Cellular Traction Research Articles

Stress-induced plasticity of dynamic collagen networks

The structure and mechanics of tissues is constantly perturbed by endogenous forces originated from cells, and at the same time regulate many important cellular functions such as migration, differentiation, and growth. Here we show that 3D collagen gels, major components of connective tissues and extracellular matrix (ECM), are significantly and irreversibly remodeled by cellular traction forces, as well as by macroscopic strains. To understand this ECM plasticity, we develop a computational model that takes into account the sliding and merging of ECM fibers. We have confirmed the model predictions with experiment. Our results suggest the profound impacts of cellular traction forces on their host ECM during development and cancer progression, and suggest indirect mechanical channels of cell-cell communications in 3D fibrous matrices.

Full L1-regularized Traction Force Microscopy over whole cells

BackgroundTraction Force Microscopy (TFM) is a widespread technique to estimate the tractions that cells exert on the surrounding substrate. To recover the tractions, it is necessary to solve an inverse problem, which is ill-posed and needs regularization to make the solution stable. The typical regularization scheme is given by the minimization of a cost functional, which is divided in two terms: the error present in the data or data fidelity term; and the regularization or penalty term. The classical approach is to use zero-order Tikhonov or L2-regularization, which uses the L2-norm for both terms in the cost function. Recently, some studies have demonstrated an improved performance using L1-regularization (L1-norm in the penalty term) related to an increase in the spatial resolution and sensitivity of the recovered traction field. In this manuscript, we present a comparison between the previous two regularization schemes (relying in the L2-norm for the data fidelity term) and the full L1-regularization (using the L1-norm for both terms in the cost function) for synthetic and real data.ResultsOur results reveal that L1-regularizations give an improved spatial resolution (more important for full L1-regularization) and a reduction in the background noise with respect to the classical zero-order Tikhonov regularization. In addition, we present an approximation, which makes feasible the recovery of cellular tractions over whole cells on typical full-size microscope images when working in the spatial domain.ConclusionsThe proposed full L1-regularization improves the sensitivity to recover small stress footprints. Moreover, the proposed method has been validated to work on full-field microscopy images of real cells, what certainly demonstrates it is a promising tool for biological applications.

Biophysical and biomolecular determination of cellular age in humans

Ageing research has focused either on assessing organ- and tissue-based changes, such as lung capacity and cardiac function, or on changes at the molecular scale such as gene expression, epigenetic modifications and metabolism. Here, by using a cohort of 32 samples of primary dermal fibroblasts collected from individuals between 2 and 96 years of age, we show that the degradation of functional cellular biophysical features-including cell mechanics, traction strength, morphology and migratory potential-and associated descriptors of cellular heterogeneity predict cellular age with higher accuracy than conventional biomolecular markers. We also demonstrate the use of high-throughput single-cell technologies, together with a deterministic model based on cellular features, to compute the cellular age of apparently healthy males and females, and to explore these relationships in cells from individuals with Werner syndrome and Hutchinson-Gilford progeria syndrome, two rare genetic conditions that result in phenotypes that show aspects of premature ageing. Our findings suggest that the quantification of cellular age may be used to stratify individuals on the basis of cellular phenotypes and serve as a biological proxy of healthspan.

Microfluidic traction force microscopy to study mechanotransduction in angiogenesis.

The formation of new blood vessels from existing vasculature, angiogenesis, is driven by coordinated endothelial cell migration and matrix remodeling in response to local signals. Recently, a growing body of evidence has shown that mechanotransduction, along with chemotransduction, is a major regulator of angiogenesis. Mechanical signals, such as fluid shear stress and substrate mechanics, influence sprouting and network formation, but the mechanisms behind this relationship are still unclear. Here, we present cellular traction forces as possible effectors activated by mechanosensing to mediate matrix remodeling, and encourage the use of TFM to study mechanotransduction in angiogenesis. We also suggest that deciphering the response of EC to mechanical signals could reveal an optimal angiogenic mechanical environment, and provide insight into development, wound healing, the initiation and growth of tumors, and new strategies for tissue engineering.

