Myosin VII, a member of the diverse myosin superfamily, is found distributed in a variety of tissues including retina and inner ear. The most intriguing finding is that myosin VIIa is responsible for three human sensory disorders, Usher syndrome 1B (causing sensorineural deafness and blindness due to retitis pigmentosa), DFNB2 and DFNA11. However, understanding of the mechanism of these diseases suffers from the lack of knowledge of the myosin VIIa function at a molecular level. The goal of the proposed project is to clarify the motor function and regulation of myosin VIIa at a molecular level. First, we will clarify the motor characteristics of myosin VIIa. We will address this objective using two approaches. First, we will analyze each elementary kinetic step of the ATPase cycle that couples with each step of the crossbridge cycle. The analysis determines the duration of the force generating state of myosin VIIa. Second, the characteristic of actin translocating activity of myosin VIIa will be studied by in vitro surface gliding assay with particular emphasis on the use of the single molecule nano-technology. Each step size and the production of successive multiple steps will be determined by use of optical tweezers and nanometry at the single molecule level. The continuous movement of myosin VIIa on actin will be visualized with the recently developed single molecule imaging system. Using these technologies, we will determine whether or not myosin VIIa moves multiple steps before dissociating from actin and the step size. Nothing is known about the regulation mechanism of myosin VIIa. We hypothesize three components to account for the regulation of myosin VIIa. First, the motor activity of myosin VIIa might be modulated by phosphorylation. Our preliminary results have indicated that myosin VIIa is phosphorylated by various protein kinases. Second, Ca binding to the calmodulin light chain directly regulates the motor activity of myosin VIIa. Third, the interaction between the heads of myosin VIIa may play a role in the regulation. The proposed project will clarify the regulatory mechanism of myosin VIIa at a molecular level. Significant numbers of missense mutations of the human sensory disorders are located in the head domain of myosin VIIa, but nothing is known about the effects of these mutations on myosin VIIa function to date. The proposal will clarify functional defects of these mutations at a molecular level. The proposed project will clarify the function and regulation of myosin VIIa, thus providing important information in understanding of the mechanism underlying the human sensory disorders of deafness and blindness.
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