Motor Function and Regulation of Myosin VII

描述（由申请人提供）：Myosin VII是多种Myosin超家族的成员，分布于视网膜和内耳等多种组织中。最有趣的发现是，肌球蛋白VIIa是三种人类感觉障碍的原因，Usher综合征1B（由色斑性视网膜炎引起感觉神经性耳聋和失明），DFNB2和DFNA11。然而，由于缺乏对肌球蛋白VIIa在分子水平上的功能的了解，对这些疾病的机制的理解受到了影响。本项目的目的是在分子水平上阐明肌凝蛋白VIIa的运动功能和调控。首先，我们将阐明肌凝蛋白via的运动特性。我们将使用两种方法来实现这一目标。首先，我们将分析与交叉桥循环的每个步骤耦合的atp酶循环的每个基本动力学步骤。分析确定了肌凝蛋白via发力状态的持续时间。其次，通过体外表面滑动实验研究myosin VIIa的肌动蛋白易位活性特征，特别强调单分子纳米技术的使用。每个步骤的大小和连续多个步骤的生产将通过使用光镊和单分子水平的纳米测量来确定。肌凝蛋白VIIa在肌动蛋白上的连续运动将通过最近开发的单分子成像系统进行可视化。利用这些技术，我们将确定肌凝蛋白VIIa在与肌动蛋白分离之前是否移动了多个步骤和步长。肌球蛋白via的调控机制尚不清楚。我们假设三个组成部分来解释肌凝蛋白via的调节。首先，肌凝蛋白VIIa的运动活性可能通过磷酸化调节。我们的初步结果表明，myosin VIIa可被多种蛋白激酶磷酸化。其次，钙与钙调蛋白轻链的结合直接调节肌凝蛋白via的运动活性。第三，myosin VIIa头间的相互作用可能在调控中发挥作用。本项目将在分子水平上阐明myosin VIIa的调控机制。人类感觉障碍的大量错义突变位于myosin VIIa的头部结构域，但迄今为止对这些突变对myosin VIIa功能的影响尚不清楚。该建议将在分子水平上阐明这些突变的功能缺陷。该项目将阐明myosin VIIa的功能和调控，从而为理解人类耳聋和失明感觉障碍的机制提供重要信息。

DESCRIPTION (provided by applicant): 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.