Mechano-responsiveness of fibrillar adhesions on stiffness-gradient gels

Abstract

Fibrillar adhesions are important structural and adhesive components in fibroblasts that are critical for fibronectin fibrillogenesis. While nascent and focal adhesions are known to respond to mechanical cues, the mechanoresponsive nature of fibrillar adhesions remains unclear. Here, we used ratiometric analysis of paired adhesion components to determine an appropriate fibrillar adhesion marker. We found that active 5{beta}1-integrin exhibits the most definitive fibrillar adhesion localisation compared to other proteins, such as tensin-1, reported to be in fibrillar adhesions. To elucidate the mechanoresponsiveness of fibrillar adhesions, we designed and fabricated thin polyacrylamide (PA) hydrogels, embedded with fluorescently labelled beads, with physiologically relevant stiffness gradients using a cost-effective and reproducible technique. We generated a correlation curve between bead density and hydrogel stiffness, thus allowing the use of bead density as a readout of stiffness, eliminating the need for specialised knowhow including atomic force microscopy (AFM). We find that stiffness promotes the growth of fibrillar adhesions in a tensin-dependent manner. Thus, the formation of these extracellular matrix-depositing structures is coupled to the mechanical parameters of the cell environment and may enable cells to fine-tune their matrix environment in response to alternating physical conditions.