggest that the level of Rap1GAP expression influences the migratory mechanisms that are operative in tumor cells.     

Rac activation by lysophosphatidic acid LPA1 receptors through the guanine nucleotide exchange factor Tiam1

Lysophosphatidic acid (LPA) is a serum-borne phospholipid that activates its own G protein-coupled receptors present in numerous cell types. In addition to stimulating cell proliferation, LPA also induces cytoskeletal changes and promotes cell migration in a RhoA- and Rac-dependent manner. Whereas RhoA is activated via Galpha(12/13)-linked Rho-specific guanine nucleotide exchange factors, it is unknown how LPA receptors may signal to Rac. Here we report that the prototypic LPA(1) receptor (previously named Edg2), when expressed in B103 neuroblastoma cells, mediates transient activation of RhoA and robust, prolonged activation of Rac leading to cell spreading, lamellipodia formation, and stimulation of cell migration. LPA-induced Rac activation is inhibited by pertussis toxin and requires phosphoinositide 3-kinase activity. Strikingly, LPA fails to activate Rac in cell types that lack the Rac-specific exchange factor Tiam1; however, enforced expression of Tiam1 restores LPA-induced Rac activation in those cells. Tiam1-deficient cells show enhanced RhoA activation, stress fiber formation, and cell rounding in response to LPA, consistent with Tiam1/Rac counteracting RhoA. We conclude that LPA(1) receptors couple to a G(i)-phosphoinositide 3-kinase-Tiam1 pathway to activate Rac, with consequent suppression of RhoA activity, and thereby stimulate cell spreading and motility.     

The N-terminal DH-PH domain of Trio induces cell spreading and migration by regulating lamellipodia dynamics in a Rac1-dependent fashion

The guanine-nucleotide exchange factor Trio encodes two DH-PH domains that catalyze nucleotide exchange on Rac1, RhoG and RhoA. The N-terminal DH-PH domain is known to activate Rac1 and RhoG, whereas the C-terminal DH-PH domain can activate RhoA. The current study shows that the N-terminal DH-PH domain, upon expression in HeLa cells, activates Rac1 and RhoG independently from each other. In addition, we show that the flanking SH3 domain binds to the proline-rich region of the C-terminus of Rac1, but not of RhoG. However, this SH3 domain is not required for Rac1 or RhoG GDP-GTP exchange. Rescue experiments in Trio-shRNA-expressing cells showed that the N-terminal DH-PH domain of Trio, but not the C-terminal DH-PH domain, restored fibronectin-mediated cell spreading and migration defects that are observed in Trio-silenced cells. Kymograph analysis revealed that the N-terminal DH-PH domain, independent of its SH3 domain, controls the dynamics of lamellipodia. Using siRNA against Rac1 or RhoG, we found that Trio-D1-induced lamellipodia formation required Rac1 but not RhoG expression. Together, we conclude that the GEF Trio is responsible for lamellipodia formation through its N-terminal DH-PH domain in a Rac1-dependent manner during fibronectin-mediated spreading and migration.     

Enhanced intrinsic migration of aggressive breast cancer cells by inhibition of Rac1 GTPase

Rac GTPases are known to play a crucial role in regulating cytoskeletal changes necessary for cell migration. Migration has been shown to be positively regulated by Rac in most cell types. However, there is also a large body of conflicting evidence in some other cell types with respect to the role of Rac in migration, suggesting that Rac GTPases regulate cell migration in a cell type-dependent manner. In the present study, we have characterized the effects of Rac1 GTPase inhibition on the migratory abilities of a number of breast cancer cell lines with differential degrees of tumorigenic and metastatic potentials. We show that Rac1 inhibition in non-metastatic (MCF-7, T-47D) or moderately metastatic (Hs578T) cell lines results in inhibition of migration, whereas in highly metastatic cell lines (MDA-MB-435, MDA-MB-231, and C3L5) Rac1 inhibition results in stimulation of migration. This stimulation of migration following Rac1 inhibition is also accompanied by the enhanced RhoA activity, suggesting a possible existence of a dominating role of RhoA over Rac1 in regulating intrinsic migration of the highly metastatic breast cancer cells.     

