Live-Cell Single-Molecule Imaging of Human Myosin IIIA

Abstract

Stereocilia is an actin-based mechanosensing structure in the inner ear and plays an important role for auditory function. Myosin IIIA (MYO3A) accumulates in the tip of stereocilia and is important for structural and functional integrity of stereocilia. Loss of function of MYO3A causes progressive hearing loss. Since MYO3A is an actin-based motor protein, it has been thought that MYO3A functions as a cellular transporter of cargo molecules that create structural and functional base of stereocilia. A critical unanswered question is how MYO3A can transport its cargo molecules to constitute the structure of stereocilia. To address this question, we established live-cell single-molecule imaging of MYO3A in living cell. Deletion of kinase domain of MYO3A (MYO3AΔK) is previously shown to localize at the tip of filopodia in Hela cells. Here, we studied the dynamics of MYO3A movement at single-molecule level in cells for the first time: Kymograph analysis revealed continuous movement of MYO3AΔK towards filopodial tips. MYO3AΔK's average velocity on the filopodia in living Hela cells was ∼120 nm/s. This velocity is consistent with previous value measured by in-vitro F-actin gliding assays. In the same system single-molecule imaging of myosin X showed the velocity of ∼370 nm/s. Thus, the velocity of MYO3A was 3 times lower than that of MYO10. According to biochemical assay MYO3A is reported to have 20 times smaller rate of ATPase cycle compared to MYO10. These results suggest that in cells motor domain of MYO3A may have much faster ATPase cycle than that determined with the isolated protein.