Mechanisms underpinning meiotic spindle formation and behavior

项目总结 减数分裂是产生单倍体配子的一组特殊的细胞分裂。在雌性减数分裂I(MI)期间， 两极纺锤体的形成和在卵母细胞内的定位必须严格控制，以确保忠实 染色体分离和适当的基因组遗传。在体细胞有丝分裂中，两极纺锤体的形成 定位依赖于一对中心体，每个中心体包含两个中心粒。有趣的是，减数分裂的卵母细胞 缺乏中心粒，因此缺乏经典的中心体。相反，减数分裂的卵母细胞含有大量微管。 (MT)组织中心(MTOC)，由基本上不为人所知的机制组织起来，以建立两个 主轴极(极MTOC)。传统观点认为，在哺乳动物卵母细胞中，MTS(及其相关的 蛋白质)是负责组织这种MTOC纺锤体的唯一细胞骨架组件。然而， 最近的数据表明，F-肌动蛋白也参与了纺锤体双极性的调节。F-肌动蛋白如何与MTS相互作用 在心肌梗死期间管理极性的MTOC组织是我们理解如何 减数分裂纺锤体已经建成。我们最近发现了一类新的、功能不同的MTOCs(McMTOCs)和 发现卵母细胞中心的纺锤体维持受到两种相反的力量(mcMTOC介导)的调节 MTS与F-肌动蛋白)。我们最近还观察到，大约50%的主轴没有集中组装。到目前为止，这样的 由于与主轴相关的技术限制，无法观察到外围主轴组件 荧光(即实时成像)。为了绕过这个问题，我们生成了一个Cep192-EGFP报告小鼠模型 无论它在哪里组装，都可以实现主轴跟踪。引人注目的是，通常遵循外周纺锤体的形成。 通过纺锤向中心移动--这是一种以前没有文献记载的现象。了解 调节这种纠正性发育事件的分子机制代表着我们在 MI期间减数分裂纺锤体时空调节的知识。这项建议为我们的 长期目标：了解MI期间的两个关键事件-双极主轴组装和定位- 在没有中心粒的情况下进行调节，以确保忠实的染色体分离。要做到这一点，我们将利用 最先进的方法，包括转基因小鼠模型、遗传结构、激光消融和 尖端成像，以解决三个关键目标：(I)确定F-肌动蛋白如何与MTS相互作用以组织 两极主轴构建过程中的两极MTOCs，(Ii)建立机制(S) 无着丝点纺锤体迁移到卵母细胞中心，以及(Iii)决定是否存在生化差异 McMTOCs与极性MTOCs的组成是其功能差异的基础。考虑到那条染色体 分离错误(在MI期间非常常见)导致非整倍体，这是发育的主要遗传原因 障碍和流产，这些研究有可能极大地促进我们对 在MI期间的两个基本过程--纺锤形成和定位--同时剥离 解释为什么MI出了名的容易出错。

Project summary Meiosis is a specialized set of cell divisions that produce haploid gametes. During meiosis I (MI) in females, bipolar spindle formation and positioning within the oocyte must be regulated tightly to ensure faithful chromosome segregation and proper genome inheritance. In somatic mitotic cells, bipolar spindle formation and positioning rely on a centrosome pair, each of which contains two centrioles. Interestingly, meiotic oocytes lack centrioles and, hence, lack classic centrosomes. Meiotic oocytes, instead, contain numerous microtubule (MT) organizing centers (MTOCs) that are organized, by largely unknown mechanisms, to establish two spindle poles (polar MTOCs). The traditional view was that, in mammalian oocytes, MTs (and their associated proteins) are the only cytoskeletal components responsible for organizing such MTOC spindles. However, recent data suggest that F-actin is also involved in spindle bipolarity regulation. How F-actin interacts with MTs to regulate polar MTOC organization during MI represents a critical gap in our understanding of how the meiotic spindle is built. We recently identified a novel, functionally different, class of MTOCs (mcMTOCs) and found that spindle maintenance at the oocyte center is regulated by two opposing forces (mcMTOC-mediated MTs vs. F-actin). We also recently observed that ~50% of spindles are not assembled centrally. To date, such peripheral spindle assembly was unobservable owing to technical limitations associated with spindle fluorescence (i.e. live imaging). To circumvent this, we generated a Cep192-eGfp reporter mouse model enabling spindle tracking wherever it is assembled. Strikingly, peripheral spindle formation is typically followed by spindle migration towards the center – a previously undocumented phenomenon. Understanding the molecular mechanisms regulating this corrective developmental event represents a major gap in our knowledge of meiotic spindle spatiotemporal regulation during MI. This proposal lays the foundations for our long-term goal: To understand how two critical events during MI — bipolar spindle assembly and positioning — are regulated, in the absence of centrioles, to ensure faithful chromosome segregation. To do so, we will utilize state-of-the-art approaches, including transgenic mouse models, genetic constructs, laser ablation, and cutting-edge imaging, to tackle three critical goals: (i) determine how F-actin interacts with MTs to organize polar MTOCs during bipolar spindle building, (ii) establish the mechanism(s) by which the peripheral acentriolar spindle migrates to the oocyte center, and (iii) determine whether differences in biochemical compositions of mcMTOCs vs. polar MTOCs underlie their functional differences. Given that chromosome segregation errors (very common during MI) lead to aneuploidy, the leading genetic cause of developmental disorders and miscarriage, these studies have the potential to significantly advance our basic understanding of two fundamental processes — spindle formation and positioning — during MI whilst simultaneously shedding light on why MI is notoriously error prone.