Role and Mechanisms of Microtubule Nucleation in Spindle Assembly

项目摘要 细胞分裂是由有丝分裂纺锤体，由成千上万的微管（MT）组成。 自从50年前发现MT结构单元微管蛋白以来，科学家们一直对有丝分裂是如何发生的感到困惑。 纺锤体通过MT组装并执行染色体分离，尽管MT周转数秒。现在我们 我们知道，主轴组件很大程度上依赖于MT成核。然而，何时、何地以及如何使MT成核， 它们随后如何结合到双极纺锤体中仍然不清楚。 基于我对分支MT成核的发现，我的实验室有助于理解几个 必需因子，即蛋白质复合物augmin、相分离蛋白TPX 2和成核剂g- TuRC，进行此反应。与此同时，已经表明，这种机制在一个特定的时间内产生了大多数MT。 纺锤体。作为这项工作的结果，我们处于一个理想的位置，以研究如何分支MT成核是 整合到纺锤体组装中以产生支持染色体分离的连续MT框架。 我们将追求三个目标：目标1：确定MT在何处、何时以及如何在 染色体我们将观察MT在非洲爪蟾卵纯化染色体上形成的确切位置和时间 提取物我们开发了一种新的检测方法来可视化来自染色体的MT成核， 在活细胞中很难做到。此外，我们将定义RanGTP对MT生成的贡献 途径，分支MT成核，染色体乘客复合物和动粒。我们假设 分支MT成核是染色体MT的主要来源。目标2：阐明重要性 调节分支微管成核的开始。RanGTP释放主轴装配系数TPX 2 从importins，然后刺激分支MT成核。以前的研究假设TPX 2存在于 单体。相比之下，我们最近发现TPX 2的活性形式经历液液相 分离（LLPS），并且输入抑制这种TPX 2冷凝物。importins如何抑制TPX 2的LLPS 不仅对MT组装很重要，而且在细胞生物学中也广泛相关，因为很少有研究描述了如何 抑制LLPS可以调节细胞功能。我们将进一步评估第二个基本分支是否 augmin因子也受RanGTP的调节，RanGTP是一种为细胞周期提供额外控制的途径 调控目的3：提供对核心分支因子augmin的机理性理解。确定 作为Augmin介导的分支MT成核的结构基础，我们将求解单粒子低温EM结构 关于Augmin这项工作将揭示augmin的八个亚基的位置和折叠。使用结构-功能 分析，我们将调查的功能接口，通过它augmin绑定到MTs和g-TuRC，此外， 研究augmin和TPX 2如何相互作用实现这些目标将有助于回答公开和紧迫的问题 在细胞生物学中，关于MT成核如何在正确的位置和正确的时间发生， 有丝分裂纺锤体

Project Summary Cell division is orchestrated by the mitotic spindle, composed of hundreds of thousands of microtubules (MT). Since the discovery of the MT building block tubulin 50 years ago, scientists have puzzled over how the mitotic spindle assembles via MTs and executes chromosome segregation despite a MT turnover of seconds. Now we know that spindle assembly relies largely on MT nucleation. Yet, when, where, and how MTs are nucleated, and how they are subsequently incorporated into the bipolar spindle, remains unclear. Based on my discovery of branching MT nucleation, my laboratory contributed to understanding how several essential factors, namely the protein complex augmin, the phase-separating protein TPX2, and the nucleator g- TuRC, conduct this reaction. Meanwhile it has been shown that this mechanism creates a majority of MTs in a spindle. As a result of this work, we are in an ideal position to investigate how branching MT nucleation is incorporated into spindle assembly to produce a continuous MT framework supporting chromosome segregation. We will pursue three aims: Aim 1: Determine where, when, and how MTs form in the vicinity of chromosomes. We will observe exactly where and when MTs form at purified chromosomes in Xenopus egg extract. We developed a novel assay to visualize MT nucleation from chromosomes, a direct visualization that is difficult to do in living cells. Further, we will define the contribution toward MT generation of the RanGTP pathway, branching MT nucleation, the chromosomal passenger complex, and the kinetochore. We hypothesize that branching MT nucleation is the main source of MTs from chromosomes. Aim 2: Elucidate how importins regulate onset of branching microtubule nucleation. RanGTP releases the spindle assembly factor TPX2 from importins, which then stimulates branching MT nucleation. Previous studies assumed that TPX2 exists as a monomer. In contrast, we recently showed that the active form of TPX2 undergoes a liquid liquid phase separation (LLPS), and importins inhibit this TPX2 condensate. How importins achieve inhibition of TPX2’s LLPS is not only important for MT assembly but also widely relevant in cell biology, as few studies have described how inhibition of LLPS can regulate cellular function. We will further assess whether the second essential branching factor, augmin, is also regulated by RanGTP, a pathway that would provide additional control for cell-cycle regulation. Aim 3: Provide mechanistic insight into the core branching factor augmin. To determine the structural basis of augmin-medidated branching MT nucleation, we will solve the single particle cryo-EM structure of augmin. This work will reveal the location and fold of augmin’s eight subunits. Using structure-function analysis, we will investigate the functional interfaces through which augmin binds to MTs and g-TuRC, besides interrogating how augmin and TPX2 interact. Achieving these aims will help answer open and pressing questions in cell biology about how MT nucleation occurs in the correct location and at the correct time to assemble the mitotic spindle.