Focal adhesions, stress fibers and mechanical tension

Stress fibers and focal adhesions are complex protein arrays that produce, transmit and sense mechanical tension. Evidence accumulated over many years led to the conclusion that mechanical tension generated within stress fibers contributes to the assembly of both stress fibers themselves and their associated focal adhesions. However, several lines of evidence have recently been presented against this model. Here we discuss the evidence for and against the role of mechanical tension in driving the assembly of these structures. We also consider how their assembly is influenced by the rigidity of the substratum to which cells are adhering. Finally, we discuss the recently identified connections between stress fibers and the nucleus, and the roles that these may play, both in cell migration and regulating nuclear function

Rho protein crosstalk: another social network?

Many fundamental processes in cell biology are regulated by Rho GTPases, including cell adhesion, migration and differentiation. While regulating cellular functions, members of the Rho protein family cooperate or antagonize each other. The resulting molecular network exhibits many levels of interaction dynamically regulated in time and space. In the first part of this review, we describe the main mechanisms of this crosstalk, which can occur at three different levels of the pathway: (1) through regulation of activity, (2) through regulation of protein expression and stability, and (3) through regulation of downstream signaling pathways. In the second part, we illustrate the importance of Rho protein crosstalk with two examples: integrin-based adhesion and cell migration

Transglutaminase-dependent RhoA Activation and Depletion by Serotonin in Vascular Smooth Muscle Cells

The small G protein RhoA plays a major role in several vascular processes and cardiovascular disorders. Here we analyze the mechanisms of RhoA regulation by serotonin (5-HT) in arterial smooth muscle. 5-HT (0.1-10 microM) induced activation of RhoA followed by RhoA depletion at 24-72 h. Inhibition of 5-HT1 receptors reduced the early phase of RhoA activation but had no effect on 5-HT-induced delayed RhoA activation and depletion, which were suppressed by the 5-HT transporter inhibitor fluoxetine and the transglutaminase inhibitor monodansylcadaverin and in type 2 transglutaminase-deficient smooth muscle cells. Coimmunoprecipitations demonstrated that 5-HT associated with RhoA both in vitro and in vivo. This association was calcium-dependent and inhibited by fluoxetine and monodansylcadaverin. 5-HT promotes the association of RhoA with the E3 ubiquitin ligase Smurf1, and 5-HT-induced RhoA depletion was inhibited by the proteasome inhibitor MG132 and the RhoA inhibitor Tat-C3. Simvastatin, the Rho kinase inhibitor Y-27632, small interfering RNA-mediated RhoA gene silencing, and long-term 5-HT stimulation induced Akt activation. In contrast, inhibition of 5-HT-mediated RhoA degradation by MG132 prevented 5-HT-induced Akt activation. Long-term 5-HT stimulation also led to the inhibition of the RhoA/Rho kinase component of arterial contraction. Our data provide evidence that 5-HT, internalized through the 5-HT transporter, is transamidated to RhoA by transglutaminase. Transamidation of RhoA leads to RhoA activation and enhanced proteasomal degradation, which in turn is responsible for Akt activation and contraction inhibition. The observation of transamidation of 5-HT to RhoA in pulmonary artery of hypoxic rats suggests that this process could participate in pulmonary artery remodeling and hypertension

Localized Tensional Forces on PECAM-1 Elicit a Global Mechanotransduction Response via the Integrin-RhoA Pathway

SummaryBackgroundMechanical forces regulate cell behavior and function during development, differentiation, and tissue morphogenesis. In the vascular system, forces produced by blood flow are critical determinants not only of morphogenesis and function, but also of pathological states such as atherosclerosis. Endothelial cells (ECs) have numerous mechanotransducers, including platelet endothelial cell adhesion molecule-1 (PECAM-1) at cell-cell junctions and integrins at cell-matrix adhesions. However, the processes by which forces are transduced to biochemical signals and subsequently translated into downstream effects are poorly understood.ResultsHere, we examine mechanochemical signaling in response to direct force application on PECAM-1. We demonstrate that localized tensional forces on PECAM-1 result in, surprisingly, global signaling responses. Specifically, force-dependent activation of phosphatidylinositol 3-kinase (PI3K) downstream of PECAM-1 promotes cell-wide activation of integrins and the small GTPase RhoA. These signaling events facilitate changes in cytoskeletal architecture, including growth of focal adhesions and adaptive cytoskeletal stiffening.ConclusionsTaken together, our work provides the first evidence of a global signaling event in response to a localized mechanical stress. In addition, these data provide a possible mechanism for the differential stiffness of vessels exposed to distinct hemodynamic force patterns in vivo