Vav2 is required for cell spreading.

Vav2 is a widely expressed Rho family guanine nucleotide exchange factor highly homologous to Vav1 and Vav3. Activated versions of Vav2 are transforming, but the normal function of Vav2 and how it is regulated are not known. We investigated the pathways that regulate Vav2 exchange activity in vivo and characterized its function. Overexpression of Vav2 activates Rac as assessed by both direct measurement of Rac-GTP and cell morphology. Vav2 also catalyzes exchange for RhoA, but does not cause morphologic changes indicative of RhoA activation. Vav2 nucleotide exchange is Src-dependent in vivo, since the coexpression of Vav2 and dominant negative Src, or treatment with the Src inhibitor PP2, blocks both Vav2-dependent Rac activation and lamellipodia formation. A mutation in the pleckstrin homology (PH) domain eliminates exchange activity and this construct does not induce lamellipodia, indicating the PH domain is necessary to catalyze nucleotide exchange. To further investigate the function of Vav2, we mutated the dbl homology (DH) domain and asked whether this mutant would function as a dominant negative to block Rac-dependent events. Studies using this mutant indicate that Vav2 is not necessary for platelet-derived growth factor- or epidermal growth factor-dependent activation of Rac. The Vav2 DH mutant did act as a dominant negative to inhibit spreading of NIH3T3 cells on fibronectin, specifically by blocking lamellipodia formation. These findings indicate that in fibroblasts Vav2 is necessary for integrin, but not growth factor-dependent activation of Rac leading to lamellipodia.     

Myosin II and Arp2/3 cross-talk governs intracellular hydraulic pressure and lamellipodia formation

Human fibroblasts can switch between lamellipodia-dependent and -independent migration mechanisms on two-dimensional surfaces and in three-dimensional (3D) matrices. RhoA GTPase activity governs the switch from low-pressure lamellipodia to high-pressure lobopodia in response to the physical structure of the 3D matrix. Inhibiting actomyosin contractility in these cells reduces intracellular pressure and reverts lobopodia to lamellipodial protrusions via an unknown mechanism. To test the hypothesis that high pressure physically prevents lamellipodia formation, we manipulated pressure by activating RhoA or changing the osmolarity of the extracellular environment and imaged cell protrusions. We find RhoA activity inhibits Rac1-mediated lamellipodia formation through two distinct pathways. First, RhoA boosts intracellular pressure by increasing actomyosin contractility and water influx but acts upstream of Rac1 to inhibit lamellipodia formation. Increasing osmotic pressure revealed a second RhoA pathway, which acts through nonmuscle myosin II (NMII) to disrupt lamellipodia downstream from Rac1 and elevate pressure. Interestingly, Arp2/3 inhibition triggered a NMII-dependent increase in intracellular pressure, along with lamellipodia disruption. Together, these results suggest that actomyosin contractility and water influx are coordinated to increase intracellular pressure, and RhoA signaling can inhibit lamellipodia formation via two distinct pathways in high-pressure cells.     

