Cellular Traction Forces Research Articles

Merging of ventral fibers at adhesions drives the remodeling of cellular contractile systems in fibroblasts.

Ventral stress fibers (VSFs) are contractile actin fibers dynamically attached to cell-matrix focal adhesions. VSFs are critical in cellular traction force production and migration. VSFs vary from randomly oriented short, thinner fibers to long, thick fibers that span along the whole long axis of a cell. De novo VSF formation was shown to occur by cortical actin mesh condensation or by crosslinking of dorsal stress fibers and transverse arcs at the cell front. However, the formation of long VSFs that extend across the whole cell axis is not well understood. Here, we report a novel phenomenon of VSF merging in migratory fibroblast cells, which is guided by mechanical force balance and contributes to VSF alignment along the long cell axis. The mechanism of VSF merging involves two steps: connection of two ventral fibers by an emerging myosin II bridge at an intervening adhesion and intervening adhesion dissolution. Our data indicate that these two steps are interdependent: slow adhesion disassembly leads to the slowing of the myosin bridge formation. Cellular data and computational modeling show that the contact angle between merging fibers decides successful merging, with shallow angles leading to merge failure. Our data and modeling further show that merging increases the share of uniformly aligned long VSFs, likely contributing to directional traction force production. Thus, we characterize merging as a process for dynamic reorganization of VSFs with functional significance for directional cell migration.

Differential Impacts on Tensional Homeostasis of Gastric Cancer Cells Due to Distinct Domain Variants of E-Cadherin.

Simple SummaryTensional homeostasis describes the ability of cells and tissues to maintain their internal mechanical tension stable at a set point value. A breakdown of tensional homeostasis is the hallmark of disease progression, including cancers. In cancers of epithelial origin, this phenomenon is closely associated with dysfunction of E-cadherin. In this study, we investigated how E-cadherin mutations identified in patients with cancer affect tensional homeostasis. Our results show that mutations affecting the juxtamembrane and intracellular domains of E-cadherin are detrimental for tensional homeostasis of gastric cancer cells.In epithelia, breakdown of tensional homeostasis is closely associated with E-cadherin dysfunction and disruption of tissue function and integrity. In this study, we investigated the effect of E-cadherin mutations affecting distinct protein domains on tensional homeostasis of gastric cancer cells. We used micropattern traction microscopy to measure temporal fluctuations of cellular traction forces in AGS cells transfected with the wild-type E-cadherin or with variants affecting the extracellular, the juxtamembrane, and the intracellular domains of the protein. We focused on the dynamic aspect of tensional homeostasis, namely the ability of cells to maintain a consistent level of tension, with low temporal variability around a set point. Cells were cultured on hydrogels micropatterned with different extracellular matrix (ECM) proteins to test whether the ECM adhesion impacts cell behavior. A combination of Fibronectin and Vitronectin was used as a substrate that promotes the adhesive ability of E-cadherin dysfunctional cells, whereas Collagen VI was used to test an unfavorable ECM condition. Our results showed that mutations affecting distinct E-cadherin domains influenced differently cell tensional homeostasis, and pinpointed the juxtamembrane and intracellular regions of E-cadherin as the key players in this process. Furthermore, Fibronectin and Vitronectin might modulate cancer cell behavior towards tensional homeostasis.

Wrinkle force microscopy: a machine learning based approach to predict cell mechanics from images

Combining experiments with artificial intelligence algorithms, we propose a machine learning based approach called wrinkle force microscopy (WFM) to extract the cellular force distributions from the microscope images. The full process can be divided into three steps. First, we culture the cells on a special substrate allowing to measure both the cellular traction force on the substrate and the corresponding substrate wrinkles simultaneously. The cellular forces are obtained using the traction force microscopy (TFM), at the same time that cell-generated contractile forces wrinkle their underlying substrate. Second, the wrinkle positions are extracted from the microscope images. Third, we train the machine learning system with GAN (generative adversarial network) by using sets of corresponding two images, the traction field and the input images (raw microscope images or extracted wrinkle images), as the training data. The network understands the way to convert the input images of the substrate wrinkles to the traction distribution from the training. After sufficient training, the network is utilized to predict the cellular forces just from the input images. Our system provides a powerful tool to evaluate the cellular forces efficiently because the forces can be predicted just by observing the cells under the microscope, which is much simpler method compared to the TFM experiment. Additionally, the machine learning based approach presented here has the profound potential for being applied to diverse cellular assays for studying mechanobiology of cells.

