Masters and Servants of the Force: Focal Adhesion Size Controls Recruitment of α-Smooth Muscle Actin to Stress Fibers

Mechanical stress is a crucial factor for modulating fibroblasts into differentiated myofibroblasts, which are characterized by expression of α-smooth muscle actin (α-SMA). Incorporation of α-SMA into stress fibers generates high contractile activity and drives the formation of “supermature” focal adhesions (FA) that are considerably longer (6–30 μm) compared with “classical” FAs (2–6 μm) of α-SMA-negative fibroblasts. We here show that in turn, supermature FAs control myofibroblast differentiation by communicating the level of extracellular matrix stress to the cytoskeleton. Culture on compliant silicone substrates reduces the size of supermature FAs to that of classical FAs and leads to a concomitant decrease of α-SMA expression. Incorporation of α-SMA into stress fibers requires the formation of FAs longer than 6 μm as demonstrated by plating myofibroblasts on arrays of adhesive islets with dimensions ranging from 1.5 × 2–20 μm and spacing between 2–6 μm, which are created on rigid culture surfaces by means of microcontact printing (μCP). Stretching 6 μm islets on flexible silicone membranes to 8 μm length induces stress fiber formation of α-SMA-EGFP transfected fibroblasts; this was not achieved by applying the same stretch (30%) to cells initially grown on 4 μm islets. By analyzing local deformations created in deformable micropatterned substrates by paxillin-EGFP transfected myofibroblasts, we determined a linear relationship between the size of supermature FAs and local force exertion; hence we determined the minimal tension at individual supermature FAs required for α-SMA recruitment.