Cell Traction Force Generation Research Articles

Inferring cellular contractile forces and work using deep morphology traction microscopy

Traction-force microscopy (TFM) has emerged as a widely used standard methodology to measure cell-generated traction forces and determine their role in regulating cell behavior. While TFM platforms have enabled many discoveries, their implementation remains limited due to complex experimental procedures, specialized substrates, and the ill-posed inverse problem whereby low-magnitude high-frequency noise in the displacement field severely contaminates the resulting traction measurements. Here, we introduce deep morphology traction microscopy (DeepMorphoTM), a deep-learning alternative to conventional TFM approaches. DeepMorphoTM first infers cell-induced substrate displacement solely from a sequence of cell shapes and subsequently computes cellular traction forces, thus avoiding the requirement of a specialized fiduciarily marked deformable substrate or force-free reference image. Rather, this technique drastically simplifies the overall experimental methodology, imaging, and analysis needed to conduct cell-contractility measurements. We demonstrate that DeepMorphoTM quantitatively matches conventional TFM results while offering stability against the biological variability in cell contractility for a given cell shape. Without high-frequency noise in the inferred displacement, DeepMorphoTM also resolves the ill-posedness of traction computation, increasing the consistency and accuracy of traction analysis. We demonstrate the accurate extrapolation across several cell types and substrate materials, suggesting robustness of the methodology. Accordingly, we present DeepMorphoTM as a capable yet simpler alternative to conventional TFM for characterizing cellular contractility in two dimensions.

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.

Piezo1 activation gains traction

A major unanswered question in biology is how mechanical forces are generated, detected and transduced by cells to impact biochemical and genetic programs. Our work is aimed at uncovering the mechanical principles at play in cells and tissues using novel molecular, imaging and bioengineering tools. Here we present insights gleaned from non-invasive approaches to measure and manipulate mechanical signals in native cellular conditions. We find that the mechanically-activated ion channel Piezo1 transduces cell-generated traction forces: actomyosin-based traction forces evoke localized Piezo1 Ca2+ flickers in the absence of externally-applied mechanical forces. We examine the functional effects of cell-generated Piezo1 activity in neural stem/progenitor cells and in epithelial keratinocytes.

A hybrid model of intercellular tension and cell-matrix mechanical interactions in a multicellular geometry.

Epithelial cells form continuous sheets of cells that exist in tensional homeostasis. Homeostasis is maintained through cell-to-cell junctions that distribute tension and balance forces between cells and their underlying matrix. Disruption of tensional homeostasis can lead to epithelial-mesenchymal transition (EMT), a transdifferentiation process in which epithelial cells adopt a mesenchymal phenotype, losing cell-cell adhesion and enhancing cellular motility. This process is critical during embryogenesis and wound healing, but is also dysregulated in many disease states. To further understand the role of intercellular tension in spatial patterning of epithelial cell monolayers, we developed a multicellular computational model of cell-cell and cell-substrate forces. This work builds on a hybrid cellular Potts model (CPM)-finite element model to evaluate cell-matrix mechanical feedback of an adherent multicellular cluster. Cellular movement is governed by thermodynamic constraints from cell volume, cell-cell and cell-matrix contacts, and durotaxis, which arises from cell-generated traction forces on a finite element substrate. Junction forces at cell-cell contacts balance these traction forces, thereby producing a mechanically stable epithelial monolayer. Simulations were compared to in vitro experiments using fluorescence-based junction force sensors in clusters of cells undergoing EMT. Results indicate that the multicellular CPM model can reproduce many aspects of EMT, including epithelial monolayer formation dynamics, changes in cell geometry, and spatial patterning of cell-cell forces in an epithelial tissue.

A computational model to predict cell traction-mediated prestretch in the mitral valve

Prestretch is observed in many soft biological tissues, directly influencing the mechanical behavior of the tissue in question. The development of this prestretch occurs through complex growth and remodeling phenomena, which yet remain to be elucidated. In the present study it was investigated whether local cell-mediated traction forces can explain the development of global anisotropic tissue prestretch in the mitral valve. Towards this end, a model predicting actin stress fiber-generated traction forces was implemented in a finite element framework of the mitral valve. The overall predicted magnitude of prestretch induced valvular contraction after release of in vivo boundary constraints was in good agreement with data reported on valvular retraction after excision from the heart. Next, by using a systematic variation of model parameters and structural properties, a more anisotropic prestretch development in the valve could be obtained, which was also similar to physiological values. In conclusion, this study shows that cell-generated traction forces could explain prestretch magnitude and anisotropy in the mitral valve.

