Focal Adhesion Redistribution Research Articles

Tracking of Endothelial Cell Migration and Stiffness Measurements Reveal the Role of Cytoskeletal Dynamics.

Cell migration is a complex, tightly regulated multistep process in which cytoskeletal reorganization and focal adhesion redistribution play a central role. Core to both individual and collective migration is the persistent random walk, which is characterized by random force generation and resistance to directional change. We first discuss a model that describes the stochastic movement of ECs and characterizes EC persistence in wound healing. To that end, we pharmacologically disrupted cytoskeletal dynamics, cytochalasin D for actin and nocodazole for tubulin, to understand its contributions to cell morphology, stiffness, and motility. As such, the use of Atomic Force Microscopy (AFM) enabled us to probe the topography and stiffness of ECs, while time lapse microscopy provided observations in wound healing models. Our results suggest that actin and tubulin dynamics contribute to EC shape, compressive moduli, and directional organization in collective migration. Insights from the model and time lapse experiment suggest that EC speed and persistence are directionally organized in wound healing. Pharmacological disruptions suggest that actin and tubulin dynamics play a role in collective migration. Current insights from both the model and experiment represent an important step in understanding the biomechanics of EC migration as a therapeutic target.

Inhibition of cell migration and invasion mediated by the TAT-RasGAP317–326 peptide requires the DLC1 tumor suppressor

TAT-RasGAP(317-326), a peptide corresponding to the 317-326 sequence of p120 RasGAP coupled with a cell-permeable TAT-derived peptide, sensitizes the death response of various tumor cells to several anticancer treatments. We now report that this peptide is also able to increase cell adherence, prevent cell migration and inhibit matrix invasion. This is accompanied by a marked modification of the actin cytoskeleton and focal adhesion redistribution. Interestingly, integrins and the small Rho GTP-binding protein, which are well-characterized proteins modulating actin fibers, adhesion and migration, do not appear to be required for the pro-adhesive properties of TAT-RasGAP(317-326). In contrast, deleted in liver cancer-1, a tumor suppressor protein, the expression of which is often deregulated in cancer cells, was found to be required for TAT-RasGAP(317-326) to promote cell adherence and inhibit migration. These results show that TAT-RasGAP(317-326), besides its ability to favor tumor cell death, hampers cell migration and invasion.

Role of Intercellular Junctions in Redistribution of Focal Adhesions and Orientation of Vascular Endothelial Cells Exposed to Cyclic Stretching

The redistribution of focal adhesions (FAs) containing integrin β1 and paxillin plays an important role in the cyclic stretching-induced morphological changes of endothelial cells (ECs). In addition to focal adhesion kinase (FAK), known to be a primary regulator for FA redistribution, intercellular junctions (IJs) have recently been reported to be involved in signaling upstream of FAs. Here, we addressed the role of IJs in the morphological changes and redistribution of FAs in ECs exposed to cyclic stretching. Both confluent and sparse ECs were oriented nearly perpendicularly to the stretch direction after 10 min of exposure. Orientation of sparse ECs, but not confluent ECs, was suppressed by treatment with a phospho-FAK inhibitor. FAK inhibitor blocked integrin β1 redistribution in ECs, which was observed in non-inhibited cells after 10-min stretch exposure. However, paxillin redistribution in confluent ECs was observed regardless of FAK inhibitor treatment after 2-min stretch exposure. When we blocked signals from IJs with an inhibitor of Src homology 2 domain-containing tyrosine phosphatase-2, the percentage of oriented ECs decreased and paxillin redistribution, but not integrin β1, was suppressed. These findings suggest that IJs are involved in the orientation of ECs subjected to cyclic stretching through signaling pathways other than FAK.

Differential Regulation of Pulmonary Endothelial Monolayer Integrity by Varying Degrees of Cyclic Stretch

Ventilator-induced lung injury is a life-threatening complication of mechanical ventilation at high-tidal volumes. Besides activation of proinflammatory cytokine production, excessive lung distension directly affects blood-gas barrier and lung vascular permeability. To investigate whether restoration of pulmonary endothelial cell (EC) monolayer integrity after agonist challenge is dependent on the magnitude of applied cyclic stretch (CS) and how these effects are linked to differential activation of small GTPases Rac and Rho, pulmonary ECs were subjected to physiologically (5% elongation) or pathologically (18% elongation) relevant levels of CS. Pathological CS enhanced thrombin-induced gap formation and delayed monolayer recovery, whereas physiological CS induced nearly complete EC recovery accompanied by peripheral redistribution of focal adhesions and cortactin after 50 minutes of thrombin. Consistent with differential effects on monolayer integrity, 18% CS enhanced thrombin-induced Rho activation, whereas 5% CS promoted Rac activation during the EC recovery phase. Rac inhibition dramatically attenuated restoration of monolayer integrity after thrombin challenge. Physiological CS preconditioning (5% CS, 24 hours) enhanced EC paracellular gap resolution after step-wise increase to 18% CS (30 minutes) and thrombin challenge. These results suggest a critical role for the CS amplitude and the balance between Rac and Rho in mechanochemical regulation of lung EC barrier.

