Cell Traction Research Articles

1C15 FRETバイオセンサーによる細胞の牽引力の可視化(OS2-1:細胞・分子のバイオメカニクス(1))

1C15 FRETバイオセンサーによる細胞の牽引力の可視化(OS2-1:細胞・分子のバイオメカニクス(1))

H2-calponin Gene Knockout Increases Traction Force of Mouse Fibroblasts in vitro

Cell traction force (CTF) plays a critical role in controlling cell shape, enabling cell motility, and maintaining cellular homeostasis during various biological processes such as wound healing, angiogenesis, and cancer metastasis. It has been demonstrated that h2-calponin, an actin binding protein found in smooth muscle and non-muscle cells including epithelial cells, endothelial cells, macrophages and fibroblasts, plays a role in regulating the actin cytoskeleton activities in cell adhesion, migration and cytokinesis. We recently found that knockout (KO) of h2-calponin gene increased cell motility when compared to wild-type (WT) cells. This finding indicated a potential involvement of h2-calponin in producing CTF. The present study investigated the role of h2-calponin in mouse fibroblast traction force. Primary fibroblasts were isolated from leg muscles of h2-calponin KO and WT mice and analyzed using CTF-microscopy. CTF-microscopy is the current state-of-the-art method to determine CTF in a cell spread on a two-dimensional substrate. Using CTF-microscopy, we determined the root-mean square traction force, the total strain energy, net contractile movement produced by mouse fibroblasts cultured on a thin layer of 8-kPa polyacrylamide gel containing fluorescent beads of 0.2 µm in diameter. The results showed that h2-calponin KO fibroblasts had greater traction force than WT control. In comparison to WT cells expressing abundant tropomyosin-2, h2-calponin KO fibroblasts lost tropomyosin-2, a phenotype mimicking that of metastatic cancer cells. H2-calponin KO fibroblasts also adhered to cultural substrate slower than WT control, had smaller cell spreading area, and rounded up faster during trypsin treatment, supporting the role of h2-calponin in stabilizing the actin cytoskeleton. Our findings indicate that h2-calponin has an inhibitory role in the production of CTF, consistent with the increased motility of h2-calponin-null cells. Further studies on the mechanisms of h2-calponin-mediated CTF regulation and cell motility are underway.

Actomyosin Dynamics in 3D Traction Force Generation

The generation and coordination of cellular traction forces plays important roles in cell adhesion, cell migration, and extracellular matrix (ECM) remodeling, and hence in the development and repair of biological tissues. Historically, models for how cells generate and sense mechanical force have been derived from observations of cells grown on hard, two-dimensional (2D) surfaces. However, the cytoskeletal geometry of cells embedded in porous, 3D environments is inherently different than those found in 2D. Here we seek to understand the spatio-temporal regulation of cellular traction forces in a 3D environment. We embed primary human fibroblasts in a fluorescently labeled fibrin matrix, and use multicolor, time-lapse confocal microscopy to simultaneously image matrix deformation induced by cellular traction and the dynamics of the acto-myosin cytoskeleton and paxillin-rich cell-matrix adhesions. We observe significant traction forces transduced to the matrix through dynamic actomyosin-rich protrusions. Flux analysis of myosin concentrations in the protrusions reveals recruitment of myosin towards the protrusion tip during extension, and localization of myosin farther from the tip during retraction. Moreover, we observe both retrograde and anterograde movement of F-actin and myosin during cell traction generation, as well as similar bi-directional dynamics for paxillin-rich adhesions. Our data suggests a model of force generation and mechanotransduction different from the canonical continuous lamellipodial retrograde flow implicated in 2D cell migration. Ongoing work examines the role of signal transduction pathways in regulating cellular force generation, with the end goal of understanding how cells couple force generation and mechanotransduction in fully 3D environments.

Measurement of cell traction forces with ImageJ.

The quantification of cell traction forces requires three key steps: cell plating on a deformable substrate, measurement of substrate deformation, and the numerical estimation of the corresponding cell traction forces. The computing steps to measure gel deformation and estimate the force field have somehow limited the adoption of this method in cell biology labs. Here we propose a set of ImageJ plug-ins so that every lab equipped with a fluorescent microscope can measure cell traction forces.