Substrate rigidity modulates traction forces and stoichiometry of cell-matrix adhesions.

In cell-matrix adhesions, integrin receptors and associated proteins provide a dynamic coupling of the extracellular matrix (ECM) to the cytoskeleton. This allows bidirectional transmission of forces between the ECM and the cytoskeleton, which tunes intracellular signaling cascades that control survival, proliferation, differentiation, and motility. The quantitative relationships between recruitment of distinct cell-matrix adhesion proteins and local cellular traction forces are not known. Here, we applied quantitative super-resolution microscopy to cell-matrix adhesions formed on fibronectin-stamped elastomeric pillars and developed an approach to relate the number of talin, vinculin, paxillin, and focal adhesion kinase (FAK) molecules to the local cellular traction force. We find that FAK recruitment does not show an association with traction-force application, whereas a ∼60 pN force increase is associated with the recruitment of one talin, two vinculin, and two paxillin molecules on a substrate with an effective stiffness of 47kPa. On a substrate with a fourfold lower effective stiffness, the stoichiometry of talin:vinculin:paxillin changes to 2:12:6 for the same ∼60 pN traction force. The relative change in force-related vinculin recruitment indicates a stiffness-dependent switch in vinculin function in cell-matrix adhesions. Our results reveal a substrate-stiffness-dependent modulation of the relationship between cellular traction-force and the molecular stoichiometry of cell-matrix adhesions.

Chemo-Mechanical Factors That Limit Cellular Force Generation

Cellular traction forces that are dependent on actin-myosin activity are necessary for numerous developmental and physiological processes. As traction force emerges as a promising cancer biomarker there is a growing need to understand force generation in response to chemical and mechanical cues. Our goal is to present a unified modeling framework that integrates actin-myosin activity, substrate stiffness, integrin bond type, and adhesion complex dynamics to explain how force develops under specific conditions. Our simulation results show that substrate stiffness and number of myosin motors contribute to the maximum actin-myosin forces that can be generated but do not solely control the force transmitted by the cells to the surface, i.e., the traction force. The kinetics of the bonds between the cell and the substrate plays an equally important role. Overall, we find that while the cell can generate large actin-myosin forces in individual stress fibers (> 300 pN), the maximum force transmitted to the surface per cell-substrate attachment only reaches a fraction of these values (approx. 50 pN). Traction stress, the sum of forces transferred by all cell-substrate attachments in a unit area, is biphasic or sigmoidal with increasing substrate stiffness depending on the number of active myosin motors generating forces. Finally, we conclude that adhesions < 1 μm2 generate widely variable traction forces and that impulse, the magnitude and duration of a force generating event, is a key limiting factor in traction stress.

Multicellular mechanochemical hybrid cellular Potts model of tissue formation during epithelial‐mesenchymal transition

AbstractEpithelial‐mesenchymal transition (EMT) is the transdifferentiation of epithelial cells to a mesenchymal phenotype, in which cells lose epithelial‐like cell–cell adhesions and gain mesenchymal‐like enhanced contractility and mobility. EMT is crucial for tissue regeneration and is also implicated in pathological conditions, such as cancer metastasis. Prior work has shown that transforming growth factor‐1 (TGF‐1) is a potent inducer of this biological process. In this study, we develop a computational model coupling mechanical and biochemical signaling in a multicellular tissue undergoing EMT. Specifically, we utilize a recently developed formulation that integrates a multicellular cellular Potts model (CPM), a lattice‐based stochastic model governing cell movement; a first moment of area model, governing cellular traction and junctional forces; a finite element model, which defines extracellular matrix (ECM) substrate strains; an intracellular signaling TGF‐1‐mediated EMT model that governs cellular phenotype; and an extracellular signaling component governing ECM and TGF‐1 signaling. In this study, we modeled the spatial cellular patterns that occur in tissue and the ECM during EMT. Our model predicts that EMT often initially occurs at a tissue boundary due to mechanochemical coupling, which results in transdifferentiation to progress inwards toward the center. Variation in model parameters demonstrated conditions enhancing and suppressing EMT, especially to drive EMT in the absence of TGF‐1 and inhibit EMT in the presence of TGF‐1. Specifically, enhancing the mechanochemical feedback typically promoted EMT, whereas greater assembled ECM degradation suppressed EMT. Simulated scratch test experiments illustrate that ECM composition can impact closure directly through EMT signaling. In conclusion, we integrated mechanical, biochemical, and extracellular signaling networks in a novel hybrid computational model to reproduce tissue formation dynamics of EMT.

