Direct Single Molecule Observations of the Unique Mechanical State of Human Myosin-6

Abstract

Myosin-6 is a molecular motor that translocates and generates force towards the pointed-end of the actin filament, and is important in a variety of cellular processes including the maintenance of the Golgi complex, exocytosis and cell migration. Myosin-6 has direct relevance to diseases such as cancer, cardiomyopathy, deafness, and neurodegenerative diseases and has thus been a focus of many investigations. Previous in vitro mechanical studies have shown that forced dimers of myosin-6 have a large step-size in comparison to its rather short lever arm, as well as a broad step-size distribution. Furthermore, a recent study has shown evidence that myosin-6 moves via both “hand-over-hand” and “inchworm-like” mechanisms. To elucidate this dualistic mechanism of myosin-6 motility we have generated a human myosin-6 (MYO6) heavy meromyosin-like construct (amino acids1-1021), with a N-terminal AviTag, a C-terminal GCN4 leucine zipper to force dimerization of two heavy-chains, a GFP moiety for fluorescence imaging and FLAG-tag for affinity purification. To examine acto-myosin-6-HMM interactions at both high spatial and temporal resolutions, we utilized negative-stained electron microscopy, fluorescence microscopy and interferometric scattering microscopy (iSCAT) assays. We present here direct evidence that the forced dimers of human myosin-6 dwell in an actin filament bound state where the two heads are directly next to each other and that the possible explanation of this mechanical state may be due to the inchworm-like mechanism. Further studies are underway to examine these dwell states, as well as the stepping mechanism, in greater detail. This work was supported by the Intramural Research Program of the NHLBI (to J.R.S.) and the NIDCD (to T.B.F.), and the ERC NanoScope (to P.K.).