Real-time single-cell response to stiffness

Abstract

Living cells adapt to the stiffness of their environment. However, cell response to stiffness is mainly thought to be initiated by the deformation of adhesion complexes under applied force. In order to determine whether cell response was triggered by stiffness or force, we have developed a unique method allowing us to tune, in real time, the effective stiffness experienced by a single living cell in a uniaxial traction geometry. In these conditions, the rate of traction force buildup dF∕dt was adapted to stiffness in less than 0.1 s. This integrated fast response was unambiguously triggered by stiffness, and not by force. It suggests that early cell response could be mechanical in nature. In fact, local force-dependent signaling through adhesion complexes could be triggered and coordinated by the instantaneous cell-scale adaptation of dF∕dt to stiffness. Remarkably, the effective stiffness method presented here can be implemented on any mechanical setup. Thus, beyond single-cell mechanosensing, this method should be useful to determine the role of rigidity in many fundamental phenomena such as morphogenesis and development.cell mechanics | mechanotransduction | dynamic stiffness control L iving cells are sensitive to their mechanical environment and adapt their activity to it. Many parameters such as forces, deformations, and the geometry and stiffness of the ECM were identified that could trigger cellular functions (1-3). In particular, it was shown that the stiffness of the ECM could influence cell spreading (4, 5), orientation (6), contractility (7,8), migration (9), and even differentiation (10, 11). These phenomena were mainly attributed to the ability of adhesion complexes to respond to applied forces (12, 13). These complexes, based on integrin transmembrane proteins, transmit forces from inside (cytoskeleton) to outside the cell (ECM) (14, 15) and were thus natural candidates for mechanosensing. On soft substrates, cell contractility could induce high substrate deformation and low generated forces. The adhesion complexes would then be weakly deformed, leading to a weak cell response. On stiff-less deformablesubstrates, cell contractility could lead to high stretching of mechanosensory molecules that would activate specific mechanochemical signaling pathways and enhance, in turn, contractility (9,16,17).Following the observation that nonmuscle myosins were needed for stem cells to feel matrix elasticity (11), we have recently investigated the specific role of actomyosin contractility in rigidity sensing. We found that cell response to the rigidity of its environment could reflect the adaptation of the actomyosin machinery to load (8). In this context, it is noteworthy that mechanosensing through adhesion-based signaling pathways and actomyosin-based sensitivity should lead to cell responses occurring at distinct characteristic time scales. At short times, an "instantaneous" actomyosin-dependent response could adapt the cytoskeletal tension, followed, at longer times, by the onset of the regulatory adhesio...

Cells as liquid motors: Mechanosensitivity emerges from collective dynamics of actomyosin cortex

“…3A) or alternatively, just as cells do when exhibiting a mechanosensitive behavior (17,19) (SI Text, S4.2). This spring-like behavior is supported by our previous report (14) that, when the external spring stiffness is instantaneously changed in experiments, cells adapt their rate of force buildup dF=dt to the new conditions within 0.1 s. This observation was repeated using an AFM-based technique (15). In ref.…”

“…(C) Instantaneous adaptation to a change of the microplate stiffness k. The red line shows stiffness imposed using a feedback loop. Black dots show the force measured (14). The blue line shows the 1D model prediction of force using the stiffness changes imposed in the experiment (red line) and the four parameters obtained in A and B without any additional adjustment.…”

“…S4). Without additional adjustment, they also lead to predictions of the dynamical adaptation of the loading rate of a cell between microplates of variable stiffness (14) (Fig. 2C) and the forcevelocity-length phase portrait of the experiments (Fig.…”

“…Cells are able to sense not only the local rigidity of the material with which they are in contact (12) but also, the one associated with distant cell substrate contacts. This ability has been demonstrated by tracking the amount of extra force needed to achieve a given displacement of microplates between which the cell is placed (13,14) (Fig. 1B), of an atomic force microscope (AFM) cantilever (15,16) or elastic micropillars (17).…”

Stiffening hydrogels to probe short- and long-term cellular responses to dynamic mechanics

“…In the last decade, matrix stiffness alone has been implicated in regulating cellular functions, such as contraction 3,4 , migration 5,6 , proliferation 7,8 and differentiation 9,10 . With this in mind, a variety of natural (for example, gelatin, collagen) and synthetic (for example, polyacrylamide) polymer systems were used in vitro to mimic the elasticity of native tissues, which varies significantly throughout the body (for example, brain: ~0.2-1 kPa (refs [11][12][13], muscle: ~10 kPa (ref.…”