Quantifying force transmission through fibroblasts: changes of traction forces under external shearing

Mammalian cells have evolved complex mechanical connections to their microenvironment, including focal adhesion clusters that physically connect the cytoskeleton and the extracellular matrix. This mechanical link is also part of the cellular machinery to transduce, sense and respond to external forces. Although methods to measure cell attachment and cellular traction forces are well established, these are not capable of quantifying force transmission through the cell body to adhesion sites. We here present a novel approach to quantify intracellular force transmission by combining microneedle shearing at the apical cell surface with traction force microscopy at the basal cell surface. The change of traction forces exerted by fibroblasts to underlying polyacrylamide substrates as a response to a known shear force exerted with a calibrated microneedle reveals that cells redistribute forces dynamically under external shearing and during sequential rupture of their adhesion sites. Our quantitative results demonstrate a transition from dipolar to monopolar traction patterns, an inhomogeneous distribution of the external shear force to the adhesion sites as well as dynamical changes in force loading prior to and after the rupture of single adhesion sites. Our strategy of combining traction force microscopy with external force application opens new perspectives for future studies of force transmission and mechanotransduction in cells.

Mechanics-driven nuclear localization of YAP can be reversed by N-cadherin ligation in mesenchymal stem cells

Mesenchymal stem cells adopt differentiation pathways based upon cumulative effects of mechanosensing. A cell’s mechanical microenvironment changes substantially over the course of development, beginning from the early stages in which cells are typically surrounded by other cells and continuing through later stages in which cells are typically surrounded by extracellular matrix. How cells erase the memory of some of these mechanical microenvironments while locking in memory of others is unknown. Here, we develop a material and culture system for modifying and measuring the degree to which cells retain cumulative effects of mechanosensing. Using this system, we discover that effects of the RGD adhesive motif of fibronectin (representative of extracellular matrix), known to impart what is often termed “mechanical memory” in mesenchymal stem cells via nuclear YAP localization, are erased by the HAVDI adhesive motif of the N-cadherin (representative of cell-cell contacts). These effects can be explained by a motor clutch model that relates cellular traction force, nuclear deformation, and resulting nuclear YAP re-localization. Results demonstrate that controlled storage and removal of proteins associated with mechanical memory in mesenchymal stem cells is possible through defined and programmable material systems.

High-Resolution, Highly-Integrated Traction Force Microscopy Software.

Accurate measurement of cellular traction force is critical for understanding physical interaction between cells and the extracellular matrix. Traction force microscopy (TFM) has become the most widely used tool for this purpose. While TFM has made continual progress in terms of resolution and accuracy, there have been challenges regarding obtaining user-friendly software and choosing the right values for parameters and sub-processes associated with the software. Here we provide step-by-step instructions for a MATLAB-based TFM software application equipped with multiple methods for image deformation quantification and force reconstruction, along with clarification on the computational meaning of the parameters within the software. We outline how to choose the optimal sub-methods and values for parameters for each process, depending on the characteristics of images and purpose of the analyses. The software's runtime is 20, 4, and 0.05 min by Fast BEM L1 (Boundary Element Method L1-regularization), Fast BEM L2 (L2-regularization), and FTTC (Fourier Transform Traction Cytometry), respectively, in addition to 7 min of particle-tracking velocimetry-based deformation tracking, for a single image (1280 × 960 pixel) on a standard workstation. Finally, the colocalization accuracies, in reference to a paxillin-GFP image, are compared between the three force reconstruction methods. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Setting up the TFM package in MATLAB Basic Protocol 2: Running the TFM package Alternate Protocol 1: Stage drift correction: Efficient subpixel registration Alternate Protocol 2: Force field calculation: FastBEM.