Interactions of Omentin and Lactotransferrin in the Progression of Metastatic Ovarian Cancer

Advanced stage serous ovarian cancer metastasizes preferentially to the omentum, which is a major site of intra‐abdominal fat accumulation and is covered by a layer of mesothelial cells (mesothelium). Ovarian cancer cells first adhere to and then penetrate through the mesothelium. It was previously shown the expression of adipokine omentin (ITLN1) in mesothelial cells was down‐regulated when mesothelial cells were co‐cultured with ovarian cancer cells. Survival correlation studies demonstrated that patients with serum ITLN1 levels of >350 ng/mL at the time of first treatment experienced longer survival times than those with lower levels of ITLN1. In addition, wound‐healing assay and invasion assay demonstrated that ovarian cancer cells (SKOV3 and A224) treated with physiological levels of ITLN1 showed a significant decrease in motility and invasion potential. However, the molecular mechanism by which ITLN1 modulates these malignant phenotypes remains unknown. Cancer cells migrate using cell‐matrix mechanocoupling mechanisms through paths generated by the degradation and remodeling of the extracellular matrix (ECM) and by force‐mediated deformation (powered by cell traction force from cancer cells). To explore whether omentin suppresses ovarian cancer invasion potential via inhibiting ECM proteolysis, RNA sequencing and pathway analyses were performed on ITLN1 treated cancer cells. Significantly lower levels of MMP1 (matrix metalloproteinase‐1) was found in cancer cells treated with ITLN1 than untreated cells, which was confirmed by qRT‐PCR analysis. Since ITLN1 has been shown to interact with LTF (lactotransferrin), and binding of LTF to its receptors LRP1 (low‐intensity lipoprotein‐receptor‐related protein 1) can transcriptionally upregulate MMP1, an in vitro pull‐down assay on purified LTF and recombinant ITLN1 in serum‐free media was first performed to confirm that ITLN1 interacts and binds with LTF. To determine whether ITLN1 can interfere with the binding of LTF to LRP1 on ovarian cancer cells, a Duolink proximity ligation assay was performed using both anti‐LTF and anti‐LRP1 antibodies. There was a significant decrease in fluorescent signals in cells that were treated with ITLN1 compared to the control. The results suggested that ITLN1 interacts with LTF and prevents LTF from binding to its receptor LRP1 on ovarian cancer cells. Furthermore, ITLN1 abrogated the effects of LTF on MMP1 expression in a dose‐dependent manner in ovarian cancer cells. LTF not only induced MMP1 expression, but also triggered an increase of cytosolic Ca2+ and cell traction force in ovarian cancer cells, which were attenuated by pre‐incubation with ITLN1. Taken together, these data suggest that ITLN1 suppressed LTF's effect on the motility and invasion potential of ovarian cancer cells by decreasing MMP1 expression and cell traction force generation through modulating a calcium dependent LTF‐LRP1 signaling pathway.Support or Funding InformationDepartment of Defense: grant W81XWH‐17‐1‐0146This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Increased Cell Traction-Induced Prestress in Dynamically Cultured Microtissues.

Prestress is a phenomenon present in many cardiovascular tissues and has profound implications on their in vivo functionality. For instance, the in vivo mechanical properties are altered by the presence of prestress, and prestress also influences tissue growth and remodeling processes. The development of tissue prestress typically originates from complex growth and remodeling phenomena which yet remain to be elucidated. One particularly interesting mechanism in which prestress develops is by active traction forces generated by cells embedded in the tissue by means of their actin stress fibers. In order to understand how these traction forces influence tissue prestress, many have used microfabricated, high-throughput, micrometer scale setups to culture microtissues which actively generate prestress to specially designed cantilevers. By measuring the displacement of these cantilevers, the prestress response to all kinds of perturbations can be monitored. In the present study, such a microfabricated tissue gauge platform was combined with the commercially available Flexcell system to facilitate dynamic cyclic stretching of microtissues. First, the setup was validated to quantify the dynamic microtissue stretch applied during the experiments. Next, the microtissues were subjected to a dynamic loading regime for 24 h. After this interval, the prestress increased to levels over twice as high compared to static controls. The prestress in these tissues was completely abated when a ROCK-inhibitor was added, showing that the development of this prestress can be completely attributed to the cell-generated traction forces. Finally, after switching the microtissues back to static loading conditions, or when removing the ROCK-inhibitor, prestress magnitudes were restored to original values. These findings show that intrinsic cell-generated prestress is a highly controlled parameter, where the actin stress fibers serve as a mechanostat to regulate this prestress. Since almost all cardiovascular tissues are exposed to a dynamic loading regime, these findings have important implications for the mechanical testing of these tissues, or when designing cardiovascular tissue engineering therapies.

