Mechanistic insights into the regulation of human myosin-7a.

Human myosin-7a (Myo7a) is an actin-based motor protein essential for vision and hearing. It plays critical roles in the development and functions of actin rich protrusions. Previous studies using Drosophila homolog showed that myosin-7a is a monomeric, high duty ratio motor and can move processively upon dimerization. However, characterization of full-length mammalian myosin-7a has been limited by the difficulty of expressing and purifying stable, intact protein. Here, we report the production of a complete human myosin-7a holoenzyme in insect cells and study its regulation by intra- and intermolecular mechanisms. We found that human myosin-7a utilizes regulatory light chain (RLC), calmodulin and calmodulin like protein 4 (CALML4) as the light chain subunits. CALML4 is recently discovered highly enriched in stereocilia and identified as a deafness candidate gene. We show that CALML4 does not bind to Ca2+ but plays a key role in regulating the dynamic binding of calmodulin to myosin-7a in response to Ca2+ signaling. The cochlea expresses two myosin-7a splicing isoforms differed by a short N-terminal extension. Using combined optical trapping, biochemistry, and in vitro motility assays, we show that the N-terminal extension greatly influences the enzymatic and mechanical behaviors of mammalian myosin-7a. We propose that the hair cell regulates its mechanosensitivity by adjusting the expression levels of the two myosin-7a isoforms. Finally, using single-molecule motility assays, we show that purified full-length myosin-7a alone does not move processively on actin in vitro, but in the presence of MyRIP, a known myosin-7a binding protein in neuroretina, it exhibits processive movements. Together, our results provide new molecular insight into how myosin-7a functions in hair cell stereocilia and neuroretina. The production of intact human myosin-7a protein enables future studies to understand the molecular details of human vision and hearing loss caused by myosin-7a defects.