The Tail Domain of Myosin-VI Ensures the Directed Processive Movement

Abstract

Myosin-VI is a motor protein that plays an important role in a large variety of cellular events such as vesicle transport and the anchoring of actin bundles to the plasma membrane. Myosin-VI is thought to move processively as a dimer along an actin filament in a hand-over-hand fashion with stepsizes similar to myosin-V.Recently we showed by using the FIONA method that myosin-VI contains two forward step types, a large forward step (72 nm) and a small forward one (44 nm), and one backward step type (−44 nm). To establish a new stepping model that includes these findings, we performed single-molecule high-resolution colocalization (SHREC), an advanced multi-color FIONA technique that involves labeling the two heads with different colored Qdots. This led to a model where for large steps, myosin-VI heads take a state where they are distant when both are bound; while for small and backward steps, the heads take a state where they are adjacent. This model, however, fails to consider the relationship between the head and tail domain during step generation, which has yet to be explored.To clarify this relationship, we here performed SHREC measurements of myosin-VI labeled at its head and tail domains with different colored Qdots at various nucleotide concentrations. These studies indicate that the lever arm swings forward following small step in < 100ms (within our frame rate) and that it does not swing backward during a backward step. These results suggest that during the adjacent heads binding state, the lever arm is directed forward. This prohibits successive backward steps, which in turns aids in ensuring directed myosin-VI processive movement.