Cell Traction Force Microscopy Research Articles

Identification of CD44 as a key mediator of cell traction force generation in hyaluronic acid-rich extracellular matrices.

Mechanical properties of the extracellular matrix (ECM) critically regulate a number of important cell functions including growth, differentiation and migration. Type I collagen and glycosaminoglycans (GAGs) are two primary components of ECMs that contribute to mammalian tissue mechanics, with the collagen fiber network sustaining tension, and GAGs withstanding compression. The architecture and stiffness of the collagen network are known to be important for cell-ECM mechanical interactions via integrin cell surface adhesion receptors. In contrast, studies of GAGs in modulating cell-ECM interactions are limited. Here, we present experimental studies on the roles of hyaluronic acid (HA, an unsulfated GAG) in single tumor cell traction force generation using a recently developed 3D cell traction force microscopy method. Our work reveals that CD44, a cell surface adhesion receptor to HA, is engaged in cell traction force generation in conjunction with β1-integrin. We find that HA significantly modifies the architecture and mechanics of the collagen fiber network, decreasing tumor cells' propensity to remodel the collagen network, attenuating traction force generation, transmission distance, and tumor invasion. Our findings point to a novel role for CD44 in traction force generation, which can be a potential therapeutic target for diseases involving HA rich ECMs such as breast cancer and glioblastoma.

Abstract P2049: Myosin Heavy Chain-7 E848G Mutation-induced Sarcopenia And Hypocontractility In Induced Pluripotent Stem Cell Model Of Hypertrophic Cardiomyopathy Are Rescued By Muscle Ring Finger 1 Ablation

Hypertrophic cardiomyopathy (HCM) presents with idiopathic left ventricular hypertrophy and occasionally systolic dysfunction. In a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of HCM, we previously reported the pathogenic MYH7 E848G variant increased hiPSC-CM size and impaired contractility. To elucidate the mechanisms by which the E848G variant causes HCM, we now examined cardiac tissue from a MYH7 wt/E848G patient. We found reduced expression of the following sarcomeric proteins: β-myosin heavy chain (βMHC) (97.8 ± 1.1% reduction), α-sarcomeric actinin (αSA) (84.8 ± 7.3%), and ventricular myosin light chain (MLC-1v) (45.9 ± 13.9%) relative to healthy patient tissue. Isogenic MYH7 wt/E848G hiPSC-CMs also had reduced protein expression of βMHC (22.8%) and αSA (24.5%) relative to MYH7 wt/wt hiPSC-CMs and unchanged mRNA expression. These results suggest MYH7 E848G HCM induces broad sarcomere component deficiency. We tested hiPSC-CM contractility using single cell traction force microscopy (TFM) and engineered heart tissues (EHT). Single isogenic MYH7 wt/E848G hiPSC-CMs had lower maximum twitch force (80 ± 12 nN) than MYH7 wt/wt hiPSC-CMs (150 ± 20 nN) by TFM. Isogenic MYH7 wt/E848G EHTs had lower maximum twitch force (118.9 ± 23.7 μN) than MYH7 wt/wt EHTs (287.4 ± 27.7 μN). Because MuRF1, an E3 ubiquitin-protein ligase, is known to target βMHC for proteasomal degradation, we hypothesized MuRF1 mediates the sarcopenic and hypocontractile phenotype. Genetic ablation of TRIM63 (encoding MuRF1) significantly reduced MYH7 wt/E848G hiPSC-CM cell size by flow cytometry (6.8 ± 1.7%). MYH7 wt/E848G TRIM63 -/- hiPSC-CMs increased protein expression of βMHC (154.7 ± 51.9%), αSA (44.9 ± 25.6%), and MLC-1v (246.9 ± 57.3%) relative to MYH7 wt/E848G TRIM63 +/+ hiPSC-CMs. MYH7 wt/E848G TRIM63 -/- had significantly higher maximum twitch force by TFM (133 ± 14 nN) and EHTs (178.2 ± 9.8 μN) than MYH7 wt/E848G TRIM63 +/+ by TFM (67 ± 8.0 nN) and EHTs (118.9 ± 23.7 μN). Our results implicate sarcopenia in the pathogenesis of MYH7 E848G HCM and identify MuRF1 as a potential novel therapeutic target for HCM. Further study will determine if these findings are generalizable to other pathogenic HCM variants in MYH7 and other sarcomeric genes.

