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Traction force microscopy has been established as the accepted method for evaluating cell-induced mechanical stresses to their microenvironments, typically using two-dimensional (2D) elastic, synthetic gel-substrates. As cells naturally experience 3D environments in vivo, traction microscopy has been adapted to 3D gels; cells can be tracked over time in 3D. Microscopy images acquired in several fields-of-view e.g. in a time series, may experience drift, which can produce artefactual results that may appear valid and lead to flawed analysis. Hence, we have developed an algorithm for 2D/3D de-drifting of cell-images on 3D gels with fiducial markers (beads) as anchor points. Both lateral and vertical de-drifting are performed using gel-internalized beads, as those used in traction microscopy experiments; this eliminates need for immobilizing beads under the gel for de-drifting, and reduces experiment time. We introduce simulations of initially grid-ordered dots (beads) that are radially displaced to experimentally observed distances, while also applying additive drift. This facilitates testing and demonstration of the de-drifting procedures in 2D/3D. We demonstrate the importance of applying de-drifting using both computer-simulated drifts and experimentally observed drifts in confocal microscopy images. We show that our de-drifting algorithm can remove lateral and/or vertical drift revealing even small, underlying signals. The 2D/3D de-drifting algorithm, crucial for accurate identification of cell-induced marker-displacement, as well as the bead simulations, will shorten traction microscopy experiments and facilitate optimization of the experimental protocols.

In native states, animal cells of many types are supported by a fibrous network that forms the main structural component of the ECM. Mechanical interactions between cells and the 3D ECM critically regulate cell function, including growth and migration. However, the physical mechanism that governs the cell interaction with fibrous 3D ECM is still not known. In this article, we present single-cell traction force measurements using breast tumor cells embedded within 3D collagen matrices. We recreate the breast tumor mechanical environment by controlling the microstructure and density of type I collagen matrices. Our results reveal a positive mechanical feedback loop: cells pulling on collagen locally align and stiffen the matrix, and stiffer matrices, in return, promote greater cell force generation and a stiffer cell body. Furthermore, cell force transmission distance increases with the degree of strain-induced fiber alignment and stiffening of the collagen matrices. These findings highlight the importance of the nonlinear elasticity of fibrous matrices in regulating cell–ECM interactions within a 3D context, and the cell force regulation principle that we uncover may contribute to the rapid mechanical tissue stiffening occurring in many diseases, including cancer and fibrosis.

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.

Non-contacting, adjacent cancer cells can mechanically interact through their substrate to increase their invasive and migratory capacities that underly metastases-formation. Such mechanical interactions may induce additive or synergistic enhancement of invasiveness, potentially indicating different underlying force-mechanisms. To identify cell–cell-gel interactions, we monitor the time-evolution of three-dimensional traction strains induced by MDA-MB-231 breast cancer cells adhering on physiological-stiffness (1.8 kPa) collagen gels and compare to simulations. Single metastatic cells apply strain energies of 0.2–2 pJ (average 0.51 ± 0.06 pJ) at all observation times (30–174 min) inducing a mechanical volume-of-effect in the collagen gel that is initially (<60 min from seeding) on the cell-volume scale (∼3000 µm3) and on average increases with time from cell seeding. When cells adhere closely adjacent, at short times (<60 min) we distinguish the additive contributions of neighboring cells to the strains, while at longer times strain fields are synergistically amplified and may facilitate increased cooperative/collective cancer-cell-invasiveness. The results of well-spaced and closely adjacent cells at short times match our simulations of additive deformations induced by radially applied strains with experimentally based inverse-distance decay. We thus reveal a time-dependent evolution from additive to synergistic interactions of adjacently adhering cells that may facilitate metastatic invasion.

Talin and vinculin are mechanosensitive proteins that are recruited early to integrin-based nascent adhesions (NAs). In two epithelial cell systems with well-delineated NA formation, we find these molecules concurrently recruited to the subclass of NAs maturing to focal adhesions. After the initial recruitment under minimal load, vinculin accumulates in maturing NAs at a ~ fivefold higher rate than in non-maturing NAs, and is accompanied by a faster traction force increase. We identify the R8 domain in talin, which exposes a vinculin-binding-site (VBS) in the absence of load, as required for NA maturation. Disruption of R8 domain function reduces load-free vinculin binding to talin, and reduces the rate of additional vinculin recruitment. Taken together, these data show that the concurrent recruitment of talin and vinculin prior to mechanical engagement with integrins is essential for the traction-mediated unfolding of talin, exposure of additional VBSs, further recruitment of vinculin, and ultimately, NA maturation.

