In Vitro Dynamics of Kinesin 1 and Myosin VI

描述（由申请人提供）：分子马达——驱动蛋白和肌球蛋白——在健康细胞的组织、运动和信号传导的维持和发展中发挥着至关重要的作用。中枢神经系统疾病，例如阿尔茨海默病和某些类型的癌症，都是由分子马达出了问题引起的。然而，关于 kinesin-1 如何移动还存在几个基本问​​题。 （Kinesin-1 是驱动蛋白的“旗手”。）同样很大程度上未知的是肌球蛋白 VI 如何移动。为了回答这些问题，我们将在体外、单分子水平上解决马达问题。我们将应用许多单分子工具，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（散焦定向和位置成像）和 SHREC（单分子高分辨率共定位）。驱动蛋白部分的主题是：驱动蛋白的各个部分（包括头部、卷曲线圈和尾部）如何影响运动？肌球蛋白VI部分的主题是：这么小的马达如何迈出这么大的步？我们将提供未发表的数据，表明驱动蛋白在跑步过程中被两个头束缚，使得脱落的可能性非常低。我们还将建议，驱动蛋白-1 卷绕区域中的大非螺旋区域允许该电机以“不对称方式”行走，从而允许货物向前移动。最后，我们建议全长驱动蛋白由于尾部区域折叠并与头部区域以及可能的微管相互作用而“暂停”。关于肌球蛋白VI，我们有大量的初步数据。它将表明头部在动力冲程期间经历 180° 摆动。此外，我们建议杠杆臂中的 3 螺旋图案展开并产生前所未有的 24 nm 延伸，这使得电机能够迈出 36 nm 的步长。公共卫生相关性 分子马达的作用是移动和组织细胞内的细胞器。电机内部的问题会导致脑癌、阿尔茨海默氏病等。我们试图了解两种重要电机驱动蛋白和肌球蛋白 VI 的基本工作原理。

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 We will present unpublished data which suggests that kinesin is bound by both heads during a run, making the probability of falling off very low.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.