Abstract
Cell mechanical activity generated from the interplay between the extracellular matrix (ECM) and the actin cytoskeleton is essential for the regulation of cell adhesion, spreading and migration during normal and cancer development. Keratins are the intermediate filament (IF) proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatic epithelial cell IFs are made solely of keratins 8/18 (K8/K18), hallmarks of all simple epithelia. Notably, our recent work on these epithelial cells has revealed a key regulatory function for K8/K18 IFs in adhesion/migration, through modulation of integrin interactions with ECM, actin adaptors and signaling molecules at focal adhesions. Here, using K8-knockdown rat H4 hepatoma cells and their K8/K18-containing counterparts seeded on fibronectin-coated substrata of different rigidities, we show that the K8/K18 IF-lacking cells lose their ability to spread and exhibit an altered actin fiber organization, upon seeding on a low-rigidity substratum. We also demonstrate a concomitant reduction in local cell stiffness at focal adhesions generated by fibronectin-coated microbeads attached to the dorsal cell surface. In addition, we find that this K8/K18 IF modulation of cell stiffness and actin fiber organization occurs through RhoA-ROCK signaling. Together, the results uncover a K8/K18 IF contribution to the cell stiffness-ECM rigidity interplay through a modulation of Rho-dependent actin organization and dynamics in simple epithelial cells.
Highlights
The ability of cells to sense and adapt to mechanical cues from the extracellular matrix (ECM) is crucial for several biological processes, including the involvement of mechanical force in dictating embryonic development [1]
Since the shape of adherent cells largely depends on an interplay between cytoskeleton and ECM [27], we first assessed to which extend a loss of the K8/K18 intermediate filament (IF) network in hepatic cells affects their shape in response to FN-coated polyacrylamide gel (FN-gel) rigidities that are representative of normal and fibrotic hepatic tissue [28,29,30]
By comparing shK8b versus H4ev cell adaptation to ECM-derived mechanical cues, using FN-gels of controlled rigidity and measuring cell stiffness with an optical tweezers, we find that a K8/K18 IF loss perturbs the mechanotransduction by: altering the cell mechanosensing, along with a marked actin fiber differential re-organization; interfering with the proper match between cell stiffness and ECM rigidity; and impairing the Rho-ROCK pathway controlling cell stiffness and actin dynamics
Summary
The ability of cells to sense and adapt to mechanical cues from the extracellular matrix (ECM) is crucial for several biological processes, including the involvement of mechanical force in dictating embryonic development [1]. Cell contractility and its associated internal stiffness can be assessed by measuring the force-induced displacement of fibronectin (FN)coated beads attached at FAs generated at the dorsal cell surface [7,8] Such measurements at the cellular level have established, for instance, that a de-polymerization of the actin cytoskeleton reduces cell stiffness, recognizing this cytoskeletal network as a prominent contributor of the cellular response to mechanical force applied at FAs [8,9]. The balance between internal stiffness and extracellular force exerted at FAs is maintained by modulating the fibrillar actin contractility [5,6,10], which occurs through activation of Rho and the effector ROCK, a regulator of the myosin light chain [11,12] Such cellgenerated Rho-dependent contractility points to a prominent actin cytoskeleton involvement in the interplay between cell stiffness and ECM rigidity
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