Elucidating the molecular determinants of force transmission in myosin ensembles using QCM-D.

Abstract

Myosin II motors collectively produce force to facilitate muscle contraction via an ATP-mediated mechanochemical process, causing the motors to step along actin filaments. Such myosin II ensembles have been shown to display environment-based mechanosensitive abilities, where ensemble motor duty ratio can change in response to external signals, including force and local rigidity. However, at the single-molecule level, myosin II is non-processive; as such, it is becoming increasingly clear that myosin II motor ensembles do not behave as the sum of their single molecule properties. Interrogation of myosin motors within cytoskeletal filament networks in vitro is needed to understand how motors that crosslink multiple filaments know whether to be in a low duty ratio, non-processive mode as an individual molecule or in a high duty ratio, processive mode within ensembles. A quartz crystal microbalance with dissipation monitoring (QCM-D) is a powerful biophysical tool that can measure the kinetics and viscoelastic properties of protein complexes, films, and surface interactions. Here, we adapt in vitro cytoskeleton reconstitution assays to QCM-D experiments to investigate the assembly kinetics and viscoelastic properties of actomyosin bundles consisting of multiple myosin II motors acting between actin filaments. Actively monitoring the binding kinetics and stiffness of such bundles in real-time with the QCM-D as each element of the bundle assembles into the higher order actomyosin architecture will provide insight into the molecular-level drivers of mechanosensation and communication within the local cytoskeletal environment.