Mechanical stability of talin rod controls cell migration and substrate sensing

Cells adhere to the surrounding tissue and probe its mechanical properties by forming cell-matrix adhesions. Talin is a critical adhesion protein and participates in the transmission of mechanical signals between extracellular matrix and cell cytoskeleton. Force induced unfolding of talin rod subdomains has been proposed to act as a cellular mechanosensor, but so far evidence linking their mechanical stability and cellular response has been lacking. Here, by utilizing computationally designed mutations, we demonstrate that stepwise destabilization of the talin rod R3 subdomain decreases cellular traction force generation, which affects talin and vinculin dynamics in cell-matrix adhesions and results in the formation of talin-rich but unstable adhesions. We observed a connection between talin stability and the rate of cell migration and also found that talin destabilization affects the usage of different integrin subtypes and sensing of extracellular matrix proteins. Experiments with truncated forms of talin confirm the mechanosensory role of the talin R3 subdomain and exclude the possibility that the observed effects are caused by the release of talin head-rod autoinhibition. In conclusion, this study provides evidence into how the controlled talin rod domain unfolding acts as a key regulator of adhesion structure and function and consequently controls central cellular processes such as cell migration and substrate sensing.

Collagen Gels with Different Fibrillar Microarchitectures Elicit Different Cellular Responses.

The extracellular matrix consists of a complex mixture of fibrillar proteins, in which the architecture and mechanical properties of the protein fibrils vary considerably in various tissues. Here, we systematically polymerized collagen gels at different temperatures, providing substrates with tunable mechanics and defined local microarchitecture. We studied the dependence of spreading dynamics, proliferation, migration, and differentiation of human mesenchymal stem cells (hMSCs) on the fibrillar properties as compared to the bulk properties of the matrix. We found that high fiber stiffness, together with shorter fiber lengths, limited the transfer of cellular traction forces to nearby fibers. As a result, cells were not able to build up sufficient tension, which suppressed cell spreading, proliferation, and migration. Cells on such fibers also showed limited focal adhesion formation and different lineage selection preferences. In contrast, cell spreading, proliferation, and migration was always associated with fiber recruitment, long-range deformations in the collagen gel networks and an increase in collagen density around cells. Typically, cells on such substrates had a preference for osteogenic differentiation and showed higher levels of focal adhesions formation. These results contribute to a further understanding of the mechanotransduction process and to the design criteria for future biomimetic materials for tissue-engineering applications.

Theory on Bending in Cantilever Beams With Adsorbed Islands

Traction between adsorbed islands and the substrate is commonly seen in both living and material systems: deposited material gathers into islands at the early stage of polycrystalline film deposition and generates stress due to lattice mismatch, cells exert cellular traction to extracellular matrix to probe their surrounding microenvironment in vivo, and so on. The traction between these islands and the substrate can result in perceivable macroscopic deformation in the substrate and may be measurable if the substrate is a cantilever beam. However, currently broadly used Stoney equation is incapable of handling such boundary condition. In this paper, we give the closed-form expression on the resulted curvature in substrate beams by distributed tractions. Such a relationship could be employed to monitor the stress evolution during thin film deposition, to quantify the stress level of cell traction as cells adhere to cantilever beams, and other related mechanical systems like charging–discharging induced stress in island-patterned electrode films. Moreover, we found that follower traction induced by an array of islands could lead to negative curvature. It shields light on the early stage compressive stress during polycrystalline film deposition.

Mechanotransduction Dynamics at the Cell-Matrix Interface

The ability of cells to sense and respond to mechanical cues from the surrounding environment has been implicated as a key regulator of cell differentiation, migration, and proliferation. The extracellular matrix (ECM) is an oft-overlooked component of the interface between cells and their surroundings. Cells assemble soluble ECM proteins into insoluble fibrils with unique mechanical properties that can alter the mechanical cues a cell receives. In this study, we construct a model that predicts the dynamics of cellular traction force generation and subsequent assembly of fibrils of the ECM protein fibronectin (FN). FN fibrils are the primary component in primordial ECM and, as such, FN assembly is a critical component in the cellular mechanical response. The model consists of a network of Hookean springs, each representing an extensible domain within an assembling FN fibril. As actomyosin forces stretch the spring network, simulations predict the resulting traction force and FN fibril formation. The model accurately predicts FN fibril morphometry and demonstrates a mechanism by which FN fibril assembly regulates traction force dynamics in response to mechanical stimuli and varying surrounding substrate stiffness.

