Role of focal adhesion formation in migration and morphogenesis of endothelial cells

Cell motility and morphogenesis are regulated by a balance between formation and disassembly of stress fibers and focal adhesions. To understand the mechanisms underlying these cellular responses in angiogenesis, we studied the Rho family protein-driven pathways in FGF-2-induced chemotaxis and capillary morphogenesis of murine brain capillary endothelial cell line, IBE cells. Cells seeded onto fibronectin-coated surface migrated toward FGF-2. Expression of dominant negative Rho A (DNRho) or kinase-dead p21-activated kinase 1 (KDPAK1), or treatment with Y27632 inhibited chemotaxis in association with the lack of FGF-2-induced decrease in focal adhesions. On Matrigel, DNRho and Y27632 induced FGF-2-independent capillary morphogenesis despite loss of stress fiber formation. KDPAK1 cells formed stress fibers and showed capillary morphogenesis in response to FGF-2. Increase in focal adhesions was closely associated with capillary morphogenesis. Our results suggest that formation or disassembly of focal adhesions seems to determine the motility or morphogenesis of endothelial cells.     

RNAi knockdown of the focal adhesion protein TES reveals its role in actin stress fibre organisation

TES was originally identified as a candidate tumour suppressor gene and has subsequently been found to encode a novel focal adhesion protein. As well as localising to cell-matrix adhesions, TES localises to cell-cell contacts and to actin stress fibres. TES interacts with a variety of cytoskeletal proteins including zyxin, mena, VASP, talin and actin. There is evidence that TES may function in actin-dependent processes as overexpression of TES results in increased cell spreading and decreased cell motility. Together with TES's interacting partners, these data suggest that TES might be involved in regulation of the actin cytoskeleton. Here, for the first time, we have used RNAi to successfully knockdown TES in HeLa cells and we demonstrate that loss of TES from focal adhesions results in loss of actin stress fibres. Similarly, and as previously reported, RNAi-mediated knockdown of zyxin results in loss of actin stress fibres. TES siRNA treated cells show reduced RhoA activity, suggesting that the Rho GTPase pathway may be involved in the TES RNAi-induced loss of stress fibres. We have also used RNAi to examine the requirement of TES and zyxin for each other's localisation at focal adhesions, and we propose a hierarchy of recruitment, with zyxin being first, followed by VASP and then TES. Cell Motil.     

Rho and Rab Small G Proteins Coordinately Reorganize Stress Fibers and Focal Adhesions in MDCK Cells

The Rho subfamily of the Rho small G protein family (Rho) regulates formation of stress fibers and focal adhesions in many types of cultured cells. In moving cells, dynamic and coordinate disassembly and reassembly of stress fibers and focal adhesions are observed, but the precise mechanisms in the regulation of these processes are poorly understood. We previously showed that 12-O-tetradecanoylphorbol-13-acetate (TPA) first induced disassembly of stress fibers and focal adhesions followed by their reassembly in MDCK cells. The reassembled stress fibers showed radial-like morphology that was apparently different from the original. We analyzed here the mechanisms of these TPA-induced processes. Rho inactivation and activation were necessary for the TPA-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. Both inactivation and activation of the Rac subfamily of the Rho family (Rac) inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly. Moreover, microinjection or transient expression of Rab GDI, a regulator of all the Rab small G protein family members, inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly, indicating that, furthermore, activation of some Rab family members is necessary for their TPA-induced reassembly. Of the Rab family members, at least Rab5 activation was necessary for the TPA-induced reassembly of stress fibers and focal adhesions. The TPA-induced, small G protein-mediated reorganization of stress fibers and focal adhesions was closely related to the TPA-induced cell motility. These results indicate that the Rho and Rab family members coordinately regulate the TPA-induced reorganization of stress fibers and focal adhesions that may cause cell motility.     

