Effects of cell tension on the small GTPase Rac

Cells in the body are subjected to mechanical stresses such as tension, compression, and shear stress. These mechanical stresses play important roles in both physiological and pathological processes; however, mechanisms transducing mechanical stresses into biochemical signals remain elusive. Here, we demonstrated that equibiaxial stretch inhibited lamellipodia formation through deactivation of Rac. Nearly maximal effects on Rac activity were obtained with 10% strain. GAP-resistant, constitutively active V12Rac reversed this inhibition, supporting a critical role for Rac inhibition in the response to stretch. In contrast, activation of endogenous Rac with a constitutively active nucleotide exchange factor did not, suggesting that regulation of GAP activity most likely mediates the inhibition. Uniaxial stretch suppressed lamellipodia along the sides lengthened by stretch and increased it at the adjacent ends. A fluorescence assay for localized Rac showed comparable changes in activity along the sides versus the ends after uniaxial stretch. Blocking polarization of Rac activity by expressing V12Rac prevented subsequent alignment of actin stress fibers. Treatment with Y-27632 or ML-7 that inhibits myosin phosphorylation and contractility increased lamellipodia through Rac activation and decreased cell polarization. We hypothesize that regulation of Rac activity by tension may be important for motility, polarization, and directionality of cell movement

A Dynamic Stochastic Model of Frequency-Dependent Stress Fiber Alignment Induced by Cyclic Stretch

BACKGROUND: Actin stress fibers (SFs) are mechanosensitive structural elements that respond to forces to affect cell morphology, migration, signal transduction and cell function. Cells are internally stressed so that SFs are extended beyond their unloaded lengths, and SFs tend to self-adjust to an equilibrium level of extension. While there is much evidence that cells reorganize their SFs in response to matrix deformations, it is unclear how cells and their SFs determine their specific response to particular spatiotemporal changes in the matrix. METHODOLOGY/PRINCIPAL FINDINGS: Bovine aortic endothelial cells were subjected to cyclic uniaxial stretch over a range of frequencies to quantify the rate and extent of stress fiber alignment. At a frequency of 1 Hz, SFs predominantly oriented perpendicular to stretch, while at 0.1 Hz the extent of SF alignment was markedly reduced and at 0.01 Hz there was no alignment at all. The results were interpreted using a simple kinematic model of SF networks in which the dynamic response depended on the rates of matrix stretching, SF turnover, and SF self-adjustment of extension. For these cells, the model predicted a threshold frequency of 0.01 Hz below which SFs no longer respond to matrix stretch, and a saturation frequency of 1 Hz above which no additional SF alignment would occur. The model also accurately described the dependence of SF alignment on matrix stretch magnitude. CONCLUSIONS: The dynamic stochastic model was capable of describing SF reorganization in response to diverse temporal and spatial patterns of stretch. The model predicted that at high frequencies, SFs preferentially disassembled in the direction of stretch and achieved a new equilibrium by accumulating in the direction of lowest stretch. At low stretch frequencies, SFs self-adjusted to dissipate the effects of matrix stretch. Thus, SF turnover and self-adjustment are each important mechanisms that cells use to maintain mechanical homeostasis

Stretch-Induced Stress Fiber Remodeling and the Activations of JNK and ERK Depend on Mechanical Strain Rate, but Not FAK

BACKGROUND: Cells within tissues are subjected to mechanical forces caused by extracellular matrix deformation. Cells sense and dynamically respond to stretching of the matrix by reorienting their actin stress fibers and by activating intracellular signaling proteins, including focal adhesion kinase (FAK) and the mitogen-activated proteins kinases (MAPKs). Theoretical analyses predict that stress fibers can relax perturbations in tension depending on the rate of matrix strain. Thus, we hypothesized stress fiber organization and MAPK activities are altered to an extent dependent on stretch frequency. PRINCIPAL FINDINGS: Bovine aortic endothelial cells and human osteosarcoma cells expressing GFP-actin were cultured on elastic membranes and subjected to various patterns of stretch. Cyclic stretching resulted in strain rate-dependent increases in stress fiber alignment, cell retraction, and the phosphorylation of the MAPKs JNK, ERK and p38. Transient step changes in strain rate caused proportional transient changes in the levels of JNK and ERK phosphorylations without affecting stress fiber organization. Disrupting stress fiber contractile function with cytochalasin D or Y27632 decreased the levels of JNK and ERK phosphorylation. Previous studies indicate that FAK is required for stretch-induced cell alignment and MAPK activations. However, cyclic uniaxial stretching induced stress fiber alignment and the phosphorylation of JNK, ERK and p38 to comparable levels in FAK-null and FAK-expressing mouse embryonic fibroblasts. CONCLUSIONS: These results indicate that cyclic stretch-induced stress fiber alignment, cell retraction, and MAPK activations occur as a consequence of perturbations in fiber strain. These findings thus shed new light into the roles of stress fiber relaxation and reorganization in maintenance of tensional homeostasis in a dynamic mechanical environment

