Organization of the Mammalian Mitotic Spindle

描述（由申请人提供）：准确的染色体分离对于物种繁殖和细胞活力至关重要，并由称为纺锤体的复杂微管结构驱动。纺锤体组织和染色体运动是由微管相关蛋白（马达和非马达）的协同作用和微管固有的动力学特性驱动的。尽管对纺锤体形态发生和染色体运动所涉及的蛋白质有广泛的了解，但对哺乳动物细胞有丝分裂期间如何确保染色体分离的准确性知之甚少。本文提出的实验目的是将生物化学方法和活细胞成像联合收割机，以鉴定蛋白质并确定人类细胞有丝分裂期间染色体分离高保真度的潜在机制。由于染色体通过动粒与纺锤体微管相连，因此重点将是定义纺锤体微管与动粒动态连接的分子和机制。本研究的具体目的是：1）将联合收割机活细胞成像技术与定量染色体分离技术相结合，以确定染色体精确分离所必需的动粒-微管附着的调控机制：2）利用活细胞成像技术研究纺锤体组装的时空顺序对染色体精确分离的贡献; 3）使用生物化学方法来确定在有丝分裂过程中驱动蛋白-13蛋白Kif 2b的激动素相关微管解聚活性是如何调节的;以及4）使用活细胞测定来确定错误分离染色体的人类细胞的命运。公共卫生相关性：染色体错误分离导致非整倍体，导致出生缺陷，通常与晚期癌症有关。本文提出的实验的目标是结合联合收割机生物化学方法和活细胞成像来鉴定蛋白质，并确定人类细胞有丝分裂期间染色体分离高保真度的潜在机制。从这项工作产生的数据将提供深入了解肿瘤细胞中的非整倍体的机制，并可能揭示染色体不稳定的非整倍体肿瘤的治疗策略。

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.