Localized zones of Rho and Rac activities drive initiation and expansion of epithelial cell-cell adhesion

Spatiotemporal coordination of cell-cell adhesion involving lamellipodial interactions, cadherin engagement, and the lateral expansion of the contact is poorly understood. Using high-resolution live-cell imaging, biosensors, and small molecule inhibitors, we investigate how Rac1 and RhoA regulate actin dynamics during de novo contact formation between pairs of epithelial cells. Active Rac1, the Arp2/3 complex, and lamellipodia are initially localized to de novo contacts but rapidly diminish as E-cadherin accumulates; further rounds of activation and down-regulation of Rac1 and Arp2/3 occur at the contacting membrane periphery, and this cycle repeats as a restricted membrane zone that moves outward with the expanding contact. The cortical bundle of actin filaments dissolves beneath the expanding contacts, leaving actin bundles at the contact edges. RhoA and actomyosin contractility are activated at the contact edges and are required to drive expansion and completion of cell-cell adhesion. We show that zones of Rac1 and lamellipodia activity and of RhoA and actomyosin contractility are restricted to the periphery of contacting membranes and together drive initiation, expansion, and completion of cell-cell adhesion.     

srGAP1 regulates lamellipodial dynamics and cell migratory behavior by modulating Rac1 activity

The distinct levels of Rac activity differentially regulate the pattern of intrinsic cell migration. However, it remains unknown how Rac activity is modulated and how the level of Rac activity controls cell migratory behavior. Here we show that Slit-Robo GAP 1 (srGAP1) is a modulator of Rac activity in locomotive cells. srGAP1 possesses a GAP activity specific to Rac1 and is recruited to lamellipodia in a Rac1-dependent manner. srGAP1 limits Rac1 activity and allows concomitant activation of Rac1 and RhoA, which are mutually inhibitory. When both GTPases are activated, the protrusive structures caused by Rac1-dependent actin reorganization are spatially restricted and periodically destabilized, causing ruffling by RhoA-induced actomyosin contractility. Depletion of srGAP1 overactivates Rac1 and inactivates RhoA, resulting in continuous spatiotemporal spreading of lamellipodia and a modal shift of intrinsic cell motility from random to directionally persistent. Thus srGAP1 is a key determinant of lamellipodial dynamics and cell migratory behavior.     

ROCK1 and 2 differentially regulate actomyosin organization to drive cell and synaptic polarity

RhoGTPases organize the actin cytoskeleton to generate diverse polarities, from front–back polarity in migrating cells to dendritic spine morphology in neurons. For example, RhoA through its effector kinase, RhoA kinase (ROCK), activates myosin II to form actomyosin filament bundles and large adhesions that locally inhibit and thereby polarize Rac1-driven actin polymerization to the protrusions of migratory fibroblasts and the head of dendritic spines. We have found that the two ROCK isoforms, ROCK1 and ROCK2, differentially regulate distinct molecular pathways downstream of RhoA, and their coordinated activities drive polarity in both cell migration and synapse formation. In particular, ROCK1 forms the stable actomyosin filament bundles that initiate front–back and dendritic spine polarity. In contrast, ROCK2 regulates contractile force and Rac1 activity at the leading edge of migratory cells and the spine head of neurons; it also specifically regulates cofilin-mediated actin remodeling that underlies the maturation of adhesions and the postsynaptic density of dendritic spines.     

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.     

