Changes in tissue and organ stiffness occur during development and are frequently symptoms of disease. Many cell types respond to the stiffness of substrates and neighboring cells in vitro and most cell types increase adherent area on stiffer substrates that are coated with ligands for integrins or cadherins. In vivo cells engage their extracellular matrix (ECM) by multiple mechanosensitive adhesion complexes and other surface receptors that potentially modify the mechanical signals transduced at the cell/ECM interface. Here we show that hyaluronic acid (also called hyaluronan or HA), a soft polymeric glycosaminoglycan matrix component prominent in embryonic tissue and upregulated during multiple pathologic states, augments or overrides mechanical signaling by some classes of integrins to produce a cellular phenotype otherwise observed only on very rigid substrates. The spread morphology of cells on soft HA-fibronectin coated substrates, characterized by formation of large actin bundles resembling stress fibers and large focal adhesions resembles that of cells on rigid substrates, but is activated by different signals and does not require or cause activation of the transcriptional regulator YAP. The fact that HA production is tightly regulated during development and injury and frequently upregulated in cancers characterized by uncontrolled growth and cell movement suggests that the interaction of signaling between HA receptors and specific integrins might be an important element in mechanical control of development and homeostasis.

Summary Actin turnover is the central driving force underlying lamellipodial motility. The molecular components involved are largely known, and their properties have been studied extensively in vitro. However, a comprehensive picture of actin turnover in vivo is still missing. We focus on fragments from fish epithelial keratocytes, which are essentially stand-alone motile lamellipodia. The geometric simplicity of fragments and the absence of additional actin structures allow us to characterize the spatiotemporal lamellipodial actin organization with unprecedented detail. We use fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and extraction experiments to show that about two thirds of the lamellipodial actin diffuses in the cytoplasm with nearly uniform density, while the rest forms the treadmilling polymer network. Roughly a quarter of the diffusible actin pool is in filamentous form as diffusing oligomers, indicating that severing and debranching are important steps in the disassembly process generating oligomers as intermediates. The remaining diffusible actin concentration is orders of magnitude higher than the in vitro actin monomer concentration required to support the observed polymerization rates, implying that the majority of monomers are transiently kept in a nonpolymerizable ‘reserve’ pool. The actin network disassembles and reassembles throughout the lamellipodium within seconds, so the lamellipodial network turnover is local. The diffusible actin transport, on the other hand, is global: actin subunits typically diffuse across the entire lamellipodium before reassembling into the network. This combination of local network turnover and global transport of dissociated subunits through the cytoplasm makes actin transport robust, yet rapidly adaptable and amenable to regulation.

Cells respond to both their chemical environment and to microenvironmental stiffness by the process of mechanotransduction. The mechanisms by which cells monitor and respond to the mechanical properties of their environment are largely unknown. Cellular response to the stiffness of the substrate is highly cell type specific and depends on the chemical composition of the substrate and therefore the type of adhesion receptors that engage it. Nearly all studies of mechanobiology in vitro employ substrates coated with protein or peptide ligands for integrins, but the native extracellular matrix (ECM) is highly enriched with glycosaminoglycans and proteoglycans that can alter cell adhesion and signaling though integrins. Hyaluronic acid (HA) is one of the major ECM components that helps maintains the viscoelasticity of connective tissues, controls tissue hydration, and organizes the supramolecular assembly of proteoglycans. In this study we investigate the role of HA together with integrin ligands in promoting hepatocellular carcinoma cell (Huh7) spreading on very soft substrates (300 Pa), resulting in morphology and motility similar to that which these cells develop only on stiff substrates (30 kPa/glass) in the absence of HA. In particular, we test the hypothesis that cell interaction with HA leads to activation of the PI3K/Akt signaling pathway, which in turn promotes actin remodeling to facilitate cell spreading without requiring high contractile forces that are generated on stiff substrates. Inhibition of polyphosphoinositide turnover whether by two different PI3kinase inhibitors or by a cell-permeant polyphosphoinositide-binding peptide causes both Huh7 cells and murine fibroblasts to decrease spreading and detach whereas cells on stiffer substrates show almost no response. Traction force microscopy (TFM) shows that the cell maintains a very low total strain energy and net contractile moment on HA substrates as compared to stiff 30 kPa substrate even though cells on both substrates have large spread areas, extensive focal adhesions, and actin bundles (generally called stress fibers). Measurements of cell membrane tension by lipid tether pulling show a similar level of membrane tension on HA substrate as on stiff substrates. These results suggest that simultaneous signaling stimulated by HA and an integrin ligand can generate PI3K-dependent signals to the cytoskeleton that mimic those generated by high cellular tension, to produce increased actin and focal adhesion assembly and large spread areas.

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Models based on statistical physics have recently appeared in the literature as an attempt to elucidate the complexities of living cell adhesion. We propose here that some basic principles of cell/substrate adhesion can be investigated using a shear-lag type mechanical model classically used in the field of composite material science. The shear stress at a given time along an adhesion site is calculated, thereby providing a "snapshot view" of the stress present along the cell/substrate interface as a result of the force exerted by internal actin bundles. The latter, also called stress fibers, are bundles of filaments in which tension is induced through myosin II molecular motors. The shape of such snapshot stress profiles suggests a likely mechanism for the biochemical feedback activity leading to a growth of the adhesion region. The role of various material and geometrical characteristics of the adhesion region is predictable in principle, using the proposed mechanical model as a guide.Live cells react to mechanical signals by producing an array of biochemical processes that in turn can generate mechanical changes in the cells, consecutively leading to additional biochemical effects. This area of biophysics is currently the subject of extensive research, [1][2][3][4][5][6][7][8][9][10][11][12][13][14] and general reviews of the field have recently appeared. [15,16] One important facet of this intricate dynamic biochemical-mechanical coupling is the ability of a cell to transfer (and receive) mechanical signals-stress or strain-via the actin bundles to (and from) an extracellular matrix (ECM, or simply "substrate"). It has become evident that the regulation of cellular behavior is a function of the extent and quality of the contact between the cell and the underlying substrate. Interestingly, the issue of stress transfer and interfacial adhesion between strong/stiff fibers and a surrounding softer matrix (a polymer in most cases) has been, and still is, the focus of enormous interest in the field of composite materials science. Indeed, most physical properties of fibrous composites are a direct function of the degree of interfacial adhesion between the fiber and the matrix, which itself depends on the geometrical and chemical characteristics of the contact area. The fascinating geometrical and mechanical similarities between the two areas (cell adhesion and composite materials mechanics) are the thrust behind the present work. The process of adhesion between a live cell and a substrate is a complex phenomenon composed of several steps. At first, transmembrane proteins from the integrin family bind to the substrate to form initial clusters; then, upon stabilization, these clusters lead to nascent 2D adhesive sites (called focal complexes) that grow to form larger, elongated adhesive entities termed focal adhesions (FAs). FAs are complex multimolecular assemblies linked on one side to the ECM via membrane-bound receptors, and on the other side to the termini of actin fibers within the cell cytoskeleton (Fig. 1). FA...