Tug of War at the Cell-Matrix Interface

Mechanical interactions between a cell and its environment, or between cells, influence key developmental and physiologic processes as well as many aspect of disease (1,2). Indeed the ability of a cell to sense, produce, and respond to mechanical cues has emerged as a fundamental regulator of cell behaviors such as differentiation, proliferation, survival, and migration. In mechanical terms the interactions governing these behaviors are regulated by intracellular and extracellular physical events that are orchestrated by complex biochemical and mechanical signals.

Zinc oxide nanorod field effect transistor for long-time cellular force measurement

Mechanical forces generated by cells are known to influence a vast range of cellular functions ranging from receptor signaling and transcription to differentiation and proliferation. We report a novel measurement approach using zinc oxide nanorods as a peeping transducer to monitor dynamic mechanical behavior of cellular traction on surrounding substrate. We develop a ZnO nanorod field effect transistor (FET) as an ultrasensitive force sensor to realize long-time, unstained, and in-situ detection of cell cycle phases, including attachment, spread, and mitosis. Excellent biocompatibility and ultra-sensitivity of the biomechanical measurement is ensured by coating a parylene film on the FET sensor as a concealment, which provides complete electronic isolation between the sensor and cell. With unique features of ultra-sensitivity, label-free, easy handling, and good biocompatibility, the force sensor allows feasible for tracking cellular dynamics in physiological contexts and understanding their contribution to biological processes.

Optogenetic control of cellular forces and mechanotransduction

Contractile forces are the end effectors of cell migration, division, morphogenesis, wound healing and cancer invasion. Here we report optogenetic tools to upregulate and downregulate such forces with high spatiotemporal accuracy. The technology relies on controlling the subcellular activation of RhoA using the CRY2/CIBN light-gated dimerizer system. We fused the catalytic domain (DHPH domain) of the RhoA activator ARHGEF11 to CRY2-mCherry (optoGEF-RhoA) and engineered its binding partner CIBN to bind either to the plasma membrane or to the mitochondrial membrane. Translocation of optoGEF-RhoA to the plasma membrane causes a rapid and local increase in cellular traction, intercellular tension and tissue compaction. By contrast, translocation of optoGEF-RhoA to mitochondria results in opposite changes in these physical properties. Cellular changes in contractility are paralleled by modifications in the nuclear localization of the transcriptional regulator YAP, thus showing the ability of our approach to control mechanotransductory signalling pathways in time and space.

Molecular Tension Sensors Reveal a Minimally Tensioned Integrin State in Living Cells

Integrins mediate cell adhesion to the extracellular matrix and enable the construction of complex, multicellular organisms. Despite this biological prominence, fundamental aspects of integrin-based adhesion remain poorly understood. Notably, the magnitude of the mechanical load experienced by individual integrins within living cells is unclear, due principally to limitations inherent to existing techniques. We used Forster resonance energy transfer (FRET)-based molecular tension sensors (MTSs) to measure the distribution of forces exerted by individual integrin heterodimers in living cells. Taking advantage of the sensors’ modular nature, we engineered MTS variants that display a minimal RGD sequence derived from fibronectin (MTSRGD) or the 9th and 10th type III domains of fibronectin (MTSFN), and are sensitive to low (0-7 pN) or intermediate (8-11 pN) loads. Using these sensors, we found that a large fraction of integrins in living cells exert forces <3 pN, while a minority subpopulation experiencing substantially higher loads was enriched in large adhesion complexes. Treatment with the filamentous (F)-actin disruptor cytochalasin D revealed that only the high-load integrin population required an intact actin cytoskeleton, and that the low-load (<3 pN) population was sufficient to mediate cell adhesion on short, ∼15 minute timescales. Integrin engagement with the fibronectin synergy site, a secondary binding site specifically for α5β1 integrin, led to increased recruitment of α5β1 integrin to adhesions, but not to an increase in overall cellular traction generation. Consistent with previous reports, the presence of the synergy site did, however, increase the resistance of cells to detachment via externally applied load. Based on these data, we suggest that a large pool of engaged, but minimally tensioned integrins may provide a synergy site-dependent adhesive reserve that imparts cells and tissues with mechanical integrity in the presence of widely varying mechanical loads. In ongoing work, we take advantage of the unique capabilities of MTSs to determine how subpopulations of load-bearing integrins are altered in response to external mechanical perturbations, and how (and whether) distinct integrin subtypes bear differing levels of mechanical load.

