Abstract
Myosin VI is a unique motor protein that moves towards the minus end of actin filaments, distinguishing it from all other myosin family members. This reverse directionality enables myosin VI to perform essential cellular functions, including endocytosis vesicle trafficking, Golgi maintenance, and stereocilia formation in inner ear hair cells. Mutations in the myosin VI gene are associated with hereditary hearing loss, but the mechanisms underlying these defects remain poorly understood. In this study, I employed single-molecule total internal reflection fluorescence microscopy (TIRFM) to investigate the motility of D179Y mutant myosin VI, which causes deafness, in living Snell’s waltzer fibroblast cells. I found that The D179Y mutation causes significantly slower movement, longer dwell times, and shorter run lengths compared to wild-type myosin VI. I also further found that omecamtiv mecarbil (OM), a cardiac myosin activator, rescues the motility of the D179Y mutant by restoring processive movement. At 10 nM OM, a 3-fold increase in stepping rate was observed compared to the untreated mutant. Interestingly, OM caused a slight slowing of wild-type (WT) myosin VI motility, consistent with its allosteric modulation of the myosin kinetic cycle. Collectively, my findings provide the first direct evidence that a deafness-causing myosin VI mutation D179Y disrupt motility of myosin VI in living cells and can be rescued by a small molecule therapeutic, which is OM, in living cells, establishing a proof of principle for therapies for myosin VI-related hearing loss.