Mechanistic Analysis Of Microtubule Based Motors

基于微管的电机的机械分析

• 批准号: 6636184
• 负责人: SUSAN P. GILBERT
• 金额: $ 25.05万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6636184
• 关键词: adenosine triphosphate; cell cycle; centrifugation; chemical kinetics; enzyme mechanism; kinesin; meiosis; microtubules; thermodynamics

Our long term goal is to establish the structural and mechanistic basis for force production by biological motors. The specific aims of this proposal are to establish the kinetic and thermodynamic basis of force generation of the Eg5 and Kar3 ATPases in direct comparison to Ncd and conventional kinesin. All four are kinesin superfamily molecular motors that use ATP to drive unidirectional microtubule-based movements. Ncd, Eg5, and Kar3 are all 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 drives movements of membranous organelles. Kinesin's motility is distinctive because of its processivity. Ncd, Kar3, and Eg5 are believed not to be processive, and their motility is significantly slower than that promoted by kinesin. 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. Because Kar3 is monomeric and can generate unidirectional movement, Kar3 is an interesting motor to study in direct comparison to the dimeric kinesins-- kinesin, Eg5, Ncd. Our 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. As a spindle motor, its mechanism may be more similar to Ncd's even though Ncd motility is directed to the minus-end of microtubules. Our studies with Eg5 in direct comparison to Ncd and kinesin will define the mechanistic features required specifically for processivity that may be distinct from those features that drive plus-end directed movements. The 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 kinetic and thermodynamic analysis of these 4 molecular motors will provide rigorous and direct information to begin to understand the structural and mechanistic requirements for the diverse movements occurring during the cell cycle where genetic alteration can result in birth defects and diseases such as cancer.

我们的长期目标是建立生物马达产生力的结构和机械基础。该提案的具体目标是建立 Eg5 和 Kar3 ATP 酶产生力的动力学和热力学基础，并与 Ncd 和传统驱动蛋白直接比较。所有四个都是驱动蛋白超家族分子马达，它们使用 ATP 驱动基于微管的单向运动。 Ncd、Eg5 和 Kar3 均参与减数分裂和/或有丝分裂期间的纺锤体动力学，因此是正确染色体分离所必需的。相反，驱动蛋白是一种驱动膜细胞器运动的神经元马达。驱动蛋白的运动性因其持续性而与众不同。 Ncd、Kar3 和 Eg5 被认为不是进行性的，它们的运动明显慢于驱动蛋白促进的运动。 Ncd 和 Kar3 均促进负端定向微管运动，而驱动蛋白和 Eg5 则促进正端定向运动。此外，Kar3 作为单体表现出单向运动。由于 Kar3 是单体并且可以产生单向运动，因此与二聚驱动蛋白（驱动蛋白、Eg5、Ncd）直接比较，Kar3 是一种值得研究的有趣马达。我们对 Ncd 和驱动蛋白的结果表明，二聚体的两个运动域都是运动所必需的。 Eg5 也是二聚体，但迄今为止的证据表明它不是持续进行的。作为主轴电机，其机制可能与 Ncd 更相似，尽管 Ncd 的运动指向微管的负端。我们对 Eg5 的研究与 Ncd 和驱动蛋白直接比较，将定义专门用于持续性的机械特征，这些特征可能与驱动正端定向运动的那些特征不同。这些实验将评估主轴电机共有的机械特征，同时解决有关二聚体电机与单体电机相比能量转换的具体问题。对这 4 个分子马达进行全面的动力学和热力学分析将提供严格和直接的信息，以开始了解细胞周期中发生的各种运动的结构和机械要求，在细胞周期中，基因改变可能导致出生缺陷和癌症等疾病。