Kinetochore Specification and Function

描述（由申请人提供）：在细胞分裂过程中，复制的基因组必须准确地分布到子细胞。这个过程中的细微错误会导致出生缺陷，并可能导致癌症的发生。基因组分布的严重缺陷导致细胞死亡，并使用靶向微管的药物诱导细胞死亡，微管是基因组分布机制的核心组成部分，是癌症化疗中广泛使用的策略。在细胞分裂过程中，基因组分布的主要参与者是着丝点，它是一种蛋白质机器，建立在有丝分裂染色体的着丝粒区域上，与纺锤体微管产生动态界面。着丝点-微管相互作用的力学特性直接影响纺锤体上染色体的排列和分离。该界面的机制与检测和纠正染色体-纺锤体微管附着体几何错误的信号通路紧密结合，并阻止细胞周期进程，直到所有染色体正确连接。这些调控途径是基因组准确遗传的核心，因为它们确保复制的染色体精确地分为两个子细胞。普遍保守的KNL-1/Mis12复合体/Ndc80复合体（KMN）蛋白网络被认为在着丝点-微管界面上起核心作用。该蛋白网络主要通过4亚基Ndc80复合物上的微管结合表面提供着丝点的核心微管结合活性。KMN网络还在纺锤体检查点中发挥重要作用，纺锤体检查点是一种着丝点锚定的调节途径，它产生“等待后期”信号，直到细胞中的所有染色体都正确地附着在纺锤体上。建议的工作将侧重于定义KMN网络的KNL-1亚基作为协调外着丝点组装，微管附着和检查点信号传导的支架的机制。KMN网络的微管结合机制，以及Ndc80复合体和KNL-1蛋白中两种不同的微管结合活性之间的功能相互作用，也将使用结构和生化方法相结合的方法进行分析。在细胞中，着丝点结合的纺锤体微管明显稳定。为了确定KMN网络对这种性质的贡献程度，将在体外研究KMN网络对微管聚合动力学的调节。KMN网络与着丝酶定位的激酶和磷酸酶密切相关，在某些情况下直接接触，这些激酶和磷酸酶与准确的染色体分离有关。这种固定在KMN网络上的局部磷酸通量在染色体分离和检查点信号传导中的作用也将得到解决。

DESCRIPTION (provided by applicant): The replicated genome must be accurately distributed to daughter cells during cell division. Subtle errors in this process lead to birth defects and likely contribute to the genesis of cancer. Severe defects in genome distribution lead to cell lethality and their induction using drugs targeting microtubules, the protein polymers that are a central component of the genome distribution machinery, is a widely used strategy in cancer chemotherapy. A major player in genome distribution during cell division is the kinetochore, a proteinaceous machine built on the centromere regions of mitotic chromosomes to generate a dynamic interface with spindle microtubules. The mechanical properties of kinetochore-microtubule interactions direct chromosome alignment and segregation on the spindle. The mechanics at this interface are tightly integrated with signaling pathways that detect and correct errors in the geometry of chromosome-spindle microtubule attachments and prevent cell cycle progression until all chromosomes are properly connected. These regulatory pathways are central to accurate inheritance of the genome as they ensure that replicated chromosomes are precisely divided into the two daughter cells. The ubiquitously conserved KNL-1/Mis12 complex/Ndc80 complex (KMN) protein network is proposed to play a central role at the kinetochore-microtubule interface. This protein network provides the core microtubule-binding activity of the kinetochore, primarily through a microtubule- binding surface on the 4-subunit Ndc80 complex. The KMN network additionally plays an important role in the spindle checkpoint-the kinetochore-anchored regulatory pathway that generates a "wait anaphase' signal until all of the chromosomes in a cell are correctly attached to the spindle. The proposed work will focus on defining the mechanisms by which the KNL-1 subunit of the KMN network functions as a scaffold coordinating outer kinetochore assembly, microtubule attachment, and checkpoint signaling. The mechanism of microtubule binding by the KMN network and the functional interactions between the two distinct microtubule-binding activities in this protein set, which reside in the Ndc80 complex and in KNL-1, will also be analyzed using a combination of structural and biochemical approaches. In cells, kinetochore-bound spindle microtubules are significantly stabilized. To determine the extent to which the KMN network contributes to this property, the regulation of microtubule polymerization dynamics by the KMN network will be investigated in vitro. The KMN network is closely associated with, and in some cases directly contacts, kinetochore-localized kinases and phosphatases that are implicated in accurate chromosome segregation. The functions of this localized phosphate flux anchored to the KMN network in chromosome segregation and checkpoint signaling will also be addressed. PUBLIC HEALTH RELEVANCE: Errors in distributing the genome during cell division lead to birth defects and contribute to the genesis of cancer. The cellular machinery used for genome distribution is a common target in cancer chemotherapy. This project will focus on understanding the mechanisms that ensure accurate distribution of the genome to daughter cells during cell division. Elucidation of these mechanisms will contribute to our understanding of the development and progression of cancer and provide new avenues for therapeutic development.