A structural bio-chemo-mechanical model for vascular smooth muscle cell traction force microscopy.

Altered vascular smooth muscle cell (VSMC) contractility is both a response to and a driver for impaired arterial function, and the leading experimental technique for quantifying VSMC contraction is traction force microscopy (TFM). TFM involves the complex interaction among several chemical, biological, and mechanical mechanisms, making it difficult to translate TFM results into tissue-scale behavior. Here, a computational model capturing each of the major aspects of the cell traction process is presented. The model incorporates four interacting components: a biochemical signaling network, individual actomyosin fiber bundle contraction, a cytoskeletal network of interconnected fibers, and elastic substrate displacement due to cytoskeletal force. The synthesis of these four components leads to a broad, flexible framework for describing TFM and linking biochemical and biomechanical phenomena on the single-cell level. The model recapitulated available data on VSMCs following biochemical, geometric, and mechanical perturbations. The structural bio-chemo-mechanical model offers a tool to interpret TFM data in new, more mechanistic ways, providing a framework for the evaluation of new biological hypotheses, interpolation of new data, and potential translation from single-cell experiments to multi-scale tissue models.

The collective mechanosensing of glioma spheroid at the cell-ECM interfaces

The extracellular matrix (ECM) serves an essential role in regulating cell physiology. At the cell-ECM interface, through various transmembrane proteins, cells can transduce the stimuli from ECM and activate the intracellular signaling accordingly. The importance of cell-ECM interactions are demonstrated in numerous studies, however, the propagation of signals from the cell-ECM interface to the whole cell collectives is not fully explored yet. In this study, the glioma spheroid with a diameter of approximately 250 µm was used as the multicellular model. While the glioma spheroid locally contacting to the substrate covered with collagen, the nano-indentation data showed an increasing stiffness of cell spheroid (∼ 100%). Interestingly, the alteration of ensemble mechanical properties occurred in few minutes. In comparison to collagen, the stiffness of glioma on hyaluronic acid only increased by 26%, and the results of cell traction force microscopy revealed higher cellular force at cell-collagen interface, than the one at cell-HA interface. The data implied a correlation exists between the cellular force and stiffness alteration. We further identified the reorganization of the cytoskeleton within the glioma spheroid using confocal microscopy. Since actin polymerization is upregulated by the applied forces, the observed denser actin network within the cell spheroid not only suggests the broadcast of signaling from local interfacial contact to the whole cell collectives, and the potential role of cellular force in the collective mechanosensing. Considering the importance of cross-talk between cells and ECM in carcinogenesis, our results highlight the potential influence of local ECM change on multicellular populations. Moreover, the fast cytoskeleton reorganization of the whole spheroid implies the complicated but transient stimulus presented in the tumor microenvironment during the disease progression.

Biomimicking the tumor microenvironment for biomechanical study of glioblastoma cells analyzed by single cell traction force microscopy

The structures of the tissue microenvironment influence cell function and behavior, both in physiological and pathological conditions. Cells experience and integrate a multitude of mechanical and physical cues from this 3D tissue microenvironment to adapt to organismal development. In turn, they respond by exerting forces, regulating their shape, internal cytoskeletal tension, and elasticmodulus. Disruption of the cellular forces, as well as variations in subcellular mechanical properties, can lead to altered pathophysiological conditions and the onset of diseases i.e., cancer. Diseases like cancer are characterized by dramatic changes in cell and tissue mechanics, and dysregulation of forcesat the cell and tissue level can activate mechanosensing to compromise tissue integrity and function and promote disease progression. Cells can move by exerting traction forces to their environment. These can be assessed by using microfabricated substrates and improved computational approaches, enabling the characterization of the biomechanical forces generated by single cells cultured in defined microenvironments. This provides valuable insights into the malignancy of cancer cells. However, the conventional microfabricated structures i.e., 2D substrates are far from matching the complexities present in vivo. Hence, there is a need for biomimicking the natural surfaces for cellular applications. We here show the traction forces induced by single glioblastoma cells in 3D tumor microenvironment-inspired scaffolds i.e., collagen in comparison with 2D collagen coating. The dimensionality of cell culture influences cell motility and cellular interaction with the surrounding cells and ECM. Cells grown on 2D scaffolds, adapt to the artificial environment and may no longer display characteristics of the original tumor. An attempt to develop 3D tumor microenvironment-inspired scaffolds and their functionality in unravelling the role of microenvironment on tumor cell behaviors are also examined. Characterizing the cues involved in glioblastoma cell migration could enable the scientific and medical community to develop better strategies to understand and treat brain cancer.

