Chromosome Movement in Meiosis and Mitosis

描述(由申请人提供)：该项目的长期目标是识别和描述在减数分裂和有丝分裂中驱动染色体运动的力量。这些力对正常的染色体分布是必不可少的，主要来自微管动力学和微管马达。一个主要的目标是了解微管马达蛋白在纺锤体和染色体动力学中在细胞分裂中的作用，包括运动方向性和力产生的分子基础，以及运动方向性和力产生在分裂中的作用。拟议的研究集中在果蝇的一种负端微管运动蛋白NCD上。NCD马达在减数分裂和有丝分裂中对纺锤体的组装和功能起着至关重要的作用。拟议的研究是为了检查基本的电机特性，目的是了解电机在细胞中是如何工作的。 拟议研究的具体目标是 1.确定NCD柄/颈旋转的核苷酸水解步骤。柄的旋转代表了一个潜在的产生力量的行程，驱动电机运动到微管减去端。我们将检验这样的假设，即当马达与微管结合并释放ADP时，茎会旋转，从而产生我们在激光陷阱分析中观察到的大位移。 2.确定马达结构变化的分子基础，这些变化对力的产生和传导是必要的。基于我们最近的低温电子显微镜结果，我们将通过突变关键残基并在体外测试突变的马达的ATPase活性和滑动速度来验证这一假设，即Switch II螺旋的融化和中央2-折叠的扭曲是运动功能所必需的。将对选定的突变体进行结构分析，以获得有关元素发生变化的信息。 3.确定马达在末期纺锤体微管成核中的作用及其对纺锤体微管动力学的影响。将分析活卵母细胞中成熟和组装的纺锤体，以确定是否存在3微管蛋白以及倒星体减数分裂1纺锤体中微管的极性。新设计的影响马达产生力能力的NCD突变体将被检测它们对减数分裂I纺锤体中微管动力学的影响，研究结果将与突变马达对纺锤体组装的影响相关联。

DESCRIPTION (provided by applicant): The long-term objective of this project is to identify and characterize the forces that drive chromosome movement in meiosis and mitosis. These forces are essential for normal chromosome distribution and arise primarily from microtubule dynamics and microtubule motors. A major goal is to understand the role of microtubule motor proteins in spindle and chromosome dynamics in cell division, including the molecular basis of motor directionality and force generation, and the role of motor directionality and force production in division. The proposed studies focus on Ncd, a minus-end microtubule motor protein of Drosophila. The Ncd motor plays an essential role in spindle assembly and function in meiosis and mitosis. The proposed studies are to examine basic motor properties with the objective of understanding how motors work in the cell. Specific aims of the proposed studies are to 1. Determine the step of nucleotide hydrolysis at which the Ncd stalk/neck rotates. The stalk rotation represents a potential force-producing stroke that drives movement of the motor to the microtubule minus end. We will test the hypothesis that the stalk rotates when the motor binds to microtubules and releases ADP, producing the large displacement we observed in laser trap assays. 2. Define the molecular basis of motor structural changes essential for force generation and transduction. We will test the hypothesis, based on our recent cryoEM results, that switch II helix melting and distortion of the central 2-sheet are essential for motor function by mutating key residues and assaying the mutated motors for ATPase activity and gliding velocity in vitro. Structural analysis of selected mutants will be performed to obtain information regarding the changes the elements undergo. 3. Determine the role of motors in microtubule nucleation in anastral meiosis I spindles and their effects on microtubule dynamics in the spindle. Mature and assembling spindles will be analyzed in live oocytes to determine whether 3tubulin is present and the polarity of microtubules in the anastral meiosis I spindle. Newly designed ncd mutants that affect the ability of the motor to produce force will be examined for their effects on microtubule dynamics in the meiosis I spindle and findings will be correlated with effects of the mutant motors on spindle assembly.