A Computational Model of Cell-Generated Traction Forces and Fibronectin Assembly

The extracellular matrix (ECM) is an assembly of proteins that surround cells and serves as the cell substrate in vivo. A primary component of newly synthesized ECM is fibronectin (FN), critical for embryonic development and wound healing. Despite years of research, the mechanism of FN assembly is still not understood. We hypothesize that FN assembly occurs through the revelation of buried FN-FN binding sites within any of the15 Type III FN domains, and that these binding sites are exposed by stretching of FN-III beta strands, allowing attachment of additional FN molecules via a beta strand addition mechanism. To investigate this hypothesis, we developed a biophysical computational model of cell-FN-substrate biomechanical-chemical interactions. In the model, FN-III domains are represented by Hookean springs with distinct stiffnesses; thus, each FN dimer is represented by 30 springs in series. Integrin binding/unbinding is represented by a stochastic first-order reversible chemical reaction with force-dependent off-rate. Model results are validated using in vitro tools: FN fibril growth and traction forces applied over time are measured using microfabricated pillar arrays in a human fibroblast cell line. Beta-strand exposure is probed using Thioflavin T, a fluorescent probe that binds to free beta strands. Results indicate that the computational model recapitulates three unique features that are observed in experimental measurements of FN fibrils: 1) FN fibrils are highly elastic, but have a maximal 4-fold elongation of their resting length; 2) FN fibril length and force are not strongly correlated; and 3) FN fibrils have discrete stable lengths, suggesting local minima of force balance within growing fibrils. These results will be discussed and compared with simulations in which FN fibril assembly is mediated by either a single cryptic binding site or a subset of cryptic binding sites.

Spatial distribution of cell-cell and cell-ECM adhesions regulates force balance while main-taining E-cadherin molecular tension in cell pairs.

Mechanical linkage between cell-cell and cell-extracellular matrix (ECM) adhesions regulates cell shape changes during embryonic development and tissue homoeostasis. We examined how the force balance between cell-cell and cell-ECM adhesions changes with cell spread area and aspect ratio in pairs of MDCK cells. We used ECM micropatterning to drive different cytoskeleton strain energy states and cell-generated traction forces and used a Förster resonance energy transfer tension biosensor to ask whether changes in forces across cell-cell junctions correlated with E-cadherin molecular tension. We found that continuous peripheral ECM adhesions resulted in increased cell-cell and cell-ECM forces with increasing spread area. In contrast, confining ECM adhesions to the distal ends of cell-cell pairs resulted in shorter junction lengths and constant cell-cell forces. Of interest, each cell within a cell pair generated higher strain energies than isolated single cells of the same spread area. Surprisingly, E-cadherin molecular tension remained constant regardless of changes in cell-cell forces and was evenly distributed along cell-cell junctions independent of cell spread area and total traction forces. Taken together, our results showed that cell pairs maintained constant E-cadherin molecular tension and regulated total forces relative to cell spread area and shape but independently of total focal adhesion area.

A Computational Model of Cell-Generated Traction Forces and Fibronectin Assembly

A Computational Model of Cell-Generated Traction Forces and Fibronectin Assembly

Cell-generated traction forces and the resulting matrix deformation modulate microvascular alignment and growth during angiogenesis.

