Efficiency and fidelity in mitotic spindle assembly

细胞分裂（有丝分裂）的目的是将遗传信息以染色体的形式平均分配到细胞中。 两个子细胞为了实现这一目标，“动粒”，专门的大分子复合物上 染色体，必须附着在“纺锤”的两极，“纺锤”是一个由动力学组装而成的大分子机器。 生物聚合物称为“微管”。附着缺陷导致染色体错误分离，这是一个标志 肿瘤的发生。我们研究的首要目标是揭示使纺锤体 快速组装，错误最少。我们以前的工作表明， 主轴组件可以通过几条可选的路线到达。有些路线是迅速的，但容易出错，其他 是准确的，但不是有效的。这种替代机制的多样性促使人们假设， 必须保持来自各个机制的贡献的平衡，以确保无错误的染色体 种族隔离我们将通过定量表征正常与对照组的纺锤体组装来检验这一假设。 染色体不稳定（CIN）细胞，其经常错误分离其染色体。在未来五年内 我们将集中研究主轴装配的四个主要方面：1）通过 其直接捕获在纺锤体极处成核的微管抑制了分离错误的数量。 虽然只有~25%的染色体通常利用直接捕获，分离错误变得很多， 这一机制的缺失。2)表征控制附着的分子机制 非中心体微管在着丝粒附近成核，这是主要的方式。 正常细胞中约75%的染色体采用附着。具体来说，我们将定位微管- 成核活性，并建立驱动蛋白CenpE的作用， 形成具有适当极性的微管束（K纤维）。3)我们将描述 动粒内的结构变化触发了“检查点蛋白”的去除。这个过程是 对控制有丝分裂的有序进程至关重要。4)最后，我们将量化染色体 在各种类型的细胞中，通过施加在短纤维远端的动力蛋白介导的拉力向极推进。 K纤维而不是更常见的机制，涉及在动粒内产生力。 由替代力产生机制运输的动粒的超微结构组织将被 比较的贡献和贡献，这些机制的无错误染色体分离将 被描述。为了实现我们的目标，我们采用先进的成像技术，如激光显微手术，精确的 染色体运动的跟踪，并进行相关的电子显微镜分析， 其行为在活细胞中被跟踪直到固定的时刻的动粒。这些方法在 结合用于灭活特定蛋白质的分子和细胞生物学技术， 对确保染色体分离的高保真度的机制的重要新见解。

The goal of cell division (mitosis) is to partition genetic information, in the form of chromosomes, equally into the two daughter cells. To achieve this goal, ‘kinetochores’, specialized macromolecular complexes on chromosomes, must attach to the poles of the ‘spindle’, a macromolecular machine assembled from dynamic biopolymers called ‘microtubules’. Attachment defects lead to chromosome mis-segregation, which is a hallmark of tumorigenesis. The overarching goal of our research is to reveal the mechanisms that allow the spindle to assemble rapidly and with minimal number of errors. Our previous work demonstrates that every major step in spindle assembly can be reached via several alternative routes. Some routes are swift but error prone, others are accurate but not efficient. This multiplicity of alternative mechanisms prompts the hypothesis that a proper balance in the contributions from individual mechanisms must be maintained to ensure error-free chromosome segregation. We will test this hypothesis by quantitatively characterizing spindle assembly in normal vs. chromosomally instable (CIN) cells that frequently mis-segregate their chromosomes. Over the next five years we will focus our studies on the four major aspects of spindle assembly: 1) Identification of the mechanism(s) by which direct capture of microtubules nucleated at the spindle poles suppresses the number of segregation errors. Although only ~25% of chromosomes normally utilize direct capture, segregation errors become numerous in the absence of this mechanism. 2) Characterization of the molecular mechanisms that govern attachment to non-centrosomal microtubules nucleated in the immediate proximity of kinetochores, which is the main mode of attachment employed by ~75% of chromosomes in normal cells. Specifically, we will localize microtubule- nucleating activities to a particular domain(s) within the kinetochore and establish the role of kinesin CenpE in the formation of microtubule bundles (K-fibers) with proper polarity of microtubules. 3) We will characterize structural changes within the kinetochore that trigger removal of the ‘checkpoint proteins’. This process is essential for controlling orderly progression through mitosis. 4) Finally, we will quantify how often chromosomes in various cell types are propelled poleward by a dynein-mediated pulling force exerted at the distal end of short K-fibers instead of the more common mechanism that involves generation of the force within the kinetochore. Ultrastructural organization of kinetochores transported by the alternative force production mechanisms will be compared contributions and the contributions of these mechanisms for error-free chromosome segregation will be characterized. To achieve our goals, we employ sophisticated imaging such as laser microsurgery, precise tracking of chromosome movements, and correlative electron-microscopy analyses conducted on the kinetochores whose behavior was followed in live cells up to the moment of fixation. These approaches in conjunction with molecular and cell-biology techniques for inactivation of specific proteins will produce a significant new insight into the mechanisms that ensure high fidelity of chromosome segregation.