Motor Function and Regulation of Myosin VII

说明(申请人提供)：肌球蛋白VII是肌球蛋白超家族的一员，广泛分布于视网膜和内耳等多种组织中。最耐人寻味的发现是，肌球蛋白VIIa导致了三种人类感觉障碍，即Usher综合征1B(因退行性色素变性导致感觉神经性耳聋和失明)、DFNB2和DFNA11。然而，对这些疾病的机制的了解受累于在分子水平上缺乏对肌球蛋白VIIa功能的了解。拟议项目的目标是在分子水平上阐明肌球蛋白VIIa的运动功能和调控。首先，我们将阐明肌球蛋白VIIa的运动特征。我们将使用两种方法来实现这一目标。首先，我们将分析ATPase循环的每个基本动力学步骤，这些步骤与CrosBridge循环的每个步骤相耦合。该分析确定了肌球蛋白VIIa的力产生状态的持续时间。其次，将通过体外表面滑动实验来研究肌球蛋白VIIa的肌动蛋白转运活性的特征，重点是单分子纳米技术的应用。每一步的大小和连续多步的生产将通过使用光学镊子和单分子水平的纳米测量来确定。肌球蛋白VIIa在肌动蛋白上的连续运动将用最近开发的单分子成像系统来可视化。使用这些技术，我们将确定肌球蛋白VIIa在与肌动蛋白分离之前是否移动了多个步骤，并确定了步长。肌球蛋白VIIa的调节机制尚不清楚。我们假设有三种成分可以解释肌球蛋白VIIa的调节。首先，肌球蛋白VIIa的运动活性可能受磷酸化的调节。我们的初步结果表明，肌球蛋白VIIa被各种蛋白激酶磷酸化。第二，钙离子与钙调蛋白轻链的结合直接调节肌球蛋白VIIa的运动活性。第三，肌球蛋白VIIa头部之间的相互作用可能在调节中发挥作用。拟议的项目将在分子水平上阐明肌球蛋白VIIa的调控机制。相当数量的人类感觉障碍错义突变位于肌球蛋白VIIa的头部区域，但到目前为止，这些突变对肌球蛋白VIIa功能的影响尚不清楚。该提案将在分子水平上澄清这些突变的功能缺陷。拟议的项目将阐明肌球蛋白VIIa的功能和调节，从而为理解人类耳聋和失明的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.