Stretch-induced Reorientation Research Articles

As the endothelial cell reorients, its tensile forces stabilize

When adherent cells are subjected to uniaxial sinusoidal stretch at frequencies close to physiological, their body and their contractile stress fibers realign nearly perpendicularly to the stretch axis. A common explanation for this phenomenon is that stress fibers reorient along the direction where they are unaffected by the applied cyclic stretch and thus can maintain optimal (homeostatic) tensile force. The ability of cells to achieve tensional homeostasis in response to external disturbances is important for normal physiological functions of cells and tissues and it provides protection against diseases, including cancer and atherosclerosis. However, quantitative experimental data that support the idea that stretch-induced reorientation is associated with tensional homeostasis are lacking. We observed previously that in response to uniaxial cyclic stretch of 10% strain amplitudes, traction forces of single endothelial cells reorient in the direction perpendicular to the stretch axis. Here we carried out a secondary analysis of those data to investigate whether this reorientation of traction forces is associated with tensional homeostasis. Our analysis showed that stretch-induced reorientation of traction forces was accompanied by attenuation of temporal variability of the traction field to the level that was observed in the absence of stretch. These findings represent a quantitative experimental evidence that stretch-induced reorientation of cellular traction forces is associated with the cell’s tendency to achieve tensional homeostasis.

Rho guanine nucleotide exchange factors involved in cyclic-stretch-induced reorientation of vascular endothelial cells.

Cyclic stretch is an artificial model of mechanical force loading, which induces the reorientation of vascular endothelial cells and their stress fibers in a direction perpendicular to the stretch axis. Rho family GTPases are crucial for cyclic-stretch-induced endothelial cell reorientation; however, the mechanism underlying stretch-induced activation of Rho family GTPases is unknown. A screen of short hairpin RNAs targeting 63 Rho guanine nucleotide exchange factors (Rho-GEFs) revealed that at least 11 Rho-GEFs – Abr, alsin, ARHGEF10, Bcr, GEF-H1 (also known as ARHGEF2), LARG (also known as ARHGEF12), p190RhoGEF (also known as ARHGEF28), PLEKHG1, P-REX2, Solo (also known as ARHGEF40) and α-PIX (also known as ARHGEF6) – which specifically or broadly target RhoA, Rac1 and/or Cdc42, are involved in cyclic-stretch-induced perpendicular reorientation of endothelial cells. Overexpression of Solo induced RhoA activation and F-actin accumulation at cell-cell and cell-substrate adhesion sites. Knockdown of Solo suppressed cyclic-stretch- or tensile-force-induced RhoA activation. Moreover, knockdown of Solo significantly reduced cyclic-stretch-induced perpendicular reorientation of endothelial cells when cells were cultured at high density, but not when they were cultured at low density or pretreated with EGTA or VE-cadherin-targeting small interfering RNAs. These results suggest that Solo is involved in cell-cell-adhesion-mediated mechanical signal transduction during cyclic-stretch-induced endothelial cell reorientation.

A mechanochemical model of cell reorientation on substrates under cyclic stretch.

We report a theoretical study on the cyclic stretch-induced reorientation of spindle-shaped cells. Specifically, by taking into account the evolution of sub-cellular structures like the contractile stress fibers and adhesive receptor-ligand clusters, we develop a mechanochemical model to describe the dynamics of cell realignment in response to cyclically stretched substrates. Our main hypothesis is that cells tend to orient in the direction where the formation of stress fibers is energetically most favorable. We show that, when subjected to cyclic stretch, the final alignment of cells reflects the competition between the elevated force within stress fibers that accelerates their disassembly and the disruption of cell-substrate adhesion as well, and an effectively increased substrate rigidity that promotes more stable focal adhesions. Our model predictions are consistent with various observations like the substrate rigidity dependent formation of stable adhesions and the stretching frequency, as well as stretching amplitude, dependence of cell realignment. This theory also provides a simple explanation on the regulation of protein Rho in the formation of stretch-induced stress fibers in cells.

