Kinetochore Specification and Function

描述（由申请人提供）：复制的基因组必须在细胞分裂期间准确分布到子细胞。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。基因组分布中的严重缺陷导致细胞致死性，并且使用靶向微管的药物对其进行诱导，微管是基因组分布机制的核心组分的蛋白质聚合物，是癌症化疗中广泛使用的策略。在细胞分裂过程中，基因组分布的一个主要参与者是动粒，一种建立在有丝分裂染色体的着丝粒区域上的蛋白质机器，用于与纺锤体微管产生动态界面。着丝粒-微管相互作用的机械性质指导纺锤体上的染色体排列和分离。这个界面的机制与信号通路紧密结合，这些信号通路检测和纠正染色体-纺锤体微管连接的几何错误，并阻止细胞周期进展，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/Mis 12复合物/Ndc 80复合物（KMN）蛋白网络被认为在着丝粒-微管界面起着核心作用。该蛋白质网络主要通过4-亚基Ndc 80复合物上的微管结合表面提供动粒的核心微管结合活性。此外，KMN网络在纺锤体检查点中起着重要作用，纺锤体检查点是一种激动剂锚定的调节途径，它产生一个“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。拟议的工作将集中于定义KMN网络的KNL-1亚基作为协调外动粒组装、微管附着和检查点信号传导的支架的机制。微管结合的KMN网络的机制和两个不同的微管结合活性之间的功能相互作用，在这个蛋白质组，这驻留在Ndc 80复合物和KNL-1，也将使用结构和生化方法的组合进行分析。在细胞中，结合有着丝粒的纺锤体微管被显著稳定。为了确定在何种程度上的KMN网络有助于这一属性，微管聚合动力学的调控KMN网络将在体外进行研究。KMN网络与涉及精确染色体分离的着丝粒定位激酶和磷酸酶密切相关，并且在某些情况下直接接触。还将讨论锚定到KMN网络的这种局部磷酸盐通量在染色体分离和检查点信号传导中的功能。 公共卫生相关性：细胞分裂期间基因组分布错误导致出生缺陷，并导致癌症的发生。用于基因组分布的细胞机器是癌症化疗中的常见靶标。该项目将侧重于了解在细胞分裂过程中确保基因组准确分布到子细胞的机制。阐明这些机制将有助于我们理解癌症的发展和进展，并为治疗开发提供新的途径。

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.