The Abl-related gene tyrosine kinase acts through p190RhoGAP to inhibit actomyosin contractility and regulate focal adhesion dynamics upon adhesion to fibronectin

In migrating cells, actin polymerization promotes protrusion of the leading edge, whereas actomyosin contractility powers net cell body translocation. Although they promote F-actin-dependent protrusions of the cell periphery upon adhesion to fibronectin (FN), Abl family kinases inhibit cell migration on FN. We provide evidence here that the Abl-related gene (Arg/Abl2) kinase inhibits fibroblast migration by attenuating actomyosin contractility and regulating focal adhesion dynamics. arg-/- fibroblasts migrate at faster average speeds than wild-type (wt) cells, whereas Arg re-expression in these cells slows migration. Surprisingly, the faster migrating arg-/- fibroblasts have more prominent F-actin stress fibers and focal adhesions and exhibit increased actomyosin contractility relative to wt cells. Interestingly, Arg requires distinct functional domains to inhibit focal adhesions and actomyosin contractility. The kinase domain-containing Arg N-terminal half can act through the RhoA inhibitor p190RhoGAP to attenuate stress fiber formation and cell contractility. However, Arg requires both its kinase activity and its cytoskeleton-binding C-terminal half to fully inhibit focal adhesions. Although focal adhesions do not turn over efficiently in the trailing edge of arg-/- cells, the increased contractility of arg-/- cells tears the adhesions from the substrate, allowing for the faster migration observed in these cells. Together, our data strongly suggest that Arg inhibits cell migration by restricting actomyosin contractility and regulating its coupling to the substrate through focal adhesions.     

Rho family GTPases and neuronal growth cone remodelling: relationship between increased complexity induced by Cdc42Hs, Rac1, and acetylcholine and collapse induced by RhoA and lysophosphatidic acid.

Rho family GTPases have been assigned important roles in the formation of actin-based morphologies in nonneuronal cells. Here we show that microinjection of Cdc42Hs and Rac1 promoted formation of filopodia and lamellipodia in N1E-115 neuroblastoma growth cones and along neurites. These actin-containing structures were also induced by injection of Clostridium botulinum C3 exoenzyme, which abolishes RhoA-mediated functions such as neurite retraction. The C3 response was inhibited by coinjection with the dominant negative mutant Cdc42Hs(T17N), while the Cdc42Hs response could be competed by coinjection with RhoA. We also demonstrate that the neurotransmitter acetylcholine (ACh) can induce filopodia and lamellipodia on neuroblastoma growth cones via muscarinic ACh receptor activation, but only when applied in a concentration gradient. ACh-induced formation of filopodia and lamellipodia was inhibited by preinjection with the dominant negative mutants Cdc42Hs(T17N) and Rac1(T17N), respectively. Lysophosphatidic acid (LPA)-induced neurite retraction, which is mediated by RhoA, was inhibited by ACh, while C3 exoenzyme-mediated neurite outgrowth was inhibited by injection with Cdc42Hs(T17N) or Rac1(T17N). Together these results suggest that there is competition between the ACh- and LPA-induced morphological pathways mediated by Cdc42Hs and/or Rac1 and by RhoA, leading to either neurite development or collapse.     

Nonpolarized signaling reveals two distinct modes of 3D cell migration

We search in this paper for context-specific modes of three-dimensional (3D) cell migration using imaging for phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and active Rac1 and Cdc42 in primary fibroblasts migrating within different 3D environments. In 3D collagen, PIP3 and active Rac1 and Cdc42 were targeted to the leading edge, consistent with lamellipodia-based migration. In contrast, elongated cells migrating inside dermal explants and the cell-derived matrix (CDM) formed blunt, cylindrical protrusions, termed lobopodia, and Rac1, Cdc42, and PIP3 signaling was nonpolarized. Reducing RhoA, Rho-associated protein kinase (ROCK), or myosin II activity switched the cells to lamellipodia-based 3D migration. These modes of 3D migration were regulated by matrix physical properties. Specifically, experimentally modifying the elasticity of the CDM or collagen gels established that nonlinear elasticity supported lamellipodia-based migration, whereas linear elasticity switched cells to lobopodia-based migration. Thus, the relative polarization of intracellular signaling identifies two distinct modes of 3D cell migration governed intrinsically by RhoA, ROCK, and myosin II and extrinsically by the elastic behavior of the 3D extracellular matrix.     

