Mechanistic Analysis Of Microtubule Based Motors

我们的长期目标是建立生物马达产生力量的结构和机械基础。本提案的具体目的是建立Eg5和Kar3 atp酶产生力的动力学和热力学基础，并与Ncd和传统的激酶进行直接比较。这四种都是超家族分子马达中的马达，它们利用ATP驱动基于微管的单向运动。Ncd， Eg5和Kar3都参与减数分裂和/或有丝分裂期间的纺锤体动力学，因此需要适当的染色体分离。相反，运动蛋白是驱动膜细胞器运动的神经元马达。运动学的运动性是独特的，因为它的进程性。Ncd， Kar3和Eg5被认为不是进程性的，它们的运动明显比运动蛋白促进的慢。Ncd和Kar3均促进负端定向微管运动，而激酶和Eg5促进正端定向微管运动。此外，Kar3作为单体表现为单向运动。因为Kar3是单体的，可以产生单向运动，所以Kar3是一个有趣的马达，可以与二聚体的运动蛋白——运动蛋白Eg5， Ncd进行直接比较。我们对Ncd和激酶的研究结果表明，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进行性的。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和激酶的直接比较将明确加工能力所需的机械特征，这些特征可能与驱动端导向运动的特征不同。实验将评估主轴电机的共同机械特性，同时解决二聚体电机与单体电机比较的能量转导的具体问题。对这4种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制要求，其中遗传改变可能导致出生缺陷和癌症等疾病。

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.