Cell mechanical activity generated from the interplay between the extracellular matrix (ECM) and the actin cytoskeleton Deltarasin HCl is essential for the regulation of cell adhesion spreading and migration during normal and cancer development. we show that this K8/K18 IF-lacking cells drop 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. Introduction The ability Deltarasin HCl 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 Deltarasin HCl dictating embryonic development . For instance embryonic stem cells progressively stiffen as they undergo differentiation and tune their stiffness to the rigidity variation of the underlying ECM . In a similar way there is compelling evidence for the involvement of increased ECM rigidity in promoting the emergence of primary tumors and the subsequent metastatic migration of escaping cells . Moreover aggressive tumorigenic cells in suspension where they are impartial from ECM conversation are more compliant than less aggressive cells which in turn are more compliant than healthy cells ; still tumorigenic cells seeded on a rigid Deltarasin HCl ECM substratum exhibit increased contractility . Such differences in cell behavior in link with changes in ECM rigidity highlight how important it is for cells to adapt to mechanical cues in order to counterbalance ECM constraints. An ECM-derived stress is perceived and integrated intracellularly through the participation of integrin receptors acting as mechanotransducers interfacing with signaling cascades and actin cytoskeleton at focal adhesions (FAs) to elicit cellular responses such as cell migration and contractility  . Experimentally 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  . 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  . At the molecular level the balance between internal stiffness and extracellular force exerted at FAs is usually maintained by modulating the fibrillar actin contractility    which occurs through activation of Rho and the effector ROCK a regulator of the myosin light chain  . Such Deltarasin HCl cell-generated Rho-dependent contractility points to a prominent actin cytoskeleton involvement in the interplay between cell stiffness and ECM rigidity. Keratins (Ks) the intermediate filament (IF) proteins of epithelial cells constitute the largest family of cytoskeletal proteins and are grouped into type I (K9-28) and type II (K1-K8 and K71-K80) subfamilies . Keratin IFs are obligate heteropolymers that include at Rabbit Polyclonal to MRPL54. least one Deltarasin HCl type I and one type II keratin and are coordinately expressed as specific pairs in a cell lineage and differentiation manner. IFs from all simple epithelial cells contain K8/K18 and most possess 2-3 other keratins as well  . Notably K8 and K18 are the ancestral genes for the multiple specialized Type II and Type I keratin classes respectively and constitute the first cytoplasmic IF genes expressed in the embryo at the time of stem cell differentiation along the different cell lineages  . With regard to cancer there is accumulating evidence showing for instance that persistence of K8/K18 IFs is usually a hallmark of invasive squamous cell carcinoma where such perturbed K8/K18 expression appears to contribute to cell invasiveness through an actin-dependent motility . In addition point mutations in K8 and K18 genes lead to IF disorganization and predispose to liver cirrhosis  and in turn cirrhosis reflects increasing hepatic tissue stiffness a ECM-linked mechanical alteration often.