The effects of xeno-free cryopreservation on the contractile properties of human iPSC derived cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have advanced our ability to study the basic function of the heart and model cardiac diseases. Due to the complexities in stem cell culture and differentiation protocols, many researchers source their hiPSC-CMs from collaborators or commercial biobanks. Generally, the field has assumed the health of frozen cardiomyocytes is unchanged if the cells adhere to the substrate and commence beating. However, very few have investigated the effects of cryopreservation on hiPSC-CM's functional and transcriptional health at the cellular and molecular level. Here we review methods and challenges associated with cryopreservation, and examine the effects of cryopreservation on the functionality (contractility and calcium handling) and transcriptome of hiPSC-CMs from six healthy stem cell lines. Utilizing protein patterning methods to template physiological cell aspect ratios (7:1, length:width) in conjunction with polyacrylamide (PA) hydrogels, we measured changes in force generation and calcium handling of single hiPSC-CMs. We observed that cryopreservation altered the functionality and transcriptome of hiPSC-CMs towards larger sizes and contractile force as assessed by increased spread area and volume, single cell traction force microscopy and delayed calcium dynamics. hiPSC-CMs are broadly used for basic science research, regenerative medicine, and testing biological therapeutics. This study informs the design of experiments utilizing hiPSC-CMs to avoid confounding functional changes due to cryopreservation with other treatments.

A novel method to make viscoelastic polyacrylamide gels for cell culture and traction force microscopy.

Polyacrylamide hydrogels are commonly used in cell biology, notably to cultivate cells on soft surfaces. Polyacrylamide gels are purely elastic and well adapted to cell culture as they are inert and can be conjugated with adhesion proteins. Here, we report a method to make viscoelastic polyacrylamide gels with mechanical properties more closely resembling biological tissues and suitable for cell culture in vitro. We demonstrate that these gels can be used for traction force microscopy experiments. We also show that multiple cell types respond to the viscoelasticity of their substrate and that viscous dissipation has an influence on cell spreading, contractility, and motility. This new material provides new opportunities for investigating how normal or malignant cells sense and respond to viscous dissipation within the extra-cellular matrix.

An optochemical tool for light-induced dissociation of adherens junctions to control mechanical coupling between cells

The cadherin-catenin complex at adherens junctions (AJs) is essential for the formation of cell-cell adhesion and epithelium integrity; however, studying the dynamic regulation of AJs at high spatio-temporal resolution remains challenging. Here we present an optochemical tool which allows reconstitution of AJs by chemical dimerization of the force bearing structures and their precise light-induced dissociation. For the dimerization, we reconstitute acto-myosin connection of a tailless E-cadherin by two ways: direct recruitment of α-catenin, and linking its cytosolic tail to the transmembrane domain. Our approach enables a specific ON-OFF switch for mechanical coupling between cells that can be controlled spatially on subcellular or tissue scale via photocleavage. The combination with cell migration analysis and traction force microscopy shows a wide-range of applicability and confirms the mechanical contribution of the reconstituted AJs. Remarkably, in vivo our tool is able to control structural and functional integrity of the epidermal layer in developing Xenopus embryos.

Kindlin-2 deficiency induces fatal intestinal obstruction in mice.

