Directed cell migration is a physical process that requires dramatic changes in cell shape and adhesion to the extracellular matrix. For efficient movement, these processes must be spatiotemporally coordinated. To a large degree, the morphological changes and physical forces that occur during migration are generated by a dynamic filamentous actin (F-actin) cytoskeleton. Adhesion is regulated by dynamic assemblies of structural and signaling proteins that couple the F-actin cytoskeleton to the extracellular matrix. Here, we review current knowledge of the dynamic organization of the F-actin cytoskeleton in cell migration and the regulation of focal adhesion assembly and disassembly with an emphasis on how mechanical and biochemical signaling between these two systems regulate the coordination of physical processes in cell migration.

Focal adhesions (FAs) are the predominant mechanism by which cells mechanically couple to and exert traction forces on their extracellular matrix (ECM). It is widely presumed that FA size is modulated by force to mediate changes in adhesion strength at different levels of cellular tension. However, previous studies seeking correlations between force and FA morphology have yielded variable and often conflicting results. Here we show that a strong correlation between adhesion size and traction force exists only during the initial stages of myosin-mediated adhesion maturation and growth. For mature adhesions, no correlation between traction stress and size is observed. Rather, the tension that is sustained at mature adhesions is more strongly influenced by proximity to the cell edge, with peripheral adhesions transmitting higher tension than adhesions near the cell center. Finally, we show that mature adhesions can withstand sixfold increases in tension without changes in size. Thus, although a strong correlation between adhesion size and mechanical tension is observed during the initial stages of myosin-mediated adhesion maturation, no correlation is observed in mature, elongated adhesions. This work places spatiotemporal constraints on the force-dependent growth of adhesions and provides insight into the mechanical regulation of cell-ECM adhesion.

Adhesion between cardiac myocytes is essential for the heart to function as an electromechanical syncytium. Although cell-matrix and cell-cell adhesions reorganize during development and disease, the hierarchical cooperation between these subcellular structures is poorly understood. We reasoned that, during cardiac development, focal adhesions mechanically stabilize cells and tissues during myofibrillogenesis and intercalated disc assembly. As the intercalated disc matures, we postulated that focal adhesions disassemble as systolic stresses are transmitted intercellularly. Finally, we hypothesized that pathological remodeling of cardiac microenvironments induces excessive mechanical loading of intercalated discs, leading to assembly of stabilizing focal adhesions adjacent to the junction. To test our model, we engineered μtissues composed of two ventricular myocytes on deformable substrates of tunable elasticity to measure the dynamic organization and functional remodeling of myofibrils, focal adhesions, and intercalated discs as cooperative ensembles. Maturing μtissues increased systolic force while simultaneously developing into an electromechanical syncytium by disassembling focal adhesions at the cell-cell interface and forming mature intercalated discs that transmitted the systolic load. We found that engineering the microenvironment to mimic fibrosis resulted in focal adhesion formation adjacent to the cell-cell interface, suggesting that the intercalated disc required mechanical reinforcement. In these pathological microenvironments, μtissues exhibited further evidence of maladaptive remodeling, including lower work efficiency, longer contraction cycle duration, and weakened relationships between cytoskeletal organization and force generation. These results suggest that the cooperative balance between cell-matrix and cell-cell adhesions in the heart is guided by an architectural and functional hierarchy established during development and disrupted during disease.adherens junctions | sarcomere | mechanotransduction | extracellular matrix I n the adult heart, healthy ventricular tissue is characterized by spatial segregation of cell-matrix adhesions to transverse myocyte borders (1) and cell-cell adhesions to longitudinal myocyte borders (2). Many cardiomyopathies are characterized by lateralization of cell-cell adhesions (3-5), increased expression of cell-matrix adhesions (6, 7), and arrhythmogenesis (8), suggesting that spatially organized adhesion is essential for effective eletromechanical coupling (9). Although in vivo studies have shown that localization of cell-matrix and cell-cell adhesions is developmentally regulated (10-13), the factors that regulate their assembly and disassembly are not well understood.Cell-matrix adhesions are especially important during organogenesis, anchoring developing cells and tissues to the extracellular matrix (ECM) (14) and directing cell migration (15-17). As development progresses, cell-matrix adhesions must selectively disassemble so that cell-cell adhesions can f...

SummaryStructural phenotyping based on classical image feature detection has been adopted to elucidate the molecular mechanisms behind genetically or pharmacologically induced changes in cell morphology. Here, we developed a set of 11 metrics to capture the increasing sarcomere organization that occurs intracellularly during striated muscle cell development. To test our metrics, we analyzed the localization of the contractile protein α-actinin in a variety of primary and stem-cell derived cardiomyocytes. Further, we combined these metrics with data mining algorithms to unbiasedly score the phenotypic maturity of human-induced pluripotent stem cell-derived cardiomyocytes.

SUMMARY Background The spatiotemporal regulation of adhesion to the extracellular matrix is important in metazoan cell migration and mechanosensation. While adhesion assembly depends on intracellular and extracellular tension, the biophysical regulation of force transmission between the actin cytoskeleton and extracellular matrix during this process remains largely unknown. Results To elucidate the nature of force transmission as myosin-II tension is applied to focal adhesions, we correlated the dynamics of focal adhesion proteins and the actin cytoskeleton to local traction stresses. We find that, under low extracellular tension, newly formed adhesions near the cell periphery undergo a transient retrograde displacement preceding elongation. We identify that myosin-II generated tension drives this mobility and determine the interface of differential motion, or ‘slip’, to be between the integrin and the ECM. We found that the magnitude and duration of both adhesion slip and associated F-actin dynamics is strongly modulated by the ECM compliance. Furthermore, traction forces are generated throughout the slip period with adhesion immobilization occurring at a constant tension. Conclusions We have identified a tension-dependent, extracellular ‘clutch’ between integrins and the extracellular matrix that is utilized to stabilize adhesions under myosin-II driven tension. This work signifies that modulating adhesion alters force transmission during focal adhesion maturation.

SummaryAdvances in stem cell manufacturing methods have made it possible to produce stem cell-derived cardiac myocytes at industrial scales for in vitro muscle physiology research purposes. Although FDA-mandated quality assurance metrics address safety issues in the manufacture of stem cell-based products, no standardized guidelines currently exist for the evaluation of stem cell-derived myocyte functionality. As a result, it is unclear whether the various stem cell-derived myocyte cell lines on the market perform similarly, or whether any of them accurately recapitulate the characteristics of native cardiac myocytes. We propose a multiparametric quality assessment rubric in which genetic, structural, electrophysiological, and contractile measurements are coupled with comparison against values for these measurements that are representative of the ventricular myocyte phenotype. We demonstrated this procedure using commercially available, mass-produced murine embryonic stem cell- and induced pluripotent stem cell-derived myocytes compared with a neonatal mouse ventricular myocyte target phenotype in coupled in vitro assays.

The dynamics and organization of the lamellar actin cytoskeleton at different levels of tension are identified. The force-dependent steps of stress fiber assembly are studied.

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties. To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces. Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.