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: 10581903
• 负责人: AHMED BALBOULA
• 金额: $ 24.92万
• 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/10581903
• 关键词: Ablation; Administrative Supplement; 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; Parents; Peripheral; Positioning Attribute; Process; Proteins; Regulation; Reporter; Spontaneous abortion; Transgenic Mice; developmental disease; egg; male; migration; mouse model; novel; spatiotemporal; sperm cell

Administrative Supplement for R35 GM142537 (PD/PI: Balboula): “Mechanisms underpinning meiotic spindle formation and behavior” From Parent R35 GM142537 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.

R35 GM 142537（PD/PI：Balboula）的管理补充：“减数分裂的基础机制 纺锤体的形成和行为” 来自母体R35 GM 142537 项目概要： 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，双极纺锤体的形成和 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微管（MT） 组织中心（MTOC），通过基本未知的机制组织起来，建立两个主轴极 （极性MTOC）。传统观点认为，在哺乳动物卵母细胞中，MT（及其相关蛋白质） 负责组织这种MTOC纺锤体的唯一细胞骨架组分。然而，最近的数据显示， F-肌动蛋白也参与纺锤体双极性调节。F-actin如何与MT相互作用以调节极性 MTOC组织在MI代表了一个关键的差距，在我们的理解如何减数分裂纺锤体建成。 我们最近发现了一类新型、功能不同的MTOCs（mcMTOCs），并发现纺锤体 卵母细胞中心的维持受两种相反的力量（mcMTOC介导的MT与F-肌动蛋白）调节。我们 最近还观察到，约50%的锭子不是集中组装的。迄今为止，这种外围主轴 由于与纺锤体荧光相关的技术限制（即活的 成像）。为了避免这一点，我们产生了Cep 192-eGfp报告小鼠模型， 无论在哪里，引人注目的是，外周纺锤体形成之后通常是纺锤体迁移 一种以前没有记录的现象。了解分子机制 调节这种纠正性发育事件代表了我们对减数分裂纺锤体知识的一个主要空白 心肌梗死时的时空调节这一提议为我们的长期目标奠定了基础： 如何在MI期间调节两个关键事件-双极主轴组装和定位， 以确保染色体的分离。为此，我们将利用最先进的方法， 包括转基因小鼠模型、基因构建、激光消融和尖端成像， 关键目标：（i）确定F-actin如何与MT相互作用，以在双极纺锤体期间组织极性MTOC 建立，（ii）建立外周无中心纺锤体迁移到卵母细胞中心的机制， 和（iii）确定mcMTOC与极性MTOC的生化组成的差异是否是其 功能差异。考虑到染色体分离错误（在MI期间非常常见）导致非整倍体， 发育障碍和流产的主要遗传原因，这些研究有可能 大大提高了我们对两个基本过程--主轴形成和定位--的基本理解 - 同时阐明为什么MI是众所周知的容易出错的。