Single Focal Adhesions Research Articles

Machine learning interpretable models of cell mechanics from protein images

Cellular form and function emerge from complex mechanochemical systems within the cytoplasm. Currently, no systematic strategy exists to infer large-scale physical properties of a cell from its molecular components. This is an obstacle to understanding processes such as cell adhesion and migration. Here, we develop a data-driven modeling pipeline to learn the mechanical behavior of adherent cells. We first train neural networks to predict cellular forces from images of cytoskeletal proteins. Strikingly, experimental images of a single focal adhesion (FA) protein, such as zyxin, are sufficient to predict forces and can generalize to unseen biological regimes. Using this observation, we develop two approaches-one constrained by physics and the other agnostic-to construct data-driven continuum models of cellular forces. Both reveal how cellular forces are encoded by two distinct length scales. Beyond adherent cell mechanics, our work serves as a case study for integrating neural networks into predictive models for cell biology.

Pattern Generation for Micropattern Traction Microscopy.

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

Label-Free Imaging of Nanoscale Displacements and Free-Energy Profiles of Focal Adhesions with Plasmonic Scattering Microscopy.

Cell adhesion plays a critical role in cell communication, cell migration, cell proliferation, and integration of medical implants with tissues. Focal adhesions physically link the cell cytoskeleton to the extracellular matrix, but it remains challenging to image single focal adhesions directly. Here, we show that plasmonic scattering microscopy (PSM) can directly image the single focal adhesions in a label-free, real-time, and non-invasive manner with sub-micrometer spatial resolution. PSM is developed based on surface plasmon resonance (SPR) microscopy, and the evanescent illumination makes it immune to the interference of intracellular structures. Unlike the conventional SPR microscopy, PSM can provide a high signal-to-noise ratio and sub-micrometer spatial resolution for imaging the analytes with size down to a single-molecule level, thus allowing both the super-resolution lateral localization for measuring the nanoscale displacement and precise tracking of vertical distances between the analyte centroid and the sensor surface for analysis of free-energy profiles. PSM imaging of the RBL-2H3 cell with temporal resolution down to microseconds shows that the focal adhesions have random diffusion behaviors in addition to their directional movements during the antibody-mediated activation process. The free-energy mapping also shows a similar movement tendency, indicating that the cell may change its morphology upon varying the binding conditions of adhesive structures. PSM provides insights into the individual focal adhesion activities and can also serve as a promising tool for investigating the cell/surface interactions, such as cell capture and detection and tissue adhesive materials screening.

Mechanical Control of Cell Migration by the Metastasis Suppressor Tetraspanin CD82/KAI1.

The plasma membrane is a key actor of cell migration. For instance, its tension controls persistent cell migration and cell surface caveolae integrity. Then, caveolae constituents such as caveolin-1 can initiate a mechanotransduction loop that involves actin- and focal adhesion-dependent control of the mechanosensor YAP to finely tune cell migration. Tetraspanin CD82 (also named KAI-1) is an integral membrane protein and a metastasis suppressor. Its expression is lost in many cancers including breast cancer. It is a strong inhibitor of cell migration by a little-known mechanism. We demonstrated here that CD82 controls persistent 2D migration of EGF-induced single cells, stress fibers and focal adhesion sizes and dynamics. Mechanistically, we found that CD82 regulates membrane tension, cell surface caveolae abundance and YAP nuclear translocation in a caveolin-1-dependent manner. Altogether, our data show that CD82 controls 2D cell migration using membrane-driven mechanics involving caveolin and the YAP pathway.

Evolving roles and dynamics for catch and slip bonds during adhesion cluster maturation.

Focal adhesions are the loci of cellular adhesion to the extracellular matrix. At these sites, various integrins forge connections between the intracellular cytoskeleton and the outside world; large patches of multiple types of integrins together grip hold of collagen, fibronectin, and other extracellular matrix components. A single focal adhesion will likely contain bonds whose lifetime increases with applied load (catch bonds), and bonds whose lifetime decreases with applied load (slip bonds). Prior work suggests that the combination of different types of integrins is essential for focal adhesion stability and mechanosensory functionality. In the present work, we investigate numerically the interplay between two distinct types of bonds, and we ask how the presence of slip bonds, in the same focal integrin cluster, augments the collective behavior of the catch bonds. We show that mixing these two components may increase the low-force mechanical integrity that may be lacking in pure-catch adhesions, while preserving the potential to strengthen the entire adhesion when a force is applied. We investigate the spatial distribution in mixed-integrin focal adhesions, and we show that the differential response to loading leads, via an excluded volume interaction, to a dependence of the individual integrin diffusivities on the applied load, an effect that has been reported in experiments.

