Cell migration plays a major role in many fundamental biological processes, such as morphogenesis, tumor metastasis, and wound healing. As they anchor and pull on their surroundings, adhering cells actively probe the stiffness of their environment. Current understanding is that traction forces exerted by cells arise mainly at mechanotransduction sites, called focal adhesions, whose size seems to be correlated to the force exerted by cells on their underlying substrate, at least during their initial stages. In fact, our data show by direct measurements that the buildup of traction forces is faster for larger substrate stiffness, and that the stress measured at adhesion sites depends on substrate rigidity. Our results, backed by a phenomenological model based on active gel theory, suggest that rigidity-sensing is mediated by a large-scale mechanism originating in the cytoskeleton instead of a local one. We show that large-scale mechanosensing leads to an adaptative response of cell migration to stiffness gradients. In response to a step boundary in rigidity, we observe not only that cells migrate preferentially toward stiffer substrates, but also that this response is optimal in a narrow range of rigidities. Taken together, these findings lead to unique insights into the regulation of cell response to external mechanical cues and provide evidence for a cytoskeleton-based rigidity-sensing mechanism.actin cytoskeleton | microfabrication | mechanobiology | cell mechanics C ell migration is not only sensitive to the biochemical composition of the environment, but also to its mechanical properties. Cells directly probe the physical properties of their environment, such as substrate stiffness, by pulling on it. Increasing evidence show that matrix or tissue elasticity has a key role in regulating numerous cell functions, such as adhesion (1), migration (2) and differentiation (3). Such functions are affected by cell-generated actomyosin forces that depend on substrate stiffness through a feedback mechanism (4). The sensitivity of cells to mechanical properties of the extracellular matrix (ECM) arises from the mechanosensitive nature of cell adhesion. Numerous plausible candidates for the transduction of mechanochemical signals have been tested (5). Among them, focal adhesions (FAs) appear to be the most prominent, as shown by the reported correlation between their area and sustained force exhibiting a constant stress (6-9). This mechanosensitivity is usually accounted for by a generic local mechanism in which a force applied to an FA induces an elastic deformation of the contact that triggers conformational and organizational changes of some of its constitutive proteins, which in turn can enhance binding with new proteins enabling growth of the contact (10-12). However, how this local mechanosensitivity can result in the ability of cells to sense and respond to the rigidity of their surroundings (2, 3, 13, 14) at a large scale remains largely unknown (5, 15). Much conflicting evidence has emerged from a variety of studi...

The role of geometrical confinement on collective cell migration has been recognized but has not been elucidated yet. Here, we show that the geometrical properties of the environment regulate the formation of collective cell migration patterns through cell-cell interactions. Using microfabrication techniques to allow epithelial cell sheets to migrate into strips whose width was varied from one up to several cell diameters, we identified the modes of collective migration in response to geometrical constraints. We observed that a decrease in the width of the strips is accompanied by an overall increase in the speed of the migrating cell sheet. Moreover, largescale vortices over tens of cell lengths appeared in the wide strips whereas a contraction-elongation type of motion is observed in the narrow strips. Velocity fields and traction force signatures within the cellular population revealed migration modes with alternative pulling and/or pushing mechanisms that depend on extrinsic constraints. Force transmission through intercellular contacts plays a key role in this process because the disruption of cell-cell junctions abolishes directed collective migration and passive cell-cell adhesions tend to move the cells uniformly together independent of the geometry. Altogether, these findings not only demonstrate the existence of patterns of collective cell migration depending on external constraints but also provide a mechanical explanation for how large-scale interactions through cell-cell junctions can feed back to regulate the organization of migrating tissues.cell traction forces | collective dynamics | madin darby canine kidney epithelial cells | particle image velocimetry C ollective behavior is a fundamental phenomenon exhibited by a wide variety of systems such as flows in granular matter (1), collective movements of animals (2), self-organization of bacteria (3), and morphogenesis of biological tissues (4). Although collective behaviors have been observed across diverse physical and biological systems, it is increasingly clear that there are broad unifying and common parameters that govern the emergence of this phenomenon such as the density of the constituent particles, the boundary conditions within which the movements occur, and the nature of coupling between the individual particles. In this context, collective behavior in migrating cells is of particular interest as a highly out-of-equilibrium process where cells passively interact with each other and exert active forces in response to their mechanical environment (4). Such collective behavior drives many biological processes such as embryonic development (5), tissue morphogenesis (6), wound healing (7), and tumor metastasis (8, 9). Although single cell dynamics has been extensively studied (10-12), the movement of multicellular structures could not be simply explained by cell autonomous behaviors (13-16). Instead, intercellular interactions and large-scale propagation of mechanical signals (over several cell sizes) are necessary to understand the emergen...

Collective behavior refers to the emergence of complex migration patterns over scales larger than those of the individual elements constituting a system. It plays a pivotal role in biological systems in regulating various processes such as gastrulation, morphogenesis and tissue organization. Here, by combining experimental approaches and numerical modeling, we explore the role of cell density ('crowding'), strength of intercellular adhesion ('cohesion') and boundary conditions imposed by extracellular matrix (ECM) proteins ('constraints') in regulating the emergence of collective behavior within epithelial cell sheets. Our results show that the geometrical confinement of cells into well-defined circles induces a persistent, coordinated and synchronized rotation of cells that depends on cell density. The speed of such rotating large-scale movements slows down as the density increases. Furthermore, such collective rotation behavior depends on the size of the micropatterned circles: we observe a rotating motion of the overall cell population in the same direction for sizes of up to 200 μm. The rotating cells move as a solid body, with a uniform angular velocity. Interestingly, this upper limit leads to length scales that are similar to the natural correlation length observed for unconfined epithelial cell sheets. This behavior is strongly altered in cells that present a downregulation of adherens junctions and in cancerous cell types. We anticipate that our system provides a simple and easy approach to investigate collective cell behavior in a well-controlled and systematic manner.

The ability of skin to act as a barrier is primarily determined by the efficiency of skin cells to maintain and restore its continuity and integrity. In fact, during wound healing keratinocytes migrate collectively to maintain their cohesion despite heterogeneities in the extracellular matrix. Here, we show that monolayers of human keratinocytes migrating along functionalized micropatterned surfaces comprising alternating strips of extracellular matrix (fibronectin) and non-adherent polymer form suspended multicellular bridges over the non-adherent areas. The bridges are held together by intercellular adhesion and are subjected to considerable tension, as indicated by the presence of prominent actin bundles. We also show that a model based on force propagation through an elastic material reproduces the main features of bridge maintenance and tension distribution. Our findings suggest that multicellular bridges maintain tissue integrity during wound healing when cell-substrate interactions are weak and may prove helpful in the design of artificial scaffolds for skin regeneration.

The closure of gaps within epithelia is crucial to maintain its integrity during biological processes such as wound healing and gastrulation. Depending on the distribution of extracellular matrix, gap closure occurs through assembly of multicellular actin-based contractile cables or protrusive activity of border cells into the gap. Here we show that the supracellular actomyosin contractility of cells near the gap edge exerts sufficient tension on the surrounding tissue to promote closure of non-adherent gaps. Using traction force microscopy, we observe that cell-generated forces on the substrate at the gap edge first point away from the centre of the gap and then increase in the radial direction pointing into the gap as closure proceeds. Combining with numerical simulations, we show that the increase in force relies less on localized purse-string contractility and more on large-scale remodelling of the suspended tissue around the gap. Our results provide a framework for understanding the assembly and the mechanics of cellular contractility at the tissue level.