High-Speed Nanometric Tracking of Myosin 5 with Interferometric Scattering Microscopy

Abstract

Myosin 5 is a molecular motor that moves along actin filaments by taking 74 nm steps. Although its enzymatic cycle is well understood, there is inconclusive and conflicting information on the motion that describes individual steps. Studies aimed at revealing the stepping mechanism have either reported periods of increased flexibility or proposed partitioning of the step into one or multiple sub-events. All resulting models involve a forward aiming power stroke followed by a Brownian search of the unbound head. We used interferometric scattering microscopy (iSCAT) to track the head of myosin 5 with simultaneous nanometer spatial and millisecond temporal precision. We observed a single, spatially constrained transient state of the detached head. Within individual traces, the position of the transient always occurred on the same side of actin filament but the sidedness could change when myosin molecule switched tracks. Simultaneous tracking of both heads revealed that myosin moves in a “spinning” hand-over-hand fashion, where both heads follow the same path and where torsional strain plays an important role. We also show that, in contradiction to the Brownian search hypothesis, the detached head reaches the desired binding site in a highly controlled manner. Improving the spatial precision to the sub-nm regime revealed a structural transition in the motor domain associated with the power stroke in the leading head, which may correspond to strain release. Taken together, our results demonstrate how the motor structure tightly controls its movement.