Mechanisms underpinning meiotic spindle formation and behavior

项目摘要 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， 卵母细胞内双极纺锤体的形成和定位必须严格调节，以确保卵母细胞的忠实性。 染色体分离和适当的基因组遗传。在体细胞有丝分裂中，双极纺锤体形成 和定位依赖于中心体对，每个中心体对包含两个中心粒。有趣的是，减数分裂卵母细胞 缺乏中心粒，因此缺乏典型的中心体。相反，减数分裂卵母细胞含有大量的微管， (MT)组织中心（MTOC），由基本上未知的机制组织起来，建立两个 纺锤极（极性MTOC）。传统观点认为，在哺乳动物卵母细胞中，MT（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分。然而，在这方面， 最近的数据表明，F-肌动蛋白也参与纺锤体双极性调节。F-actin如何与MT相互作用 在MI期间调节极性MTOC组织代表了我们对如何理解的关键差距， 减数分裂纺锤体形成。我们最近发现了一种新的、功能不同的MTOC（mcMTOC）， 发现卵母细胞中心纺锤体的维持受两种相反的力量（mcMTOC介导的 MT相对于F-肌动蛋白）。我们最近还观察到，约50%的锭子不是集中组装的。迄今为止， 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）。为了避免这一点，我们产生了Cep 192-eGfp报告基因小鼠模型， 使得主轴能够在任何装配位置进行跟踪。引人注目的是，外周纺锤体的形成通常遵循 纺锤体向中心迁移--这是一种以前没有记录的现象。了解 调节这种纠正性发育事件的分子机制代表了我们在这方面的一个主要空白。 MI期间减数分裂纺锤体时空调控的知识。这一建议为我们的 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- 在中心粒不存在的情况下，被调节以确保忠实的染色体分离。为此，我们将利用 最先进的方法，包括转基因小鼠模型，遗传构建，激光消融， 尖端成像，以解决三个关键目标：（i）确定F-肌动蛋白如何与MT相互作用，以组织 （ii）建立外周MTOC在双极纺锤体构建过程中的机制， 无中心纺锤体迁移到卵母细胞中心，和（iii）确定生物化学差异是否 mcMTOC与极性MTOC的组成是它们功能差异的基础。考虑到这个染色体 分离错误（在MI期间非常常见）导致非整倍体，这是发育不良的主要遗传原因。 疾病和流产，这些研究有可能显着推进我们的基本理解， 两个基本过程--锭子形成和定位--在MI期间，同时开口 为什么MI是出了名的容易出错。

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.