Direct Observation of Nanometer-Sized Steps of Single Myosin VI Molecules in Living Cells

TL;DR

Researchers directly observed the tiny steps of myosin VI motor proteins in living cells, revealing how they move and function.

Contribution

The study directly measured nanometer-sized steps of myosin VI in living cells using advanced labeling and microscopy techniques.

Findings

Wild-type myosin VI steps were larger than expected based on its lever arm structure.

A mutation in the ATP-binding pocket caused longer dwell times, slower movement, and shorter run lengths.

The ATP-binding pocket is critical for myosin VI motility.

Abstract

Living cells undergo dynamic biological processes. For
example,
motor proteins transport cargos by taking nanometer-sized steps. However,
it is challenging to measure nanometer-sized steps in living cells.
Using cell-permeable, extremely bright, and photostable deuterium
congeners of tetramethyl­(silicon)­rhodamine (SiR-d12) connected chloroalkane
linker to label single HaloTag-fused myosin VI in living cells and
total internal reflection fluorescence microscopy (TIRFM), we measured
nanometer-sized steps of single myosin VI in living cells. The measured
step size of wild-type myosin VI was larger than that predicted from
its short-lever arms. Furthermore, myosin VI harboring a mutation
in the ATP-binding pocket exhibited longer dwell times between steps,
reduced velocity, and shorter run lengths than wild-type myosin VI,
underscoring the critical role of the ATP-binding pocket in motility.
Therefore, our direct measurements of nanometer-sized steps of single
motor proteins in living cells provide mechanistic insights into the
dynamics and biological processes of motor proteins in living cells.

Figures