Requirement for Rho in Integrin Signalling

Overnight culture of Swiss 3T3 cells in serum-free medium leads to loss of focal adhesions and associated actin stress fibres, although the cells remain well spread. The small GTP-binding protein Rho is required for the formation of stress fibres and focal adhesions induced by growth factors such as lysophosphatidic acid (LPA) in serum-starved Swiss 3T3 cells, and for the LPA-induced tyrosine phosphorylation of several focal adhesion proteins. Plating of cells on extracellular matrix proteins also stimulates protein tyrosine phosphorylation and the formation of stress fibres and focal adhesions in the absence of added growth factors. These responses were inhibited in cells scrape-loaded with the Rho inhibitor C3 transferase. Focal adhesion and stress fibre formation was also triggered by addition of a peptide GRGDS, which is recognised by a number of integrins and is contained within the cell binding domain of a variety of extracellular matrix proteins. The activity of the GRGDS peptide was blocked by microinjecting cells with C3 transferase, suggesting that peptide binding to integrins stimulates a Rho-dependent assembly of focal adhesions. These experiments indicate that Rho is involved in signalling downstream of integrins.     

Requirement for Rho in Integrin Signalling

Overnight culture of Swiss 3T3 cells in serum-free medium leads to loss of focal adhesions and associated actin stress fibres, although the cells remain well spread. The small GTP-binding protein Rho is required for the formation of stress fibres and focal adhesions induced by growth factors such as lysophosphatidic acid (LPA) in serum-starved Swiss 3T3 cells, and for the LPA-induced tyrosine phosphorylation of several focal adhesion proteins. Plating of cells on extracellular matrix proteins also stimulates protein tyrosine phosphorylation and the formation of stress fibres and focal adhesions in the absence of added growth factors. These responses were inhibited in cells scrape-loaded with the Rho inhibitor C3 transferase. Focal adhesion and stress fibre formation was also triggered by addition of a peptide GRGDS, which is recognised by a number of integrins and is contained within the cell binding domain of a variety of extracellular matrix proteins. The activity of the GRGDS peptide was blocked by microinjecting cells with C3 transferase, suggesting that peptide binding to integrins stimulates a Rho-dependent assembly of focal adhesions. These experiments indicate that Rho is involved in signalling downstream of integrins.     

Model of coupled transient changes of Rac, Rho, adhesions and stress fibers alignment in endothelial cells responding to shear stress

Interactions of cell adhesions, Rho GTPases and actin in the endothelial cells' response to external forces are complex and not fully understood, but a qualitative understanding of the mechanosensory response begins to emerge. Here, we formulate a mathematical model of the coupled dynamics of cell adhesions, small GTPases Rac and Rho and actin stress fibers guiding a directional reorganization of the actin cytoskeleton. The model is based on the assumptions that the interconnected cytoskeleton transfers the shear force to the adhesion sites, which in turn transduce the force into a chemical signal that activates integrins at the basal surface of the cell. Subsequently, activated and ligated integrins signal and transiently de-activate Rho, causing the disassembly of actin stress fibers and inhibiting the maturation of focal complexes into focal contacts. Focal complexes and ligated integrins activate Rac, which in turn enhances focal complex assembly. When Rho activity recovers, stress fibers re-assemble and promote the maturation of focal complexes into focal contacts. Merging stress fibers self-align, while the elevated level of Rac activity at the downstream edge of the cell is translated into an alignment of the cells and the newly forming stress fibers in the flow direction. Numerical solutions of the model equations predict transient changes in Rac and Rho that compare well with published experimental results. We report quantitative data on early alignment of the stress fibers and its dependence on cell shape that agrees with the model.     

Rho family GTPases regulate VEGF-stimulated endothelial cell motility

Migration of endothelial cells induced by vascular endothelial growth factor (VEGF) is a critical step in angiogenesis. Stimulation of motility by growth factors such as VEGF requires interaction with the signal transduction pathways activated by the extracellular matrix (ECM). Here we demonstrate that the Rac GTPase is the critical intersection activated by type 1 collagen ECM and VEGF during stimulation of endothelial cell motility. To analyze the role of the Rho family GTPases in VEGF-stimulated endothelial cell chemotaxis and ECM-stimulated haptotaxis, we transduced the respective fusion proteins in human foreskin dermal endothelial cells using a Tat peptide from the human immunodeficiency virus Tat protein. VEGF signaling required Rac activation during chemotaxis, and Rac and Cdc42 were activated during haptotaxis on type I collagen. Similar to VEGF, Rac activation induced an increase in endothelial cell stress fiber and focal adhesion. Surprisingly, Rho activation was not present in collagen-induced haptotaxis or stimulation of chemotaxis by VEGF, although Rho induced stress fibers and focal adhesions similar to Rac activation. The result of constitutive Rho activation was an inhibition of haptotaxis. Thus, Rac is required and sufficient for the activation of endothelial cell haptotaxis and VEGF-stimulated chemotaxis.     

