Organization of the Mammalian Mitotic Spindle

哺乳动物有丝分裂纺锤体的组织

• 批准号: 7931624
• 负责人: Duane A. Compton
• 金额: $ 7.54万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7931624
• 关键词: Aneuploidy; Biochemical; Biological Assay; Cataloging; Catalogs; Cell Survival; Cells; Chromosome Segregation; Chromosomes; Complex; Congenital Abnormality; Data; Diagnostic Neoplasm Staging; Diploid Cells; Diploidy; Disabled Persons; Ensure; Fluorescence; Frequencies; Funding; Goals; Human; Immunoprecipitation; Kinesin; Kinetochores; Knowledge; Life; Link; Mammalian Cell; Maps; Mass Spectrum Analysis; Metaphase; Methods; Microtubule-Associated Proteins; Microtubules; Mitosis; Mitotic; Mitotic spindle; Morphogenesis; Motor; Mutation; Pathway interactions; Phosphorylation Site; Property; Proteins; Proteomics; Regulation; Research; Site; Structure; System; Testing; Tubulin; Work; base; cellular imaging; chromosome movement; insight; neoplastic cell; photoactivation; public health relevance; research study; segregation; tumor

DESCRIPTION (provided by applicant): Accurate chromosome segregation is essential for the propagation of species and the viability of cells, and is driven by a complex microtubule-based structure called the spindle. Spindle organization and chromosome movement are driven by the concerted actions of microtubule-associated proteins (motor and non-motor) and the inherent dynamic properties of microtubules. Despite extensive knowledge of the proteins involved in spindle morphogenesis and chromosome movement, very little is known about how the accuracy of chromosome segregation is ensured during mitosis in mammalian cells. The purpose of the experiments proposed here is to combine biochemical methods and live cell imaging to identify the proteins and determine the mechanisms underlying the high fidelity of chromosome segregation during mitosis in human cells. Because chromosomes are linked to spindle microtubules through the kinetochore, a focus will be on defining the molecules and mechanisms that govern the dynamic attachment of spindle microtubules to kinetochores. The specific aims of this research are to: 1) combine live cell imaging with quantitative chromosome segregation assays to define the mechanisms regulating kinetochore- microtubule attachment necessary for accurate chromosome segregation; 2) use live cell imaging to examine how the spatial and temporal sequence of spindle assembly contributes to the accuracy of chromosome segregation; 3) use biochemical methods to determine how the kinetochore-associated microtubule depolymerizing activity of the kinesin-13 protein Kif2b is regulated during mitosis; and 4) use live cell assays to determine the fate of human cells that mis-segregate chromosomes. PUBLIC HEALTH RELEVANCE: Chromosome mis-segregation causes aneuploidy that causes birth defects and is commonly associated with advanced stage cancer. The goal of the experiments proposed here is to combine biochemical methods and live cell imaging to identify the proteins and determine the mechanisms underlying the high fidelity of chromosome segregation during mitosis in human cells. Data generated from this work will provide insight into mechanisms of aneuploidy in tumor cells and may reveal strategies for therapy of chromosomally unstable aneuploid tumors.

描述（由申请人提供）：准确的染色体分离对于物种的传播和细胞的生存能力至关重要，并且由复杂的基于微管的结构驱动，称为纺锤体。主轴组织和染色体运动是由微管相关蛋白（运动和非运动型）和微管的固有动态特性的一致作用驱动的。尽管广泛了解参与纺锤形态发生和染色体运动的蛋白质，但对于如何在哺乳动物细胞中有丝分裂过程中确保染色体分离的准确性知之甚少。这里提出的实验的目的是结合生化方法和活细胞成像，以鉴定蛋白质并确定人类细胞中有丝分裂期间染色体隔离的高忠诚度的机制。由于染色体通过动力学与纺锤微管有关，因此将重点放在定义控制纺锤微管动态附着在动力学上的分子和机制。这项研究的具体目的是：1）将活细胞成像与定量染色体隔离测定法相结合，以定义调节动力学微管附着的机制，以精确的染色体隔离； 2）使用活细胞成像检查纺锤体组件的空间和时间序列如何有助于染色体分离的准确性； 3）使用生化方法来确定在有丝分裂期间调节驱动蛋白13蛋白KIF2B的动力学相关的微管解聚活性； 4）使用活细胞测定法确定脱离染色体的人类细胞的命运。公共卫生相关性：染色体错误分离会导致非整倍性引起先天缺陷，并且通常与晚期阶段癌症有关。这里提出的实验的目的是结合生化方法和活细胞成像，以识别蛋白质并确定人类细胞中有丝分裂期间染色体隔离的高保真度的机制。这项工作产生的数据将为肿瘤细胞中非整倍性的机理提供深入的了解，并可能揭示了染色体不稳定的非蛋白酶肿瘤治疗的策略。