High Speed Imaging For Myosin VI

Abstract

Myosin-family molecular motors have been a subject of extensive research recently. While there is a general consensus on how do these motor molecules perform their function, myosin VI presents a serious challenge to a conventional view. It has been reported that myosin VI moves processively in a hand-over-hand manner toward the minus end of actin filament, opposite to other classes of myosin. The head takes large steps (50 - 70 nm) relative to its short lever arms (7.2 nm), and frequently moves backward with half the size of a forward step. Using a video rate FIONA technique, we observed the above features under the several ATP concentrations (10 uM ATP, ∼67±19 nm, N=206; 100 uM ATP, ∼73±13 nm, N=362; 1000 uM ATP, ∼73±19 nm, N=105). The back-steps frequently occur (∼8%), and its size is half of the forward step (42 ±14 nm, N=57). The mystifying observation of a large step size and its large distribution has been explained in terms of the proximal tail domain to unwind which allows the myosin VI molecule to stretch out. However, this hypothesis does not support the exerted mechanical force, which was measured in myosin VI to be 2 pN without slowing. We believe that the major action takes place during the rapid stepping transition (<30 msec) between a quick release of a rear head from the actin molecule and a strong rebinding event. To address all the above questions, we are currently working on the direct observation of the one head dynamics using a dark-field imaging microscopy with much-improved microsecond time resolution.