Chromosome Movement in Meiosis and Mitosis

减数分裂和有丝分裂中的染色体运动

• 批准号: 7872868
• 负责人: Sharyn A. Endow
• 金额: $ 30.89万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7872868
• 关键词: ATP phosphohydrolase; Address; Affect; Aneuploidy; Appearance; Binding; Binding Proteins; Biological Assay; Cell division; Cells; Centrosome; Chromosomes; Defect; Drosophila Proteins; Drosophila genus; Elements; Embryo; Fiber; Fluorescence; Generations; Genetic Nondisjunction; Goals; Green Fluorescent Proteins; Head; Homologous Gene; Human; Hydrolysis; In Vitro; Kinesin; Laboratories; Lasers; Life; Mediating; Meiosis; Microtubules; Minus End of the Microtubule; Mitosis; Molecular; Molecular Abnormality; Motor; Movement; Mus; Mutate; Neck; Nucleotides; Oocytes; Organism; Pathway interactions; Play; Production; Property; Protein Binding; Proteins; Reporting; Research; Resolution; Role; Rotation; Stroke; Structure; Testing; Tubulin; Work; base; chromosome movement; design; driving force; melting; mutant; tumorigenesis

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.

描述（由申请人提供）：该项目的长期目标是识别和表征减数分裂和有丝分裂中驱动染色体运动的力量。这些力对于正常的染色体分布至关重要，主要来自微管动力学和微管马达。一个主要目标是了解微管运动蛋白在细胞分裂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。拟议的研究重点是 Ncd，果蝇的负端微管运动蛋白。 Ncd 马达在纺锤体组装以及减数分裂和有丝分裂的功能中发挥着重要作用。拟议的研究旨在检查基本的运动特性，目的是了解运动在细胞中的工作原理。 拟议研究的具体目标是 1. 确定 Ncd 茎/颈旋转的核苷酸水解步骤。柄旋转代表潜在的产生力的行程，驱动电机向微管负端移动。我们将测试以下假设：当电机与微管结合并释放 ADP 时，杆会旋转，从而产生我们在激光陷阱测定中观察到的大位移。 2. 定义对于力的产生和传导至关重要的运动结构变化的分子基础。根据我们最近的冷冻电镜结果，我们将通过突变关键残基并在体外分析突变马达的 ATP 酶活性和滑动速度来检验这一假设，即开关 II 螺旋熔化和中央 2 片层的变形对于运动功能至关重要。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. 确定电机在星体减数分裂 I 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。将在活卵母细胞中分析成熟和组装的纺锤体，以确定是否存在 3 微管蛋白以及前体减数分裂 I 纺锤体中微管的极性。新设计的影响马达产生力的能力的 ncd 突变体将检查它们对减数分裂 I 纺锤体微管动力学的影响，并将结果与​​突变体马达对纺锤体组装的影响相关联。