Xavier Trepat

Universal physical responses to stretch in the living cell.

With every beat of the heart, inflation of the lung or peristalsis of the gut, cell types of diverse function are subjected to substantial stretch. Stretch is a potent stimulus for growth, differentiation, migration, remodelling and gene expression. Here, we report that in response to transient stretch the cytoskeleton fluidizes in such a way as to define a universal response class. This finding implicates mechanisms mediated not only by specific signalling intermediates, as is usually assumed, but also by non-specific actions of a slowly evolving network of physical forces. These results support the idea that the cell interior is at once a crowded chemical space and a fragile soft material in which the effects of biochemistry, molecular crowding and physical forces are complex and inseparable, yet conspire nonetheless to yield remarkably simple phenomenological laws. These laws seem to be both universal and primitive, and thus comprise a striking intersection between the worlds of cell biology and soft matter physics.

Collective cell guidance by cooperative intercellular forces

Cells comprising a tissue migrate as part of a collective. How collective processes are coordinated over large multi-cellular assemblies has remained unclear, however, because mechanical stresses exerted at cell-cell junctions have not been accessible experimentally. We report here maps of these stresses within and between cells comprising a monolayer. Within the cell sheet there arise unanticipated fluctuations of mechanical stress that are severe, emerge spontaneously, and ripple across the monolayer. This stress landscape becomes increasingly rugged, sluggish, and cooperative with increasing system density. Within that landscape, local cellular migrations follow local orientations of maximal principal stress. Migrations of both endothelial and epithelial monolayers conform to this behavior, as do breast cancer cell lines before but not after the epithelial-mesenchymal transition. Collective migration in these diverse systems is seen to be governed by a simple but unifying physiological principle: neighboring cells join forces to transmit appreciable normal stress across the cell-cell junction, but migrate along orientations of minimal intercellular shear stress.

Glass-like dynamics of collective cell migration

Collective cell migration in tissues occurs throughout embryonic development, during wound healing, and in cancerous tumor invasion, yet most detailed knowledge of cell migration comes from single-cell studies. As single cells migrate, the shape of the cell body fluctuates dramatically through cyclic processes of extension, adhesion, and retraction, accompanied by erratic changes in migration direction. Within confluent cell layers, such subcellular motions must be coupled between neighbors, yet the influence of these subcellular motions on collective migration is not known. Here we study motion within a confluent epithelial cell sheet, simultaneously measuring collective migration and subcellular motions, covering a broad range of length scales, time scales, and cell densities. At large length scales and time scales collective migration slows as cell density rises, yet the fastest cells move in large, multicell groups whose scale grows with increasing cell density. This behavior has an intriguing analogy to dynamic heterogeneities found in particulate systems as they become more crowded and approach a glass transition. In addition we find a diminishing self-diffusivity of short-wavelength motions within the cell layer, and growing peaks in the vibrational density of states associated with cooperative cell-shape fluctuations. Both of these observations are also intriguingly reminiscent of a glass transition. Thus, these results provide a broad and suggestive analogy between cell motion within a confluent layer and the dynamics of supercooled colloidal and molecular fluids approaching a glass transition.

Reinforcement versus Fluidization in Cytoskeletal Mechanoresponsiveness

Every adherent eukaryotic cell exerts appreciable traction forces upon its substrate. Moreover, every resident cell within the heart, great vessels, bladder, gut or lung routinely experiences large periodic stretches. As an acute response to such stretches the cytoskeleton can stiffen, increase traction forces and reinforce, as reported by some, or can soften and fluidize, as reported more recently by our laboratory, but in any given circumstance it remains unknown which response might prevail or why. Using a novel nanotechnology, we show here that in loading conditions expected in most physiological circumstances the localized reinforcement response fails to scale up to the level of homogeneous cell stretch; fluidization trumps reinforcement. Whereas the reinforcement response is known to be mediated by upstream mechanosensing and downstream signaling, results presented here show the fluidization response to be altogether novel: it is a direct physical effect of mechanical force acting upon a structural lattice that is soft and fragile. Cytoskeletal softness and fragility, we argue, is consistent with early evolutionary adaptations of the eukaryotic cell to material properties of a soft inert microenvironment.

Substrate stiffening promotes endothelial monolayer disruption through enhanced physical forces

A hallmark of many, sometimes life-threatening, inflammatory diseases and disorders is vascular leakage. The extent and severity of vascular leakage is broadly mediated by the integrity of the endothelial cell (EC) monolayer, which is in turn governed by three major interactions: cell-cell and cell-substrate contacts, soluble mediators, and biomechanical forces. A potentially critical but essentially uninvestigated component mediating these interactions is the stiffness of the substrate to which the endothelial monolayer is adherent. Accordingly, we investigated the extent to which substrate stiffening influences endothelial monolayer disruption and the role of cell-cell and cell-substrate contacts, soluble mediators, and physical forces in that process. Traction force microscopy showed that forces between cell and cell and between cell and substrate were greater on stiffer substrates. On stiffer substrates, these forces were substantially enhanced by a hyperpermeability stimulus (thrombin, 1 U/ml), and gaps formed between cells. On softer substrates, by contrast, these forces were increased far less by thrombin, and gaps did not form between cells. This stiffness-dependent force enhancement was associated with increased Rho kinase activity, whereas inhibition of Rho kinase attenuated baseline forces and lessened thrombin-induced inter-EC gap formation. Our findings demonstrate a central role of physical forces in EC gap formation and highlight a novel physiological mechanism. Integrity of the endothelial monolayer is governed by its physical microenvironment, which in normal circumstances is compliant but during pathology becomes stiffer.