Two-Layer Elastographic 3-D Traction Force Microscopy

Cellular traction force microscopy (TFM) requires knowledge of the mechanical properties of the substratum where the cells adhere to calculate cell-generated forces from measurements of substratum deformation. Polymer-based hydrogels are broadly used for TFM due to their linearly elastic behavior in the range of measured deformations. However, the calculated stresses, particularly their spatial patterns, can be highly sensitive to the substratum’s Poisson’s ratio. We present two-layer elastographic TFM (2LETFM), a method that allows for simultaneously measuring the Poisson’s ratio of the substratum while also determining the cell-generated forces. The new method exploits the analytical solution of the elastostatic equation and deformation measurements from two layers of the substratum. We perform an in silico analysis of 2LETFM concluding that this technique is robust with respect to TFM experimental parameters, and remains accurate even for noisy measurement data. We also provide experimental proof of principle of 2LETFM by simultaneously measuring the stresses exerted by migrating Physarum amoeboae on the surface of polyacrylamide substrata, and the Poisson’s ratio of the substrata. The 2LETFM method could be generalized to concurrently determine the mechanical properties and cell-generated forces in more physiologically relevant extracellular environments, opening new possibilities to study cell-matrix interactions.

Cellular traction forces increase during consecutive mechanical stretching following traction force attenuation

Cellular traction forces increase during consecutive mechanical stretching following traction force attenuation

スループットの高い細胞トラクションフォースアッセイの開発

スループットの高い細胞トラクションフォースアッセイの開発

Substrate stiffness promotes latent TGF-β1 activation in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) was usually coupled with increased stiffness of the extracellular matrix (ECM) and elevated level of transforming growth factor-β1 (TGF-β1). However, the mechanism by which substrate rigidity modulated TGF-β1 signaling transduction remained unknown. This paper investigated the molecular mechanism of how matrix stiffness regulating TGF-β1 signaling in HCC cells. By means of stiffness tunable collagen I-coated polyacrylamide (PA) gels, we found that the expressions of β1 integrin, p-FAK Y397 and p-Smad2 upregulated on stiffer gels as well as the content of TGF-β1 in culture media of HCC cells, which were inhibited by RGD blocking peptides, Y-27632 (ROCK inhibitor) or Blebbistatin (myosin II inhibitor). Cellular traction force was also significantly higher when plated on stiffer substrates but dramatically decreased after treatment with Y-27632 or Blebbistatin. Furthermore, the upregulation of p-Smad2 in the HCC cells on stiffer PA gels induced by exogenetic latent TGF-β1 was downregulated in the presence of RGD peptides. The nuclear translocation of Smad2 induced by latent TGF-β1 was inhibited by Y-27632 or Blebbistatin. Our results suggested that the extracellular matrix stiffness regulated latent TGF-β1 activation by cytoskeletal tension in HCC cells, showing that matrix stiffness was a key regulator involving the TGF-β1 activity in HCC cells. The current study presented a mechanism of how hepatocirrhosis developed into liver cancer.

Single Molecule Force Measurements in Living Cells Reveal a Minimally Tensioned Integrin State.