The Direction of Stretch-Induced Cell and Stress Fiber Orientation Depends on Collagen Matrix Stress

<div><p>Cell structure depends on both matrix strain and stiffness, but their interactive effects are poorly understood. We investigated the interactive roles of matrix properties and stretching patterns on cell structure by uniaxially stretching U2OS cells expressing GFP-actin on silicone rubber sheets supporting either a surface-adsorbed coating or thick hydrogel of type-I collagen. Cells and their actin stress fibers oriented perpendicular to the direction of cyclic stretch on collagen-coated sheets, but oriented parallel to the stretch direction on collagen gels. There was significant alignment parallel to the direction of a steady increase in stretch for cells on collagen gels, while cells on collagen-coated sheets did not align in any direction. The extent of alignment was dependent on both strain rate and duration. Stretch-induced alignment on collagen gels was blocked by the myosin light-chain kinase inhibitor ML7, but not by the Rho-kinase inhibitor Y27632. We propose that active orientation of the actin cytoskeleton perpendicular and parallel to direction of stretch on stiff and soft substrates, respectively, are responses that tend to maintain intracellular tension at an optimal level. Further, our results indicate that cells can align along directions of matrix stress without collagen fibril alignment, indicating that matrix stress can directly regulate cell morphology.</p></div

Cyclic stretch-induced cell and SF alignment on soft collagen gels depends on stretch frequency.

<p>Representative images of non-confluent U2OS cells adhered on a soft collagen matrix subjected to 3 h of 10% cyclic stretch at frequencies of 0.01 (A), 0.1 (B) and 1 Hz (C). Order parameters for cells (D) and SFs (E) were computed for each cell to quantify the extents of alignment and the results were summarized (mean ± SEM; n = 90). * indicates significant differences between groups as determined by ANOVA followed by Student-Neuman–Keuls post-hoc multiple comparison testing (P<0.01). Scale bar, 50 µm.</p

Cyclic stretch-induced SF alignment on soft collagen gels and stiff silicone rubber sheets.

<p>A–D: Representative images and circular histograms depicting SF angular distributions of non-confluent U2OS cells (A, B) (n = 90 for each condition) and hMSCs (C, D) (n = 60 for each condition) subjected to 3 h of 10% cyclic uniaxial stretch at frequency of 1 Hz on collagen-coated rubber sheets (A, C) and collagen gels (B, D). Scale bar, 50 µm. E, F: Representative confocal reflectance images of collagen fibrils (red) in regions containing a U2OS cell (E) and devoid of cells (F) and circular histograms depicting collagen fibril alignment after 3 h of 10% cyclic stretching at 1 Hz.; Scale bar, 5 µm.</p

Effects of pre-stretch and strain rate on steady stretch-induced cell and SF alignment.

<p>Representative images of non-confluent U2OS cells seeded on 10% pre-stretched collagen gel (A) or adhered onto collagen gels (that were not pre-stretched) subjected to 10% stretch at ramp rates of 20%/s (B) and 0.2%/s (C). Representative image of U2OS cells adhered onto collagen-coated silicone rubber sheets subjected to 10% stretch at 20%/s (D). Cell (E) and SF (F) order parameters (n = 90) are summarized. * indicates significant differences between groups as determined by ANOVA followed by Student-Neuman–Keuls post-hoc multiple comparison testing (P<0.01). Scale bar, 50 µm.</p

Roles of Rho-kinase and MLCK on cyclic stretch-induced SF alignment in cells on 3-D collagen gels.

<p>Representative images of non-confluent U2OS cells (n = 60) adhered on soft collagen gels subjected to 3 h of 10% cyclic uniaxial stretch at 1 Hz after treatment with 30 µM ML7 (A) or 10 µM Y27632 (B). Scale bar, 50 µm.</p