Plithotaxis and emergent dynamics in collective cellular migration

For a monolayer sheet to migrate cohesively, it has long been suspected that each constituent cell must exert physical forces not only upon its extracellular matrix but also upon neighboring cells. The first comprehensive maps of these distinct force components reveal an unexpected physical picture. Rather than showing smooth and systematic variation within the monolayer, the distribution of physical forces is dominated by heterogeneity, both in space and in time, which emerges spontaneously, propagates over great distances, and cooperates over the span of many cell bodies. To explain the severe ruggedness of this force landscape and its role in collective cell guidance, the well known mechanisms of chemotaxis, durotaxis, haptotaxis are clearly insufficient. In a broad range of epithelial and endothelial cell sheets, collective cell migration is governed instead by a newly discovered emergent mechanism of innately collective cell guidance - plithotaxis.

Chase-and-run between adjacent cell populations promotes directional collective migration

Collective cell migration in morphogenesis and cancer progression often involves the coordination of multiple cell types. How reciprocal interactions between adjacent cell populations lead to new emergent behaviours remains unknown. Here we studied the interaction between neural crest (NC) cells, a highly migratory cell population, and placodal cells, an epithelial tissue that contributes to sensory organs. We found that NC cells chase placodal cells by chemotaxis, and placodal cells run when contacted by NC. Chemotaxis to Sdf1 underlies the chase, and repulsion involving PCP and N-cadherin signalling is responsible for the run. This chase-and-run requires the generation of asymmetric forces, which depend on local inhibition of focal adhesions. The cell interactions described here are essential for correct NC migration and for segregation of placodes in vivo and are likely to represent a general mechanism of coordinated migration.

Control of cell–cell forces and collective cell dynamics by the intercellular adhesome

Dynamics of epithelial tissues determine key processes in development, tissue healing and cancer invasion. These processes are critically influenced by cell–cell adhesion forces. However, the identity of the proteins that resist and transmit forces at cell–cell junctions remains unclear, and how these proteins control tissue dynamics is largely unknown. Here we provide a systematic study of the interplay between cell–cell adhesion proteins, intercellular forces and epithelial tissue dynamics. We show that collective cellular responses to selective perturbations of the intercellular adhesome conform to three mechanical phenotypes. These phenotypes are controlled by different molecular modules and characterized by distinct relationships between cellular kinematics and intercellular forces. We show that these forces and their rates can be predicted by the concentrations of cadherins and catenins. Unexpectedly, we identified different mechanical roles for P-cadherin and E-cadherin; whereas P-cadherin predicts levels of intercellular force, E-cadherin predicts the rate at which intercellular force builds up.

Mechanical regulation of a molecular clutch defines force transmission and transduction in response to matrix rigidity

Cell function depends on tissue rigidity, which cells probe by applying and transmitting forces to their extracellular matrix, and then transducing them into biochemical signals. Here we show that in response to matrix rigidity and density, force transmission and transduction are explained by the mechanical properties of the actin–talin–integrin–fibronectin clutch. We demonstrate that force transmission is regulated by a dynamic clutch mechanism, which unveils its fundamental biphasic force/rigidity relationship on talin depletion. Force transduction is triggered by talin unfolding above a stiffness threshold. Below this threshold, integrins unbind and release force before talin can unfold. Above the threshold, talin unfolds and binds to vinculin, leading to adhesion growth and YAP nuclear translocation. Matrix density, myosin contractility, integrin ligation and talin mechanical stability differently and nonlinearly regulate both force transmission and the transduction threshold. In all cases, coupling of talin unfolding dynamics to a theoretical clutch model quantitatively predicts cell response.

Forces driving epithelial wound healing

A fundamental feature of multicellular organisms is their ability to self-repair wounds through the movement of epithelial cells into the damaged area. This collective cellular movement is commonly attributed to a combination of cell crawling and “purse-string” contraction of a supracellular actomyosin ring. Here we show by direct experimental measurement that these two mechanisms are insufficient to explain force patterns observed during wound closure. At early stages of the process, leading actin protrusions generate traction forces that point away from the wound, showing that wound closure is initially driven by cell crawling. At later stages, we observed unanticipated patterns of traction forces pointing towards the wound. Such patterns have strong force components that are both radial and tangential to the wound. We show that these force components arise from tensions transmitted by a heterogeneous actomyosin ring to the underlying substrate through focal adhesions. The structural and mechanical organization reported here provides cells with a mechanism to close the wound by cooperatively compressing the underlying substrate.