Regulation of Rho GTPase crosstalk, degradation and activity by RhoGDI1

At steady state, most Rho GTPases are bound in the cytosol to Rho Guanine nucleotide Dissociation Inhibitors (RhoGDI) 1. RhoGDIs have generally been considered to passively hold Rho proteins in an inactive state within the cytoplasm. Here we describe an evolutionarily conserved mechanism by which RhoGDI1 controls the homeostasis of Rho proteins in eukaryotic cells. We found that depletion of RhoGDI1 promotes misfolding and degradation of the cytosolic geranylgeranylated pool of Rho GTPases while unexpectedly activating the remaining membrane-bound fraction. Since RhoGDI1 levels are limiting, and Rho proteins compete for binding to RhoGDI1, overexpression of an exogenous Rho GTPase displaces endogenous Rho proteins bound to RhoGDI1, inducing their degradation and inactivation. These results raise important questions about the conclusions drawn from studies that manipulate Rho protein levels. In many cases the response observed may arise not simply from the overexpression per se, but from additional effects on the levels and activity of other Rho GTPases due to competition for binding to RhoGDI1, and may require a re-evaluation of previously published studies that rely exclusively on these techniques

Isolated nuclei adapt to force and reveal a mechanotransduction pathway in the nucleus

Mechanical forces influence many aspects of cell behavior. Forces are detected and transduced into biochemical signals by force bearing molecular elements located at the cell surface, in adhesion complexes or in cytoskeletal structures1. The nucleus is physically connected to the cell surface through the cytoskeleton and the linker of nucleoskeleton and cytoskeleton (LINC) complex, allowing rapid mechanical stress transmission from adhesions to the nucleus2. Whereas it has been demonstrated that nuclei experience force3, the direct effect of force on the nucleus is not known. Here we show that isolated nuclei are able to respond to force by adjusting their stiffness to resist the applied tension. Using magnetic tweezers, we found that applying force on nesprin-1 triggers nuclear stiffening that does not involve chromatin or nuclear actin, but requires an intact nuclear lamina and emerin, a protein of the inner nuclear membrane. Emerin becomes tyrosine phosphorylated in response to force and mediates the nuclear mechanical response to tension. Our results demonstrate that mechanotransduction is not restricted to cell surface receptors and adhesions but can occur within the nucleus

The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins

How individual cells respond to mechanical forces is of considerable interest to biologists as force affects many aspects of cell behavior1. Application of force on integrins triggers cytoskeletal rearrangements and growth of the associated adhesion complex, resulting in increased cellular stiffness2,3, also known as reinforcement4. While RhoA has been shown to play a role during reinforcement3, the molecular mechanisms that regulate its activity are unknown. By combining biochemical and biophysical approaches, we identified two guanine nucleotide exchange factors (GEFs), LARG and GEF-H1, as key molecules that regulate the cellular adaptation to force. We show that stimulation of integrins with tensional force triggers activation of these two GEFs and their recruitment to adhesion complexes. Surprisingly, activation of LARG and GEF-H1 involves distinct signaling pathways. Our results reveal that LARG is activated by the Src family tyrosine kinase Fyn, whereas GEF-H1 catalytic activity is enhanced by ERK downstream of a signaling cascade that includes FAK and Ras

The Vinculin C-terminal Hairpin Mediates F-actin Bundle Formation, Focal Adhesion, and Cell Mechanical Properties

Vinculin is an essential and highly conserved cell adhesion protein, found at both focal adhesions and adherens junctions, where it couples integrins or cadherins to the actin cytoskeleton. Vinculin is involved in controlling cell shape, motility, and cell survival, and has more recently been shown to play a role in force transduction. The tail domain of vinculin (Vt) contains determinants necessary for binding and bundling of actin filaments. Actin binding to Vt has been proposed to induce formation of a Vt dimer that is necessary for cross-linking actin filaments. Results from this study provide additional support for actin-induced Vt self-association. Moreover, the actin-induced Vt dimer appears distinct from the dimer formed in the absence of actin. To better characterize the role of the Vt strap and carboxyl terminus (CT) in actin binding, Vt self-association, and actin bundling, we employed smaller amino-terminal (NT) and CT deletions that do not perturb the structural integrity of Vt. Although both NT and CT deletions retain actin binding, removal of the CT hairpin (1061–1066) selectively impairs actin bundling in vitro. Moreover, expression of vinculin lacking the CT hairpin in vinculin knock-out murine embryonic fibroblasts affects the number of focal adhesions formed, cell spreading as well as cellular stiffening in response to mechanical force

Mechanically Induced Focal Adhesion Assembly Amplifies Anti-Adipogenic Pathways in Mesenchymal Stem Cells

The fate of pluripotent mesenchymal stem cells (MSC) is determined through integration of chemical, spatial, and physical signals. The suppression of MSC adipogenesis by mechanical stimuli, which requires Akt-induced inhibition of glycogen synthase kinase 3β (GSK3β) with β-catenin activation, can be enhanced by repetitive dosing within a single day. Here, we demonstrate that reapplication of cyclic strain within a 24-hour period leads to amplification of both Akt activation and its subsequent inhibition of GSK3β, such that total cycle number can be reduced while still inhibiting adipogenesis. Amplification of Akt signaling is facilitated by a dynamic restructuring of the cell in response to mechanical signals, as evidenced by a transient increase in focal adhesion (FA) number and increased RhoA activity. Preventing FA assembly or development of tension blocks activation of Akt by mechanical signals, but not by insulin. This indicates that the FA infrastructure is essential to the physical, but not necessarily the chemical, sensitivity, and responsiveness of the cell. Exploiting the transient nature of cytoskeletal remodeling may represent a process to enhance cell responsiveness to mechanical input and ultimately define the fate of MSCs with a minimal input