Organization of the Mammalian Mitotic Spindle

描述(申请人提供)：准确的染色体分离对于物种的繁殖和细胞的存活至关重要，它是由一种称为纺锤体的复杂的微管结构驱动的。纺锤体的组织和染色体的运动是由微管相关蛋白(马达和非马达)和微管固有的动态特性共同驱动的。尽管对参与纺锤体形态发生和染色体运动的蛋白质有广泛的了解，但对哺乳动物细胞有丝分裂过程中如何确保染色体分离的准确性知之甚少。这些实验的目的是结合生化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过程中染色体分离高保真的机制。因为染色体通过着丝点与纺锤体微管相连，所以重点将放在确定控制纺锤体微管与着丝点的动态连接的分子和机制上。本研究的具体目标是：1)将活细胞成像与定量染色体分离分析相结合，以确定调控着丝粒-微管附着的机制，这对于准确的染色体分离是必要的；2)利用活细胞成像检测纺锤体组装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Kinesin-13蛋白Kif2b的动粒相关微管解聚活性在有丝分裂过程中如何受到调节；以及4)利用活细胞分析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。本文提出的实验目的是结合生化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过程中染色体分离高保真的机制。这项工作产生的数据将提供对肿瘤细胞非整倍体机制的洞察，并可能揭示治疗染色体不稳定的非整倍体肿瘤的策略。

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.