Mechanisms underpinning meiotic spindle formation and behavior

减数分裂纺锤体形成和行为的基础机制

• 批准号: 10468208
• 负责人: AHMED BALBOULA
• 金额: $ 37.92万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10468208
• 关键词: Ablation; Aneuploidy; Behavior; Biochemical; Biological Models; Bipolar I; Cell division; Cells; Centrioles; Centrosome; Chromosome Segregation; Data; Development; Down Syndrome; Ensure; Event; F-Actin; Female; Fluorescence; Foundations; Genetic; Genome; Germ Cells; Goals; Haploidy; Image; Infertility; Knowledge; Lasers; Lead; Light; Maintenance; Mediating; Meiosis; Microtubule-Organizing Center; Microtubules; Mitotic; Molecular; Mus; Oocytes; Peripheral; Positioning Attribute; Process; Proteins; Public Health; Regulation; Reporter; Spontaneous abortion; Transgenic Mice; developmental disease; egg; male; migration; mouse model; novel; spatiotemporal; sperm cell

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.

项目摘要 减数分裂是一组专业的细胞分裂，产生单倍体游戏。在减数分裂时我（MI）在女性中， 必须严格调节双极主轴形成和卵母细胞内的位置 染色体分离和适当的基因组遗传。在体细胞有丝分裂细胞中，双极纺锤体形成 定位依赖于一个中心体对，每对都包含两个中心。有趣的是，减数分裂卵母细胞 减数分裂卵母细胞含有许多微管 （MT）通过在很大程度上未知的机制组织的组织中心（MTOC）建立两个 主轴杆（极性mTOC）。传统观点是，在哺乳动物的卵母细胞中，MTS（及其相关的 蛋白质）是唯一负责组织此类纺锤体的细胞骨架成分。然而， 最近的数据表明，F-肌动蛋白也参与了双极性调节。 F-肌动蛋白如何与MTS相互作用 在MI期间调节极地MTOC组织是我们对如何理解如何的关键差距 减数分裂纺锤体是建造的。我们最近确定了一种新颖的，功能上不同的MTOC类（MCMTOC）和 发现卵母细胞中心的主轴维护受两个相对力（MCMTOC介导的）调节 MTS与F-actin）。我们最近还观察到，约有50％的纺锤体不是集中组装的。迄今为止，这样 由于纺锤的技术限制，外围主轴组件无法观察 荧光（即实时成像）。为了避免这种情况，我们生成了一个CEP192-EGFP记者鼠标模型 在何处启用主轴跟踪。令人惊讶的是，通常遵循周围主轴形成 通过纺锤体向中心迁移 - 以前无证的现象。了解 调查这种纠正性发育事件的分子机制代表了我们的主要差距 MI期间的减数分裂纺锤时空调节的知识。该提议为我们的基础奠定了基础 长期目标：了解MI期间的两个关键事件如何 - 双极主轴组件和定位 - 在缺乏中心元素的情况下，受到调节，以确保忠实的染色体隔离。为此，我们将使用 最先进的方法，包括转基因小鼠模型，遗传构建体，激光消融和 尖端的成像，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 双极主轴建筑期间的极性mTOC，（ii）建立了外围的机制 acentriolar主轴迁移到卵母细胞中心，（iii）确定生化的差异是否存在 MCMTOC与极地MTOC的组成是其功能差异的基础。鉴于染色体 隔离误差（在MI期间非常常见）导致非整倍性，这是发展的主要遗传原因 疾病和流产，这些研究有可能显着提高我们对 在MI中，两个基本过程 - 主轴形成和定位 明白了为什么MI臭名昭著的错误。