Rho GTPases as targets of bacterial protein toxins

Several bacterial toxins target Rho GTPases, which constitute molecular switches in several signaling processes and master regulators of the actin cytoskeleton. The biological activities of Rho GTPases are blocked by C3-like transferases, which ADP-ribosylate Rho at Asn41, but not Rac or Cdc42. Large clostridial cytotoxins (e. g., Clostridium difficile toxin A and B) glucosylate Rho GTPases at Thr37 (Rho) or Thr35 (Rac/Cdc42), thereby inhibiting Rho functions by preventing effector coupling. The 'injected' toxins ExoS, YopE and SptP from Pseudomonas aeruginosa, Yersinia and Salmonella ssp., respectively, which are transferred into the eukaryotic target cells by the type-III secretion system, inhibit Rho functions by acting as Rho GAP proteins. Rho GTPases are activated by the cytotoxic necrotizing factors CNF1 and CNF2 from Escherichia coli and by the dermonecrotizing toxin DNT from B. bronchiseptica. These toxins deamidate/transglutaminate Gln63 of Rho to block the intrinsic and GAP-stimulated GTP hydrolysis, thereby constitutively activating the GTPases. Rho GTPases are also activated by SopE, a type-III system injected protein from Salmonella ssp., that acts as a GEF protein.     

Cytotoxic necrotizing factor 1 hinders skeletal muscle differentiation in vitro by perturbing the activation/deactivation balance of Rho GTPases

The current knowledge assigns a crucial role to the Rho GTPases family (Rho, Rac, Cdc42) in the complex transductive pathway leading to skeletal muscle cell differentiation. Their exact function in myogenesis, however, remains largely undefined. The protein toxin CNF1 was herein employed as a tool to activate Rho, Rac and Cdc42 in the myogenic cell line C2C12. We demonstrated that CNF1 impaired myogenesis by affecting the muscle regulatory factors MyoD and myogenin and the structural protein MHC expressions. This was principally driven by Rac/Cdc42 activation whereas Rho apparently controlled only the fusion process. More importantly, we proved that a controlled balance between Rho and Rac/Cdc42 activation/deactivation state was crucial for the correct execution of the differentiation program, thus providing a novel view for the role of Rho GTPases in muscle cell differentiation. Also, the use of Rho hijacking toxins can represent a new strategy to pharmacologically influence the differentiative process.     

Rnd1, a novel rho family GTPase, induces the formation of neuritic processes in PC12 cells

Rho family GTPases have been shown to be involved in the regulation of neuronal cell morphology, including neurite extension and retraction. Rho activation leads to neurite retraction and cell rounding, whereas Rac and Cdc42 are implicated in the promotion of filopodia and lamellipodia formation in growth cones and, therefore, in neurite extension. In this study, we examined the morphological role of Rnd1, a new member of Rho family GTPases, in PC12 cells, and found that expression of Rnd1 by itself caused the formation of many neuritic processes from the cell body with disruption of the cortical actin filaments, the processes having microtubules but few filamentous actin and neurofilaments. Treatment with cytochalasin D, an inhibitor of actin polymerization, could mimic the effects of expression of Rnd1, in that this inhibitor disrupted the cortical actin filaments and induced the formation of many thin processes containing microtubules. The process formation induced by Rnd1 was inhibited by dominant negative Rac1. These results suggest that Rnd1 induces the Rac-dependent neuritic process formation in part by disruption of the cortical actin filaments.     

Identification of the region of rho involved in substrate recognition by Escherichia coli cytotoxic necrotizing factor 1 (CNF1).