Quantification of Cell Traction Force of Osteoblast Cells Using Si Nanopillar-Based Mechanical Sensor

Quantification of Cell Traction Force of Osteoblast Cells Using Si Nanopillar-Based Mechanical Sensor

A Hybrid Model to Test the Importance of Mechanical Cues Driving Cell Migration in Angiogenesis

Many studies are stressing the crucial importance of the mechanical component in angiogenesis, but still, very few models really integrate mechanics. In this paper, we propose to investigate the importance of mechanical cues for cell migration in the context of angiogenesis. We propose a hybrid continuous-discrete model that describes the individual migration of contracting cells on an elastic matrix of fibres. The matrix is described as a continuum whereas the cells are described as discrete elements. We also take into account the degradation of the matrix by proteases. The Young’s modulus characterizing the matrix rigidity depends on the local and time-dependent density of matrix fibres. Our results show that acting on the mechanics, specifically on the cell traction force intensity and on the matrix rigidity, can significantly alter cell migration and angiogenesis. First, there is a limited range of traction force intensities for which a vascular network can be obtained. Second, the matrix rigidity plays a role, but only in a very specific range, compatible with the underlying biological process. Alteration of the matrix due to cell degradation appears too small to induce significant changes in cell migration trajectories.

Viscoelastic Effects of Silicone Gels at the Micro- and Nanoscale

There has been an increased use of silicone gel for applications such as cell traction force measurements as well as lab- and organ-on-chip systems. However, silicone gel is a viscoelastic material which tends to undergo non-elastic deformation and displays time-dependent and strain rate-dependent responses. Here, we evaluated the mechanical responses of two types of commonly used silicone gels, Sylgard-184 and CY52-276, when subjected to nanoNewton force and micrometer displacement length scales. Using different mechanical characterization tools and theoretical models, we characterized and quantified the viscoelastic parameters of these substrates. Our experimental results showed that silicone substrates with high stiffness and elasticity and negligible strain rate-dependency and creep responses will be most suited for use at the nanoNewton force and micrometer displacement length scales such as that encountered in cell traction force assays.

A tissue adaptation model based on strain-dependent collagen degradation and contact-guided cell traction

Soft biological tissues adapt their collagen network to the mechanical environment. Collagen remodeling and cell traction are both involved in this process. The present study presents a collagen adaptation model which includes strain-dependent collagen degradation and contact-guided cell traction. Cell traction is determined by the prevailing collagen structure and is assumed to strive for tensional homeostasis. In addition, collagen is assumed to mechanically fail if it is over-strained. Care is taken to use principally measurable and physiologically meaningful relationships. This model is implemented in a fibril-reinforced biphasic finite element model for soft hydrated tissues.The versatility and limitations of the model are demonstrated by corroborating the predicted transient and equilibrium collagen adaptation under distinct mechanical constraints against experimental observations from the literature. These experiments include overloading of pericardium explants until failure, static uniaxial and biaxial loading of cell-seeded gels in vitro and shortening of periosteum explants. In addition, remodeling under hypothetical conditions is explored to demonstrate how collagen might adapt to small differences in constraints.Typical aspects of all essentially different experimental conditions are captured quantitatively or qualitatively. Differences between predictions and experiments as well as new insights that emerge from the present simulations are discussed. This model is anticipated to evolve into a mechanistic description of collagen adaptation, which may assist in developing load-regimes for functional tissue engineered constructs, or may be employed to improve our understanding of the mechanisms behind physiological and pathological collagen remodeling.

Glycolysis is the primary bioenergetic pathway for cell motility and cytoskeletal remodeling in human prostate and breast cancer cells

The ability of a cancer cell to detach from the primary tumor and move to distant sites is fundamental to a lethal cancer phenotype. Metabolic transformations are associated with highly motile aggressive cellular phenotypes in tumor progression. Here, we report that cancer cell motility requires increased utilization of the glycolytic pathway. Mesenchymal cancer cells exhibited higher aerobic glycolysis compared to epithelial cancer cells while no significant change was observed in mitochondrial ATP production rate. Higher glycolysis was associated with increased rates of cytoskeletal remodeling, greater cell traction forces and faster cell migration, all of which were blocked by inhibition of glycolysis, but not by inhibition of mitochondrial ATP synthesis. Thus, our results demonstrate that cancer cell motility and cytoskeleton rearrangement is energetically dependent on aerobic glycolysis and not oxidative phosphorylation. Mitochondrial derived ATP is insufficient to compensate for inhibition of the glycolytic pathway with regard to cellular motility and CSK rearrangement, implying that localization of ATP derived from glycolytic enzymes near sites of active CSK rearrangement is more important for cell motility than total cellular ATP production rate. These results extend our understanding of cancer cell metabolism, potentially providing a target metabolic pathway associated with aggressive disease.