Somatostatin,acting at receptor subtype 1, inhibits Rho activity,the assembly of actin stress fibers,and cell migration

Somatostatin regulates multiple biological functions by acting through a family of five G protein-coupled receptors, somatostatin receptors (SSTRs) 1-5. Although all five receptor subtypes inhibit adenylate cyclase activity and decrease intracellular cAMP levels, specific receptor subtypes also couple to additional signaling pathways. In CCL39 fibroblasts expressing either human SSTR1 or SSTR2, we demonstrate that activation of SSTR1 (but not SSTR2) attenuated both thrombin- and integrin-stimulated Rho-GTP complex formation. The reduction in Rho-GTP formation in the presence of somatostatin was associated with decreased translocation of Rho and LIM kinase to the plasma membrane and fewer focal contacts. Activation of Rho resulted in the formation of intracellular actin stress fibers and cell migration. In CCL39-R1 cells, somatostatin treatment prevented actin stress fiber assembly and attenuated thrombin-stimulated cell migration through Transwell membranes to basal levels. To show that native SSTR1 shares the ability to inhibit Rho activation, we demonstrated that somatostatin treatment of human umbilical vein endothelial cells attenuated thrombin-stimulated Rho-GTP accumulation. These data show for the first time that a G protein-coupled receptor, SSTR1, inhibits the activation of Rho, the assembly of focal adhesions and actin stress fibers, and cell migration.     

Signaling adaptor protein v-Crk activates Rho and regulates cell motility in 3Y1 rat fibroblast cell line.

The adaptor protein Crk has been reported to associate with focal adhesions and is thought to be involved in integrin-mediated signaling pathway. However, the precise mechanism of Crk-dependent regulation of cytoskeleton still remains under investigation. In this study, we have established a v-Crk-inducible cell line in rat fibroblasts 3Y1 cells and found that v-Crk activated Rho and induced actin stress fiber formation. In addition to the induction of tyrosine-phosphorylation of p130(Cas) and paxillin, we demonstrated that v-Crk induced threonine-phosphorylated bands sized at 72/78 kDa found specifically in 3Y1 cells. Both of the inhibitors of Rho and Rho-associated kinase, C3 and Y27632, respectively, inhibited these v-Crk-induced biochemical effects. Although v-Crk-induced cells exhibited a decrease of cell motility, integrin stimulation recovered the suppression of motility. Furthermore, v-Crk enhanced motility in chemotactic assay toward fibronectin with additional activation of Rho and the increase of levels of CD44 cleavage. These results suggest that v-Crk activated Rho and induced actin stress fiber formation and CD44 cleavage leading to the regulation of cell motility.     

Control of the direction of lamellipodia extension through changes in the balance between Rac and Rho activities

The direction in which cells extend new motile processes, such as lamellipodia and filopodia, can be controlled by altering the geometry of extracellular matrix adhesive islands on which individual cells are cultured, thereby altering mechanical interactions between cells and the adhesive substrate [Parker (2002)]. Here we specifically investigate the intracellular molecular signals that mediate the mechanism by which cells selectively extend these processes from the corners of polygonal-shaped adhesive islands. Constitutive activation of the small GTPase Rac within cells cultured on square-shaped islands of fibronectin resulted in the elimination of preferential extension from corners. This loss of directionality was accompanied by a re-distribution of focal adhesions: the large focal adhesions normally found within the corner regions of square cells were lost and replaced by many smaller focal contacts that were distributed along the entire cell perimeter. Inhibition of the small GTPase, Rho, using C3 exoenzyme blocked lamellipodia extension entirely. However, inhibition of Rho signaling in combination with ectopic Rac activation rescued the corner localization of motile processes and focal adhesions. These results suggest that the ability of cells to sense their physical surroundings and respond by moving in a spatially oriented manner is mediated by a balance between Rho and Rac activities.     