The Escherichia coli cytotoxic necrotizing factor 1 (CNF1) and the Bordetella dermonecrotic toxin (DNT) activate Rho GTPases by deamidation of Gln(63) of RhoA (Gln(61) of Cdc42 and Rac). In addition, both toxins possess in vitro transglutaminase activity in the presence of primary amines. Here we characterized the region of Rho essential for substrate recognition by the toxins using Rho/Ras chimeras as protein substrates. The chimeric protein Ras55Rho was deamidated or transglutaminated by CNF1. Rat pheochromocytoma PC12 cells microinjected with Ras55Rho developed formation of neurite-like structures after treatment with the CNF1 holotoxin indicating activation of the Ha-Ras chimera and Ras-like effects in intact cells. The Ras59Rho78Ras chimera protein contained the minimal Rho sequence allowing deamidation or transglutamination by CNF1. A peptide covering mainly the switch II region and consisting of amino acid residues Asp(59) through Asp(78) of RhoA was substrate for CNF1. Changes of amino acid residues Arg(68) or Leu(72) of RhoA into the corresponding residues of Ras (R68ARhoA and L72QRhoA) inhibited deamidation and transglutamination of the mutants by CNF1. In contrast to CNF1, DNT did not modify Rho/Ras chimeras or the switch II peptide (Asp(59) through Asp(78)). Glucosylation of RhoA at Thr(37) blocked deamidation by DNT but not by CNF. The data indicate that CNF1 recognizes Rho GTPases exclusively in the switch II region, whereas the substrate recognition by DNT is characterized by additional structural requirements.     

Inactivation of small Rho GTPases by the multifunctional RTX toxin from Vibrio cholerae

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.     

Structure of the Rho-activating domain of Escherichia coli cytotoxic necrotizing factor 1.

Certain uropathogenic and neonatal meningitis-causing strains of Escherichia coli express a 114 kDa protein toxin called cytotoxic necrotizing factor 1 (CNF1). The toxin causes alteration of the host cell actin cytoskeleton and promotes bacterial invasion of blood-brain barrier endothelial cells. CNF1 belongs to a unique group of large cytotoxins that cause constitutive activation of Rho guanosine triphosphatases (GTPases), which are key regulators of the actin cytoskeleton. This group also includes E. coli cytotoxic necrotizing factor 2 (CNF2, 114 kDa) and dermonecrotic toxins (DNT, 159 kDa) of Bordetella spp. with related sequences occurring in Yersinia spp. Here we show that the catalytic region of CNF1 exhibits a novel protein fold as determined by its 1.83 A resolution crystal structure. The structure reveals that CNF1 has a Cys-His-main chain oxygen catalytic triad reminiscent of enzymes belonging to the catalytic triad superfamily. The position of the catalytic Cys residue at the base of a deep pocket restricts access to potential substrates and helps explain the high specificity of this and related toxins.     

CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion

CNF1 toxin is a virulence factor produced by uropathogenic Escherichia coli. Upon cell binding and introduction into the cytosol, CNF1 deamidates glutamine 63 of RhoA (or 61 of Rac and Cdc42), rendering constitutively active these GTPases. Unexpectedly, we measured in bladder cells a transient CNF1-induced activation of Rho GTPases, maximal for Rac. Deactivation of Rac correlated with the increased susceptibility of its deamidated form to ubiquitin/proteasome-mediated degradation. Sensitivity to ubiquitylation could be generalized to other permanent-activated forms of Rac and to its sustained activation by Dbl. Degradation of the toxin-activated Rac allowed both host cell motility and efficient cell invasion by uropathogenic bacteria. CNF1 toxicity thus results from a restricted activation of Rho GTPases through hijacking the host cell proteasomal machinery.     

Activation of Rho GTPases by Cytotoxic Necrotizing Factor 1 Induces Macropinocytosis and Scavenging Activity in Epithelial Cells

Macropinocytosis, a ruffling-driven process that allows the capture of large material, is an essential aspect of normal cell function. It can be either constitutive, as in professional phagocytes where it ends with the digestion of captured material, or induced, as in epithelial cells stimulated by growth factors. In this case, the internalized material recycles back to the cell surface. We herein show that activation of Rho GTPases by a bacterial protein toxin, the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), allowed epithelial cells to engulf and digest apoptotic cells in a manner similar to that of professional phagocytes. In particular, we have demonstrated that 1) the activation of all Rho, Rac, and Cdc42 by CNF1 was essential for the capture and internalization of apoptotic cells; and 2) such activation allowed the discharge of macropinosomal content into Rab7 and lysosomal associated membrane protein-1 acidic lysosomal vesicles where the ingested particles underwent degradation. Taken together, these findings indicate that CNF1-induced "switching on" of Rho GTPases may induce in epithelial cells a scavenging activity, comparable to that exerted by professional phagocytes. The activation of such activity in epithelial cells may be relevant, in mucosal tissues, in supporting or integrating the scavenging activity of resident macrophages.     

