Kinetochore Specification and Function

项目总结 为了使生物体从单个细胞发育起来，基因组必须复制并精确分布。 在每一次细胞分裂中。考虑到建筑和建筑过程中涉及的细胞分裂事件的天文数字 维持生物体(据估计，成人人体由40万亿个细胞组成，至少有一个 每天分裂数万亿)，将复制的基因组分配给女儿变得至关重要 细胞，一个被称为染色体分离的过程，以极高的准确性发生。染色体中的错误 隔离是导致出生缺陷/不育的基础，并足以引发基因组浩劫，这是 癌症。因此，了解细胞确保准确的染色体分离的机制 细胞分裂既具有基础意义，又具有治疗意义。我们的工作重点是动素，蛋白质 在有丝分裂染色体上组装的机器来协调它们的分离。动心坐标 多个微管接口活动，集体定位和分离染色体，同时也 将机械事件与控制决定留在或退出的信号机制相结合 有丝分裂。我们的早期工作确定了一组保守的蛋白质，称为KMN网络(对于KnL1复合体， Mis12复合体、NDC80复合体)，位于这些协调着丝粒功能的核心。在目标1中，我们 重点关注动粒加速和延迟有丝分裂退出的能力，我们建议优化这一点 有丝分裂持续时间允许所有染色体有足够的时间连接到纺锤体，同时也最大限度地减少时间 处于脆弱阶段，此时主要细胞功能，如转录、翻译和分泌 监管下调。我们提出的工作解决了与这种有丝分裂定时控制的二重性相关的主要悬而未决的问题 通过运动琐事。着丝粒的主要机械活动是与动态纺锤体微管连接。 几种不同的微管接口活动集中在动粒和有丝分裂染色质上， 这使得对染色体-微管相互作用的分析在细胞背景下具有挑战性。在目标2中，我们 描述一种“白板加回”的方法，在这种方法中，我们消除了有丝分裂上的所有微管接口活动。 染色体，然后把它们孤立地加回去。这种方法是对传统功能丧失的补充 分析，并将用于研究在运动中枢定向和中心的微管接口活动 纺锤体上的染色体和阻止有丝分裂退出直到微管附着的信号的关闭 都是制造出来的。最后，为了实现准确的分离，动粒-微管的相互作用必须紧密 受监管的。在目标3中，我们将重点放在鲜为人知的调节机制上，通过这种调节机制，纺锤体极的产生 有丝分裂激酶Aurora A的梯度控制着动粒-微管的附着。随着极光A被放大 在特定的癌症背景下，这一努力有可能揭示癌症特有的染色体失调。 并提出了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.