The biochemical composition of the actomyosin network sets the magnitude of cellular traction forces.

The regulation of cellular force production relies on the complex interplay between a well-conserved set of proteins of the cytoskeleton: actin, myosin, and α-actinin. Despite our deep knowledge of the role of these proteins in force production at the molecular scale, our understanding of the biochemical regulation of the magnitude of traction forces generated at the entire-cell level has been limited, notably by the technical challenge of measuring traction forces and the endogenous biochemical composition in the same cell. In this study, we developed an alternative Traction-Force Microscopy (TFM) assay, which used a combination of hydrogel micropatterning to define cell adhesion and shape and an intermediate fixation/immunolabeling step to characterize strain energies and the endogenous protein contents in single epithelial cells. Our results demonstrated that both the signal intensity and the area of the Focal Adhesion (FA)–associated protein vinculin showed a strong positive correlation with strain energy in mature FAs. Individual contents from actin filament and phospho-myosin displayed broader deviation in their linear relationship to strain energies. Instead, our quantitative analyzes demonstrated that their relative amount exhibited an optimum ratio of phospho-myosin to actin, allowing maximum force production by cells. By contrast, although no correlation was identified between individual α-actinin content and strain energy, the ratio of α-actinin to actin filaments was inversely related to strain energy. Hence, our results suggest that, in the cellular model studied, traction-force magnitude is dictated by the relative numbers of molecular motors and cross-linkers per actin filament, rather than the amounts of an individual component in the cytoskeletal network. This assay offers new perspectives to study in more detail the complex interplay between the endogenous biochemical composition of individual cells and the force they produce.

A possible molecular mechanism for mechanotransduction at cellular focal adhesion complexes

Mechanotransduction at focal adhesion complexes is key for various cellular events. Theoretical analyses were performed to predict a potential role of lipid membranes in modulating mechanotransduction at focal adhesions. Calculations suggested that the size of nanostructural constraints and mechanical pulling applied on lipid membranes affect the generation of cellular traction forces and signaling transduction at focal adhesions. This work provides predictions on how lipid membranes contribute to mechanotransduction at cellular focal adhesions.

Correlation of cellular traction forces and dissociation kinetics of adhesive protein zyxin revealed by multi-parametric live cell microscopy.

Cells exert traction forces on the extracellular matrix to which they are adhered through the formation of focal adhesions. Spatial-temporal regulation of traction forces is crucial in cell adhesion, migration, cellular division, and remodeling of the extracellular matrix. By cultivating cells on polyacrylamide hydrogels of different stiffness we were able to investigate the effects of substrate stiffness on the generation of cellular traction forces by Traction Force Microscopy (TFM), and characterize the molecular dynamics of the focal adhesion protein zyxin by Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery After Photobleaching (FRAP). As the rigidity of the substrate increases, we observed an increment of both, cellular traction generation and zyxin residence time at the focal adhesions, while its diffusion would not be altered. Moreover, we found a positive correlation between the traction forces exerted by cells and the residence time of zyxin at the substrate elasticities studied. We found that this correlation persists at the subcellular level, even if there is no variation in substrate stiffness, revealing that focal adhesions that exert greater traction present longer residence time for zyxin, i.e., zyxin protein has less probability to dissociate from the focal adhesion.

Hypergravity affects cell traction forces of fibroblasts

Cells sense and react on changes of the mechanical properties of their environment and, likewise, respond to external mechanical stress applied to them. However, whether the gravitational field as overall body force modulates cellular behavior is unclear. Different studies demonstrated that micro- and hypergravity influences the shape and elasticity of cells, initiate cytoskeleton reorganization, and influence cell motility. All these cellular properties are interconnected and contribute to forces that cells apply on their surrounding microenvironment. Yet, studies that investigated changes of cell traction forces under hypergravity conditions are scarce. Here, we performed hypergravity experiments on 3T3 fibroblast cells using the large-diameter centrifuge at the European Space Agency - European Space Research and Technology Centre. Cells were exposed to hypergravity of up to 19.5 g for 16 h in both the upright and the inverted orientation with respect to the g-force vector. We observed a decrease in cellular traction forces when the gravitational field was increased up to 5.4 g, followed by an increase of traction forces for higher gravity fields up to 19.5 g independent of the orientation of the gravity vector. We attribute the switch in cellular response to shear thinning at low g-forces, followed by significant rearrangement and enforcement of the cytoskeleton at high g-forces.