The Rho-mDia1 pathway regulates cell polarity and focal adhesion turnover in migrating cells through mobilizing Apc and c-Src

Directed cell migration requires cell polarization and adhesion turnover, in which the actin cytoskeleton and microtubules work critically. The Rho GTPases induce specific types of actin cytoskeleton and regulate microtubule dynamics. In migrating cells, Cdc42 regulates cell polarity and Rac works in membrane protrusion. However, the role of Rho in migration is little known. Rho acts on two major effectors, ROCK and mDia1, among which mDia1 produces straight actin filaments and aligns microtubules. Here we depleted mDia1 by RNA interference and found that mDia1 depletion impaired directed migration of rat C6 glioma cells by inhibiting both cell polarization and adhesion turnover. Apc and active Cdc42, which work together for cell polarization, localized in the front of migrating cells, while active c-Src, which regulates adhesion turnover, localized in focal adhesions. mDia1 depletion impaired localization of these molecules at their respective sites. Conversely, expression of active mDia1 facilitated microtubule-dependent accumulation of Apc and active Cdc42 in the polar ends of the cells and actin-dependent recruitment of c-Src in adhesions. Thus, the Rho-mDia1 pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and delivering Apc/Cdc42 and c-Src to their respective sites of action.     

An Escherichia coli cytotoxin increases superoxide anion generation via rac in epithelial cells

Cytotoxic Necrotizing Factor 1 (CNF1) is a protein toxin from Escherichia coli that induces the activation of Rho, Rac, and Cdc42 GTPases, all involved in actin reorganization. Rac plays a further role in oxidase function. In epithelial cells, CNF1 has been reported to induce a phagocytic-like behavior in terms of a ruffle-driven ingestion of large material. We herein show that CNF1-activated epithelial cells may exert additional cell responses typical of professional phagocytes following stimulation, i.e., an increase in oxygen consumption and the generation of superoxide anions. Such effects were triggered by the contact of latex beads with epithelial cells and were significantly augmented by CNF1-induced Rac activation. Altogether our data indicate that Rac, one of the targets of CNF1, plays a pivotal role in these phenomena, suggesting the involvement in epithelial cells of a Rac-dependent NADPH-oxidase complex similar to that employed by professional phagocytes.     

Ultrastructural study of Rac1 and its effectors beneath the substratum-facing membrane

The cytoskeletal architecture and adhesion apparatus are tightly controlled during embryogenesis, tissue development, and carcinogenesis. The Rho family GTPases play central roles in regulation of the cytoskeleton and adhesions. Rac1, one of the Rho family GTPases, appears to be activated at the plasma membrane and exert its functions through its effectors. However, where Rac1 and its effectors function at the molecular level remains to be determined. In this study, we examined the molecular organization on the cytoplasmic surface of the substratum-facing plasma membrane, focusing on Rac1 and its effectors, IQGAP1 and Sra-1, by electron microscopy. We employed deep-etch immunoreplica methods to observe the membrane cytoskeletal architecture while determining molecular locations. Beneath the plasma membrane, Rac1 and its effectors showed similar, but distinct, destinations. Rac1 localized on the membrane and associated with the membrane cytoskeleton. IQGAP1 predominantly localized beside actin filaments and occasionally near microtubules together with Rac1. On the other hand, Sra-1 localized at actin filaments, microtubules, and the plasma membrane. Sra-1 colabeled with Rac1 was mainly found at the membrane and actin filaments. These results suggest that IQGAP1 and Sra-1 colocalize with Rac1 at distinct places, including the plasma membrane and cytoskeletal architecture, for their specific functions.     