Rationale: Smooth muscle-motility disorders are mainly characterized by impaired contractility and functional intestinal obstruction. Some of these cases are caused by genetic mutations of smooth muscle genes ACTA2, ACTG2, MYH11, MYLK and LMOD1. Still the etiology is complex and multifactorial and the underlying pathology is poorly understood. Integrin interaction protein Kindlin-2 is widely expressed in striated and smooth muscle cells (SMC). However, the function of Kindlin-2 in the smooth muscle remains elusive.Methods: We generated two mouse models using different cre promoter transgenic mice, Kindlin-2fl/fl SM22α-cre+ (cKO mice) and Kindlin-2fl/fl; MYH-cre+ (iKO mice). Embryos and adult tissues were prepared for hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) apoptosis assay. We investigated ultrastructure changes of mouse smooth muscle using transmission electron microscopy (TEM) and measured smooth muscle contractile force in mounting aortic and intestinal rings using the multiwire myograph system (DMT 620M). In addition, cell traction force microscopy (CTFM) was applied to observe the functional change of primary SMC after Kindlin-2 depletion by RNAi.Results: Depletion of Kindlin-2 encoding gene Fermt2 in embryonic smooth muscles leads to apoptosis, downregulates the key components of SMC, impairs smooth muscle development, and finally causes embryonic death at E14.5. Tamoxifen-induced Kindlin-2-specific knockout in adult mouse smooth muscle showed decreased blood pressure, intestinal hypoperistalsis, and eventually died of intestinal obstruction. Kindlin-2 depletion also leads to downregulated Myh11, α-SMA, and CNN, shortened myofilament, broken myofibrils, and impaired contractility of the smooth muscles in iKO mice. Mechanistically, loss of Kindlin-2 decreases Ca2+ influx in primary vascular smooth muscle cells (PVSMC) by downregulating the expression of calcium-binding protein S100A14 and STIM1.Conclusion: We demonstrated that Kindlin-2 is essential for maintaining the normal structure and function of smooth muscles. Loss of Kindlin-2 impairs smooth muscle formation during embryonic development by inducing apoptosis and jeopardizes the contraction of adult smooth muscle by blocking Ca2+ influx that leads to intestinal obstruction. Mice with Kindlin-2 depletion in adult smooth muscle could be a potent animal model of intestinal obstruction for disease research, drug treatment and prognosis.

Single cell force profiling of human myofibroblasts reveals a biophysical spectrum of cell states.

ABSTRACTMechanical force is a fundamental regulator of cell phenotype. Myofibroblasts are central mediators of fibrosis, a major unmet clinical need characterised by the deposition of excessive matrix proteins. Traction forces of myofibroblasts play a key role in remodelling the matrix and modulate the activities of embedded stromal cells. Here, we employ a combination of unsupervised computational analysis, cytoskeletal profiling and single cell traction force microscopy as a functional readout to uncover how the complex spatiotemporal dynamics and mechanics of living human myofibroblast shape sub-cellular profiling of traction forces in fibrosis. We resolve distinct biophysical communities of myofibroblasts, and our results provide a new paradigm for studying functional heterogeneity in human stromal cells.

Inverse Estimation of 3-D Traction Stress Field of Adhered Cell based on Optimal Control Technique using Image Intensities

Cells adhere to a substrate and generate traction forces in focal adhesions that enable them to apprehend extracellular mechanical properties [1]. Current concerns are focused on mechanisms how the mechanical balances hold in the cell and affect the cell behavior, and therefore non-invasive measurement techniques for the cell traction forces are required. The cell traction force microscopy (TFM) generalized by Dembo and Wang [2] is an attractive approach to non-invasively estimate cell traction force fields, in which an inverse problem is solved using a mechanical model of the substrate and displacement fields from fluorescent images of immersed beads in the substrate. So far, a wide variety of TFMs has been developed [3], and the authors have also proposed a novel TFM approach, which can offer the 3-D cell traction force field using in-plane displacements on multiple slices [4]. In these TFMs, since partial information of displacement fields is used as observation data, one of the component to determine the estimation accuracy is an image processing technique to evaluate the displacement field from the observation images for fluorescent beads. In addition, if the number density of the image-based displacements is not enough, an ill-posed condition occurs in solving the inverse problem. In this study, the authors propose a novel TFM approach which directly uses image intensities as the observation data without pre-processing the beads’ displacement. The 3-D traction stress field is inversely estimated by solving an optimization problem, which minimizes a L2 norm of the difference of image intensities between the numerical model and observation, by an optimal control technique. A validity of the proposed approach has been confirmed though a numerical experiment using pseudo-observation data w/ and w/o data noise.

