Dynactin/microtubule interactions in dynein motility

描述(由申请人提供)： 细胞质动力蛋白和动力肌动蛋白复合体产生力量，为一系列细胞功能提供运动。在神经元的轴突过程中，远距离运动尤其重要，动力蛋白和动力蛋白在那里逆行移动货物，距离从微米到几米不等。动力蛋白和动力蛋白运动机械组件中的遗传损伤会改变轴突运输，并可能导致严重的神经元障碍。动力蛋白在不脱离微管的情况下采取多个步骤的能力被称为过程性，动力蛋白增强了这种能力。为了实现这种加工性增强，dynactin必须能够结合微管。这一建议的目的是阐明动力蛋白/微管相互作用增加细胞质动力蛋白加工能力的机制。我们的假设是，加工能力的增强是因为动力蛋白作为动力蛋白、货物和微管细胞骨架之间的分子纽带。作为一种分子系绳，dynactin阻止发动机/货物复合体与微管的完全解离，并偏向于Dynein与微管的重新结合。 这项建议的第一个具体目标是描述动态肌动蛋白和微管之间发生的相互作用，目的是测试动态肌动蛋白拴系模型的有效性，并区分可能的作用机制。这将通过A)确定动力蛋白的哪些部分在微管结合中发挥作用并表征相互作用的表观结合强度，B)分析动力蛋白/微管相互作用的单分子行为，以及C)表征动力蛋白/微管相互作用在前进运动中的作用。第二个具体目的是阐明动力蛋白/微管相互作用的调节机制。这将通过A)检测dynactin分子异质性和B)检测Lis1功能来完成。 细胞质动力蛋白和动力蛋白运动复合体的异常功能可能引起或加剧广泛的人类健康问题，这增加了动力蛋白和动力蛋白成分可能成为治疗干预的有用靶点的可能性。然而，我们需要更好地了解基于动力蛋白的运动是如何发生的，以及动力蛋白和动力蛋白在这些障碍中可能扮演的角色，然后才能尝试干预。

DESCRIPTION (provided by applicant): The cytoplasmic dynein and dynactin motor complex generates force to provide movement for a vast array of cellular functions. Long-range movements are especially important in the axonal processes of neurons where dynein and dynactin move retrograde cargo over distances ranging from microns to meters. Genetic lesions in components of the dynein and dynactin motor machinery alter axonal transport and can result in severe neuronal disorders. The ability of dynein to take multiple steps without dissociating from the microtubule, called processivity, is enhanced by dynactin. For this processivity enhancement to take place, dynactin must be able to bind microtubules. The goal of this proposal is to elucidate the mechanism by which the dynactin/microtubule interaction increases cytoplasmic dynein processivity. Our hypothesis is that processivity enhancement occurs because dynactin acts as a molecular tether between dynein, cargo, and the microtubule cytoskeleton. As a molecular tether, dynactin prevents the complete dissociation of the motor/cargo complex from the microtubule and biases the rebinding of dynein to the microtubule. The first specific aim of this proposal is to characterize the interactions that occur between dynactin and microtubules with the goal of testing the validity of the dynactin tethering model and discriminating between possible mechanisms of action. This will be done by A) determining what portions of dynactin play a role in microtubule binding and characterizing the apparent binding strengths of the interactions, B) analyzing the single-molecule behavior of the dynactin/microtubule interaction, and C) characterizing the role of the dynactin/microtubule interaction in processive motility. The second specific aim is to elucidate regulatory mechanisms of the dynactin/microtubule interaction. This will be done by A) examining dynactin molecular heterogeneity and B) examining lis1 function. The wide range of human health problems that may be caused or compounded through the aberrant function of the cytoplasmic dynein and dynactin motor complex raises the possibility that dynein and dynactin components may be useful targets for therapeutic intervention. However, we require a better understanding of how dynein-based motility occurs and the role that dynein and dynactin may play in these disorders before intervention can be attempted.