A novel method for localizing reporter fluorescent beads near the cell culture surface for traction force microscopy.

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 µm in diameter, in PA gels, within 1.6 μm of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

3D Viscoelastic traction force microscopy.

Native cell-material interactions occur on materials differing in their structural composition, chemistry, and physical compliance. While the last two decades have shown the importance of traction forces during cell-material interactions, they have been almost exclusively presented on purely elastic in vitro materials. Yet, most bodily tissue materials exhibit some level of viscoelasticity, which could play an important role in how cells sense and transduce tractions. To expand the realm of cell traction measurements and to encompass all materials from elastic to viscoelastic, this paper presents a general, and comprehensive approach for quantifying 3D cell tractions in viscoelastic materials. This methodology includes the experimental characterization of the time-dependent material properties for any viscoelastic material with the subsequent mathematical implementation of the determined material model into a 3D traction force microscopy (3D TFM) framework. Utilizing this new 3D viscoelastic TFM (3D VTFM) approach, we quantify the influence of viscosity on the overall material traction calculations and quantify the error associated with omitting time-dependent material effects, as is the case for all other TFM formulations. We anticipate that the 3D VTFM technique will open up new avenues of cell-material investigations on even more physiologically relevant time-dependent materials including collagen and fibrin gels.

FAK is required for tension-dependent organization of collective cell movements in Xenopus mesendoderm

Collective cell movements are integral to biological processes such as embryonic development and wound healing and also have a prominent role in some metastatic cancers. In migrating Xenopus mesendoderm, traction forces are generated by cells through integrin-based adhesions and tension transmitted across cadherin adhesions. This is accompanied by assembly of a mechanoresponsive cadherin adhesion complex containing keratin intermediate filaments and the catenin-family member plakoglobin. We demonstrate that focal adhesion kinase (FAK), a major component of integrin adhesion complexes, is required for normal morphogenesis at gastrulation, closure of the anterior neural tube, axial elongation and somitogenesis. Depletion of zygotically expressed FAK results in disruption of mesendoderm tissue polarity similar to that observed when expression of keratin or plakoglobin is inhibited. Both individual and collective migrations of mesendoderm cells from FAK depleted embryos are slowed, cell protrusions are disordered, and cell spreading and traction forces are decreased. Additionally, keratin filaments fail to organize at the rear of cells in the tissue and association of plakoglobin with cadherin is diminished. These findings suggest that FAK is required for the tension-dependent assembly of the cadherin adhesion complex that guides collective mesendoderm migration, perhaps by modulating the dynamic balance of substrate traction forces and cell cohesion needed to establish cell polarity.

Cell force mapping using a double-sided micropillar array based on the moiré fringe method

The mapping of traction forces is crucial to understanding the means by which cells regulate their behavior and physiological function to adapt to and communicate with their local microenvironment. To this end, polymeric micropillar arrays have been used for measuring cell traction force. However, the small scale of the micropillar deflections induced by cell traction forces results in highly inefficient force analyses using conventional optical approaches; in many cases, cell forces may be below the limits of detection achieved using conventional microscopy. To address these limitations, the moiré phenomenon has been leveraged as a visualization tool for cell force mapping due to its inherent magnification effect and capacity for whole-field force measurements. This Letter reports an optomechanical cell force sensor, namely, a double-sided micropillar array (DMPA) made of poly(dimethylsiloxane), on which one side is employed to support cultured living cells while the opposing side serves as a reference pattern for generating moiré patterns. The distance between the two sides, which is a crucial parameter influencing moiré pattern contrast, is predetermined during fabrication using theoretical calculations based on the Talbot effect that aim to optimize contrast. Herein, double-sided micropillar arrays were validated by mapping mouse embryo fibroblast contraction forces and the resulting force maps compared to conventional microscopy image analyses as the reference standard. The DMPA-based approach precludes the requirement for aligning two independent periodic substrates, improves moiré contrast, and enables efficient moiré pattern generation. Furthermore, the double-sided structure readily allows for the integration of moiré-based cell force mapping into microfabricated cell culture environments or lab-on-a-chip devices.