The multiscale stiffness of electrospun substrates and aspects of their mechanical biocompatibility

In contrast to homogeneous materials, the mechanical properties of fibrous substrates depend on the probing lengthscale. This suggests that cells feel very different mechanical cues than expected from the macroscale characterisation of the substrate materials. By means of multiscale computational analyses we study here the mechanical environment of cells adhering to typical electrospun networks used in biomedical applications, with comparable macroscopic stiffness but different fibre diameters. The stiffness evaluated at the level of focal adhesions varies significantly, and the overall magnitude is strongly affected by the fibre diameter. The microscopic stiffness evaluated at cell scale depends substantially on the network topology and is about one order of magnitude lower than the macroscopic stiffness of the substrate, and two to three orders of magnitude below the fibres’ elastic modulus. Moreover, the translation of stiffness over the scales is modulated by global deformations of the scaffold. In particular, uniaxial or biaxial stretching of the substrate induces nonlinear microscopic stiffening. Finally, although electrospun networks allow long-range transmission of cell-induced deformations, the comparison between the range of forces measured in cell traction force microscopy and those required to markedly deform typical electrospun networks reveals an order of magnitude difference, suggesting that these scaffolds provide a rather rigid environment for cells. All these results underline that the achievement of mechanical biocompatibility at all relevant lengthscales, and over the whole range of physiological loading states is extremely challenging. At the same time, the study shows that the diameter, length and curvature of fibre segments might be tunable towards achieving this goal. Statement of SignificanceElectrospun fabrics have growing use as substrates and scaffolds in tissue engineering and other biomedical applications. Based on multiscale computational analyses, this study shows that substrates of comparable macroscopic stiffness can provide tremendously different mechanical micro-environments, and that cells adhering to fibrous substrates may thus experience by orders of magnitude different mechanical cues than it would be expected from macroscale material characterisation. The simulations further reveal that the transfer of stiffness over the length scales changes with macroscopic deformation, and identify some key parameters that govern the transfer ratio. We believe that such refined understanding of the multiscale aspects of mechanical biocompatibility is key to the development of successful scaffold materials.

Investigation of human iPSC-derived cardiac myocyte functional maturation by single cell traction force microscopy.

Recent advances have made it possible to readily derive cardiac myocytes from human induced pluripotent stem cells (hiPSC-CMs). HiPSC-CMs represent a valuable new experimental model for studying human cardiac muscle physiology and disease. Many laboratories have devoted substantial effort to examining the functional properties of isolated hiPSC-CMs, but to date, force production has not been adequately characterized. Here, we utilized traction force microscopy (TFM) with micro-patterning cell printing to investigate the maximum force production of isolated single hiPSC-CMs under varied culture and assay conditions. We examined the role of length of differentiation in culture and the effects of varied extracellular calcium concentration in the culture media on the maturation of hiPSC-CMs. Results show that hiPSC-CMs developing in culture for two weeks produced significantly less force than cells cultured from one to three months, with hiPSC-CMs cultured for three months resembling the cell morphology and function of neonatal rat ventricular myocytes in terms of size, dimensions, and force production. Furthermore, hiPSC-CMs cultured long term in conditions of physiologic calcium concentrations were larger and produced more force than hiPSC-CMs cultured in standard media with sub-physiological calcium. We also examined relationships between cell morphology, substrate stiffness and force production. Results showed a significant relationship between cell area and force. Implementing directed modifications of substrate stiffness, by varying stiffness from embryonic-like to adult myocardium-like, hiPSC-CMs produced maximal forces on substrates with a lower modulus and significantly less force when assayed on increasingly stiff adult myocardium-like substrates. Calculated strain energy measurements paralleled these findings. Collectively, these findings further establish single cell TFM as a valuable approach to illuminate the quantitative physiological maturation of force in hiPSC-CMs.