Integrin engagement suppresses RhoA activity via a c-Src-dependent mechanism

The Rho family GTPases Cdc42, Rac1 and RhoA control many of the changes in the actin cytoskeleton that are triggered when growth factor receptors and integrins bind their ligands [1] [2]. Rac1 and Cdc42 stimulate the formation of protrusive structures such as membrane ruffles, lamellipodia and filopodia. RhoA regulates contractility and assembly of actin stress fibers and focal adhesions. Although prolonged integrin engagement can stimulate RhoA [3] [4] [5], regulation of this GTPase by early integrin-mediated signals is poorly understood. Here we show that integrin engagement initially inactivates RhoA, in a c-Src-dependent manner, but has no effect on Cdc42 or Rac1 activity. Additionally, early integrin signaling induces activation and tyrosine phosphorylation of p190RhoGAP via a mechanism that requires c-Src. Dynamic modulation of RhoA activity appears to have a role in motility, as both inhibition and activation of RhoA hinder migration [6] [7] [8]. Transient suppression of RhoA by integrins may alleviate contractile forces that would otherwise impede protrusion at the leading edge of migrating cells.     

Involvement of Local Lamellipodia in Endothelial Barrier Function

Recently we observed that endothelial cells cultured in tightly confluent monolayers display frequent local lamellipodia, and that thrombin, an agent that increases endothelial permeability, reduces lamellipodia protrusions. This led us to test the hypothesis that local lamellipodia contribute to endothelial barrier function. Movements of subcellular structures containing GFP-actin or VE-cadherin-GFP expressed in endothelial cells were recorded using time-lapse microscopy. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function. Changes in both lamellipodia dynamics and TER were assessed during baseline and after cells were treated with either the barrier-disrupting agent thrombin, or the barrier-stabilizing agent sphingosine-1-phosphate (S1P). The myosin II inhibitor blebbistatin was used to selectively block lamellipodia formation, and was used to test their role in the barrier function of endothelial cell monolayers and isolated, perfused rat mesenteric venules. Myosin light chain (MLC) phosphorylation was assessed by immunofluorescence microscopy. Rac1 and RhoA activation were evaluated using G-LISA assays. The role of Rac1 was tested with the specific inhibitor NSC23766 or by expressing wild-type or dominant negative GFP-Rac1. The results show that thrombin rapidly decreased both TER and the lamellipodia protrusion frequency. S1P rapidly increased TER in association with increased protrusion frequency. Blebbistatin nearly abolished local lamellipodia protrusions while cortical actin fibers and stress fibers remained intact. Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules. Both thrombin and S1P increased MLC phosphorylation and activation of RhoA. However, thrombin and S1P had differential impacts on Rac1, correlating with the changes in TER and lamellipodia protrusion frequency. Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity. Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.     

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.     

Distinct functions of Rho and Rac are required for convergent extension during Xenopus gastrulation

We have undertaken the first detailed analysis of Rho GTPase function during vertebrate development by analyzing how RhoA and Rac1 control convergent extension of axial mesoderm during Xenopus gastrulation. Monitoring of a number of parameters in time-lapse recordings of mesoderm explants revealed that Rac and Rho have both distinct and overlapping roles in regulating the motility of axial mesoderm cells. The cell behaviors revealed by activated or inhibitory versions of these GTPases in native tissue were clearly distinct from those previously documented in cultured fibroblasts. The dynamic properties and polarity of protrusive activity, along with lamellipodia formation, were controlled by the two GTPases operating in a partially redundant manner, while Rho and Rac contributed separately to cell shape and filopodia formation. We propose that Rho and Rac operate in distinct signaling pathways that are integrated to control cell motility during convergent extension.     

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.     