Activation of myosin in HeLa cells causes redistribution of focal adhesions and F-actin from cell center to cell periphery

Activation of actomyosin II by phosphorylation of its regulatory light chain is one of the main factors involved in the regulation of cytoskeletal dynamics. Phosphorylation of myosin regulatory light chain may be mediated directly and indirectly by several kinases including myosin light chain kinase (MLCK) and kinases activated by small GTP-binding proteins. Most of the myosin kinases, including PAK, can also interact with other proteins through binding sites located outside of their catalytic domains. In an attempt to study the effects due only to phosphorylation of myosin light chain, we expressed the constitutively active catalytic domain of ameba PAK in HeLa cells. The catalytic domain phosphorylates myosin light chain in vitro with high specific activity but has none of the sequences that target mammalian PAK to other proteins and membranes. Expression of the catalytic domain caused disassembly of focal adhesions and stress fibers in the cell center and accumulation of focal adhesions and F-actin at the cell periphery. There was a twofold increase in the phosphorylation level of endogenous myosin light chain and changes in cell shape consistent with enhanced cell contractility. The phenotype was independent of MLCK, ROCK, MEK, Rac, and Rho activities but was abolished by blebbistatin, a specific inhibitor of myosin II activity. Our data are consistent with myosin being directly phosphorylated by the expressed catalytic domain of ameba PAK with the induced phenotype resulting from cell retraction driven by contraction of peripheral actomyosin. The phenotype induced by expression of the catalytic domain is reminiscent of that caused by expression of active mammalian PAK, suggesting that myosin phosphorylation may play an important role in PAK-induced cytoskeletal changes. The catalytic domain of ameba PAK may be a useful tool for studying the effects of myosin light chain phosphorylation in other cells.

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.

Differential effects of shear stress and cyclic stretch on focal adhesion remodeling, site-specific FAK phosphorylation, and small GTPases in human lung endothelial cells

Regulation of endothelial cell (EC) permeability by bioactive molecules is associated with specific patterns of cytoskeletal and cell contact remodeling. A role for mechanical factors such as shear stress (SS) and cyclic stretch (CS) in cytoskeletal rearrangements and regulation of EC permeability becomes increasingly recognized. This paper examined redistribution of focal adhesion (FA) proteins, site-specific focal adhesion kinase (FAK) phosphorylation, small GTPase activation and barrier regulation in human pulmonary EC exposed to laminar shear stress (15 dyn/cm 2) or cyclic stretch (18% elongation) in vitro. SS caused peripheral accumulation of FAs, whereas CS induced randomly distributed FAs attached to the ends of newly formed stress fibers. SS activated small GTPase Rac without effects on Rho, whereas 18% CS activated without effect on Rac. SS increased transendothelial electrical resistance (TER) in EC monolayers, which was further elevated by barrier-protective phospholipid sphingosine 1-phosphate. Finally, SS induced FAK phosphorylation at Y 576, whereas CS induced FAK phosphorylation at Y 397 and Y 576. These results demonstrate for the first time differential effects of SS and CS on Rho and Rac activation, FA redistribution, site-specific FAK phosphorylation, and link them with SS-mediated barrier enhancement. Thus, our results suggest common signaling and cytoskeletal mechanisms shared by mechanical and chemical factors involved in EC barrier regulation.

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.

Roles of Rho-associated Kinase and Myosin Light Chain Kinase in Morphological and Migratory Defects of Focal Adhesion Kinase-null Cells

Fibroblasts derived from focal adhesion kinase (FAK)-null mouse embryos have a reduced migration rate and an increase in the number and size of peripherally localized adhesions (Ilic, D., Furuta, Y., Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., Nomura, S., Fujimoto, J., Okada, M., and Yamamoto, T. (1995) Nature 377, 539-544). In this study, we have found that Y27632, a specific inhibitor for Rho-associated kinase (Rho-kinase), dramatically reversed the round cell morphology of FAK(-/-) cells to a spread fibroblast-like shape in 30 min and significantly enhanced their motility. The effects of Y27632 on the FAK(-/-) cell morphology and motility were concomitant with reorganization of the actin cytoskeleton and redistribution of focal adhesions. Conversely, the expression of the constitutively active Rho-kinase in FAK(+/+) cells led to round cell shape and inhibition of cell motility. Furthermore, coincident with the formation of cortical actin filaments, myosin light chain (MLC), Ser-19-phosphorylated MLC, and MLC kinase mainly accumulated at the FAK(-/-) cell periphery. We found that the disruption of actin filaments by cytochalasin D prevented the peripheral accumulation of MLC kinase and that inhibition of myosin-mediated contractility by 2,3-butanedione monoxime induced FAK(-/-) cells to spread. Taken together, our results suggest that Rho-kinase may mediate the formation of cortical actomyosin filaments at the FAK(-/-) cell periphery, which further recruits MLC kinase to the cell periphery and generates a non-polar contractile force surrounding the cell, leading to cell rounding and decreased motility.