Actin-based mechanisms of chromosome segregation in mammalian oocytes and embryos

项目摘要 染色体分离是由一个纺锤体机制驱动的， 子细胞之间的基因组。在卵子和它们的祖卵母细胞中， 专门的减数分裂程序。卵母细胞和卵子非常容易受到染色体 导致胚胎非整倍体的分离错误，这是自然流产的主要原因 和发育障碍。胚胎非整倍体仍然可以出现，即使在无误完成后 减数分裂染色体分离。然而，我们对胚胎特异性的潜在原因的理解 非整倍性的研究受到仅关注减数分裂衍生的非整倍性的倾向的限制。 直到最近，人们才相信微管是细胞骨架的唯一组成部分 染色体分离所必需的。我们发现纺锤体成功地挑战了这一观点 卵母细胞和卵子中促进染色体-纺锤体附着和防止非整倍体的肌动蛋白。怎么样 纺锤体F-actin的组装以及它如何在染色体-微管界面发挥其功能？ 这些都是我们对细胞分裂理解的范式转变所提出的悬而未决的问题。 我的实验室的一个主要目标是了解保护准确的 哺乳动物卵母细胞和胚胎中的染色体分离。我们发现纺锤体F-肌动蛋白 构成了这样一种保护机制，我们正在结合先进的显微镜检测， 快速蛋白质降解工具，以确定纺锤体F-肌动蛋白组装和功能所需的蛋白质， 卵母细胞这种方法揭示了控制卵母细胞的关键肌动蛋白和微管结合蛋白 染色体分离，其中一些独立地涉及零星流产， 在最近的不孕症患者的遗传研究发育障碍。我们建议以此为基础， 通过1）扩大我们的候选人为基础的快速 蛋白质降解筛选出更大的肌动蛋白-微管串扰蛋白亚群，和2）开发 一个新的生物化学和蛋白质组学耦合的实验管道，用于无偏见地鉴定新的 卵母细胞和胚胎中的纺锤体F-actin组装蛋白。此外，我们将采取直接实验 使用显微注射和激光显微手术工具添加或去除中心体的方法， 解决为什么纺锤形的F-肌动蛋白结构是唯一的acentrosomal纺锤体。小鼠早期胚胎 没有典型中心体的有丝分裂，将为我们提供一个有吸引力的 一个实验模型来回答这个长期存在的细胞生物学问题。 当这项研究完成后，我们将发现并功能性地描述 卵母细胞和胚胎中的纺锤体F-actin组装蛋白。总的来说，这项研究将揭示 细胞骨架系统在早期发育中协同驱动精确的染色体分离。

PROJECT SUMMARY Chromosome segregation is driven by a spindle machinery that distributes copies of the genome between daughter cells. In eggs and their progenitor oocytes, chromosomes are segregated in a specialized meiotic division program. Oocytes and eggs are remarkably vulnerable to chromosome segregation errors that give rise to aneuploidy in embryos, a leading cause of spontaneous miscarriages and developmental disorders. Embryo aneuploidy can nonetheless arise even after error-free completion of meiotic chromosome segregation. However, our understanding of the underlying causes of embryo-specific aneuploidies has been restricted by a tendency to focus only on meiosis-derived aneuploidies. Until recently, it was believed that microtubules are the only cytoskeletal components required for chromosome segregation. This view was successfully challenged by our discovery of spindle F-actin in oocytes and eggs that boost chromosome-spindle attachments and prevent aneuploidy. How is spindle F-actin assembled and how does it exert its function at the chromosome-microtubule interface? These are among outstanding questions raised by this paradigm shift in our understanding of cell division. A major goal of my lab is to understand the mechanisms that safeguard accurate chromosome segregation in mammalian oocytes and embryos. Driven by our discovery that spindle F-actin constitutes one such protection mechanism, we are combining advanced microscopy assays with rapid protein degradation tools to identify proteins required for spindle F-actin assembly and function in oocytes. This approach has revealed key actin- and microtubule-binding proteins that govern oocyte chromosome segregation, some of which were independently implicated in sporadic miscarriages and developmental disorders in recent genetic studies of infertility patients. We propose to build on this progress and study the origins of embryo-specific aneuploidy by 1) expanding our candidate-based rapid protein degradation screens to a larger subset of actin-microtubule crosstalk proteins, and 2) developing a new biochemical and proteomics-coupled experimental pipeline for unbiased identification of novel spindle F-actin assembly proteins in oocytes and embryos. Furthermore, we will take direct experimental approaches of adding or removing centrosomes using microinjection and laser microsurgery tools to address why spindle-shaped F-actin structures are unique to acentrosomal spindles. Early mouse embryo mitotic divisions, which are executed without canonical centrosomes, will provide us with an attractive experimental model in which to answer this long-standing question in cell biology. When this research is completed, we will have discovered and functionally characterized spindle F-actin assembly proteins in oocytes and embryos. Overall, this study will reveal how distinct cytoskeletal systems cooperate to drive accurate chromosome segregation in early development.