Myosin Steps Symmetrically along Actin

Abstract

Myosin 5a is a homodimer belonging to the family of processive molecular motors that transform chemical energy into directional motion along the cytoskeleton network. Despite a well understood kinetic cycle, the type of hand-over-hand motion describing each step remains unknown due to the intrinsic short lifetime of the unbound head state. Here, we differentially label the two motor domains of myosin 5a, one with a fluorescent quantum dot and the other with a small gold nanoparticle and simultaneously trace the three dimensional motion of each domain by combining single molecule fluorescence with interferometric scattering microscopy (iSCAT).Within individual trajectories, we observed an intermediate state for each labelled unbound head that is located to one and the same side of the actin filament from which myosin repeatedly sampled the desired actin site until strong binding occurred. Analysis over several hundred molecules showed no preference in the relative position of the transient state with respect to actin. The observation that the transient state orientation can change for the same molecule by binding to another actin filament shows that the directionality of the transient is not an intrinsic property of the motor. Instead the decision is made upon binding to actin, with both cases equally probable. Our work provides critical insight on the highly efficient mechano-chemical cycle of myosin 5a and provides strong evidence for a symmetric hand-over-hand model in which the trailing head swings by the leading head on the same side repeatedly.