Myosin-10 Produces its Power-Stroke in Two Phases and Moves Processively along a Single Actin Filament under Low-Load

Abstract

Myosin-10 is an actin-activated ATPase that participates in essential intracellular processes such as filopodia formation/extension, phagocytosis, cell migration and mitotic spindle maintenance. The myosin-10 duty-cycle ratio - i.e. the fraction of time the motor remains tightly bound to actin during its total ATPase cycle - from previous biochemical studies are inconclusive, thus whether this myosin displays intermediate or high duty ratio is still under debate. To study this motor protein's mechano-chemical properties we have used a recombinant, truncated form of myosin-10 consisting of the first 940 amino acids, followed by a GCN4 leucine zipper motif to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules (∼87%) are dimeric with a head-to-head contour distance of ∼50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of 150 - 400 nm s−1. Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (∼13 s−1) is similar to the maximum ATPase activity (∼12 - 14 s−1) and, therefore, contributes to rate-limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load (∼0.5 pN) myosin-10 interacts intermittently with actin and produces a working stroke of ∼17 nm, composed of an initial 15 nm and subsequent 2 nm movement. At low optical trap loads, we observed staircase-like processive movements of myosin-10 interacting with the actin filament, consisting of up to six, ∼35 nm, steps per binding interaction. Here we describe the kinetics and mechanics of myosin-10, interrogating bulk and single molecule biophysical/biochemical properties to further our understanding of its ATP-driven, motor mechanism and how this relates to its cellular functions.