Some basic questions on mechanosensing in cell–substrate interaction

Cells constantly probe their surrounding microenvironment by pushing and pulling on the extracellular matrix (ECM). While it is widely accepted that cell induced traction forces at the cell–matrix interface play essential roles in cell signaling, cell migration and tissue morphogenesis, a number of puzzling questions remain with respect to mechanosensing in cell–substrate interactions. Here we show that these open questions can be addressed by modeling the cell–substrate system as a pre-strained elastic disk attached to an elastic substrate via molecular bonds at the interface. Based on this model, we establish analytical and numerical solutions for the displacement and stress fields in both cell and substrate, as well as traction forces at the cell–substrate interface. We show that the cell traction generally increases with distance away from the cell center and that the traction-distance relationship changes from linear on soft substrates to exponential on stiff substrates. These results indicate that cell adhesion and migration behaviors can be regulated by cell shape and substrate stiffness. Our analysis also reveals that the cell traction increases linearly with substrate stiffness on soft substrates but then levels off to a constant value on stiff substrates. This biphasic behavior in the dependence of cell traction on substrate stiffness immediately sheds light on an existing debate on whether cells sense mechanical force or deformation when interacting with their surroundings. Finally, it is shown that the cell induced deformation field decays exponentially with distance away from the cell. The characteristic length of this decay is comparable to the cell size and provides a quantitative measure of how far cells feel into the ECM.

Discussion of “Cytoskeletal Mechanics Regulating Amoeboid Cell Locomotion” (Álvarez-González, B., Bastounis, E., Meili, R., del Alamo, J. C., Firtel, R. A., and Lasheras, J. C., 2014, ASME Appl. Mech. Rev., 66(5), p. 050804)

A virtually universal feature of adherent cells is their ability to exert traction forces. To measure these forces, several methods have been developed over the past 15 years. In this issue of Applied Mechanics Reviews, Álvarez-González and co-workers review their own traction force microscopy approach and its application to the study of amoeboid cell locomotion. They show that the cycle of cell motility is exquisitely synchronized by a cycle of traction forces. In addition, they show how traction forces and cell cycle synchronization are affected by myosin and SCAR/WAVE mutants. Here, I discuss some open questions that derive from the work of the authors and other laboratories as regards the relationship between cell motility and traction forces.

A novel cell traction force microscopy to study multi-cellular system.

Traction forces exerted by adherent cells on their microenvironment can mediate many critical cellular functions. Accurate quantification of these forces is essential for mechanistic understanding of mechanotransduction. However, most existing methods of quantifying cellular forces are limited to single cells in isolation, whereas most physiological processes are inherently multi-cellular in nature where cell-cell and cell-microenvironment interactions determine the emergent properties of cell clusters. In the present study, a robust finite-element-method-based cell traction force microscopy technique is developed to estimate the traction forces produced by multiple isolated cells as well as cell clusters on soft substrates. The method accounts for the finite thickness of the substrate. Hence, cell cluster size can be larger than substrate thickness. The method allows computing the traction field from the substrate displacements within the cells' and clusters' boundaries. The displacement data outside these boundaries are not necessary. The utility of the method is demonstrated by computing the traction generated by multiple monkey kidney fibroblasts (MKF) and human colon cancerous (HCT-8) cells in close proximity, as well as by large clusters. It is found that cells act as individual contractile groups within clusters for generating traction. There may be multiple of such groups in the cluster, or the entire cluster may behave a single group. Individual cells do not form dipoles, but serve as a conduit of force (transmission lines) over long distances in the cluster. The cell-cell force can be either tensile or compressive depending on the cell-microenvironment interactions.

How do cells produce and regulate the driving force in the process of migration?

