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...

An electrostatic force microscope (EFM) and a Kelvin probe are used to characterize the charges embedded in thin anodic alumina layers of thickness ranging from 100 to 400 nm. Introducing a method for obtaining self-supported alumina layers, we exhibit the presence of positive charges at the metal/oxide interface of anodic alumina layers. These positive charges, together with the negative charges present at the surface of the anodic layer, induce a true polarization of the layer. The magnitude of this polarization depends on the conditions of preparation of the layers and can be well controlled. As a second step, we show the influence of this polarization on charge injection in these layers with EFM: charges of both signs may be injected in unpolarized layers whereas one cannot inject negative charges in polarized layers, which thus exhibit a diode-like behavior.

We shall demonstrate in this article that characterization of the charge distribution by electrostatic force microscopy is not straightforward: we will stress the important role played by the electrostatic images of the scanned charge distribution in the tip and by the operating mode in the formation of extra features in images obtained with this instrument. To illustrate this, we will describe two models that correspond to the scanning of small and extended charge distributions. These models will be compared with experimental images.