Single-molecule characterization of Cytoplasmic Dynein

描述(由申请人提供)： 细胞质动力蛋白在体外的研究很少，这使得我们很难理解它在体内的功能和调节。例如，因为它的单分子属性尚不清楚，所以还不清楚各种辅助蛋白可能需要发挥什么作用才能实现正确的体内功能。此外，尽管一些体内证据表明，多个动力蛋白马达一起工作(并与动力蛋白)，但使用多个马达的功能意义尚不清楚。我们通过在受控环境中研究动力蛋白的功能来解决这些问题，首先是在单分子水平上，然后是在日益复杂的情况下。我们将采用体外微珠试验，使用光学陷阱来量化单个或多个动力蛋白分子如何沿着微管移动，无论是否存在可能改变动力蛋白功能的蛋白质，如动力蛋白和MAP。最近发表的一篇论文的初步结果表明，Dynein的工作方式与Kinesin完全不同，并且似乎具有一种齿轮机构，使其能够调整力产生以满足外部施加的负载。此外，我们未发表的初步结果表明，与动蛋白不同，在单分子水平上，动力蛋白并不是一种非常有效的货物运输器。这项研究将全面研究动力蛋白的单分子功能(目标1)，阐明如何改变这一功能(目标2和3)，并进一步研究拟议的齿轮可能如何发挥作用(目标4)。 我们提出了以下具体目标： 目的1：结合测力-速度曲线、过程性与负荷、功能对三磷酸腺苷的依赖性等测量与建模相结合，阐明动力蛋白的单分子功能。目标2：确定多个动力蛋白如何共同发挥作用 目的3：确定一些辅助蛋白在动态蛋白功能改变中的调节作用。研究动力蛋白复合体和/或MAPs存在的功能分支。单一的动力蛋白-动力蛋白对是一种有效的运输系统吗？地图会损害机动交通工具吗？ 目标4：测试我们解释齿轮功能的假设 了解动力蛋白直接关系到公众健康：正确的动力蛋白功能和调控对发育至关重要，动力蛋白功能的改变可能发生在许多癌症中，动力蛋白和动力蛋白突变会导致神经元退化。

DESCRIPTION (provided by applicant): Cytoplasmic dynein is poorly understood in vitro, which makes it hard to understand its function and regulation in vivo. For instance, because its single-molecule properties are not known, it is unclear what roles various accessory proteins might need to play in order to achieve correct in vivo function. Further, although some in vivo evidence suggests that multiple dynein motors work together (and with dynactin), the functional significance of employing multiple motors is unknown. We address these questions by studying dynein function in a controlled environment, first at the single-molecule level and then in increasingly complex situations. We will employ an in vitro bead assay that uses an optical trap to quantify how single---or multiple--dynein molecules move along microtubules, both in the presence or absence of proteins that might alter dynein function such as dynactin and MAPs. Initial results published in a recent paper showed that Dynein functions in a fundamentally different manner than kinesin, and appears to have a gear mechanism enabling it to adjust force production to meet external applied load. Further, our unpublished preliminary results suggest that unlike kinesin, at the single molecule level dynein is not a very good efficient cargo transporter. The research will fully investigate dynein's single-molecule function (aim 1), clarify how this function can be altered (aims 2 and 3), and further study how the proposed gear might function (aim 4). We proposed the following specific aims: Aim 1: Elucidate the single-molecule functions of dynein, combining measurements including force-velocity curve, processivity vs load, dependence of function on ATP, etc. with modeling. Aim 2: Determine how multiple dyneins function together Aim 3: Determination of the role of some accessory proteins in regulating altering dynein function. Investigate the functional ramifications of the presence of the dynactin complex and or MAPs. Is a single dynein-dynactin pair an effective transport system ? Do MAPs impair motor-driven transport? Aim 4: Test our hypothesis explaining the gear's function Understanding dynein is directly related to public health: correct dynein function and regulation is essential for development, alteration of dynein function likely occurs in many cancers, and dynein and dynactin mutations cause neuronal degeneration.