Cell migration behaviors have been studied from various aspects and at different length scales (molecular, subcellular and cellular scales), however, the mechanisms of how cell produces and controls the driving force for its migration have not been fully understood. Here for the first time we draw a more unified picture of driving force production that integrates the mechanisms from molecular to subcellular and cellular levels to show how cell produces and regulates the driving force and thus control its motility. We suggest that although the external mechanical and chemical factors can influence cell migration, the cell is able to actively control and regulate its driving force for its motility through controlling the stability of cell adhesion via actively regulating its spreading shape. To demonstrate this picture of regulation of the driving force, a FEM-based simulation framework is developed by modeling the dynamics of adhesion at cell front, de-adhesion at cell rear, and forward motion of cell body under cell traction force for different cell shape. The migration of keratocyte and fibroblast cells is simulated for different matrix rigidity and rigidity gradient. We show that the cell migration speed biphasically depends on the matrix rigidity. The mechanism is that the variation of matrix rigidity tunes the balance of competition between stability of cell adhesion at cell front and instability of adhesion at cell rear, which consequently controls the driving force of cell migration. We further propose a parameter called motility factor for a quantitative description of impact of mechanical properties of matrix and cell shape on the driving force of cell migration.

HIP-55 negatively regulates myocardial contractility at the single-cell level

Myocardial contractility is crucial for cardiac output and heart function. But the detailed mechanisms of regulation remain unclear. In the present study, we found that HIP-55, an actin binding protein, negatively regulates myocardial contractility at the single-cell level. HIP-55 was overexpressed and knocked down in cardiomyocytes with an adenovirus infection. The traction forces exerted by single cardiomyocyte were measured using cell traction force microscopy. The results showed that HIP-55 knockdown significantly increased the contractility of the cardiomyocytes and HIP-55 overexpression could markedly reverse this process. Furthermore, HIP-55 was obviously co-localized with F-actin in cardiomyocytes, suggesting that HIP-55 regulated cardiac contractile function through the interaction between HIP-55 and F-actin. This study reveals the regulatory mechanisms of myocardial contractility and provides a new target for preventing and treating cardiovascular disease.

Critical Surface Tension of Cholesteryl Ester Liquid Crystal

Cholesteryl ester liquid crystal was found to be non-toxic and it was recently applied as a cell traction force sensor. The reason for the affinity of the cells to this liquid crystal is unclear and required further investigation. This paper focused on determining the surface energy of the liquid crystals. A custom built contact angle measurement system and Fox-Zisman theory was applied to determine the critical surface tension of the cholesteryl ester liquid crystal. Eight different polar probe liquids were selected to determine the contact angle of the glass slides coated with cholesteryl ester liquid crystals. We found that the critical surface tension of the liquid crystal at 37.5 mN/m characterized the surface of the liquid crystal to be moderately hydrophobic. However, as reported in our previous work that the interaction of the liquid crystal and the cell culture media could re-orientate the amphiphilic molecules of the liquid crystals leading to the formation of lyotropic layers on the bulk cholesteric phase, therefore, making the surface to be hydrophilic. This then supported the formation of the hydrophilic layers that favors cell adhesion.

Engineering cellular response using nanopatterned bulk metallic glass.

Nanopatterning of biomaterials is rapidly emerging as a tool to engineer cell function. Bulk metallic glasses (BMGs), a class of biocompatible materials, are uniquely suited to study nanopattern–cell interactions as they allow for versatile fabrication of nanopatterns through thermoplastic forming. Work presented here employs nanopatterned BMG substrates to explore detection of nanopattern feature sizes by various cell types, including cells that are associated with foreign body response, pathology, and tissue repair. Fibroblasts decreased in cell area as the nanopattern feature size increased, and fibroblasts could detect nanopatterns as small as 55 nm in size. Macrophages failed to detect nanopatterns of 150 nm or smaller in size, but responded to a feature size of 200 nm, resulting in larger and more elongated cell morphology. Endothelial cells responded to nanopatterns of 100 nm or larger in size by a significant decrease in cell size and elongation. On the basis of these observations, nondimensional analysis was employed to correlate cellular morphology and substrate nanotopography. Analysis of the molecular pathways that induce cytoskeletal remodeling, in conjunction with quantifying cell traction forces with nanoscale precision using a unique FIB-SEM technique, enabled the characterization of underlying biomechanical cues. Nanopatterns altered serum protein adsorption and effective substrate stiffness, leading to changes in focal adhesion density and compromised activation of Rho-A GTPase in fibroblasts. As a consequence, cells displayed restricted cell spreading and decreased collagen production. These observations suggest that topography on the nanoscale can be designed to engineer cellular responses to biomaterials.