Regulation of Mammalian Class VI Myosin

描述（由申请人提供）：拟议项目的目标是阐明哺乳动物VI类肌球蛋白功能和调节的分子机制。我们的初步研究表明，Ca 2+和磷酸化的小G蛋白下游蛋白激酶调节肌球蛋白VI的运动活性。我们将首先检测肌球蛋白VI的特异性磷酸化是否发生在细胞中，然后研究Ca 2+和/或磷酸化介导的肌球蛋白VI运动活性调节的机制。我们和其他人最近的一项研究表明，VI类肌球蛋白是一种在肌动蛋白丝上长距离运动而不与肌动蛋白分离的进行性马达。然而，肌球蛋白VI沿沿着肌动蛋白丝运动的机制尚不清楚。我们将通过使用各种生物物理和电子显微镜技术来解决这个问题。显示肌球蛋白VI进行性运动的最佳方法是使用单分子分析。我们将采用两种技术，即，机械测量与光学陷阱纳米，和直接可视化的运动，全内反射（TIRF）显微镜。将通过观察附着于肌动蛋白丝上的肌球蛋白VI的珠子的运动来监测肌球蛋白VI在肌动蛋白上的旋转运动。机械循环过程中肌球蛋白VI的构象变化将通过测量肌球蛋白头部角度变化的单分子偏振TIRF显微镜进行研究。将通过X射线溶液散射监测肌球蛋白VI的总体结构变化（分辨率为0.1 nm）。肌动蛋白丝上双头肌球蛋白VI的结构将通过冷冻电子显微镜对肌球蛋白VI修饰的肌动蛋白丝进行三维图像重建来研究。我们还将确定负责肌球蛋白VI的持续合成性和反向性的结构基序，通过分析每个结构基序通过基因工程技术改变的变体的运动特性。为了实现这一目标，我们将利用重组DNA技术来生产工程化的肌球蛋白VI分子。假设对运动功能的独特性和/或调节至关重要的肌球蛋白VI分子的特定区域将被修饰和功能性表达。然后通过酶分析、生物物理分析和体外运动测定来分析运动功能，特别强调单分子测定系统。具体目标如下：1.明确肌球蛋白VI运动功能的调控机制; 2）明确肌球蛋白VI在ATP水解周期中的结构变化; 3）明确肌球蛋白VI沿沿着肌动蛋白丝运动的机制; 4）确定肌球蛋白VI方向性的分子决定因素。

DESCRIPTION (provided by applicant): The goal of the proposed project is to clarify the molecular mechanism of both function and regulation of mammalian class VI myosin. Our preliminary studies suggest that Ca 2+and phosphorylation by small G-protein down-stream protein kinases regulate the motor activity of myosin VI. We will first examine whether the specific phosphorylation of myosin VI occurs in cells, and then study the mechanism of Ca 2+ and/or phosphorylation mediated regulation of myosin VI motor activity. A recent study by others and us has revealed that class VI myosin is a processive motor that travels on actin filaments for a long distance without dissociating from actin. However, the mechanism by which myosin VI moves processively along actin filaments is not understood. We will address this problem by using various biophysical and electron microscopy techniques. The best approach to show the processive movement of myosin VI is the use of single molecule analysis. We will employ two techniques, i.e., mechanical measurement with optical trap nanometry, and direct visualization of the movement by total internal reflection (TIRF) microscopy. The rotational motion of myosin VI on actin will be monitored by visualizing the movement of beads attached to myosin VI on actin filament. The conformational changes of myosin VI during the mechanical cycle will be studied by single molecule polarization TIRF microscopy that measures the angular change of myosin head. The overall structural change of myosin VI with 0.1 nm resolution will be monitored by X-ray solution scattering. The structure of the two-headed myosin VI on actin filament will be studied by 3D image reconstitution of the myosin VI decorated actin filaments with cryo-electron microscopy. We will also determine the structural motifs responsible for the processivity and reverse directionality of myosin VI by analyzing the motor properties of the variants in which each structural motif is changed by genetic engineering technology. In order to achieve this goal, we will use recombinant DNA technology to produce engineered myosin VI molecules. Particular regions of the myosin VI molecule that are hypothesized to be critical for the uniqueness and/or regulation of motor function will be modified and functionally expressed. The motor function will then be analyzed by enzymatic analysis, biophysical analysis and in vitro motility assay, ,with a particular emphasis on the single molecule assay system. The itemized specific aims are: 1. To determine the regulatory mechanisms of myosin VI motor function; 2) To define the structural changes of myosin VI during the ATP hydrolysis cycle; 3) To define the mechanism by which myosin VI moves processively along actin filaments; 4) To identify the molecular determinant of the directionality of myosin VI.