Study on the Expansion Dynamics of MDCK Epithelium by Interstitial Flow Using a Traction Force-Measurable Microfluidic Chip.

The movement of collective cells is affected through changes in physical interactions of cells in response to external mechanical stimuli, including fluid flow. Most tissues are affected by fluid flow at the interstitial level, but few studies have investigated the physical effects in collective cells affected by a low flow rate. In this study, collective cell migration of Madin–Darby canine kidney (MDCK) epithelial cells was investigated under static or interstitial flow (0, 0.1, and 1 μL/min) using a traction microfluidic device. The optimization of calculation of cellular traction forces was first achieved by changing interrogation window size from the fluorescent bead images. Migration analysis of cell collectives patterned with a 700 μm circular shape reveals that cells under the slow flow (0.1 and 1 μL/min) showed the inhibitory migration by decreasing cell island size and cellular speed compared to that of static condition. Analysis of cellular forces shows that level of traction forces was lower in the slow flow condition (~20 Pa) compared to that of static condition (~50 Pa). Interestingly, the standard deviation of traction force of cells was dramatically decreased as the flow rate increased from 0 to 1 μL/min, which indicates that flow affects the distribution of cellular traction forces among cell collectives. Cellular tension was increased by 50% in the cells under the fluid flow rate of 1 μL/min. Treatment of calcium blocker increased the migratory speed of cells under the flow condition, whereas there is little change of cellular forces. In conclusion, it has been shown that the interstitial flow inhibited the collective movement of epithelial cells by decreasing and re-distributing cellular forces. These findings provide insights into the study of the effect of interstitial flow on cellular behavior, such as development, regeneration, and morphogenesis.

Computational 4D-OCM for label-free imaging of collective cell invasion and force-mediated deformations in collagen

Traction force microscopy (TFM) is an important family of techniques used to measure and study the role of cellular traction forces (CTFs) associated with many biological processes. However, current standard TFM methods rely on imaging techniques that do not provide the experimental capabilities necessary to study CTFs within 3D collective and dynamic systems embedded within optically scattering media. Traction force optical coherence microscopy (TF-OCM) was developed to address these needs, but has only been demonstrated for the study of isolated cells embedded within optically clear media. Here, we present computational 4D-OCM methods that enable the study of dynamic invasion behavior of large tumor spheroids embedded in collagen. Our multi-day, time-lapse imaging data provided detailed visualizations of evolving spheroid morphology, collagen degradation, and collagen deformation, all using label-free scattering contrast. These capabilities, which provided insights into how stromal cells affect cancer progression, significantly expand access to critical data about biophysical interactions of cells with their environment, and lay the foundation for future efforts toward volumetric, time-lapse reconstructions of collective CTFs with TF-OCM.

Viscoelastic substrate decouples cellular traction force from other related phenotypes

Survival and maintenance of normal physiological functions depends on continuous interaction of cells with its microenvironment. Cells sense the mechanical properties of underlying substrate by applying force and modulate their behaviour in response to the resistance offered by the substrate. Most of the studies addressing cell-substrate mechanical interactions have been carried out using elastic substrates. Since tissues within our body are viscoelastic in nature, here we explore the effect of substrate’s viscoelasticity on various properties of mesenchymal stem cells. Here, we used two sets of polyacrylamide substrates having similar storage modulus (G’ = 1.1–1.6 kPa) but different loss modulus (G” = 45 Pa and 300 Pa). We report that human mesenchymal stem cells spread more but apply less force on the viscoelastic substrate (substrate with higher loss modulus). We further investigated the effect of substrate viscoelasticity on the expression of other contractility-associated proteins such as focal adhesion (FA) proteins (Vinculin, Paxillin, Talin), cytoskeletal proteins (actin, mysion, intermediate filaments, and microtubules) and mechano-sensor protein Yes-Associated Protein (YAP). Our results show that substrate viscoelasticity decouples cellular traction from other known traction related phenotypes.

