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 动素超家族成员力量生产的基础。建议进行的研究 反映了对细胞内运输的长期兴趣，并致力于 生物医学研究。这项研究提案的具体目的是 建立了EGS力产生的动力学和热力学基础 和Kar3ATPase，直接与NCD和传统的Kinesin进行比较。全部使用 ATP驱动基于微管的单向运动。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 正常的染色体分离所必需的。相反，激动素是一种神经元 促进膜性细胞器运动的马达。运动蛋白的运动性是 因为它的过程性而与众不同。NCD、Kar3和EG5被认为不是 要循序渐进。NCD和Kar3均促进负端定向微管 运动，而激动素和EG5促进正端定向运动。 此外，作为单体的Kar3表现出单向运动；因此， Kar3是一种有趣的运动，可以直接与登山者相比较 Kinesins kinesin、EG5、NCD。NCD和Kinesin的结果表明， 二聚体的运动域是运动所必需的。EG5也是二聚体，但 到目前为止的证据表明，它不是进行性的。EGS的研究，在 直接与NCD和Kinesin进行比较，旨在定义 处理能力特别需要的功能，可能不同于 驱动加末端定向移动的功能。此外，这些实验 通过突变的运动蛋白马达将探索ATPase交叉桥循环的各个方面 通过研究野生型马达是无法获得的。建议数 实验将评估主轴电机具有的机械特性。 共同的，同时解决关于能源的具体问题 二聚马达与单体马达的换能器比较。一个 对这4个分子马达的综合分析将为 开始了解多样化的结构和机械要求 在细胞周期和神经肌肉发育期间发生的运动 以及基因改变可能导致出生缺陷、退行性疾病的功能 疾病和癌症。