Single Molecule Movement of Full-Length Myosin X

Abstract

Myosin X is an unconventional myosin that is critical for cargo transportation to filopodia, and is associated with the mechanism underlying filopodia formation and extension. We previously reported that myosin X-HMM is a processive motor with large ∼34 nm step size. Our myosin X-HMM dominantly shows the large step size on both single actin filament and fascin-actin bundle although a minor ∼18 nm step size can be seen on fascin-actin bundle [Sun et al. (2010) Nat. Struct. Mol. Biol., 17, 485-491]. On the other hands, Rock and his colleagues recently reported that their myosin X-HMM only showed ∼18 nm step size [Nagy et al. (2010) J. Biol. Chem., 285, 26608-26617; Kapitein et al. (2010) Biophys. J., 99, 2143-2152]. Thus, the actual step size of myosin X remains to be elucidated. To address the question of innate step size of myosin X molecules, we produced the full-length myosin X construct with a leucine zipper motif at the C-terminal end (M10FullLZ) to ensure the dimer formation. We first compared the localization of M10FullLZ with the wild type myosin X full-length (M10Full) in COS7 cells, and confirmed that M10FullLZ localized at the tip of filopodia, which is similar to M10Full. We next expressed M10FullLZ in Sf9 cells and purified for motility assay. The M10FullLZ could move actin filaments in PIP3 dependent manner by in vitro multi-molecule motility assay, consistent with the result of M10Full. We then analyzed the single molecular step size of M10FullLZ using a total internal reflection fluorescence microscope. The mean step size of M10FullLZ was the large ∼34 nm on single actin filaments. This result suggests that the innate step size of myosin X is ∼34 nm. The step size of M10FullLZ on actin bundles in filopodia is now underway.