Strain waveform dependence of stress fiber reorientation in cyclically stretched osteoblastic cells: effects of viscoelastic compression of stress fibers

Actin stress fibers (SFs) of cells cultured on cyclically stretched substrate tend to reorient in the direction in which a normal strain of substrate becomes zero. However, little is known about the mechanism of this reorientation. Here we investigated the effects of cyclic stretch waveform on SF reorientation in osteoblastic cells. Cells adhering to silicone membranes were subjected to cyclic uniaxial stretch, having one of the following waveforms with an amplitude of 8% for 24 h: triangular, trapezoid, bottom hold, or peak hold. SF reorientation of these cells was then analyzed. No preferential orientation was observed for the triangular and the peak-hold waveforms, whereas SFs aligned mostly in the direction with zero normal strain (~55°) with other waveforms, especially the trapezoid waveform, which had a hold time both at loaded and unloaded states. Viscoelastic properties of SFs were estimated in a quasi-in situ stress relaxation test using intact and SF-disrupted cells that maintained their shape on the substrate. The dynamics of tension F(SFs) acting on SFs during cyclic stretching were simulated using these properties. The simulation demonstrated that F(SFs) decreased gradually during cyclic stretching and exhibited a compressive value (F(SFs) < 0). The magnitude and duration time of the compressive forces were relatively larger in the group with a trapezoid waveform. The frequency of SF orientation had a significant negative correlation with the applied compressive forces integrated with time in a strain cycle, and the integrated value was largest with the trapezoid waveform. These results may indicate that the applied compressive forces on SFs have a significant effect on the stretch-induced reorientation of SFs, and that SFs realigned to avoid their compression. Stress relaxation of SFs might be facilitated during the holding period in the trapezoid waveform, and depolymerization and reorientation of SFs were significantly accelerated by their viscoelastic compression.

The kinetics of force‐induced cell reorganization depend on microtubules and actin

The cytoskeleton is an important factor in the functional and structural adaption of cells to mechanical forces. In this study we investigated the impact of microtubules and the acto-myosin machinery on the kinetics of force-induced reorientation of NIH3T3 fibroblasts. These cells were subjected to uniaxial stretching forces that are known to induce cellular reorientation perpendicular to the stretch direction. We found that disruption of filamentous actin using cytochalasin D and latrunculin B as well as an induction of a massive unpolarized actin polymerization by jasplakinolide, inhibited the stretch-induced reorientation. Similarly, blocking of myosin II activity abolished the stretch-induced reorientation of cells but, interestingly, increased their motility under stretching conditions in comparison to myosin-inhibited nonstretched cells. Investigating the contribution of microtubules to the cellular reorientation, we found that, although not playing a significant role in reorientation itself, microtubule stability had a significant impact on the kinetics of this event. Overall, we conclude that acto-myosin, together with microtubules, regulate the kinetics of force-induced cell reorientation. © 2010 Wiley-Liss, Inc.

Resveratrol Affects Stretch-Induced Reorientation of A7r5 Cells in Culture

Resveratrol Affects Stretch-Induced Reorientation of A7r5 Cells in Culture

Dynamic Reorientation of Cultured Cells and Stress Fibers under Mechanical Stress from Periodic Stretching

Cell lines derived from rat aorta and frog kidney were cultured on elastic membrane, and mechanical stress was given to the cells by stretching the membrane periodically. Cell reorientation oblique to the direction of stretching occurred as a result of the rapid withdrawal of cell periphery located along the direction of stretching and gradual extension of the cell membrane toward the direction oblique to the direction of stretching. Dynamic reorganization of stress fibers in living cells was visualized by labeling stress fibers with TRITC3–actin or EGFP-tagged moesin fragments with actin-binding ability. Stress fibers aligned in the direction of stretching disappeared soon after the start of stretching and then obliquely reoriented stress fibers appeared. The stretch-induced reorientation of cultured cells was suppressed by an inhibitor of stretch-activated (SA) cation channels and by a Ca2+ chelator. However, the rearrangement of stress fibers was not affected by these agents. From these results, we suggest that Ca2+ influx via SA channels is involved in stretch-induced cell reorientation but stress fiber rearrangement is independent of SA channels. Therefore, cell reorientation does not simply depend on the arrangement of stress fibers but may be controlled by some additional mechanism(s) which is regulated by calcium signaling.