Regulator of G-protein signalling 3 redirects prototypical Gi-coupled receptors from Rac1 to RhoA activation

The small GTPases, Rac1 and RhoA, are pivotal regulators of several essential, but distinct cellular processes. Numerous G-protein-coupled receptors signal to these GTPases, but with different specificities. Specifically, Gi-coupled receptors (GiPCRs) are generally believed to activate Rac1, but not RhoA, a process involving Gbetagamma-dimers and phosphatidylinositol 3-kinase (PI3K). Here we show that, depending on the expression level of the 519 amino acid isoform of regulator of G-protein signalling 3 (RGS3L), prototypical GiPCRs, like M2 muscarinic, A1 adenosine, and alpha2-adrenergic receptors, activate either Rac1 or RhoA in human embryonic kidney cells and neonatal rat cardiomyocyte-derived H10 cells. The switch from Rac1 to RhoA activation in H10 cells was controlled by fibroblast growth factor-2 (FGF-2), lowering the expression of RGS3L. Activation of both, Rac1 and RhoA, seen at low and high expression levels of RGS3L, respectively, was sensitive to pertussis toxin and the PI3K inhibitor LY294002 and mediated by Gbetagamma-dimers. We conclude that RGS3L functions as a molecular switch, redirecting GiPCRs via Gbetagamma-dimers and PI3K from Rac1 to RhoA activation. Considering the essential roles of Rac1 and RhoA in many signalling pathways, this additional function of RGS3L indicates a specific role of this protein in cellular signalling networks.     

Spike formation by fibroblasts adhering to fibrillar collagen I gel

The fibrillar collagen I gel induced the formation of numerous dendritic cell-like protrusions (cell spikes) from the cell body, whereas monomeric collagen I induced typical cell spreading with filopodia and lamellipodia in skin fibroblasts. Peripheral, not central stress fibers appeared upon adhesion to fibrillar collagen gel, whereas both types of fibers were evident upon adhesion to monomeric collagen. Microtubules and vimentin filaments were elongated inside stress fibers along the terminal tip of cell spikes. Spike formation was totally inhibited by nocodazole and severely delayed by cytochalasin D. This suggests that cell spike formation is dependent on microtubules rather than on F-actin. We then investigated the intracellular signaling responsible for cytoskeleton organization to identify the key factor that induces cell spike morphology. During cell spike formation, FAK and CAS were activated. More CAS was activated in cells on fibrillar collagen gel than on the monomeric form, whereas FAK was activated to the same level on either. At 90 min of culture, Rac1 was activated in cells on monomeric collagen I, whereas Cdc42, Rac1 and RhoA were activated in cells on fibrillar collagen gel. These results suggest that microtubule organization via CAS and small GTPases is important for the cell spike formation that is involved in collagen gel contraction and in wound retraction in skin.     

RhoA inhibits the nerve growth factor-induced Rac1 activation through Rho-associated kinase-dependent pathway

The Rho family of small GTPases has been shown to be involved in the regulation of neuronal morphology, and Rac and Rho exert antagonistic actions in neurite formation. In this study, we have examined the cross-talk between Rac and Rho in relation to the nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. NGF induced a rapid activation of Rac1 and suppression of RhoA activity. Constitutively active RhoA, RhoA(V14), or constitutively active Galpha(12)-induced endogenous RhoA activation inhibited the NGF-induced Rac1 activation without any effect on the NGF-induced extracellular signal-regulated kinase activation. Moreover, Y-27632, an inhibitor of Rho-associated kinase, completely abolished the RhoA-induced down-regulation of the NGF-induced Rac1 activation. We also revealed that NGF induced a rapid recruitment of Rac1 to the cell surface protrusion sites and formed filamentous actin-rich protrusions. Activation of RhoA and Rho-associated kinase formed a thick ringlike structure of cortical actin filaments at the cell periphery and then inhibited the NGF-induced recruitment of Rac1 to protrusions. These results indicate that RhoA down-regulates the NGF- induced Rac1 activation through Rho-associated kinase, inhibiting the neurite formation.     