Stochastic resetting and the mean-field dynamics of focal adhesions

In this paper we investigate the effects of diffusion on the dynamics of a single focal adhesion at the leading edge of a crawling cell by considering a simplified model of sliding friction. Using a mean-field approximation, we derive an effective single-particle system that can be interpreted as an overdamped Brownian particle with spatially dependent stochastic resetting. We then use renewal and path-integral methods from the theory of stochastic resetting to calculate the mean sliding velocity under the combined action of diffusion, active forces, viscous drag, and elastic forces generated by the adhesive bonds. Our analysis suggests that the inclusion of diffusion can sharpen the response to changes in the effective stiffness of the adhesion bonds. This is consistent with the hypothesis that force fluctuations could play a role in mechanosensing of the local microenvironment.

Mechanical Responses of Breast Cancer Cells to Substrates of Varying Stiffness Revealed by Single-Cell Measurements.

How cancer cells respond to different mechanical environments remains elusive. Here, we investigated the tension in single focal adhesions of MDA-MB-231 (metastatic breast cancer cells) and MCF-10A (normal human breast cells) cells on substrates of varying stiffness using single-cell measurements. Tension measurements in single focal adhesions using an improved FRET-based tension sensor showed that the tension in focal adhesions of MDA-MB-231 cells increased on stiffer substrates while the tension in MCF-10A cells exhibited no apparent change against the substrate stiffness. Viscoelasticity measurements using magnetic tweezers showed that the power-law exponent of MDA-MB-231 cells decreased on stiffer substrates whereas MCF-10A cells had similar exponents throughout the whole stiffness, indicating that MDA-MB-231 cells change their viscoelasticity on stiffer substrates. Such changes in tension in focal adhesions and viscoelasticity against the substrate stiffness represent an adaptability of cancer cells in mechanical environments, which can facilitate the metastasis of cancer cells to different tissues.

Mechanical Control of Cell Migration by the Metastasis Suppressor Tetraspanin CD82/KAI1

The plasma membrane is a key actor of cell migration. For instance, its tension controls persistent cell migration and cell surface caveolae integrity. Then, caveolae constituents like caveolin-1 can initiate a mechano-tranduction loop that involves actin-and focal adhesions- dependent control of the mechanosensor YAP to finely tune cell migration. Tetraspanin CD82 (also named KAI-1) is an integral membrane protein and a metastasis suppressor. Its expression is lost in many cancers including breast cancer. It is a strong inhibitor of cell migration by a not well-known mechanism. We demonstrated here that CD82 controls persistent 2D migration of EGF-induced single cell, stress fibers and focal adhesion sizes and dynamics. In addition, CD82 regulates cell mechanics e.g. membrane tension, cell surface caveolae abundance and YAP nuclear translocation in a caveolin-1 dependent manner. Altogether, our data strongly suggest that CD82 controls 2D cell migration using a membrane-driven mechanical loop involving caveolin and the YAP pathway.

In Situ Subcellular Detachment of Cells Using a Cell-Friendly Photoresist and Spatially Modulated Light.

Dynamic adhesion and detachment of subcellular regions occur during cell migration, thus a technique allowing precise control of subcellular detachment of cells will be useful for cell migration study. Previous methods for cell detachment were developed either for harvesting cells or cell sheets attached on surfaces with low resolution patterning capability, or for detaching subcellular regions located on predefined electrodes. In this paper, a method that allows in situ subcellular detachment of cells with ≈1.5 µm critical feature size while observing cells under a fluorescence microscope is introduced using a cell‐friendly photoresist and spatially modulated light. Using this method, a single cell, regions in cell sheets, and a single focal adhesion complex within a cell are successfully detached. Furthermore, different subcellular regions of migrating cells are detached and changes in cell polarity and migration direction are quantitatively analyzed. This method will be useful for many applications in cell detachment, in particular when subcellular resolution is required.

Cellular dynamics of bovine aortic smooth muscle cells measured using MEMS force sensors

Adhesive cells perceive the mechanical properties of the substrates to which they adhere, adjusting their cellular mechanical forces according to their biological characteristics. This mechanical interaction subsequently affects the growth, locomotion, and differentiation of the cell. However, little is known about the detailed mechanism that underlies this interaction between adherent cells and substrates because dynamically measuring mechanical phenomena is difficult. Here, we utilize microelectromechamical systems force sensors that can measure cellular traction forces with high temporal resolution (~2.5 µs) over long periods (~3 h). We found that the cellular dynamics reflected physical phenomena with time scales from milliseconds to hours, which contradicts the idea that cellular motion is slow. A single focal adhesion (FA) generates an average force of 7 nN, which disappears in ms via the action of trypsin-ethylenediaminetetraacetic acid. The force-changing rate obtained from our measurements suggests that the time required for an FA to decompose was nearly proportional to the force acting on the FA.