The TSC1 tumour suppressor hamartin regulates cell adhesion through ERM proteins and the GTPase Rho

Loss of the tumour-suppressor gene TSC1 is responsible for hamartoma development in tuberous sclerosis complex (TSC), which renders several organs susceptible to benign tumours. Hamartin, the protein encoded by TSC1, contains a coiled-coil domain and is expressed in most adult tissues, although its function is unknown. Here we show that hamartin interacts with the ezrin-radixin-moesin (ERM) family of actin-binding proteins. Inhibition of hamartin function in cells containing focal adhesions results in loss of adhesion to the cell substrate, whereas overexpression of hamartin in cells lacking focal adhesions results in activation of the small GTP-binding protein Rho, assembly of actin stress fibres and formation of focal adhesions. Interaction of endogenous hamartin with ERM-family proteins is required for activation of Rho by serum or by lysophosphatidic acid (LPA). Our data indicate that disruption of adhesion to the cell matrix through loss of hamartin may initiate the development of TSC hamartomas and that a Rho-mediated signalling pathway regulating cell adhesion may constitute a rate-limiting step in tumour formation.     

Focal adhesion assembly

The GTP-binding protein Rho regulates the assembly of focal adhesions and their associated bundles of actin filaments. Two different lines of research have converged to reveal how Rho might regulate assembly of these structures. One approach has been the identification of downstream effectors of Rho, whereas the other has been the exploration of the role of contractility in promoting assembly. It is now apparent that Rho is a key regulator of actomyosin-based contractility in nonmuscle cells and that contractility, combined with adhesion to a rigid substrate, leads to the formation of both stress fibres and focal adhesions.     

Constitutive p21-activated kinase (PAK) activation in breast cancer cells as a result of mislocalization of PAK to focal adhesions

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.     

The apolipoprotein(a) component of lipoprotein(a) stimulates actin stress fiber formation and loss of cell-cell contact in cultured endothelial cells

Elevated plasma concentrations of lipoprotein(a) (Lp(a)) are a risk factor for a variety of atherosclerotic disorders including coronary heart disease. In the current study, we report that incubation of cultured human umbilical vein or coronary artery endothelial cells with Lp(a) elicits a dramatic rearrangement of the actin cytoskeleton characterized by increased central stress fiber formation and redistribution of focal adhesions. These effects are mediated by the apolipoprotein(a) (apo(a)) component of Lp(a) since incubation of apo(a) with the cells evoked similar cytoskeletal rearrangements, while incubation with low density lipoprotein had no effect. Apo(a) also produced a time-dependent increase in transendothelial permeability. The cytoskeletal rearrangements evoked by apo(a) were abolished by C3 transferase, which inhibits Rho, and by Y-27632, an inhibitor of Rho kinase. In addition to actin cytoskeleton remodeling, apo(a) was found to cause VE-cadherin disruption and focal adhesion molecule reorganization in a Rho- and Rho kinase-dependent manner. Cell-cell contacts were found to be regulated by Rho and Rac but not Cdc42. Apo(a) caused a transient increase in the extent of myosin light chain phosphorylation. Finally apo(a) did not evoke increases in intracellular calcium levels, although the effects of apo(a) on the cytoskeleton were found to be calcium-dependent. We conclude that the apo(a) component of Lp(a) activates a Rho/Rho kinase-dependent intracellular signaling cascade that results in increased myosin light chain phosphorylation with attendant rearrangements of the actin cytoskeleton. We propose that the resultant increase in endothelial permeability caused by Lp(a) may help explain the atherosclerotic risk posed by elevated concentrations of this lipoprotein.     

The coordination between actin filaments and adhesion in mesenchymal migration

Mesenchymal cell motility is characterized by a polarized distribution of actin filaments, with a network of short branched actin filaments at the leading edge, and polymers of actin filaments arranged into distinct classes of actin stress fibres behind the leading edge. Importantly, the distinct actin filaments are characteristically associated with discrete adhesion structures and both the adhesions and the actin filaments are co-ordinately regulated during cell migration. While it has long been known that these macromolecular structures are intimately linked in cells, precisely how they are co-ordinately regulated is presently unknown. Live imaging data now suggests that the focal adhesions may act as sites of actin polymerization resulting in the generation of tension-bearing actin bundles of actin filaments (stress fibres). Moreover, a picture is emerging to suggest that the tropomyosin family of proteins that can determine actin filament dynamics may also play a key role in determining the transition between adhesion states. Molecules such as the tropomyosins are therefore tantalizing candidates to orchestrate the coordination of actin and adhesion dynamics during mesenchymal cell migration.     

Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism

The transition of cell-matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II-driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein-tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136-143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.     

