Mechanistic Analysis of Microtubule Based Motors

描述（由申请人提供）：驱动蛋白分子马达是多种细胞功能所必需的，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 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。该提案的目标是建立 Eg5 和 Kar3Cik1 驱动蛋白 ATP 酶与传统驱动蛋白和 Ned 相比产生力的动力学和热力学基础。 Eg5、Ned 和 Kar3 参与减数分裂和/或有丝分裂期间的纺锤体动力学，而 Kar3 对于接合期间的核配（核融合）至关重要。体内的功能作用需要微管之间的相对运动，Kar3 还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 和驱动蛋白驱动正端定向运动。 Eg5 作为同四聚体发挥作用，而 Kar3 与非运动蛋白 Cik1 结合形成异二聚体。驱动蛋白是一种处理马达，但 Eg5、Ned 和 Kar3 被认为不是处理马达。具体目标是确定 Cik1 在 Kar3Cik1 机械化学中的作用，在电机相对于另一个微管滑动的条件下检查 Kar3Cik1 的机械化学和结构转变，与 Kar3Cik1 促进微管分解的条件进行直接比较，并确定 Cik1 与独立于 Kar3 的微管的相互作用。对于 Eg5，拟议的研究将定义二聚体运动域之间的协作相互作用，并将解决 ATP 酶循环期间如何协调结构变化。对这 4 种驱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。

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.