Progress in Integrative Biomaterial Systems to Approach Three-Dimensional Cell Mechanotransduction.

Mechanotransduction between cells and the extracellular matrix regulates major cellular functions in physiological and pathological situations. The effect of mechanical cues on biochemical signaling triggered by cell–matrix and cell–cell interactions on model biomimetic surfaces has been extensively investigated by a combination of fabrication, biophysical, and biological methods. To simulate the in vivo physiological microenvironment in vitro, three dimensional (3D) microstructures with tailored bio-functionality have been fabricated on substrates of various materials. However, less attention has been paid to the design of 3D biomaterial systems with geometric variances, such as the possession of precise micro-features and/or bio-sensing elements for probing the mechanical responses of cells to the external microenvironment. Such precisely engineered 3D model experimental platforms pave the way for studying the mechanotransduction of multicellular aggregates under controlled geometric and mechanical parameters. Concurrently with the progress in 3D biomaterial fabrication, cell traction force microscopy (CTFM) developed in the field of cell biophysics has emerged as a highly sensitive technique for probing the mechanical stresses exerted by cells onto the opposing deformable surface. In the current work, we first review the recent advances in the fabrication of 3D micropatterned biomaterials which enable the seamless integration with experimental cell mechanics in a controlled 3D microenvironment. Then, we discuss the role of collective cell–cell interactions in the mechanotransduction of engineered tissue equivalents determined by such integrative biomaterial systems under simulated physiological conditions.

High-Resolution Integrin Molecular Tension Dynamics during Platelet Adhesion and Activation

Platelets are small disc-shaped cell fragments circulating in the bloodstream. Upon injury, platelets adhere and aggregate on injured subendothelium to form blood clots to stop bleeding. Irregular platelet adhesion and aggregation may cause acute coronary syndrome or other cardiovascular diseases. Membrane protein integrins mediate platelet adhesion and transmit tensions to activate platelets, therefore playing important roles in platelet functions. Nevertheless, cellular force study in platelets is very limited and integrin molecular tensions were never calibrated in platelets, presumably due to the 2∼3 µm size of platelets which is at the resolution limit of conventional cell traction force microscopy. Here we applied a novel molecular tension sensor and modulator named TGT (tension gauge tether) to study integrin tensions in platelets. TGT is a rupturable molecular linker with a programmable tension tolerance (Ttol). As a tension modulator, TGT globally restricts integrin tensions under the designed level of Ttol, enabling the study of integrin-tension dependency for a certain cellular function. As a tension sensor, TGT maps integrin tensions by fluorescence with a spatial resolution of 0.2 µm, ten times higher than conventional methods. Using TGT, we determined that platelet adhesion and spreading requires integrin tension lower than 12 pN, a force much less than the ∼40 pN integrin tension required by regular mammalian cell adhesion. This surprising result suggests that platelets have extraordinary adhesion and spreading ability which might be beneficial for the rapid response of platelets to injuries. We also monitored calcium oscillation in platelets to confirm that platelets are activated at this low integrin tension level. Next, using TGT as a tension sensor, we provided the high-resolution (0.2 µm) integrin tension map for platelet adhesion for the first time. Real-time tension mapping shows that integrin tensions at a high level (>54 pN) are initially concentrated in one or two micron-sized punctate regions upon platelet adhesion. After full platelet spreading, these integrin tensions spread out to the entire platelet-substrate interface and the tension level decreases into the range of 12∼54 pN. These high integrin tensions are not required for platelet adhesion and activation, but may be required for platelet contraction, as these tensions were abolished by myosin II inhibition without compromising platelet adhesion and activation. Overall, our research has initiated the biomechanical study of platelets at the molecular tension level, and revealed a rich dynamics of integrin tensions in platelets.

Deletion of Calponin 2 in Mouse Fibroblasts Increases Myosin II-Dependent Cell Traction Force.

