Chromosome Movement in Prometaphase

描述(由申请人提供)：我们试图在细胞生物学和分子水平上了解染色体如何移动，它们如何被引导到中期板，以及微管如何与着丝粒和其他有丝分裂细胞器相互作用，调节有丝分裂纺锤体检查点。前中期的染色体运动主要是由有丝分裂纺锤体微管和着丝点的动态相互作用驱动的。同时，动点在微管上缺乏稳定的双极连接，作为纺锤体检查点的信号，该检查点阻止后期开始，直到中期排列完成。当所有姐妹运动中枢建立稳定的双极微管连接时，这一检查点被沉默。因此，染色体在有丝分裂纺锤体上的附着和运动与纺锤体检查点的调节密切相关。此前，该实验室提供的证据表明，动点沿微管的移位是有丝分裂中染色体运动的主要媒介。后来，我们发现有丝分裂细胞中的各个动点在生物化学上是不同的，并发展了动点模型，作为纺锤体检查点信号的催化来源。我们现在已经组装了一套独特的工具来剖析脊椎动物细胞中几个最重要的染色体运动调节因子和纺锤体检查点的分子作用。我们将确定微管附着和机械张力是否以及如何调节动粒蛋白动力学和信号。我们专注于微管-动粒相互作用的一组关键调节因子：Ndc80蛋白复合体、Polo-like Kinase1、Aurora B激酶、dynein/dynactin复合体以及相关的蛋白质复合体ZW10/Rod复合体。我们将分析这些蛋白质在非洲爪哇和哺乳动物活细胞中的功能。我们将概述裂解细胞系统中的动粒调节，有丝分裂细胞的部分，以及体外纯化的蛋白质。我们将合作进行非洲爪哇鸡蛋提取物的补充研究。分析几种动态蛋白质复合体的作用是一个重大的挑战。然而，只有结合体内和体外的各种方法来研究动粒和微管蛋白的动力学，我们才能开始了解活细胞中有丝分裂的调节。

DESCRIPTION (provided by applicant): We seek to understand, at the cell biological and molecular levels, how chromosomes move, how they are guided to the metaphase plate, and how microtubule interactions with the kinetochore and other mitotic organelles regulate the mitotic spindle checkpoint. Chromosome movements during prometaphase are driven primarily by dynamic interactions of the mitotic spindle microtubules with the kinetochores. At the same time, kinetochores lacking stable bipolar attachment to microtubules serve to signal the spindle checkpoint that blocks anaphase onset until metaphase alignment is achieved. This checkpoint is silenced when the all sister kinetochores establish stable bipolar microtubule attachments. Thus, chromosome attachment and movement on the mitotic spindle are intimately intertwined with the regulation of the spindle checkpoint. Previously, this lab provided evidence that translocation of the kinetochores along microtubules is the prime mediator of chromosome movement in mitosis. We later discovered that individual kinetochores within a mitotic cell were biochemically distinct and developed the model of kinetochores as catalytic sources for spindle checkpoint signaling. We have now assembled a unique set of tools to dissect the molecular roles of several of the most important regulators of chromosome movement and the spindle checkpoint in vertebrate cells. We will determine if and how microtubule attachment and mechanical tension regulate kinetochore protein dynamics and signaling. We focus on a set of key regulators of microtubule-kinetochore interactions: the Ndc80 protein complex, Polo-like kinase1, the Aurora B kinase, the dynein/dynactin complex and an associated protein complex, the ZW10/Rod complex. We will analyze the functions of these proteins in living Xenopus and mammalian cells. We will recapitulate kinetochore regulation in lysed cell systems, in fractions from mitotic cells, and with purified proteins in vitro. We will collaborate to conduct complementary studies in Xenopus egg extracts. Analyzing the roles of several dynamic protein complexes is a significant challenge. However, only by approaching kinetochore and microtubule protein dynamics in concert with a variety of in vivo and in vitro approaches can we begin to understand the regulation of mitosis in living cells.