Kinetochore Specification and Function

项目摘要 为了从单个细胞发育成有机体，基因组必须复制并精确分布 每一次细胞分裂。考虑到细胞分裂事件的天文数字， 维持生物体（据估计，成年人的身体由40万亿个细胞组成，其中至少有一个 每天有数万亿的细胞分裂），复制的基因组在子代中的分布变得至关重要。 细胞，一个被称为染色体分离的过程，以极高的准确性发生。染色体错误 隔离是出生缺陷/不育症的基础，足以引发基因组破坏，这是一个标志， 癌因此，了解细胞在染色体分离过程中确保准确染色体分离的机制， 细胞分裂具有基础和治疗意义。我们的工作集中在动粒蛋白质 在有丝分裂染色体上组装以协调分离的机器。动粒坐标 多个微管接口活动，共同定向和分离染色体，同时也 将机械事件与信号机制相结合，信号机制控制留在或退出的决定， 分裂。我们的早期工作鉴定了一组保守的蛋白质，称为KMN网络（对于Knl 1复合物， Mis 12复合物，Ndc 80复合物），位于这些协调动粒功能的核心。目标1： 重点是着丝粒的能力，既加速和延迟退出有丝分裂，我们建议优化 有丝分裂持续时间，以允许所有染色体有足够的时间连接到纺锤体，同时也尽量减少时间 当主要的细胞功能如转录、翻译和分泌被破坏时， 下调。我们提出的工作解决了与有丝分裂时间控制的二元性相关的主要开放问题 由kinetochores。动粒的主要机械活动是与动态纺锤体微管偶联。 几种不同的微管接口活动集中在动粒以及有丝分裂染色质上， 这使得染色体-微管相互作用的分析在细胞环境中具有挑战性。在目标2中， 描述了一种“空白板加回”的方法，在这种方法中，我们消除了有丝分裂细胞上所有的微管界面活动， 然后将它们单独放回去。这种方法补充了传统的功能丧失 分析，并将用于研究微管接口活动在着丝粒的方向和中心 纺锤体上的染色体，并关闭阻止有丝分裂退出的信号，直到微管附着 都是制造出来的最后，为了实现精确的分离，着丝粒-微管的相互作用必须紧密 监管.在目标3中，我们关注的是一个纺锤体极点产生的调控机制， 有丝分裂激酶Aurora A的梯度控制着着丝粒-微管的附着。当极光A被放大 在特定的癌症背景下，这项工作有可能揭示癌症特异性染色体调节异常， 分离和提出潜在的新的治疗途径。

PROJECT SUMMARY For the development of an organism from a single cell, the genome must be duplicated and precisely distributed during every cell division. Given the astronomical number of cell division events involved in building and maintaining organisms (by one estimate, the adult human body is made up of 40 trillion cells, with at least a couple of trillion dividing every day), it becomes critical that the distribution of the replicated genome to daughter cells, a process known as chromosome segregation, occurs with extremely high accuracy. Errors in chromosome segregation underlie birth defects/infertility and are sufficient to trigger the genomic havoc that is a hallmark of cancer. Thus, understanding the mechanisms by which cells ensure accurate chromosome segregation during cell division has both fundamental and therapeutic significance. Our work is focused on kinetochores, protein machines that assemble on mitotic chromosomes to orchestrate their segregation. Kinetochores coordinate multiple microtubule-interfacing activities that collectively orient and segregate chromosomes, while also integrating mechanical events with signaling mechanisms that control the decision to either remain in or exit from mitosis. Our early work identified a conserved set of proteins, referred to as the KMN network (for Knl1 complex, Mis12 complex, Ndc80 complex) that lies at the heart of these coordinated kinetochore functions. In Aim 1, we focus on the ability of kinetochores to both accelerate and delay exit from mitosis, which we propose optimizes mitotic duration to allow sufficient time for all chromosomes to connect to the spindle, while also minimizing time spent in a vulnerable phase when major cellular functions such as transcription, translation and secretion are downregulated. The work we propose tackles major open questions related to this duality of mitotic timing control by kinetochores. The primary mechanical activity of the kinetochore is to couple to dynamic spindle microtubules. Several distinct microtubule-interfacing activities concentrate at kinetochores as well as on mitotic chromatin, which makes analysis of chromosome-microtubule interactions challenging in a cellular context. In Aim 2, we describe a "blank slate–add back" approach, in which we eliminate all microtubule-interfacing activities on mitotic chromosomes and then add them back in isolation. This approach complements traditional loss-of-function analysis and will be used to study the microtubule-interfacing activities at kinetochores that orient and center chromosomes on the spindle and that shut of the signal that prevents mitotic exit until microtubule attachments are made. Finally, to achieve accurate segregation, kinetochore-microtubule interactions must be tightly regulated. In Aim 3, we focus on the poorly understood regulatory mechanism by which a spindle pole-generated gradient of the mitotic kinase Aurora A controls kinetochore-microtubule attachments. As Aurora A is amplified in specific cancer contexts, this effort has the potential to reveal cancer-specific dysregulation of chromosome segregation and suggest potential new therapeutic avenues.