The details of the mechanical factors that modulate angiogenesis remain poorly understood. Previous in vitro studies of angiogenesis using microvessel fragments cultured within collagen constructs demonstrated that neovessel alignment can be induced via mechanical constraint of the boundaries (i.e., boundary conditions). The objective of this study was to investigate the role of mechanical boundary conditions in the regulation of angiogenic alignment and growth in an in vitro model of angiogenesis. Angiogenic microvessels within three-dimensional constructs were subjected to different boundary conditions, thus producing different stress and strain fields during growth. Neovessel outgrowth and orientation were quantified from confocal image data after 6 days. Vascularity and branching decreased as the amount of constraint imposed on the culture increased. In long-axis constrained hexahedral constructs, microvessels aligned parallel to the constrained axis. In contrast, constructs that were constrained along the short axis had random microvessel orientation. Finite element models were used to simulate the contraction of gels under the various boundary conditions and to predict the local strain field experienced by microvessels. Results from the experiments and simulations demonstrated that microvessels aligned perpendicular to directions of compressive strain. Alignment was due to anisotropic deformation of the matrix from cell-generated traction forces interacting with the mechanical boundary conditions. These findings demonstrate that boundary conditions and thus the effective stiffness of the matrix regulate angiogenesis. This study offers a potential explanation for the oriented vascular beds that occur in native tissues and provides the basis for improved control of tissue vascularization in both native tissues and tissue-engineered constructs.

In situ mechanotransduction via vinculin regulates stem cell differentiation.

Human mesenchymal stem cell (hMSC) proliferation, migration, and differentiation have all been linked to extracellular matrix stiffness, yet the signaling pathway(s) that are necessary for mechanotransduction remain unproven. Vinculin has been implicated as a mechanosensor in vitro, but here we demonstrate its ability to also regulate stem cell behavior, including hMSC differentiation. RNA interference-mediated vinculin knockdown significantly decreased stiffness-induced MyoD, a muscle transcription factor, but not Runx2, an osteoblast transcription factor, and impaired stiffness-mediated migration. A kinase binding accessibility screen predicted a cryptic MAPK1 signaling site in vinculin which could regulate these behaviors. Indeed, reintroduction of vinculin domains into knocked down cells indicated that MAPK1 binding site-containing vinculin constructs were necessary for hMSC expression of MyoD. Vinculin knockdown does not appear to interfere with focal adhesion assembly, significantly alter adhesive properties, or diminish cell traction force generation, indicating that its knockdown only adversely affected MAPK1 signaling. These data provide some of the first evidence that a force-sensitive adhesion protein can regulate stem cell fate.

Activation of beta 1 but not beta 3 integrin increases cell traction forces

Cell-generated traction forces induce integrin activation, leading to focal adhesion growth and cell spreading. It remains unknown, however, whether integrin activation feeds back to impact the generation of cytoskeletal tension. Here, we used elastomeric micropost arrays to measure cellular traction forces in wildtype and integrin-null cells. We report that activation of β1 but not β3 integrin, by either increasing density of immobilized fibronectin or treating with manganese, elicited fibroblast spreading and cytoskeletal tension. Furthermore, this force generation required Rho kinase and myosin activity. These findings suggest that integrin activation and cell traction forces comprise a bi-directional signaling unit of cell adhesion.

Mechanosensitive Vinculin Signaling Regulates Stem Cell Fate

Human mesenchymal stem cell (hMSC) proliferation, migration, and differentiation have all been linked to extracellular matrix stiffness, but despite serious scientific inquiry, a consensus on the signaling pathways that are necessary and sufficient for this mechanosensitive ability has yet to be reached. An analysis of kinase binding site accessibility revealed MAPK1 to be both prevalent and inaccessible in many focal adhesion/mechanosensing candidate proteins. One binding partner for MAPK1, vinculin, is activated upon binding to talin in a force-sensitive manner. A Cysteine Shotgun/Western Blot was used to confirm the unfolding of talin in response to changes in substrate stiffness. RNA interference was used to knock down vinculin, resulting in an 80% decrease in stiffness-induced MyoD, a muscle transcription factor; control cultures that induce Runx2 expression, an osteoblast transcription factor, were insensitive to vinculin knockdown. Vinculin knockdown was not observed to interfere with focal adhesion assembly, alter adhesive properties, or diminish cell traction force generation, indicating that its deletion only adversely affected MAPK1 signaling. In addition, vinculin domains were also selectively deleted and added back into knocked-down cells to assess which domains of the protein were sufficient for stiffness-induced differentiation. Together, these data provide some of the first in situ evidence that force-sensitive focal adhesion proteins can activate stem cell differentiation signals.