Bi-modal regulation of a formin by srGAP2

The maintenance of rapid and efficient actin dynamics in vivo requires coordination of filament assembly and disassembly. This regulation requires temporal and spatial integration of signaling pathways by protein complexes. However, it remains unclear how these complexes form and then regulate the actin cytoskeleton. Here, we identify a srGAP2 and formin-like 1 (FMNL1, also known as FRL1 or FRLα) complex whose assembly is regulated by Rac signaling. Our data suggest srGAP2 regulates FMNL1 in two ways; 1) Rac-mediated activation of FMNL1 leads to the recruitment of srGAP2, which contains a Rac-specific GAP domain; 2) the SH3 domain of srGAP2 binds the formin homology 1 domain of FMNL1 to inhibit FMNL1-mediated actin severing. Thus, srGAP2 can efficiently terminate the upstream activating Rac signal while also opposing an important functional output of FMNL1, namely actin severing. We also show that FMNL1 and srGAP2 localize to the actin-rich phagocytic cup of macrophage-derived cells, suggesting the complex may regulate this Rac- and actin-driven process in vivo. We propose that after Rac-dependent activation of FMNL1, srGAP2 mediates a potent mechanism to limit the duration of Rac action and inhibit formin activity during rapid actin dynamics.     

The GTPase-activating protein n-chimaerin cooperates with Rac1 and Cdc42Hs to induce the formation of lamellipodia and filopodia.

n-Chimaerin is a GTPase-activating protein (GAP) mainly for Rac1 and less so for Cdc42Hs in vitro. The GAP activity of n-chimaerin is regulated by phospholipids and phorbol esters. Microinjection of Rac1 and Cdc42Hs into mammalian cells induces formation of the actin-based structures lamellipodia and filopodia, respectively, with the former being prevented by coinjection of the chimaerin GAP domain. Strikingly, microinjection of the full-length n-chimaerin into fibroblasts and neuroblastoma cells induces the simultaneous formation of lamellipodia and filopodia. These structures undergo cycles of dissolution and formation, resembling natural morphological events occurring at the leading edge of fibroblasts and neuronal growth cones. The effects of n-chimaerin on formation of lamellipodia and filopodia were inhibited by dominant negative Rac1(T17N) and Cdc42Hs(T17N), respectively. n-Chimaerin's effects were also inhibited by coinjection with Rho GDP dissociation inhibitor or by treatment with phorbol ester. A mutant n-chimaerin with no GAP activity and impaired p21 binding was ineffective in inducing morphological changes, while a mutant lacking GAP activity alone was effective. Microinjected n-chimaerin colocalized in situ with F-actin. Taken together, these results suggest that n-chimaerin acts synergistically with Rac1 and Cdc42Hs to induce actin-based morphological changes and that this action involves Rac1 and Cdc42Hs binding but not GAP activity. Thus, GAPs may have morphological functions in addition to downregulation of GTPases.     

A Highlights from MBoC Selection: MgcRacGAP restricts active RhoA at the cytokinetic furrow and both RhoA and Rac1 at cell–cell junctions in epithelial cells

Localized activation of Rho GTPases is essential for multiple cellular functions, including cytokinesis and formation and maintenance of cell–cell junctions. Although MgcRacGAP (Mgc) is required for spatially confined RhoA-GTP at the equatorial cortex of dividing cells, both the target specificity of Mgc''s GAP activity and the involvement of phosphorylation of Mgc at Ser-386 are controversial. In addition, Mgc''s function at cell–cell junctions remains unclear. Here, using gastrula-stage Xenopus laevis embryos as a model system, we examine Mgc''s role in regulating localized RhoA-GTP and Rac1-GTP in the intact vertebrate epithelium. We show that Mgc''s GAP activity spatially restricts accumulation of both RhoA-GTP and Rac1-GTP in epithelial cells—RhoA at the cleavage furrow and RhoA and Rac1 at cell–cell junctions. Phosphorylation at Ser-386 does not switch the specificity of Mgc''s GAP activity and is not required for successful cytokinesis. Furthermore, Mgc regulates adherens junction but not tight junction structure, and the ability to regulate adherens junctions is dependent on GAP activity and signaling via the RhoA pathway. Together these results indicate that Mgc''s GAP activity down-regulates the active populations of RhoA and Rac1 at localized regions of epithelial cells and is necessary for successful cytokinesis and cell–cell junction structure.     

srGAP2 Arginine Methylation Regulates Cell Migration and Cell Spreading through Promoting Dimerization

