In Vitro Dynamics of Kinesin 1 and Myosin VI

描述（由申请人提供）：分子马达——运动蛋白和肌球蛋白——在健康细胞的组织、运动和信号传导的维持和发展中起着至关重要的作用。中枢神经系统紊乱，如阿尔茨海默病和某些类型的癌症，都是由分子马达出错引起的。然而，关于运动蛋白-1是如何运动的，有几个基本的问题。（驱动蛋白-1是驱动蛋白的“旗手”。）肌凝蛋白VI是如何移动的也是很大程度上未知的。为了回答这些问题，我们将在体外的单分子水平上研究马达。我们将应用一些单分子工具，包括在本提案的前4年开发的一些工具。我们将使用FIONA（一纳米精度荧光成像），DOPI（离焦取向和位置成像）和SHREC（单分子高分辨率共定位）。运动蛋白部分的主题是：运动是如何受到每个运动蛋白部分的影响的，包括头部、盘绕线圈和尾部？肌凝蛋白VI部分的主题是：这么小的马达是如何迈出这么大的一步的？我们将展示未发表的数据，这些数据表明，运动蛋白在跑步过程中受到两个头部的束缚，这使得脱落的可能性非常低。我们还将提出，在驱动蛋白-1的盘绕区域中，大的非螺旋区域允许这个马达以“不对称的方式”行走，允许货物指向前方。最后，我们将提出全长运动蛋白需要“暂停”，因为尾部区域折叠并与头部区域和可能的微管相互作用。关于肌凝蛋白VI，我们有大量的初步数据。这将表明，在动力冲程中，头部经历了180度的摆动。此外，我们建议杠杆臂中的3-螺旋基序展开并创建一个前所未有的24纳米延伸，这使得电机可以采取36纳米的步骤。分子马达的作用是移动和组织细胞内的细胞器。马达内部的问题会导致脑癌、阿尔茨海默病等。我们试图了解运动蛋白和肌球蛋白VI的基本工作原理，这两个重要的马达。

DESCRIPTION (provided by applicant): Molecular motors--kinesin and myosins--play a crucial role in the maintenance and development of the organization, motility, and signaling of healthy cells. Central nervous-system disorders, such as Alzheimer's disease and certain types of cancer, all arise from molecular motors gone awry. There are, however, several fundamental questions about how kinesin-1 moves. (Kinesin-1 is the "standard-bearer" of kinesins.) Also largely unknown is how Myosin VI moves. To answer these questions we will address the motors at the in vitro, single molecule level. We will apply a number of single molecule tools, including some that have been developed in the first 4 years of this proposal. We will use FIONA (Fluorescence Imaging with One Nanometer Accuracy), DOPI (Defocused Orientation and Position Imaging), and SHREC (Single Molecule High REsolution Colocalization). The theme of the kinesin part is: how is motility affected by each of kinesin's parts, including the head, the coiled-coil, and the tail? The theme of the myosin VI part is: how does such a small motor take such a large step? We will present unpublished data which suggests that kinesin is bound by both heads during a run, making the probability of falling off very low. We will also suggest that the large non-helical region in kinesin-1's coiled-coiled region allows this motor to walk in an "asymmetric fashion," allowing the cargo to point forward. Finally, we will suggest that full-length kinesin takes "pauses" due to the tail-region folding over and interacting with the head region and possibly the microtubule. With regards to myosin VI, we have an enormous amount of preliminary data. It will suggest that the head undergoes a 180¿ swing during the powerstroke. Furthermore, we suggest that a 3-helix motif in the lever arm uncoils and creates an unprecedented 24 nm extension, which allows the motor to take a 36 nm step. PUBLIC HEALTH RELEVANCE Molecular motors have the job of moving and organizing organelles within a cell. Problems within the motors cause brain cancer, Alzheimer's disease, etc. We seek to understand the basic workings of kinesins and myosin VI, two important motors.