Localized RhoA activation as a requirement for the induction of membrane ruffling

We examined the spatio-temporal activity of RhoA in migrating cells and growth factor-stimulated cells by using probes based on the principle of fluorescence resonance energy transfer. In HeLa cells migrating at a low cell density, RhoA was activated both at the contractile tail and at the leading edge. However, RhoA was activated only at the leading edge in MDCK cells migrating as a monolayer sheet. In growth factor-stimulated Cos1 and NIH3T3 cells, the activity of RhoA was greatly decreased at the plasma membrane, but remained high at the membrane ruffles in nascent lamellipodia. These observations are in agreement with the proposed role played by RhoA in stress fiber formation, but they also implicated RhoA in the regulation of membrane ruffling, the induction of which is a typical phenotype of activated Rac. In agreement with this view, dominant negative RhoA was found to inhibit membrane ruffling induced by active Rac. Furthermore, we found that Cdc42 activity was also required for high RhoA activity in membrane ruffles. Finally, we found that mDia1, but not ROCK, was stably associated with membrane ruffles. In conclusion, these results suggested that RhoA cooperates with Rac1 and Cdc42 to induce membrane ruffles via the recruitment of mDia.     

Interplay between Rac and Rho in the control of substrate contact dynamics.

BACKGROUND: Substrate anchorage and cell locomotion entail the initiation and development of different classes of contact sites, which are associated with the different compartments of the actin cytoskeleton. The Rho-family GTPases are implicated in the signalling pathways that dictate contact initiation, maturation and turnover, but their individual roles in these processes remain to be defined. RESULTS: We monitored the dynamics of peripheral, Rac-induced focal complexes in living cells in response to perturbations of Rac and Rho activity and myosin contractility. We show that focal complexes formed in response to Rac differentiated into focal contacts upon upregulation of Rho. Focal complexes were dissociated by inhibitors of myosin-II-dependent contractility but not by an inhibitor of Rho-kinase. The downregulation of Rac promoted the enlargement of focal contacts, whereas a block in the Rho pathway not only caused a dissolution of focal contacts but also stimulated membrane ruffling and formation of new focal complexes, which were associated with the advance of the cell front. CONCLUSIONS: Rac functions to signal the creation of new substrate contacts at the cell front, which are associated with the induction of ruffling lamellipodia, whereas Rho serves in the maturation of existing contacts, with both contact types requiring contractility for their formation. The transition from a focal complex to a focal contact is associated with a switch to Rho-kinase dependence. Rac and Rho also influence the development of focal contacts and focal complexes, respectively, through mutually antagonistic pathways.     

A quantitative description of the extension and retraction of surface protrusions in spreading 3T3 mouse fibroblasts.

We suggest a method of quantitating the motile actions of surface protrusions in spreading animal cells in culture. Its basis is the determination of the percentage of freshly plated cells which produce particle-free areas around them on a gold particle-coated glass cover slip within 50 min. Studying 3T3 cells with this assay, we found that the presence of Na+, K+, Cl-, and Mg++ or Ca++ in a neutral or slightly alkaline phosphate or bicarbonate buffered solution is sufficient to support the optimal particle removal by the cells for at least 50 min. Two metabolic inhibitors, 2,4-dinitrophenol and Na-azide, inhibit the particle removal. If D-glucose is added along with the inhibitors, particle removal can be restored, whereas the addition of three glucose analogues which are generally believed to be nonmetabolizable cannot restore the activity. Serum is not required for the mechanism(s) of the motile actions of surface protrusions in spreading 3T3 cells. However, it contains components which can neutralize the inhibitory actions of bovine serum albumin and several amino acids, particularly L-cystine or L-cystein and L-methionine. Furthermore, serum codetermines which of the major surface extension, filopodia, lamellipodia, or lobopodia, is predominantly active. We found three distinct classes of extracellular conditions under which the active surface projections are predominantly either lamellipodia, (sheetlike projections), lobopodia (blebs), or filopodia (microspikes). The quantitated dependencies on temperature, pH and the inhibition by cytochalasin B or the particle removal are very similar in all three cases. Preventing the cells from anchoring themselves for 15-20 min before plating in serum-free medium seems to stimulate particle removal threefold.