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

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.