Mechanistic Analysis of Microtubule-Based Motors

基于微管的电机的机械分析

• 批准号: 6935248
• 负责人: SUSAN P. GILBERT
• 金额: $ 10.19万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6935248
• 关键词: SDS polyacrylamide gel electrophoresis; adenosinetriphosphatase; biomechanics; centrifugation; chromosome movement; computer simulation; enzyme activity; intracellular transport; kinesin; microtubules; mitotic spindle apparatus; physical model; protein purification; sedimentation equilibrium; stop flow technique; thermodynamics

DESCRIPTION: The long term goal is to establish the structural and mechanistic basis for force production by kinesin superfamily members. The proposed studies reflect a longstanding interest in intracellular transport and a commitment to biomedical research. The specific aims of this research proposal are to establish the kinetic and thermodynamic basis of force generation of the EgS and Kar3 ATPases in direct comparison to Ncd and conventional kinesin. All use ATP to drive unidirectional microtubule based movements. Ncd, Eg5, and Kar3 are involved in spindle dynamics during meiosis and/or mitosis and therefore are required for proper chromosome segregation. In contrast, kinesin is a neuronal motor that promotes movement of membranous organelles. Kinesins motility is distinctive because of its processivity. Ncd, Kar3 and Eg5 are believed not to be processive. Both Ncd and Kar3 promote minus end directed microtubule movements, yet kinesin and Eg5 promote plus end directed movements. Furthermore, Kar3 as a monomer exhibits unidirectional movement; therefore, Kar3 is an interesting motor to study in direct comparison to the climeric kinesins kinesin, Eg5, Ncd. The results with Ncd and kinesin indicate that both motor domains of the dimer are required for movement. Eg5 is also dimeric, yet evidence to date indicates it is not processive. The studies with EgS, in direct comparison to Ncd and kinesin, are intended to define the mechanistic features required specifically for processivity that may be distinct from those features that drive plus end directed movements. In addition, the experiments with mutant kinesin motors will explore aspects of the ATPase crossbridge cycle that are not accessible by studying the wildtype motor. The proposed experiments will evaluate the mechanistic features that spindle motors have in common, and at the same time address specific questions about energy transduction for dimeric motors in comparison to monomeric motors. A comprehensive analysis of these 4 molecular motors will provide information to begin to understand the structural and mechanistic requirements for the diverse movements occurring during the cell cycle and during neuromuscular development and function where genetic alteration can result in birth defects, degenerative diseases, and cancer.

描述：长期目标是建立结构和机制 驱动蛋白超家族成员产生力量的基础。拟议的研究 反映了对细胞内运输的长期兴趣和承诺 生物医学研究。本研究计划的具体目标是 建立 EgS 力产生的动力学和热力学基础 和 Kar3 ATP 酶与 Ncd 和传统驱动蛋白直接比较。全部使用 ATP 驱动基于单向微管的运动。 Ncd、Eg5 和 Kar3 是 参与减数分裂和/或有丝分裂期间的纺锤体动力学，因此 正确的染色体分离所必需的。相反，驱动蛋白是一种神经元 促进膜细胞器运动的马达。驱动蛋白运动是 由于其持续性而与众不同。 Ncd、Kar3 和 Eg5 被认为不会 积极进取。 Ncd 和 Kar3 均促进负端定向微管 运动，而驱动蛋白和 Eg5 则促进正向运动。 此外，Kar3作为单体表现出单向运动；所以， Kar3 是一个有趣的电机，可以直接与 climeric 进行比较来研究 驱动蛋白 驱动蛋白，Eg5，Ncd。 Ncd 和驱动蛋白的结果表明，两者 二聚体的运动结构域是运动所必需的。 Eg5 也是二聚体，但 迄今为止的证据表明它不是进行性的。 EgS 的研究， 与 Ncd 和驱动蛋白直接比较，旨在定义其机制 专门用于处理能力的功能可能与那些不同 驱动加上末端定向运动的功能。此外，实验 突变驱动蛋白马达将探索 ATP 酶跨桥循环的各个方面 这是通过研究野生型马达无法获得的。拟议的 实验将评估主轴电机的机械特性 共同点，同时解决有关能源的具体问题 与单体马达相比，二聚体马达的转导。一个 对这 4 种分子马达的综合分析将为 开始了解不同的结构和机械要求 细胞周期和神经肌肉发育过程中发生的运动 基因改变可能导致出生缺陷、退化的功能和功能 疾病和癌症。