Novel reverse genetics approach to probe cytoskeletal functions in mammalian oocytes

项目摘要 基于肌动蛋白的细胞分裂过程在哺乳动物卵母细胞减数分裂中起关键作用，包括 减数分裂纺锤体的定位和不对称的细胞分裂。卵母细胞中的这些事件对于 生殖成功，因为基因敲除会损害这些过程，导致女性不育。整体 本文提出的研究目标是分析假设调节功能的关键蛋白质的作用 肌动蛋白细胞骨架在女性减数分裂。这里要追求的假设是基于我们的 公布的数据和新的未公布的数据在这里。该项目的第二个目标是技术 一个-提供有价值的原理证明，证明一个新的系统的实用性， 翻译蛋白质消耗，作为卵母细胞生物学研究社区的资产。标准 用于卵母细胞的蛋白质消耗方法（即，击倒，击倒）相对较少 用于感兴趣的靶的耗尽的时间精度。相反，考虑以下示例，从 研究芽殖酵母。通过无效突变的蛋白质的慢性消耗仅仅产生了缓慢的生长 表型，而在减数分裂的特定阶段的消耗提供了更清晰的见解， 更具体和有趣的表型。这就是这里提出的项目的灵感。这 研究将使用同样的创新方法-生长素诱导降解（AID）系统-获得 哺乳动物卵母细胞减数分裂进程的新见解。我们在这里展示了 小鼠卵母细胞中的AID系统启动和运行。这些研究的总体概念是表达功能- 改变野生型中感兴趣的蛋白质的变体（显性阴性[DN]或组成型活性[CA]） 卵母细胞干扰特定途径的功能。AID系统允许降低这一功能- 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]， 早期M期、晚期M期等）。本质上，这将卵母细胞从突变状态转化为野生型 在我们选择的时间，允许评估蛋白质功能的时间特异性。我们将使用 AID系统分析卵母细胞减数分裂的不同阶段，从进入减数分裂I的M期到减数分裂的第二阶段。 减数分裂结束II。因此，该项目的价值不仅在于它将提供的发现，而且 也用于证明AID系统在减数分裂的多个阶段的效用。Aim 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cofilin的作用，通过 研究何时何地需要cofilin用于主轴定位。目的2检查减数分裂I，中期 II停滞，并完成减数分裂II，并将测试的假设，不适当的活动的肌动蛋白- 称为ERM的膜连接蛋白家族损害纺锤体功能和极体发射。采取 总之，这些研究将提供重要的见解，以促进对这些关键肌动蛋白的理解， 卵母细胞中的依赖过程，包括导致未来的工作在体内模型。

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 The hypotheses to be pursued here are based on our published data and on new unpublished data presented here.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.