Diverse patterns of molecular changes in the mechano-responsiveness of focal adhesions

Focal adhesions anchor contractile actin fibers with the extracellular matrix, sense the generated tension and respond to it by changing their morphology and composition. Here we ask how this mechanosensing is enabled at the protein-network level, given the modular assembly and multitasking of focal adhesions. To address this, we applied a sensitive 4-color live cell imaging approach, enabling monitoring patterns of molecular changes in single focal adhesions. Co-imaging zyxin, FAK, vinculin and paxillin revealed heterogeneities in their responses to Rho-associated kinase (ROCK)-mediated perturbations of actomyosin contractility. These responses were rather weakly correlated between the proteins, reflecting diverse compositional changes in different focal adhesions. This diversity is partially attributable to the location of focal adhesions, their area, molecular content and previous contractility perturbations, suggesting that integration of multiple local cues shapes differentially focal adhesion mechano-responsiveness. Importantly, the compositional changes upon ROCK perturbations exhibited distinct paths in different focal adhesions. Moreover, the protein exhibiting the strongest response to ROCK perturbations varied among different focal adhesions. The diversity in response patterns is plausibly enabled by the modular mode of focal adhesions assembly and can provide them the needed flexibility to perform multiple tasks by combining optimally a common set of multifunctional components.

Effects of substrate stiffness and actomyosin contractility on coupling between force transmission and vinculin-paxillin recruitment at single focal adhesions.

Focal adhesions (FAs) regulate force transfer between the cytoskeleton and ECM-integrin complexes. We previously showed that vinculin regulates force transmission at FAs. Vinculin residence time in FAs correlated with applied force, supporting a mechanosensitive model in which forces stabilize vinculin's active conformation to promote force transfer. In the present study, we examined the relationship between traction force and vinculin-paxillin localization to single FAs in the context of substrate stiffness and actomyosin contractility. We found that vinculin and paxillin FA area did not correlate with traction force magnitudes at single FAs, and this was consistent across different ECM stiffness and cytoskeletal tension states. However, vinculin residence time at FAs varied linearly with applied force for stiff substrates, and this was disrupted on soft substrates and after contractility inhibition. In contrast, paxillin residence time at FAs was independent of local applied force and substrate stiffness. Paxillin recruitment and residence time at FAs, however, were dependent on cytoskeletal contractility on lower substrate stiffness values. Finally, substrate stiffness and cytoskeletal contractility regulated whether vinculin and paxillin turnover dynamics are correlated to each other at single FAs. This analysis sheds new insights on the coupling among force, substrate stiffness, and FA dynamics.

Highly Multiplexed Imaging Uncovers Changes in Compositional Noise within Assembling Focal Adhesions.

Integrin adhesome proteins bind each other in alternative manners, forming within the cell diverse cell-matrix adhesion sites with distinct properties. An intriguing question is how such modular assembly of adhesion sites is achieved correctly solely by self-organization of their components. Here we address this question using high-throughput multiplexed imaging of eight proteins and two phosphorylation sites in a large number of single focal adhesions. We found that during the assembly of focal adhesions the variances of protein densities decrease while the correlations between them increase, suggesting reduction in the noise levels within these structures. These changes correlate independently with the area and internal density of focal adhesions, but not with their age or shape. Artificial neural network analysis indicates that a joint consideration of multiple components improves the predictability of paxillin and zyxin levels in internally dense focal adhesions. This suggests that paxillin and zyxin densities in focal adhesions are fine-tuned by integrating the levels of multiple other components, thus averaging-out stochastic fluctuations. Based on these results we propose that increase in internal protein densities facilitates noise suppression in focal adhesions, while noise suppression enables their stable growth and further density increase—hence forming a feedback loop giving rise to a quality-controlled assembly.

In-situ coupling between kinase activities and protein dynamics within single focal adhesions.

The dynamic activation of oncogenic kinases and regulation of focal adhesions (FAs) are crucial molecular events modulating cell adhesion in cancer metastasis. However, it remains unclear how these events are temporally coordinated at single FA sites. Therefore, we targeted fluorescence resonance energy transfer (FRET)-based biosensors toward subcellular FAs to report local molecular events during cancer cell adhesion. Employing single FA tracking and cross-correlation analysis, we quantified the dynamic coupling characteristics between biochemical kinase activities and structural FA within single FAs. We show that kinase activations and FA assembly are strongly and sequentially correlated, with the concurrent FA assembly and Src activation leading focal adhesion kinase (FAK) activation by 42.6 ± 12.6 sec. Strikingly, the temporal coupling between kinase activation and individual FA assembly reflects the fate of FAs at later stages. The FAs with a tight coupling tend to grow and mature, while the less coupled FAs likely disassemble. During FA disassembly, however, kinase activations lead the disassembly, with FAK being activated earlier than Src. Therefore, by integrating subcellularly targeted FRET biosensors and computational analysis, our study reveals intricate interplays between Src and FAK in regulating the dynamic life of single FAs in cancer cells.