The Slit-Robo GTPase-activating proteins (srGAPs) are critical for neuronal migration through inactivation of Rho GTPases Cdc42, Rac1, and RhoA. Here we report that srGAP2 physically interacts with protein arginine methyltransferase 5 (PRMT5). srGAP2 localizes to the cytoplasm and plasma membrane protrusion. srGAP2 knockdown reduces cell adhesion spreading and increases cell migration, but has no effect on cell proliferation. PRMT5 binds to the N terminus of srGAP2 (225–538 aa) and methylates its C-terminal arginine residue Arg-927. The methylation mutant srGAP2-R927A fails to rescue the cell spreading rate, is unable to localize to the plasma membrane leading edge, and perturbs srGAP2 homodimer formation mediated by the F-BAR domain. These results suggest that srGAP2 arginine methylation plays important roles in cell spreading and cell migration through influencing membrane protrusion.     

Concerted regulation of cell dynamics by BNIP-2 and Cdc42GAP homology/Sec14p-like,proline-rich,and GTPase-activating protein domains of a novel Rho GTPase-activating protein,BPGAP1

RhoA, Cdc42, and Rac1 are small GTPases that regulate cytoskeletal reorganization leading to changes in cell morphology and cell motility. Their signaling pathways are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs). We have identified a novel RhoGAP, BPGAP1 (for BNIP-2 and Cdc42GAP Homology (BCH) domain-containing, Proline-rich and Cdc42GAP-like protein subtype-1), that is ubiquitously expressed and shares 54% sequence identity to Cdc42GAP/p50RhoGAP. BP-GAP1 selectively enhanced RhoA GTPase activity in vivo although it also interacted strongly with Cdc42 and Rac1. "Pull-down" and co-immunoprecipitation studies indicated that it formed homophilic or heterophilic complexes with other BCH domain-containing proteins. Fluorescence studies of epitope-tagged BPGAP1 revealed that it induced pseudopodia and increased migration of MCF7 cells. Formation of pseudopodia required its BCH and GAP domains but not the proline-rich region, and was differentially inhibited by coexpression of the constitutively active mutant of RhoA, or dominant negative mutants of Cdc42 and Rac1. However, the mutant without the proline-rich region failed to confer any increase in cell migration despite the induction of pseudopodia. Our findings provide evidence that cell morphology changes and migration are coordinated via multiple domains in BPGAP1 and present a novel mode of regulation for cell dynamics by a RhoGAP protein.     

Genetic deletion of Rac1 GTPase reveals its critical role in actin stress fiber formation and focal adhesion complex assembly

Rac1 is an intracellular signal transducer regulating a variety of cell functions. Previous studies by overexpression of dominant-negative or constitutively active mutants of Rac1 in clonal cell lines have established that Rac1 plays a key role in actin lamellipodia induction, cell-matrix adhesion, and cell anoikis. In the present studies, we have examined the cellular behaviors of Rac1 gene-targeted primary mouse embryonic fibroblasts (MEFs) after Cre recombinase-mediated deletion of Rac1 gene. Rac1-null MEFs became contracted and elongated in morphology and were defective in lamellipodia formation, cell spreading, cell-fibronectin adhesion, and focal contact formation in response to platelet-derived growth factor or serum. Unexpectedly, deletion of Rac1 also abolished actin stress fibers in the cells without detectable alteration of endogenous RhoA activity. Although the expression and/or activation status of focal adhesion complex components such as Src, FAK, and vinculin were not affected by Rac1 deletion, the number and size of adhesion plaques were significantly reduced, and the molecular complex between Src, FAK, and vinculin was dissembled in Rac1-null cells. Overexpression of an active RhoA mutant or ROK failed to rescue the stress fiber and adhesion plaque defects of the Rac1-null cells. Although Rac1 deletion caused a significant reduction in phospho-PAK1, -AKT, and -ERK under serum stimulation, reconstitution of active PAK1, but not AKT or MEK1, was able to rescue the actin cytoskeleton and adhesion phenotypes of the Rac1-deficient cells. Furthermore, Rac1 deletion led to a marked increase in spontaneous apoptosis that could be rescued by active PAK1, AKT, or MEK1 expression. Our results obtained from gene-targeted primary MEFs indicate that Rac1 is essential not only for lamellipodia induction but also for the RhoA-regulated actin stress fiber and focal adhesion complex formation and that Rac1 is involved in cell survival regulation through anoikis-dependent as well as -independent mechanisms.     

RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells

Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.     

p190RhoGAP has cellular RacGAP activity regulated by a polybasic region

p190RhoGAP is a GTPase-activating protein (GAP) known to regulate actin cytoskeleton dynamics by decreasing RhoGTP levels through activation of the intrinsic GTPase activity of Rho. Although the GAP domain of p190RhoGAP stimulates the intrinsic' GTPase activity of several Rho family members (Rho, Rac, Cdc42) under in vitro conditions, p190RhoGAP is generally regarded as a GAP for RhoA in the cell. The cellular RacGAP activity of the protein has not been proven directly. We have previously shown that the in vitro RacGAP and RhoGAP activity of p190RhoGAP was inversely regulated through a polybasic region of the protein. Here we provide evidence that p190RhoGAP shows remarkable GAP activity toward Rac also in the cell. The cellular RacGAP activity of p190RhoGAP requires an intact polybasic region adjacent to the GAP domain whereas the RhoGAP activity is inhibited by the same domain. Our data indicate that through its alternating RacGAP and RhoGAP activity, p190RhoGAP plays a more complex role in the Rac–Rho antagonism than it was realized earlier.     

Coordination of cell polarization and migration by the Rho family GTPases requires Src tyrosine kinase activity.

BACKGROUND: The ability of a cell to polarize and move is governed by remodeling of the cellular adhesion/cytoskeletal network that is in turn controlled by the Rho family of small GTPases. However, it is not known what signals lie downstream of Rac1 and Cdc42 during peripheral actin and adhesion remodeling that is required for directional migration. RESULTS: We show here that individual members of the Rho family, RhoA, Rac1, and Cdc42, direct the specific intracellular targeting of c-Src tyrosine kinase to focal adhesions, lamellipodia, or filopodia, respectively, and that the adaptor function of c-Src (the combined SH3/SH2 domains coupled to green fluorescent protein) is sufficient for targeting. Furthermore, Src's catalytic activity is absolutely required at these peripheral cell-matrix attachment sites for remodeling that converts RhoA-dependent focal adhesions into smaller focal complexes along Rac1-induced lamellipodia (or Cdc42-induced filopodia). Consequently, cells in which kinase-deficient c-Src occupies peripheral adhesion sites exhibit impaired polarization toward migratory stimuli and reduced motility. Furthermore, phosphorylation of FAK, an Src adhesion substrate, is suppressed under these conditions. CONCLUSIONS: Our findings demonstrate that individual Rho GTPases specify Src's exact peripheral localization and that Rac1- and Cdc42-induced adhesion remodeling and directed cell migration require Src activity at peripheral adhesion sites.     

Tetraspanin CD82 Inhibits Protrusion and Retraction in Cell Movement by Attenuating the Plasma Membrane-Dependent Actin Organization