The Small GTPases Rho and Rac Are Required for the Establishment of Cadherin-dependent Cell–Cell Contacts

Cadherins are calcium-dependent cell–cell adhesion molecules that require the interaction of the cytoplasmic tail with the actin cytoskeleton for adhesive activity. Because of the functional relationship between cadherin receptors and actin filament organization, we investigated whether members of the Rho family of small GTPases are necessary for cadherin adhesion. In fibroblasts, the Rho family members Rho and Rac regulate actin polymerization to produce stress fibers and lamellipodia, respectively. In epithelial cells, we demonstrate that Rho and Rac are required for the establishment of cadherin-mediated cell–cell adhesion and the actin reorganization necessary to stabilize the receptors at sites of intercellular junctions. Blocking endogenous Rho or Rac selectively removed cadherin complexes from junctions induced for up to 3 h, while desmosomes were not perturbed. In addition, withdrawal of cadherins from intercellular junctions temporally precedes the removal of CD44 and integrins, other microfilament-associated receptors. Our data showed that the concerted action of Rho and Rac modulate the establishment of cadherin adhesion: a constitutively active form of Rac was not sufficient to stabilize cadherindependent cell–cell contacts when endogenous Rho was inhibited. Upon induction of calcium-dependent intercellular adhesion, there was a rapid accumulation of actin at sites of cell–cell contacts, which was prevented by blocking cadherin function, Rho or Rac activity. However, if cadherin complexes are clustered by specific antibodies attached to beads, actin recruitment to the receptors was perturbed by inhibiting Rac but not Rho. Our results provide new insights into the role of the small GTPases in the cadherin-dependent cell– cell contact formation and the remodelling of actin filaments in epithelial cells.     

Escherichia coli producing CNF1 toxin hijacks Tollip to trigger Rac1-dependent cell invasion

Rho GTPases, which are master regulators of both the actin cytoskeleton and membrane trafficking, are often hijacked by pathogens to enable their invasion of host cells. Here we report that the cytotoxic necrotizing factor-1 (CNF1) toxin of uropathogenic Escherichia coli (UPEC) promotes Rac1-dependent entry of bacteria into host cells. Our screen for proteins involved in Rac1-dependent UPEC entry identifies the Toll-interacting protein (Tollip) as a new interacting protein of Rac1 and its ubiquitinated forms. We show that knockdown of Tollip reduces CNF1-induced Rac1-dependent UPEC entry. Tollip depletion also reduces the Rac1-dependent entry of Listeria monocytogenes expressing InlB invasion protein. Moreover, knockdown of Tollip, Tom1 and clathrin, decreases CNF1 and Rac1-dependent internalization of UPEC. Finally, we show that Tollip, Tom1 and clathrin associate with Rac1 and localize at the site of bacterial entry. Collectively, these findings reveal a new link between Rac1 and Tollip, Tom1 and clathrin membrane trafficking components hijacked by pathogenic bacteria to allow their efficient invasion of host cells.     

FHOD1 coordinates actin filament and microtubule alignment to mediate cell elongation

Diaphanous-related formins (DRFs) are actin nucleators that mediate rearrangements of the actin cytoskeleton downstream of specific Rho GTPases. The DRF Formin Homology 2 Domain containing 1 (FHOD1) interacts with the Rac1 GTPase and induces the formation of and associates with bundled actin stress fibers. Here we report that active FHOD1 also coordinates microtubules with these actin stress fibers. Expression of a constitutive active FHOD1 variant in HeLa cells not only resulted in pronounced formation of FHOD1-actin fibers but also caused marked cell elongation and parallel alignment of microtubules without affecting cytokinesis of these cells. The analysis of deletions in the FH1 and FH2 functional regions revealed that the integrity of both domains was strictly required for FHOD1's effects on the cytoskeleton. Dominant-negative approaches demonstrated that filament coordination and cell elongation depended on the activity of the Rho-ROCK cascade, but did not involve Rac or Cdc42 activity. Experimental depolymerization of actin filaments or microtubules revealed that the formation of FHOD1-actin fibers was a prerequisite for the polarization of microtubules. However, only simultaneous disruption of both filament systems reversed the cell elongation induced by activated FHOD1. Thus, sustained cell elongation was a consequence of FHOD1-mediated actin-microtubule coordination. These results suggest filament coordination as a conserved function of mammalian DRFs.     

