Keratin contribution to cellular mechanical stress response at focal adhesions as assayed by laser tweezers.

The ability of adherent cells to sense and adapt to a mechanical stress generated at focal adhesions (FAs) largely occurs through the integrin-mediated interaction between the cytoskeleton, namely actin microfilaments, and extracellular matrix elements, like fibronectin. Here we assessed the contribution of keratin 8 and 18 (K8/K18) intermediate filaments (IFs) in simple epithelial cells in response to a mechanical stress applied on integrins at FAs. To this end, we used monolayer cultures of K8-knockdown H4-II-E-C3 (shK8b1) rat hepatoma cells and their K8/K18-containing counterparts (H4ev). The stress was generated with a laser tweezers mediated force applied on a fibronectin-coated polystyrene bead attached to integrins alpha5/beta1 forming FAs. Measurement of the bead displacement allowed assessment of the viscoelastic response at FAs and the associated surface membrane stiffness. Notably, the loss of K8/K18 IFs in shK8b1 cells revealed an immediate reduction in bead displacements characteristic of a sudden increased in the FA elastic stiffness, incompatible with the K8/K18 IF intrinsic viscoelastic features, but in line with an induced perturbation of the mechanotransduction signals triggered at integrins. In addition, actin microfilament disruption, and to a lesser extent microtubule disruption, led to prominent decreases in the elastic stiffness of FAs, thus identifying actin-MFs and MTs as modulators of the time-dependent FA stiffening in both H4ev cells and shK8b1 cells, in response to mechanical stress. On technical ground, the laser tweezers offer a tool of choice to delineate the K8/K18 IF-mediated modulation of cytoskeletal versus signaling activities at FAs in epithelial cells in response to mechanical stress.