Chromosome Movement in Meiosis and Mitosis

描述（由申请人提供）：该项目的长期目标是确定和描述在减数分裂和有丝分裂中驱动染色体运动的力量。这些力是正常染色体分布所必需的，主要来自微管动力学和微管马达。主要目标是了解微管运动蛋白在细胞分裂过程中纺锤体和染色体动力学中的作用，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。拟研究的重点是果蝇的负端微管运动蛋白Ncd。Ncd马达在纺锤体组装和减数分裂和有丝分裂中起重要作用。拟议的研究是为了检查基本的运动特性，目的是了解运动如何在细胞中工作。

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.