Stick–slip model for actin-driven cell protrusions, cell polarization, and crawling

Cell crawling requires the generation of intracellular forces by the cytoskeleton and their transmission to an extracellular substrate through specific adhesion molecules. Crawling cells show many features of excitable systems, such as spontaneous symmetry breaking and crawling in the absence of external cues, and periodic and propagating waves of activity. Mechanical instabilities in the active cytoskeleton network and feedback loops in the biochemical network of activators and repressors of cytoskeleton dynamics have been invoked to explain these dynamical features. Here, I show that the interplay between the dynamics of cell-substrate adhesion and linear cellular mechanics is sufficient to reproduce many nonlinear dynamical patterns observed in spreading and crawling cells. Using an analytical formalism of the molecular clutch model of cell adhesion, regulated by local mechanical forces, I show that cellular traction forces exhibit stick-slip dynamics resulting in periodic waves of protrusion/retraction and propagating waves along the cell edge. This can explain spontaneous symmetry breaking and polarization of spreading cells, leading to steady crawling or bipedal motion, and bistability, where persistent cell motion requires a sufficiently strong transient external stimulus. The model also highlights the role of membrane tension in providing the long-range mechanical communication across the cell required for symmetry breaking.

Live-cell super-resolved PAINT imaging of piconewton cellular traction forces.

Despite the vital role of mechanical forces in biology, it still remains a challenge to image cellular force with sub-100-nm resolution. Here, we present tension points accumulation for imaging in nanoscale topography (tPAINT), integrating molecular tension probes with the DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) technique to map piconewton mechanical events with ~25-nm resolution. To perform live-cell dynamic tension imaging, we engineered reversible probes with a cryptic docking site revealed only when the probe experiences forces exceeding a defined mechanical threshold (~7-21 pN). Additionally, we report a second type of irreversible tPAINT probe that exposes its cryptic docking site permanently and thus integrates force history over time, offering improved spatial resolution in exchange for temporal dynamics. We applied both types of tPAINT probes to map integrin receptor forces in live human platelets and mouse embryonic fibroblasts. Importantly, tPAINT revealed a link between platelet forces at the leading edge of cells and the dynamic actin-rich ring nucleated by the Arp2/3 complex.

Slippery and magnetically responsive micropillared surfaces for manipulation of droplets and beads

Stimuli-responsive surfaces are of practical importance for applications ranging from enhanced mixing of reagents in lab-on-a-chip systems until probing cellular traction forces. Non-destructive reversible bending of cilia-inspired magnetic pillars can be used for controlled transportation of non-magnetic objects and bio-inspired sensing. Magnetic actuation of micropillars suspended in liquids allows controlled mixing, propelling, and stirring of fluids as well as droplet manipulation, which are important for various applications including generation of cell spheroids and droplet coalescence in microfluidic systems. In order to expand their practical applications, fabrication processes capable of rapid prototyping have to be developed. Inspired by biological cilia and their functionalities, actuating hairy surfaces are herein fabricated and implemented to manipulate both microbeads and droplets. The artificial cilia are based on microscale magnetic pillar arrays made of flexible polydimethylsiloxane functionalized with magnetic microparticles. The arrays are fabricated by a new method using patterned molds that relies on cryogenic separation to produce transparent cilia-inspired arrays without requiring manual interference to clean the templates during the process. Magnetic actuation of the pillar arrays is demonstrated in isopropanol and silicone oil. Filling with oil yields magnetically responsive slippery lubricated surfaces allowing directional motion of droplets by repetitive bending and recovery of the flexible magnetic pillars. The achieved structures allow manipulation of microbeads and droplets which is uncommon even at the sub-mm scale; directional motion is demonstrated for 250 μm–550 μm sized droplets. Droplet transportation is facilitated by extremely low hysteresis and a high degree of omnidirectional bending of the pillar array.

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.