Fibroblast polarization is a matrix-rigidity-dependent process controlled by focal adhesion mechanosensing

Cell elongation and polarization are basic morphogenetic responses to extracellular matrix adhesion. We demonstrate here that human cultured fibroblasts readily polarize when plated on rigid, but not on compliant, substrates. On rigid surfaces, large and uniformly oriented focal adhesions are formed, whereas cells plated on compliant substrates form numerous small and radially oriented adhesions. Live-cell monitoring showed that focal adhesion alignment precedes the overall elongation of the cell, indicating that focal adhesion orientation may direct cell polarization. siRNA-mediated knockdown of 85 human protein tyrosine kinases (PTKs) induced distinct alterations in the cell polarization response, as well as diverse changes in cell traction force generation and focal adhesion formation. Remarkably, changes in rigidity-dependent traction force development, or focal adhesion mechanosensing, were consistently accompanied by abnormalities in the cell polarization response. We propose that the different stages of cell polarization are regulated by multiple, PTK-dependent molecular checkpoints that jointly control cell contractility and focal-adhesion-mediated mechanosensing.

Abstract 435: MT regulation of tumor-dependent microenvironment remodeling

Abstract Microtubules (MTs) represent one of the most effective targets in cancer chemotherapy. However, drugs that target MTs, like the taxanes, often fail in the metastatic setting. Most studies are performed in a two-dimensional monoculture thus, overlook the complexity of the tumor microenvironment. Therefore, to gain a better understanding of tumor MT biology within a physiological context we studied the role of MT dynamics in 3D co-cultures closer mimicking the tumor microenvironment. Since tumor cells need to remodel their ECM in order to metastasize we hypothesized that the interphase MT cytoskeleton, through the coordination of cellular signaling regulates this process. To test this hypothesis we first assessed the MT-dependence of tumor-mediated ECM remodeling, using a floating collagen gel contraction assay. In this assay, breast cancer cells were embedded into a floating collagen gel and monitored for their ability to contract the gel as a result of cell generated traction force. We observed that the MDA-MB-231 metastatic breast cancer cells exhibited greater contractility as compared with the MCF-7 non-metastatic breast cancer cells or the non-tumorigenic human breast epithelial cell line MCF10A. However upon disruption of MTs with paclitaxel (PTX), tumor cell contractility was severely inhibited at drug concentrations that did not affect cell number, suggesting that dynamic MTs are essential for cell traction force generation. In addition, we observed that human mammary fibroblasts when co-cultured with breast cancer cells in 3D exhibited increased motility as compared to 3D fibroblast monocultures. Interestingly, fibroblast activation was inhibited in co-cultures with breast cancer cells previously treated with a taxane, suggesting that tumor MT stabilization negatively affects fibroblast activation. To investigate the factors that mediated the tumor cell communication with surrounding fibroblasts we monitored fibroblast motility in 3D monocultures following incubation with conditioned medium collected from either untreated or Taxol pre-treated breast cancer cells. In agreement with our 3D co-culture results, fibroblast 3D motility was inhibited following exposure to conditioned medium from the Taxol-pretreated cells. These results suggest that the composition of the tumor secretome changes upon MT stabilization, which in turn affects the ability of tumor cells to communicate with stromal fibroblasts. Currently, we are performing proteomic analysis of the conditioned media to identify soluble factors whose secretion is dependent on dynamic microtubules. In addition, to determine whether the effects of taxane treatment is solely due to microtubule stabilization we are performing the 3D co-culture experiments using a Taxol-resistant β-tubulin mutant breast cancer cell line. These findings point to a novel role of interphase MTs in tumor biology and can possibly lead to new avenues for taxane clinical efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 435. doi:10.1158/1538-7445.AM2011-435

Mapping traction forces generated by motile cells

In a continuing effort to understand how cell-generated traction forces are utilised for locomotion, we have applied our modified silicone rubber traction force assay to rapidly locomoting fish epidermal keratocytes executing turns and shape changes, and negotiating obstacles. The resulting maps show that these cells can redistribute tractions from the “steady-state” pattern (previously observed during unobstructed, gliding locomotion), into a variety of transient patterns, with lifetimes of less than 1 minute (Figs. 1-4). The map for a “steady state” locomoting keratocyte shows a maximum traction force density of ~5x10-5 dynes/μm (data not shown). This value was derived from cell-free experiments in which elastic films were manipulated with a pair of micronneedles. Such experiments, in which all forces were known, showed that both the magnitude and direction of traction forces applied to the film could be closely predicted, and that the Young’s modulus of elasticity for the silicone substratum could be calculated. The consequences for understanding the underlying molecular basis for shape change and cell motility from this type of analysis will be discussed.