Monocyte adhesion and spreading on human endothelial cells is dependent on Rho-regulated receptor clustering.

The GTPase Rho is known to mediate the assembly of integrin-containing focal adhesions and actin stress fibers. Here, we investigate the role of Rho in regulating the distribution of the monocyte-binding receptors E-selectin, ICAM-1, and VCAM-1 in human endothelial cells. Inhibition of Rho activity with C3 transferase or N19RhoA, a dominant negative RhoA mutant, reduced the adhesion of monocytes to activated endothelial cells and inhibited their spreading. Similar effects were observed after pretreatment of endothelial cells with cytochalasin D. In contrast, dominant negative Rac and Cdc42 proteins did not affect monocyte adhesion or spreading. C3 transferase and cytochalasin D did not alter the expression levels of monocyte-binding receptors on endothelial cells, but did inhibit clustering of E-selectin, ICAM-1, and VCAM-1 on the cell surface induced by monocyte adhesion or cross-linking antibodies. Similarly, N19RhoA inhibited receptor clustering. Monocyte adhesion and receptor cross-linking induced stress fiber assembly, and inhibitors of myosin light chain kinase prevented this response but did not affect receptor clustering. Finally, receptor clusters colocalized with ezrin/moesin/ radixin proteins. These results suggest that Rho is required in endothelial cells for the assembly of stable adhesions with monocytes via the clustering of monocyte-binding receptors and their association with the actin cytoskeleton, independent of stress fiber formation.     

Distinct Actions and Cooperative Roles of ROCK and mDia in Rho Small G Protein-induced Reorganization of the Actin Cytoskeleton in Madin–Darby Canine Kidney Cells

Rho, a member of the Rho small G protein family, regulates the formation of stress fibers and focal adhesions in various types of cultured cells. We investigated here the actions of ROCK and mDia, both of which have been identified to be putative downstream target molecules of Rho, in Madin-Darby canine kidney cells. The dominant active mutant of RhoA induced the formation of parallel stress fibers and focal adhesions, whereas the dominant active mutant of ROCK induced the formation of stellate stress fibers and focal adhesions, and the dominant active mutant of mDia induced the weak formation of parallel stress fibers without affecting the formation of focal adhesions. In the presence of C3 ADP-ribosyltransferase for Rho, the dominant active mutant of ROCK induced the formation of stellate stress fibers and focal adhesions, whereas the dominant active mutant of mDia induced only the diffuse localization of actin filaments. These results indicate that ROCK and mDia show distinct actions in reorganization of the actin cytoskeleton. The dominant negative mutant of either ROCK or mDia inhibited the formation of stress fibers and focal adhesions, indicating that both ROCK and mDia are necessary for the formation of stress fibers and focal adhesions. Moreover, inactivation and reactivation of both ROCK and mDia were necessary for the 12-O-tetradecanoylphorbol-13-acetate-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. The morphologies of stress fibers and focal adhesions in the cells expressing both the dominant active mutants of ROCK and mDia were not identical to those induced by the dominant active mutant of Rho. These results indicate that at least ROCK and mDia cooperatively act as downstream target molecules of Rho in the Rho-induced reorganization of the actin cytoskeleton.     

Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton.

Soluble factors from serum such as lysophosphatidic acid (LPA) are thought to activate the small GTP-binding protein Rho based on their ability to induce actin stress fibers and focal adhesions in a Rho-dependent manner. Cell adhesion to extracellular matrices (ECM) has also been proposed to activate Rho, but this point has been controversial due to the difficulty of distinguishing changes in Rho activity from the structural contributions of ECM to the formation of focal adhesions. To address these questions, we established an assay for GTP-bound cellular Rho. Plating Swiss 3T3 cells on fibronectin-coated dishes elicited a transient inhibition of Rho, followed by a phase of Rho activation. The activation phase was greatly enhanced by serum. In serum-starved adherent cells, LPA induced transient Rho activation, whereas in suspended cells Rho activation was sustained. Furthermore, suspended cells showed higher Rho activity than adherent cells in the presence of serum. These data indicate the existence of an adhesion-dependent negative-feedback loop. We also observed that both cytochalasin D and colchicine trigger Rho activation despite their opposite effects on stress fibers and focal adhesions. Our results show that ECM, cytoskeletal structures and soluble factors all contribute to regulation of Rho activity.