Novel reverse genetics approach to probe cytoskeletal functions in mammalian oocytes

项目总结 基于肌动蛋白细胞骨架的过程在哺乳动物卵母细胞的减数分裂中起着至关重要的作用，包括 减数分裂纺锤体的定位和细胞的不对称分裂。卵母细胞中的这些事件对于 生殖成功，因为破坏这些过程的基因敲除会导致女性不育。整体而言 这项研究的目的是分析被假设为调节功能的关键蛋白质的作用。 在雌性减数分裂过程中肌动蛋白细胞骨架的变化。这里将要进行的假设是基于我们的 已发布的数据和此处提供的新的未发布的数据。这个项目的第二个目标是技术上的 一是提供有价值的原则证明，证明一种新的邮寄系统的实用性。 翻译蛋白耗竭，作为卵母细胞生物学研究社区的一项资产。标准 用于卵母细胞的蛋白质耗竭方法(即基因敲除、基因敲除)相对较少。 目标耗尽的时间精度。相比之下，请考虑下面的示例，其中 芽殖酵母的研究。蛋白质通过零突变的慢性耗尽只会产生缓慢的生长 表型，而减数分裂特定阶段的耗竭提供了更敏锐的洞察力 更具体和有趣的表型。这就是这里提出的项目的灵感来源。这 研究将使用同样的创新方法-生长素诱导降解(AID)系统-来获得 对哺乳动物卵母细胞减数分裂过程的新见解。我们在这里展示了我们拥有 小鼠卵母细胞中辅助系统的启动和运行。这些研究的总体概念是表达函数- 改变野生型中感兴趣的蛋白质的变体(显性负性[dN]或结构性活性[CA]) 卵母细胞扰乱特定途径的功能。AID系统允许该功能的降级- 在减数分裂成熟的不同时间改变蛋白质(例如，M期进入[核膜破裂]， 早M期、晚M期等)。从本质上讲，这将卵母细胞从突变状态转变为野生型 在我们选择的时间，允许在评估蛋白质功能时具有时间特异性。我们将使用 用于分析卵母细胞减数分裂不同阶段的AID系统，从进入减数分裂I的M期到 减数分裂的结论二。因此，这个项目不仅有价值，因为它将提供的发现，而且 也用于演示AID系统在减数分裂多个阶段的实用性。目标1专注于主轴 定位在减数分裂I，并将确定肌动蛋白解聚蛋白的作用通过 研究何时何地需要胶原蛋白来定位主轴。目的2检查减数分裂I、中期 II停止，并完成减数分裂II，并将检验以下假设：不适当的肌动蛋白到- 膜连接蛋白家族被称为ERMS，会损害纺锤体功能和极体发射。已被占用 总之，这些研究将为促进对这些关键肌动蛋白的理解提供重要的见解。 卵母细胞中的依赖过程，包括在活体模型中的未来工作。

PROJECT SUMMARY Actin cytoskeleton-based processes play crucial roles in meiosis in mammalian oocytes, including positioning of the meiotic spindle and asymmetric cell division. These events in oocytes are essential for reproductive success, as gene knockouts that impair these processes cause female infertility. The overall goal of the research proposed here is to analyze the roles of key proteins hypothesized to modulate function of the actin cytoskeleton during female meiosis. The hypotheses to be pursued here are based on our published data and on new unpublished data presented here. A second goal of this project is a technical one – to provide valuable proof-of-principle demonstration of the utility of a novel system for post- translational protein depletion, as an asset to the oocyte biology research community. Standard approaches for protein depletion that are used in oocytes (i.e., knockout, knockdown) have relatively little temporal precision for depletion of the target of interest. In contrast, consider the following example, from study in budding yeast. Chronic depletion of a protein via a null mutation merely produced a slow growth phenotype, whereas depletion at a specific stage of meiosis provided much sharper insights with a much more specific and interesting phenotype. This is the inspiration for the project proposed here. This research will use this same innovative approach – the auxin-inducible degradation (AID) system – to gain new insights into the mammalian oocyte's progression through meiosis. We show here that we have the AID system up and running in mouse oocytes. The overall concept for these studies is to express function- altering variants of proteins of interest (dominant-negative [DN] or constitutively-active [CA]) in wild-type oocytes to perturb function of a particular pathway. The AID system allows for degradation of this function- altering protein at different times of meiotic maturation (e.g., M-phase entry [nuclear envelope breakdown], early M-phase, late M-phase, etc.). In essence, this converts oocytes from a mutant state to wild-type at a time of our choosing, allowing temporal specificity in assessing protein function. We will use the AID system to analyze different stages of oocyte meiosis, from entry into M-phase of meiosis I to the conclusion of meiosis II. Thus, this project will be valuable not only for the discoveries it will provide, but also for the demonstration of AID system utility at multiple stages of meiosis. Aim 1 focusses on spindle positioning in meiosis I, and will determine the role of the actin-depolymerization protein cofilin through studies of when and where cofilin is required for spindle positioning. Aim 2 examines meiosis I, metaphase II arrest, and completion of meiosis II, and will test the hypothesis that inappropriate activity of the actin-to- membrane linker protein family known as ERMs impairs spindle function and polar body emission. Taken together, these studies will provide important insights to advance understanding of these crucial actin- dependent processes in oocytes, including leading to future work in in vivo models.