Integrins mediate cell adhesion to the extracellular matrix and enable the construction of complex, multicellular organisms, yet fundamental aspects of integrin-based adhesion remain poorly understood. Notably, the magnitude of the mechanical load experienced by individual integrins within living cells is unclear, due principally to limitations inherent to existing techniques. Here we use Förster resonance energy transfer-based molecular tension sensors to directly measure the distribution of loads experienced by individual integrins in living cells. We find that a large fraction of integrins bear modest loads of 1-3 pN, while subpopulations bearing higher loads are enriched within adhesions. Further, our data indicate that integrin engagement with the fibronectin synergy site, a secondary binding site specifically for α5β1 integrin, leads to increased levels of α5β1 integrin recruitment to adhesions but not to an increase in overall cellular traction generation. The presence of the synergy site does, however, increase cells' resistance to detachment by externally applied loads. We suggest that a substantial population of integrins experiencing loads well below their peak capacities can provide cells and tissues with mechanical integrity in the presence of widely varying mechanical loads.

New wrinkling substrate assay reveals traction force fields of leader and follower cells undergoing collective migration

Physical forces play crucial roles in coordinating collective migration of epithelial cells, but details of such force-related phenomena remain unclear partly due to the lack of robust methodologies to probe the underlying force fields. Here we develop a method for fabricating silicone substrates that detect cellular traction forces with a high sensitivity. Specifically, a silicone elastomer is exposed to oxygen plasma under heating. Removal of the heat shrinks the substrate so as to reduce its critical buckling strain in a spatially uniform manner. Thus, even small cellular traction forces can be visualized as micro-wrinkles that are reversibly emerged on the substrate in a direction orthogonal to the applied forces. Using this technique, we show that so-called leader cells in MDCK-II cell clusters exert significant magnitudes of traction forces distinct from those of follower cells. We reveal that the direction of traction forces is highly correlated with the long axis of the local, individual cells within clusters. These results suggest that the force fields in collective migration of MDCK-II cells are predominantly determined locally at individual cell scale rather than globally at the whole cell cluster scale.

Measuring cellular traction forces on non-planar substrates.

Animal cells use traction forces to sense the mechanics and geometry of their environment. Measuring these traction forces requires a workflow combining cell experiments, image processing and force reconstruction based on elasticity theory. Such procedures have already been established mainly for planar substrates, in which case one can use the Green's function formalism. Here we introduce a workflow to measure traction forces of cardiac myofibroblasts on non-planar elastic substrates. Soft elastic substrates with a wave-like topology were micromoulded from polydimethylsiloxane and fluorescent marker beads were distributed homogeneously in the substrate. Using feature vector-based tracking of these marker beads, we first constructed a hexahedral mesh for the substrate. We then solved the direct elastic boundary volume problem on this mesh using the finite-element method. Using data simulations, we show that the traction forces can be reconstructed from the substrate deformations by solving the corresponding inverse problem with an L1-norm for the residue and an L2-norm for a zeroth-order Tikhonov regularization. Applying this procedure to the experimental data, we find that cardiac myofibroblast cells tend to align both their shapes and their forces with the long axis of the deformable wavy substrate.

Recent advances in biological uses of traction force microscopy

Cell traction forces (CTF) generated by the actomyosin cytoskeleton onto a substrate or extracellular matrix (ECM) are essential for many biological processes, including developmental morphogenesis, tissue homeostasis, and cancer metastasis. Because the cellular physical properties are closely related to the pathological states of the cells, affected by various physicochemical stimuli from their neighboring cells or surrounding environments, it is crucial to develop a quantitative measure for cellular responses to these external stimuli. Since the pioneering work of Harris et al. in 1980s1, traction force microscopy (TFM) has been widely used as a standard tool that allows the optical measurement of cellular tractions exerted on 2- and 3-dimensional soft elastic substrates. Recently, there have been many technical advances in conventional TFM to enhance its spatial and temporal resolutions as well as the range of applicability. In this review, we provide a survey on the recent advancement in TFM, especially with a special emphasis on platforms that can externally apply various stimuli such as fluid shear, mechanical tension or compression, biochemical factors, and electric field in a physiologically relevant regime.