Efficiency and fidelity in mitotic spindle assembly

细胞分裂(有丝分裂)的目标是以染色体的形式将遗传信息平等地划分到 两个子细胞。为了实现这一目标，专门的大分子络合物 染色体，必须附着在两极的“纺锤”上，一个由动态组装而成的大分子机器 被称为“微管”的生物聚合物。附着缺陷导致染色体错误分离，这是一个标志 关于肿瘤发生的研究。我们研究的首要目标是揭示允许纺锤体 快速组装，错误次数最少。我们之前的工作表明，每一个主要步骤 主轴组件可以通过几种替代路线到达。一些路线很快，但容易出错，另一些路线 是准确的，但效率不高。这种多种多样的替代机制促使人们假设，一个适当的 必须保持来自各个机制的贡献的平衡，以确保没有错误的染色体 种族隔离。我们将通过定量描述正常和正常的主轴装配来检验这一假说。 染色体不稳定(CIN)细胞，经常错误分离它们的染色体。在未来五年内 我们将重点研究主轴装配的四个主要方面：1)通过以下方式确定机构(S) 直接捕获在纺锤极成核的微管抑制了偏析错误的数量。 虽然只有25%的染色体通常使用直接捕获，但分离错误在 这一机制的缺失。2)描述支配依恋的分子机制 非中心体微管在动点附近成核，这是主要的方式。 正常细胞中约75%的染色体使用附着。具体地说，我们将定位微管- 对着丝点内的特定结构域(S)的成核活性，并建立动蛋白CenpE在 具有适当的微管极性的微管束(K纤维)的形成。3)我们将描述 动粒内部的结构变化，引发“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，我们将量化染色体的频率 在不同类型的细胞中，动力蛋白介导的拉力在短端施加，推动细胞向极地移动 K纤维取代了更常见的机制，即在动粒内产生力。 动点的超微结构组织将由替代力生产机制运输 比较的贡献和这些机制对无错误染色体分离的贡献将 被刻画出来。为了实现我们的目标，我们使用了先进的成像技术，如激光显微手术，精确 对染色体运动的跟踪，以及对 运动中枢，其行为在活细胞中一直被跟踪到固定的时刻。这些方法在 结合分子和细胞生物学技术来灭活特定蛋白质将产生一种 对确保染色体分离高保真的机制有了重大的新见解。

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.