No abstract

Cells assemble numerous types of actomyosin bundles that generate contractile forces for biological processes, such as cytokinesis and cell migration. One example of contractile bundles is a transverse arc that forms via actomyosin-driven condensation of actin filaments in the lamellipodia of migrating cells and exerts significant forces on the surrounding environments. Structural reorganization of a network into a bundle facilitated by actomyosin contractility is a physiologically relevant and biophysically interesting process. Nevertheless, it remains elusive how actin filaments are reoriented, buckled, and bundled as well as undergo tension buildup during the structural reorganization. In this study, using an agent-based computational model, we demonstrated how the interplay between the density of myosin motors and cross-linking proteins and the rigidity, initial orientation, and turnover of actin filaments regulates the morphological transformation of a cross-linked actomyosin network into a bundle and the buildup of tension occurring during the transformation.

Following injury and refractive surgery, corneal wound healing can initiate a protracted fibrotic response that interferes with ocular function. This fibrosis is related, in part, to the myofibroblast differentiation of corneal keratocytes in response to transforming growth factor beta 1 (TGF-β1). Previous studies have shown that changes in the mechanical properties of the extracellular matrix (ECM) can regulate this process, but the mechanotransductive pathways that govern stiffness-dependent changes in keratocyte differentiation remain unclear. Here, we used a polyacrylamide (PA) gel system to investigate how mechanosensing via focal adhesions (FAs) regulates the stiffness-dependent myofibroblast differentiation of primary corneal keratocytes treated with TGF-β1. Soft (1 kPa) and stiff (10 kPa) PA substrata were fabricated on glass coverslips, plated with corneal keratocytes, and cultured in defined serum free media with or without exogenous TGF-β1. In some experiments, an inhibitor of focal adhesion kinase (FAK) activation was also added to the media. Cells were fixed and stained for F-actin, as well as markers for myofibroblast differentiation (α-SMA), actomyosin contractility phosphorylated myosin light chain (pMLC), focal adhesions (vinculin), or Smad activity (pSmad3). We also used traction force microscopy (TFM) to quantify cellular traction stresses. Treatment with TGF-β1 elicited stiffness-dependent differences in the number, size, and subcellular distribution of FAs, but not in the nuclear localization of pSmad3. On stiff substrata, cells exhibited large FAs distributed throughout the entire cell body, while on soft gels, the FAs were smaller, fewer in number, and localized primarily to the distal tips of thin cellular extensions. Larger and increased numbers of FAs correlated with elevated traction stresses, increased levels of α-SMA immunofluorescence, and more prominent and broadly distributed pMLC staining. Inhibition of FAK disrupted stiffness-dependent differences in keratocyte contractility, FA patterning, and myofibroblast differentiation in the presence of TGF-β1. Taken together, these data suggest that signaling downstream of FAs has important implications for the stiffness-dependent myofibroblast differentiation of corneal keratocytes.

No abstract

Approaches to control the microstructure of hydrogels enable the control of cell–material interactions and the design of stimuli-responsive materials.