Cell traction force (CTF) plays a critical role in controlling cell shape, permitting cell motility, and maintaining cellular homeostasis in many biological processes such as angiogenesis, development, wound healing, and cancer metastasis. Calponin is an actin filament-associated cytoskeletal protein in smooth muscles and multiple types of non-muscle cells. An established biochemical function of calponin is the inhibition of myosin ATPase in smooth muscle cells. Vertebrates have three calponin isoforms. Among them, calponin 2 is expressed in epithelial cells, endothelial cells, macrophages, myoblasts, and fibroblasts and plays a role in regulating cytoskeleton activities such as cell adhesion, migration, and cytokinesis. Knockout (KO) of the gene encoding calponin 2 (Cnn2) in mice increased cell motility, suggesting a function of calponin 2 in modulating CTF. In this study, we examined fibroblasts isolated from Cnn2 KO and wild-type (WT) mice using CTF microscopy. Primary mouse fibroblasts were cultured on polyacrylamide gel substrates embedded with fluorescent beads to measure root-mean-square traction, total strain energy, and net contractile movement. The results showed that calponin 2-null fibroblasts exhibit traction force greater than that of WT cells. Adherent calponin 2-null fibroblasts de-adhered faster than the WT control during mild trypsin treatment, consistent with an increased CTF. Blebbistatin, an inhibitor of myosin II ATPase, is more effective upon an alteration in cell morphology when calponin 2 is present in WT fibroblasts than that on Cnn2 KO cells, indicating their additive effects in inhibiting myosin motor activity. The novel finding that calponin 2 regulates myosin-dependent CTF in non-muscle cells demonstrates a mechanism for controlling cell motility-based functions.

Biomechanistic Study of Smooth Muscle Cell Sheet during Circumferential Alignment in Circular Micropatterns.

Engineered functional blood vessels play an important role in tissue regeneration and wound healing, but they remain far from clinical practices because of the complex three-layer capillary histology. In general, the reconstruction of tunica media from vascular smooth muscle cells (SMCs) that mediate vasoconstriction and vasodilation of circulatory system is critical for engineering functional blood vessels. To date, the mechanotransduction during circumferential alignment of SMC layer in tunica media remains to be elucidated. In this study, concentric microwalls fabricated on a polydimethylsiloxane (PDMS) substrate were developed for cell traction force microscopy (CTFM) of SMC layer. First, the cell shape, density, and alignment orientation of SMCs during circumferential cell sheet formation were measured during 7-day cell culture. Second, a collective CTFM assay was carried out to measure the traction forces exerted by the confluent cell layer at various stages of circumferential alignment. It was found that the mechanotransduction of SMC layer grown between the circular microwalls was significantly altered compared to the SMC layer grown on a flat substrate. The results indicated the presence of an optimal region for formation of circumferentially aligned SMC layer verified by both quantitative analysis and biomechanical study. Moreover, the distributions of traction forces and von Mises stresses during cell alignment were affected by the cellular mechanotransduction from the extracellular physical constraints and cell-cell interactions. The immunofluorescence staining and real time quantitative polymerase chain reaction (RT-qPCR) analyses of actin filaments further revealed the relation between the monolayer-generated traction forces and the direction of aligned actin filaments.

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.

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.

Modeling crawling cell movement on soft engineered substrates

Self-propelled motion, emerging spontaneously or in response to external cues, is a hallmark of living organisms. Systems of self-propelled synthetic particles are also relevant for multiple applications, from targeted drug delivery to the design of self-healing materials. Self-propulsion relies on the force transfer to the surrounding. While self-propelled swimming in the bulk of liquids is fairly well characterized, many open questions remain in our understanding of self-propelled motion along substrates, such as in the case of crawling cells or related biomimetic objects. How is the force transfer organized and how does it interplay with the deformability of the moving object and the substrate? How do the spatially dependent traction distribution and adhesion dynamics give rise to complex cell behavior? How can we engineer a specific cell response on synthetic compliant substrates? Here we generalize our recently developed model for a crawling cell by incorporating locally resolved traction forces and substrate deformations. The model captures the generic structure of the traction force distribution and faithfully reproduces experimental observations, like the response of a cell on a gradient in substrate elasticity (durotaxis). It also exhibits complex modes of cell movement such as "bipedal" motion. Our work may guide experiments on cell traction force microscopy and substrate-based cell sorting and can be helpful for the design of biomimetic "crawlers" and active and reconfigurable self-healing materials.