Mechanistic Analysis of Microtubule Based Motors

描述(申请人提供)：动蛋白分子马达是广泛的细胞功能所必需的，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ATPase可以沿着微管拉动细胞，滑动一个微管相对于另一个微管，甚至通过促进微管分解来重塑微管细胞骨架。这项提议的目的是与传统的激动素和NED相比，建立EG5和Kar3Cik1激动素ATPase的动力产生的动力学和热力学基础。EG5、Ned和Kar3参与减数分裂和/或有丝分裂过程中的纺锤体动力学，而Kar3在接合过程中对核受精(核融合)是必不可少的。体内的功能角色需要微管相对运动，而Kar3也表现出微管解聚的活性。Ned和Kar3促进微管负端定向力的产生，而EG5和Kinesin驱动正端定向力的产生。EG5作为同源四聚体发挥作用，而Kar3与非运动蛋白Cik1结合形成异源二聚体。Kinesin是一种进行性马达，而EG5、Ned和Kar3被认为不是进行性的。具体目的是确定Cik1在Kar3Cik1机械力化学中的作用，检查在发动机滑动一个微管相对于另一个微管的条件下Kar3Cik1的机械力化学和结构转变，并与Kar3Cik1促进微管分解的条件进行直接比较，以及确定不依赖于Kar3的Cik1与微管的相互作用。对于EG5，拟议的研究将定义二聚体运动域之间的协同作用，并将解决如何在ATPase周期中协调结构变化。对这4种动蛋白的全面分析将提供新的信息，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，在细胞周期中，基因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.