To determine how tetraspanin KAI1/CD82, a tumor metastasis suppressor, inhibits cell migration, we assessed which cellular events critical for motility are altered by KAI1/CD82 and how KAI1/CD82 regulates these events. We found that KAI1/CD82-expressing cells typically exhibited elongated cellular tails and diminished lamellipodia. Live imaging demonstrated that the polarized protrusion and retraction of the plasma membrane became deficient upon KAI1/CD82 expression. The deficiency in developing these motility-related cellular events was caused by poor formations of actin cortical network and stress fiber and by aberrant dynamics in actin organization. Rac1 activity was reduced by KAI1/CD82, consistent with the diminution of lamellipodia and actin cortical network; while the growth factor-stimulated RhoA activity was blocked by KAI1/CD82, consistent with the loss of stress fiber and attenuation in cellular retraction. Upon KAI1/CD82 expression, Rac effector cofilin was not enriched at the cell periphery to facilitate lamellipodia formation while Rho kinase exhibited a significantly lower activity leading to less retraction. Phosphatidylinositol 4, 5-biphosphate, which initiates actin polymerization from the plasma membrane, became less detectable at the cell periphery in KAI1/CD82-expressing cells. Moreover, KAI1/CD82-induced phenotypes likely resulted from the suppression of multiple signaling pathways such as integrin and growth factor signaling. In summary, at the cellular level KAI1/CD82 inhibited polarized protrusion and retraction events by disrupting actin reorganization; at the molecular level, KAI1/CD82 deregulated Rac1, RhoA, and their effectors cofilin and Rho kinase by perturbing the plasma membrane lipids.     

Desmoglein 3 acting as an upstream regulator of Rho GTPases, Rac-1/Cdc42 in the regulation of actin organisation and dynamics

Desmoglein 3 (Dsg3), a member of the desmoglein sub-family, serves as an adhesion molecule in desmosomes. Our previous study showed that overexpression of human Dsg3 in several epithelial lines induces formation of membrane protrusions, a phenotype suggestive of Rho GTPase activation. Here we examined the interaction between Dsg3 and actin in detail and showed that endogenous Dsg3 colocalises and interacts with actin, particularly the junctional actin in a Rac1-dependent manner. Ablation of Rac1 activity by dominant negative Rac1 mutant (N17Rac1) or the Rac1 specific inhibitor (NSC23766) directly disrupts the interaction between Dsg3 and actin. Assembly of the junctional actin at the cell borders is accompanied with enhanced levels of Dsg3, while inhibition of Dsg3 by RNAi results in profound changes in the organisation of actin cytoskeleton. In accordance, overexpression of Dsg3 results in a remarkable increase of Rac1 and Cdc42 activities and to a lesser extent, RhoA. The enhancements in Rho GTPases are accompanied by the pronounced actin-based membrane structures such as lamellipodia and filopodia, enhanced rate of actin turnover and cell polarisation. Together, our results reveal an important novel function for Dsg3 in promoting actin dynamics through regulating Rac1 and Cdc42 activation in epithelial cells.     

Functional analysis of the YopE GTPase-activating protein (GAP) activity of Yersinia pseudotuberculosis

YopE of Yersinia pseudotuberculosis inactivates three members of the small RhoGTPase family (RhoA, Rac1 and Cdc42) in vitro and mutation of a critical arginine abolishes both in vitro GTPase-activating protein (GAP) activity and cytotoxicity towards HeLa cells, and renders the pathogen avirulent in a mouse model. To understand the functional role of YopE, in vivo studies of the GAP activity in infected eukaryotic cells were conducted. Wild-type YopE inactivated Rac1 as early as 5 min after infection whereas RhoA was down regulated about 30 min after infection. No effect of YopE was found on the activation state of Cdc42 in Yersinia-infected cells. Single-amino-acid substitution mutants of YopE revealed two different phenotypes: (i) mutants with significantly lowered in vivo GAP activity towards RhoA and Rac1 displaying full virulence in mice, and (ii) avirulent mutants with wild-type in vivo GAP activity towards RhoA and Rac1. Our results show that Cdc42 is not an in vivo target for YopE and that YopE interacts preferentially with Rac1, and to a lesser extent with RhoA, during in vivo conditions. Surprisingly, we present results suggesting that these interactions are not a prerequisite to establish infection in mice. Finally, we show that avirulent yopE mutants translocate YopE in about sixfold higher amount compared with wild type. This raises the question whether YopE's primary function is to sense the level of translocation rather than being directly involved in downregulation of the host defence.