Exploiting Cell Death Pathways by an E. coli Cytotoxin: Autophagy as a Double-Edged Sword for the Host

Cytotoxic necrotizing factor 1 is a bacterial protein toxin from Escherichia coli that is able to activate the Rho GTPases and to hinder apoptosis and mitotic catastrophe. Upon exposure to toxin, cells undergo a complex framework of changes, including cytoskeleton remodeling and multinucleation. These cells also show a high survival rate for long periods of time and improve both their macropinocytotic scavenging activities and microautophagy. Only at the very end, probably when “feeding” materials are exhausted, they do these cells die by autophagy. Taking into account the complex role of bacterial protein toxins in the infectious processes, we indicate the CNF1 activity as a Janus-faced paradigm of those bacteria that hijack cell fate to their own benefit. This could somehow be linked to the hypothesized connection between certain bacterial toxins and cancer onset.

Addendum to:

Is the Rac GTPase-Activating Toxin CNF1 a Smart Hijacker of Host Cell Fate?

W. Malorni and C. Fiorentini

FASEB J 2006; 20:606-9     

Functional proteomics identifies protein-tyrosine phosphatase 1B as a target of RhoA signaling

Rho GTPases are signal transduction effectors that control cell motility, cell attachment, and cell shape by the control of actin polymerization and tyrosine phosphorylation. To identify cellular targets regulated by Rho GTPases, we screened global protein responses to Rac1, Cdc42, and RhoA activation by two-dimensional gel electrophoresis and mass spectrometry. A total of 22 targets were identified of which 19 had never been previously linked to Rho GTPase pathways, providing novel insight into pathway function. One novel target of RhoA was protein-tyrosine phosphatase 1B (PTP1B), which catalyzes dephosphorylation of key signaling molecules in response to activation of diverse pathways. Subsequent analysis demonstrated that RhoA enhances post-translational modification of PTP1B, inactivates phosphotyrosine phosphatase activity, and up-regulates tyrosine phosphorylation of p130Cas, a key mediator of focal adhesion turnover and cell migration. Thus, protein profiling reveals a novel role for PTP1B as a mediator of RhoA-dependent phosphorylation of p130Cas.     

The RhoGAP activity of the Yersinia pseudotuberculosis cytotoxin YopE is required for antiphagocytic function and virulence

A variety of pathogenic bacteria use type III secretion pathways to translocate virulence proteins into host eukaryotic cells. YopE is an important virulence factor that is translocated into mammalian cells via a plasmid-encoded type III system in Yersinia spp. YopE action in mammalian cells promotes the disruption of actin filaments, cell rounding and blockage of phagocytosis. It was reported recently that two proteins with sequence similarity to YopE, SptP of Salmonella typhimurium and ExoS of Pseudomonas aeruginosa, function as GTPase-activating proteins (GAPs) for Rho GTPases. YopE contains an 'arginine finger' motif that is present in SptP, ExoS and other Rho GAPs and is essential for catalysis by this class of proteins. We show here that a GST-YopE fusion protein stimulated in vitro GTP hydrolysis by the Rho family members Cdc42, RhoA and Rac1, but not by Ras. Conversion of the essential arginine in the arginine finger motif to alanine (R144A) eliminated the in vitro GAP activity of GST-YopE. Infection assays carried out with a Yersinia pseudotuberculosis strain producing YopER144A demonstrated that GAP function was essential for the disruption of actin filaments, cell rounding and inhibition of phagocytosis by YopE in HeLa cells. Furthermore, the GAP function of YopE was important for Y. pseudotuberculosis pathogenesis in a mouse infection assay. Transfection of HeLa cells with a vector that produces a constitutively active form of RhoA (RhoA-V14) prevented the disruption of actin filaments and cell rounding by YopE. Production of an activated form of Rac1 (Rac1-V12), but not RhoA-V14, in HeLa cells interfered with YopE antiphagocytic activity. These results demonstrate that YopE functions as a RhoGAP to downregulate multiple Rho GTPases, leading to the disruption of actin filaments and inhibition of bacterial uptake into host cells.     

