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: 7367341
• 负责人: Sharyn A. Endow
• 金额: $ 30.77万
• 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/7367341
• 关键词: ATP phosphohydrolase; Address; Affect; Aneuploidy; Appearance; Binding; Binding Proteins; Biological Assay; Cell division; Cells; Centrosome; Chromosomes; Defect; Drosophila genus; Drosophila held out wings protein; Elements; Embryo; Fiber; Fluorescence; Generations; Genetic Nondisjunction; Goals; Green Fluorescent Proteins; Head; Helix (Snails); 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; ear helix; 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）：本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。这些力对于正常的染色体分布是必不可少的，并且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马达蛋白在细胞分裂中的纺锤体和染色体动力学中的作用，包括马达方向性和力产生的分子基础，以及马达方向性和力产生在分裂中的作用。本文拟对果蝇负端微管运动蛋白Ncd进行研究。Ncd马达在纺锤体组装中起重要作用，并在减数分裂和有丝分裂中起作用。拟议的研究是为了研究基本的电机特性，目的是了解电机如何在细胞中工作。 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。柄旋转代表了一个潜在的产生力的冲程，驱动电机的运动到微管负端。我们将测试的假设，柄旋转时，电机结合到微管和释放ADP，产生大位移，我们在激光陷阱检测中观察到的。 2.定义对力的产生和传递至关重要的马达结构变化的分子基础。根据我们最近的cryoEM结果，我们将通过突变关键残基并在体外测定突变的马达的ATP酶活性和滑行速度来验证这一假设，即开关II螺旋的融化和中央2片层的扭曲对于马达功能至关重要。将对选定的突变体进行结构分析，以获得有关元件发生变化的信息。 3.确定在无星减数分裂I纺锤体中微管成核中马达的作用及其对纺锤体中微管动力学的影响。将在活卵母细胞中分析成熟和组装纺锤体，以确定是否存在3微管蛋白以及无星减数分裂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突变体将被检查其对减数分裂I纺锤体中微管动力学的影响，并且发现将与突变马达对纺锤体组装的影响相关。