Kinetochore Specification and Function

描述(申请人提供)：复制的基因组必须在细胞分裂过程中准确地分配给子细胞。这一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会导致细胞死亡，使用靶向微管的药物诱导细胞死亡是癌症化疗中广泛使用的一种策略。微管是基因组分布机制的核心组成部分。在细胞分裂过程中基因组分布的一个主要参与者是动粒，这是一种建立在有丝分裂染色体着丝粒区域的蛋白质机器，以产生与纺锤体微管的动态界面。着丝粒-微管相互作用的力学性质直接决定着染色体在纺锤体上的排列和分离。这个界面上的机制与信号通路紧密结合在一起，信号通路可以检测和纠正染色体纺锤体微管连接的几何错误，并防止细胞周期进展，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被精确地划分为两个子细胞。普遍保守的Knl-1/Mis12复合体/Ndc80复合体(KMN)蛋白网络被认为在动粒-微管界面上发挥核心作用。这个蛋白质网络提供了动粒的核心微管结合活性，主要是通过4亚单位NDC80复合体上的微管结合表面。此外，KMN网络在纺锤体检查点中也扮演着重要的角色--动粒锚定的调节通路，在细胞中的所有染色体都正确地连接到纺锤体之前，它会产生一个“等待后期”的信号。拟议的工作将集中于定义KMN网络的KN1-1亚单位作为协调外部动粒组装、微管附着和检查点信号转导的支架的机制。还将使用结构和生化方法相结合的方法分析KMN网络与微管结合的机制以及该蛋白集中两个不同的微管结合活性之间的功能相互作用，这两个微管结合活性位于Ndc80复合体和Knl-1中。在细胞中，动粒结合的纺锤体微管显著稳定。为了确定KMN网络对这一特性的贡献程度，我们将在体外研究KMN网络对微管聚合动力学的调节。KMN网络与涉及精确染色体分离的动粒定位的激酶和磷酸酶密切相关，在某些情况下还与它们直接接触。这一定位于KMN网络的磷酸盐通量在染色体分离和检查点信号传递中的功能也将被讨论。 与公共卫生相关：在细胞分裂过程中错误地分配基因组会导致出生缺陷，并导致癌症的发生。用于基因组分配的细胞机制是癌症化疗中的常见靶点。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了解癌症的发展和进展，并为治疗发展提供新的途径。

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.