Many biological motor molecules move within cells using stepsizes predictable from their structures. Myosin VI, however, has much larger and more broadly distributed stepsizes than those predicted from its short lever arms. We explain the discrepancy by monitoring Qdots and gold nanoparticles attached to the myosin-VI motor domains using high-sensitivity nanoimaging. The large stepsizes were attributed to an extended and relatively rigid lever arm; their variability to two stepsizes, one large (72 nm) and one small (44 nm). These results suggest that there exist two tilt angles during myosin-VI stepping, which correspond to the pre- and postpowerstroke states and regulate the leading head. The large steps are consistent with the previously reported hand-over-hand mechanism, while the small steps follow an inchworm-like mechanism and increase in frequency with ADP. Switching between these two mechanisms in a strain-sensitive, ADP-dependent manner allows myosin VI to fulfill its multiple cellular tasks including vesicle transport and membrane anchoring.

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transition state of the force dependent step) to be 12 nm by a fit of the force dependence of actin attachment times in the presence of 50 micromolar ATP. In this study, we examine the force dependence of a myo1b truncation mutant that contains only the IQ motif closest to the motor domain. Although the unloaded biochemical kinetics of this mutant are nearly identical to the 5 IQ construct, we found the actin detachment rate to be substantially less force sensitive. These experiments suggest that the length of the regulatory domain modulates force sensitivity.

VIUnlike other molecular motors, myosin VI has much larger and broadly distributed step sizes than those predicted from its structure. Here, this discrepancy was consistently elucidated by highly sensitive single molecule imaging technique. The large step sizes and its variability were attributed to an extended rigid lever arm and two distinct tilt angles which causes newly found large and small step size.Myosin VI / Lever arm hypothesis / Single molecule / TIRF microcopy / Dark field microscopy