Stability of focal adhesion enhanced by its inner force fluctuation**Project supported by the National Natural Science Foundation of China (Grant No.*11372279).

Cells actively sense and respond to mechanical signals from the extracellular matrix through focal adhesions. By representing a single focal adhesion as a cluster of slip bonds, it has been demonstrated that the cluster often became unstable under fluctuated forces. However, an unusual case was also reported, where the stability of the cluster might be substantially enhanced by a fluctuated force with a relatively low fluctuation frequency and high fluctuation amplitude. Such an observation cannot be explained by the conventional fracture theory of fatigue. Here, we intensively investigate this intriguing observation by carrying out systematic parametric studies. Our intensive simulation results indicate that stability enhancement of this kind is in fact quite robust, which can be affected by the stochastic features of a single bond and the profile of the fluctuated forces such as the average value of bond force. We then suggest that the fluctuation of traction force within a focal adhesion might enhance its stability in a certain way.

Super‐resolution Imaging Shows Spatial Regulation of p130Cas Phosporylation within Focal Adhesions

Cell motility is critical for physiological processes such as tissue development and remodeling as well as pathologies such as cancer metastasis. Cell motility is influenced by its surrounding matrix through biochemical and mechanical stimuli. p130Cas is a signaling molecule that has been proposed to be a mechanosensor, which responds to mechanical stimuli and mediates cell motility during the early stage of lamellipodial protrusions. p130Cas localizes to focal adhesions (FAs) and binds with its effectors via tyrosine phosphorylation of its substrate domain. Previous studies have suggested that mechanical stimuli increase p130Cas tyrosine phosphorylation and these phosphotyrosines recruit SH2‐domain containing effectors like Crk, that initiate a signaling cascade activating the small Rho‐GTPase Rac1 leading to lamellipodial protrusions.Due to spatial heterogeneity of stresses within FAs, we hypothesized that p130Cas phosphorylation is not uniform across a single FA. Spatial differences across such small structures as FAs cannot be resolved using biochemical assays or standard microscopy. We used a super resolution imaging technique, PALM (photo‐activated localization microscopy) to study the differences in p130Cas phosphorylation within individual FAs using SH2 domains of Crk as probes. Our results show punctate localization of SH2 probes at single FAs compared to uniform distribution of p130Cas.Our data suggest that p130Cas activity is spatially regulated. In the future we will look at correlations between mechanical stress within FAs and p130Cas activity. Our results will help elucidate the mechanism of mechanical regulation of p130Cas phosphorylation, and may help understand how cells sense and respond to external mechanical stimuli.

A Critical Pull to Polarize the Cell

A Critical Pull to Polarize the Cell

Catching up on Slip: Focal Adhesion Composition and Mechanosensing

Unlike slip bonds, catch bonds experience reinforcement under tension. Cell adheres to the surface, using integrins forming both catch- and slip- bonds with the surface receptors. How will the catch and slip bonds interact with each other on a single adhesion scale? How does the intracellular structure vary depending on the extracellular matrix stiffness? I discuss the implications of single catch-bond characteristics for the behavior of a load-sharing cluster of such bonds: these are shown to possess a regime of strengthening with increasing applied force, similar to the manner in which focal adhesions become selectively reinforced. In addition, I present numerical simulations of mixtures of catch and slip bonds within single focal adhesion, and propose a model of how they can influence cytoskeletal reorganization, force generation and adhesion growth, interacting indirectly through applied force. Our results may shed new light on the fundamental processes that allow cells to sense the mechanical properties of their environment and in particular show how single focal adhesions may act, autonomously, as local rigidity sensors.

Contractile Fibers and Catch-Bond Clusters: a Biological Force Sensor?

Catch bonds are cellular receptor-ligand pairs whose lifetime, counterintuitively, increases with increasing load. Although their existence was initially pure theoretical speculation, recent years have seen several experimental demonstrations of catch-bond behavior in biologically relevant and functional protein-protein bonds. Particularly notable among these established catch-bond formers is the integrin α5β1, the primary receptor for fibronectin and, as such, a crucial determinant for the characteristics of the mechanical coupling between cell and matrix. In this work, we explore the implications of single catch-bond characteristics for the behavior of a load-sharing cluster of such bonds: These clusters are shown to possess a regime of strengthening with increasing applied force, similar to the manner in which focal adhesions become selectively reinforced. Our results may shed new light on the fundamental processes that allow cells to sense and respond to the mechanical properties of their environment and in particular show how single focal adhesions may act, autonomously, as local rigidity sensors.

Catch Me Because You Can: A Mathematical Model for Mechanosensing

Catch Me Because You Can: A Mathematical Model for Mechanosensing