Kaposi's sarcoma-associated herpesvirus modulates microtubule dynamics via RhoA-GTP-diaphanous 2 signaling and utilizes the dynein motors to deliver its DNA to the nucleus

Human herpesvirus 8 (HHV-8; also called Kaposi's sarcoma-associated herpesvirus), which is implicated in the pathogenesis of Kaposi's sarcoma (KS) and lymphoproliferative disorders, infects a variety of target cells both in vivo and in vitro. HHV-8 binds to several in vitro target cells via cell surface heparan sulfate and utilizes the alpha3beta1 integrin as one of its entry receptors. Interactions with cell surface molecules induce the activation of host cell signaling cascades and cytoskeletal changes (P. P. Naranatt, S. M. Akula, C. A. Zien, H. H. Krishnan, and B. Chandran, J. Virol. 77:1524-1539, 2003). However, the mechanism by which the HHV-8-induced signaling pathway facilitates the complex events associated with the internalization and nuclear trafficking of internalized viral DNA is as yet undefined. Here we examined the role of HHV-8-induced cytoskeletal dynamics in the infectious process and their interlinkage with signaling pathways. The depolymerization of microtubules did not affect HHV-8 binding and internalization, but it inhibited the nuclear delivery of viral DNA and infection. In contrast, the depolymerization of actin microfilaments did not have any effect on virus binding, entry, nuclear delivery, or infection. Early during infection, HHV-8 induced the acetylation of microtubules and the activation of the RhoA and Rac1 GTPases. The inactivation of Rho GTPases by Clostridium difficile toxin B significantly reduced microtubular acetylation and the delivery of viral DNA to the nucleus. In contrast, the activation of Rho GTPases by Escherichia coli cytotoxic necrotizing factor significantly augmented the nuclear delivery of viral DNA. Among the Rho GTPase-induced downstream effector molecules known to stabilize the microtubules, the activation of RhoA-GTP-dependent diaphanous 2 was observed, with no significant activation in the Rac- and Cdc42-dependent PAK1/2 and stathmin molecules. The nuclear delivery of viral DNA increased in cells expressing a constitutively active RhoA mutant and decreased in cells expressing a dominant-negative mutant of RhoA. HHV-8 capsids colocalized with the microtubules, as observed by confocal microscopic examination, and the colocalization was abolished by the destabilization of microtubules with nocodazole and by the phosphatidylinositol 3-kinase inhibitor affecting the Rho GTPases. These results suggest that HHV-8 induces Rho GTPases, and in doing so, modulates microtubules and promotes the trafficking of viral capsids and the establishment of infection. This is the first demonstration of virus-induced host cell signaling pathways in the modulation of microtubule dynamics and in the trafficking of viral DNA to the infected cell nucleus. These results further support our hypothesis that HHV-8 manipulates the host cell signaling pathway to create an appropriate intracellular environment that is conducive to the establishment of a successful infection.     

Regulation of Cadherin Function by Rho and Rac: Modulation by Junction Maturation and Cellular Context

Cadherins are cell–cell adhesion receptors whose adhesive function requires their association with the actin cytoskeleton via proteins called catenins. The small guanosine triphosphatases (GTPases), Rho and Rac, are intracellular proteins that regulate the formation of distinct actin structures in different cell types. In keratinocytes and in other epithelial cells, Rho and Rac activities are required for E-cadherin function. Here we show that the regulation of cadherin adhesiveness by the small GTPases is influenced by the maturation status of the junction and the cellular context. E-cadherin localization was disrupted in mature keratinocyte junctions after inhibition of Rho and Rac. However, an incubation of 2 h was required after GTPase inhibition, when compared with newly established E-cadherin contacts (30 min). Regarding other cadherin receptors, P-cadherin was effectively removed from mature keratinocytes junctions by blocking Rho or Rac. In contrast, VE-cadherin localization at endothelial junctions was independent of Rho/Rac activity. We demontrate that the insensitivity of VE-cadherin to inhibition of Rho and Rac was not due to the maturation status of endothelial junction, but rather the cellular background: when transfected into CHO cells, the localization of VE-cadherin was perturbed by inhibition of Rho proteins. Our results suggest that the same stimuli may have different activity in regulating the paracellular activity in endothelial and epithelial cells. In addition, we uncovered possible roles for the small GTPases during the establishment of E-cadherin–dependent contacts. In keratinocytes, Rac activation by itself cannot promote accumulation of actin at the cell periphery in the absence of cadherin-dependent contacts. Moreover, neither Rho nor Rac activation was sufficient to redistribute cadherin molecules to cell borders, indicating that redistribution results mostly from the homophilic binding of the receptors. Our results point out the complexity of the regulation of cadherin-mediated adhesion by the small GTPases, Rho and Rac.