Mechanistic Analysis of Microtubule Based Motors

描述（由申请人提供）：kinesin分子马达用于广泛的细胞功能，包括囊泡运输，纺锤体组装和染色体分离，以及信号转导。这些atp酶可以沿着微管拉动细胞货物，使一个微管相对于另一个微管滑动，甚至通过促进微管分解来重塑微管细胞骨架。本提案的目标是建立Eg5和Kar3Cik1激酶atp酶与传统的激酶和Ned相比产生力的动力学和热力学基础。Eg5， Ned和Kar3在减数分裂和/或有丝分裂期间参与纺锤体动力学，Kar3在接合过程中对核缩制（核融合）至关重要。在体内的功能作用需要微管相互之间的运动，并且Kar3也表现出微管解聚活性。Ned和Kar3促进微管负端定向力的产生，而Eg5和kineins则驱动正端定向运动。Eg5的功能是同型四聚体，而Kar3与非运动蛋白Cik1结合形成异源二聚体。肌动蛋白是一种进程马达，然而Eg5、Ned和Kar3被认为不是进程马达。具体目标是确定Cik1对Kar3Cik1机械化学的作用，在电机滑动一个微管相对于另一个微管的情况下，与Kar3Cik1促进微管分解的情况直接比较，检查Kar3Cik1的机械化学和结构转变，并确定独立于Kar3的Cik1与微管的相互作用。对于Eg5，拟建的研究将定义二聚体运动域之间的合作相互作用，并将解决在atp酶周期中结构变化是如何协调的。对这4种驱动蛋白的全面分析将提供新的信息，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。

DESCRIPTION (provided by applicant): The kinesin molecular motors are required for a wide range of cellular functions including vesicle trafficking, spindle assembly and chromosome segregation, as well as signal transduction. These ATPases can pull a cellular cargo along a microtubule, slide one microtubule relative to another, or even remodel the microtubule cytoskeleton by promoting microtubule disassembly. The goal of this proposal is to establish the kinetic and thermodynamic basis of force generation for the Eg5 and Kar3Cik1 kinesin ATPases in comparison to conventional kinesin and Ned. Eg5, Ned, and Kar3 are involved in spindle dynamics during meiosis and/or mitosis, and Kar3 is essential for karyogamy (nuclear fusion) during conjugation. Functional roles in vivo require movement of microtubules relative to each other, and Kar3 also exhibits a microtubule depolymerizing activity. Ned and Kar3 promote microtubule minus-end directed force generation, yet Eg5 and kinesin drive plus-end directed motion. Eg5 functions as a homotetramer, yet Kar3 associates with Cik1, a non-motor protein, to form a heterodimer. Kinesin is a processive motor, yet Eg5, Ned, and Kar3 are believed not to be processive. The specific aims are directed to determine the role of Cik1 for Kar3Cik1 mechanochemistry, to examine the mechanochemistry and structural transitions of Kar3Cik1 at conditions where the motor slides one microtubule relative to another in direct comparison to conditions where Kar3Cik1 promotes microtubule disassembly, and to determine the interactions of Cik1 with the microtubule independent of Kar3. For Eg5, the proposed research will define the cooperative interactions between the motor domains of a dimer and will address how the structural changes are coordinated during the ATPase cycle. A comprehensive analysis of these 4 kinesins will provide new 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.