Tension-Dependent Myosin Dynamics on Contractile Actomyosin Structures

Abstract

Dynamic cellular processes, such as cell migration and division, are powered by mechanical tension generated by the actomyosin cytoskeleton, where myosin molecular motors pull on actin filaments. To maintain proper mechanical tension, myosin dynamics is regulated by several factors, such as phosphorylation on the regulatory light chain. Mechanical tension is also proposed to regulate myosin dynamics. In vitro studies have shown that myosin detaches slower from F-actin under resisting load. However, it is unclear whether mechanical tension plays a role in regulating myosin dynamics in cells. In this work, we assess the role of mechanical tension in myosin turnover dynamics on stress fibers by conducting fluorescence recovery after photobleaching (FRAP) experiments. To perturb cellular tension, cells are treated with non-saturating levels of the Rho-kinase inhibitor Y27632, where the total traction force decreases while the overall actomyosin architecture is preserved. Consistent with past literature, myosin recovers much faster after photobleaching, which is thought to be due to an increase in diffusive myosin monomers. However, the recovery profile suggests a reaction-dominant process, suggesting that myosin dissociates faster due to decreased tension. Furthermore, super-resolution live-cell imaging revealed quasi-sarcomeric myosin bands on individual stress fibers recovering after photobleaching. Under normal levels of cellular tension, myosin always recovers on established myosin bands. In constrast, under reduced tension, myosin bands become more disordered and dynamic. This suggests that myosin turnover by monomers associating with or dissociating from an existing minifilament. The core of myosin bands is stable under tension, resulting in the immobile fraction in FRAP experiments. Upon tension reduction, these cores become mobile and can turn over. Together with experiments that directly modulates tension on stress fibers, such as stress fiber rupturing, our data sheds light on the role of mechanical tension regulating myosin dynamics.