| Abstract | Force interplay between the extracellular environment and the actin cytoskeleton at the surface membrane regulates several key biological processes, such as the ability of cells to sense and react to mechanical stimuli as they progress through differentiation during development. Mechanical cues largely result from external forces applied by deformation of the extracellular matrix (ECM) or internal forces generated through actin-myosin contraction, which in turn exerts a traction force on ECM. In both cases, the mechanical stimuli converge on integrins, which are transmembrane receptors composed of two subunits, α and β, that cluster within specialized focal adhesions (FAs) at the surface membrane and link the ECM and the actin cytoskeleton. Hepatocyte and H4-II-E-C3 (H4) hepatoma cell intermediate filaments (IFs) consist of the keratin 8/18 pair (K8/K18) only, and since the loss of one keratin leads to the degradation of its partner, these hepatic cells provide unique models to address K8/K18 functions in simple epithelial cells. Using cultured K8-knockdown H4-(shK8b) cells and their K8/K18-containing counterparts (H4ev), we assessed the contribution of K8/K18 in their response to mechanical stress generated either at the dorsal or ventral cell surface. In the first case, the stress was generated with a laser tweezer-mediated force applied on a fibronectin-coated polystyrene microbead attached to integrins on the surface, and the cell response was assessed by the bead displacement. Of particular interest, the loss of K8/K18 IFs in shK8b cells revealed an immediate reduction in bead displacements characteristic of a sudden increased in the FA elastic stiffness. In addition, the use of pharmacological inhibitors point to a key role for “novel” PKC as intermediary in the K8/K18 IFs contribution. In the second case, cells were seeded on fibronectin-coated polyacrylamide gel substrata of different rigidity to generate a variable mechanical stress at the basal interface. It involved the addition o f 0.03, 0.05 and 0.08 % bisacrylamide to 5 % acrylamide, which provided substrata exhibiting a Young modulus of 800, 1400 and 3000 Pa, respectively. This rigidity range mimicked the in vivo situation for tissues like liver, and was very much lower than the 3 GPa for a polystyrene substratum. Notably, shK8b cells exhibited a prominent spreading impairment for substratum rigidity below 1400 Pa. Moreover, as the substratum rigidity was decreased, a comparable reduction of FA components, like talin, was found in both H4ev and shK8b cells. In addition, the 3D arrangements of the actin cytoskeleton and the cytolinker plectin were differentially perturbed following the K8/K18 loss in shk8b cells. Overall, K8/K18 IFs appear to be important regulatory players in epithelial cell sensing and reaction to mechanical stress. Work supported by NSERC and CIHR |
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