The mechanical forces exerted by cells on their surrounding microenvironment are known as cellular traction forces. These forces play crucial roles in various biological processes, such as tissue development, wound healing and cell functions. However, it is hard for traditional techniques to measure cellular traction forces accurately because their magnitude (from pN to nN) and the length scales over which they occur (from nm to μm) are extremely small. In order to fully understand mechanotransduction, highly sensitive tools for measuring cellular forces are needed. Current powerful techniques for measuring traction forces include traction force microscopy (TFM) and fluorescent molecular force sensors (FMFS). In this review, we elucidate the force imaging principles of TFM and FMFS. Then we highlight the application of FMFS in a variety of biological processes and offer our perspectives and insights into the potential applications of FMFS.

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.

The cytoskeleton of eukaryotic cells is primarily composed of networks of filamentous proteins, F-actin, microtubules, and intermediate filaments. Interactions among the cytoskeletal components are important in determining cell structure and in regulating cell functions. For example, F-actin and microtubules work together to control cell shape and polarity, while the subcellular organization and transport of vimentin intermediate filament (VIF) networks depend on their interactions with microtubules. However, it is generally thought that F-actin and VIFs form two coexisting but separate networks that are independent due to observed differences in their spatial distribution and functions. In this paper, we present a closer investigation of both the structural and functional interplay between the F-actin and VIF cytoskeletal networks. We characterize the structure of VIFs and F-actin networks within the cell cortex using structured illumination microscopy and cryo-electron tomography. We find that VIFs and F-actin form an interpenetrating network (IPN) with interactions at multiple length scales, and VIFs are integral components of F-actin stress fibers. From measurements of recovery of cell contractility after transient stretching, we find that the IPN structure results in enhanced contractile forces and contributes to cell resilience. Studies of reconstituted networks and dynamic measurements in cells suggest direct and specific associations between VIFs and F-actin. From these results, we conclude that VIFs and F-actin work synergistically, both in their structure and in their function. These results profoundly alter our understanding of the contributions of the components of the cytoskeleton, particularly the interactions between intermediate filaments and F-actin.

Motile cells can use and switch between different modes of migration. Here, we use traction force microscopy and fluorescent labeling of actin and myosin to quantify and correlate traction force patterns and cytoskeletal distributions in Dictyostelium discoideum cells that move and switch between keratocyte‐like fan‐shaped, oscillatory, and amoeboid modes. We find that the wave dynamics of the cytoskeletal components critically determine the traction force pattern, cell morphology, and migration mode. Furthermore, we find that fan‐shaped cells can exhibit two different propulsion mechanisms, each with a distinct traction force pattern. Finally, the traction force patterns can be recapitulated using a computational model, which uses the experimentally determined spatiotemporal distributions of actin and myosin forces and a viscous cytoskeletal network. Our results suggest that cell motion can be generated by friction between the flow of this network and the substrate. SYNOPSIS A combination of imaging and computational modeling is used to investigate the traction force patterns and the distribution of actin and myosin in three different modes of migration in Dictyostelium discoideum cells. The wave dynamics of actin and myosin critically determine the traction force patterns, cell morphology, and migration modes. Two types of keratocyte‐like motion are observed, consistent with two different propulsion mechanisms. A computational model reveals that cell motion can be generated by friction between the flow of the cytoskeletal network and the substrate. Graphical Abstract A combination of imaging and computational modeling is used to investigate the traction force patterns and the distribution of actin and myosin in three different modes of migration in Dictyostelium discoideum cells.

While cells within tissues generate and sense 3D states of strain, the current understanding of the mechanics of fibrous extracellular matrices (ECMs) stems mainly from uniaxial, biaxial, and shear tests. Here, we demonstrate that the multiaxial deformations of fiber networks in 3D cannot be inferred solely based on these tests. The interdependence of the three principal strains gives rise to anomalous ratios of biaxial to uniaxial stiffness between 8 and 9 and apparent Poisson’s ratios larger than 1. These observations are explained using a microstructural network model and a coarse-grained constitutive framework that predicts the network Poisson effect and stress–strain responses in uniaxial, biaxial, and triaxial modes of deformation as a function of the microstructural properties of the network, including fiber mechanics and pore size of the network. Using this theoretical approach, we found that accounting for the Poisson effect leads to a 100-fold increase in the perceived elastic stiffness of thin collagen samples in extension tests, reconciling the seemingly disparate measurements of the stiffness of collagen networks using different methods. We applied our framework to study the formation of fiber tracts induced by cellular forces. In vitro experiments with low-density networks showed that the anomalous Poisson effect facilitates higher densification of fibrous tracts, associated with the invasion of cancerous acinar cells. The approach developed here can be used to model the evolving mechanics of ECM during cancer invasion and fibrosis.