Regulation of Mammalian Class VI Myosin

描述(申请人提供)：该项目的目标是阐明哺乳动物第VI类肌球蛋白的功能和调控的分子机制。我们的初步研究表明，钙和小G蛋白下游蛋白激酶的磷酸化调节肌球蛋白VI的运动活性。我们将首先检测细胞中是否发生肌球蛋白VI的特异性磷酸化，然后研究钙和/或磷酸化介导的肌球蛋白VI运动活性的调节机制。最近，其他人和我们的一项研究表明，VI类肌球蛋白是一种前进的马达，它依靠肌动蛋白细丝进行长距离旅行，而不会与肌动蛋白解离。然而，肌球蛋白VI沿着肌动蛋白细丝连续运动的机制尚不清楚。我们将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进行性运动的最好方法是使用单分子分析。我们将使用两种技术，即利用光学陷阱纳米技术进行机械测量，以及通过全内反射(TIRF)显微镜直接显示运动。肌球蛋白VI在肌动蛋白上的旋转运动将通过观察附着在肌球蛋白VI上的微珠在肌动蛋白细丝上的运动来监测。肌球蛋白VI在机械循环过程中的构象变化将通过单分子极化TIRF显微镜来研究，该显微镜测量肌球蛋白头部的角度变化。肌球蛋白VI在0.1 nm分辨率下的整体结构变化将通过X射线溶液散射进行监测。通过冷冻电子显微镜对肌球蛋白VI修饰的肌动蛋白细丝进行三维图像重建，研究肌球蛋白VI在肌动蛋白细丝上的结构。我们还将通过分析每个结构基序被基因工程技术改变的变体的马达特性来确定与肌球蛋白VI的加工性和反向有关的结构基序。为了实现这一目标，我们将使用重组DNA技术来生产工程肌球蛋白VI分子。肌球蛋白VI分子中被认为对运动功能的独特性和/或调节至关重要的特定区域将被修饰并在功能上表达。然后将通过酶分析、生物物理分析和体外运动分析来分析运动功能，特别是单分子检测系统。具体目标是：1.确定肌球蛋白VI运动功能的调节机制；2)确定肌球蛋白VI在ATP水解循环中的结构变化；3)确定肌球蛋白VI沿肌动蛋白细丝连续运动的机制；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.