Novel reverse genetics approach to probe cytoskeletal functions in mammalian oocytes

项目概要 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发挥着至关重要的作用，包括 减数分裂纺锤体的定位和不对称细胞分裂。卵母细胞中的这些事件对于 生殖成功，因为损害这些过程的基因敲除会导致女性不孕。整体 这里提出的研究目标是分析假设的调节功能的关键蛋白质的作用 雌性减数分裂期间肌动蛋白细胞骨架的变化。这里要追求的假设是基于我们的 已发布的数据以及此处提供的新的未发布数据。该项目的第二个目标是技术 一 - 为后期的新型系统的实用性提供有价值的原理验证演示 翻译蛋白消耗，作为卵母细胞生物学研究界的资产。标准型 用于卵母细胞的蛋白质消耗方法（即敲除、敲低）相对较少 感兴趣目标耗尽的时间精度。相反，请考虑以下示例，来自 芽殖酵母的研究。通过无效突变慢性消耗蛋白质只会导致生长缓慢 表型，而减数分裂特定阶段的耗竭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。这个 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进展的新见解。我们在这里表明我们有 AID 系统在小鼠卵母细胞中启动并运行。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（显性失活 [DN] 或组成型活性 [CA]） 卵母细胞扰乱特定途径的功能。 AID 系统允许此功能退化- 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变为野生型 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 II 的结论。因此，这个项目的价值不仅在于它所提供的发现，而且在于它所提供的发现。 还用于演示 AID 系统在减数分裂多个阶段的实用性。目标 1 聚焦主轴 定位于减数分裂 I，并将通过确定肌动蛋白解聚蛋白丝切蛋白的作用 研究何时何地需要 cofilin 来进行主轴定位。目标 2 检查减数分裂 I，中期 II 停滞，并完成减数分裂 II，并将检验肌动蛋白不适当活性的假设 称为 ERM 的膜连接蛋白家族会损害纺锤体功能和极体发射。采取 总之，这些研究将为增进对这些关键作用的理解提供重要的见解。 卵母细胞中的依赖性过程，包括导致体内模型中的未来工作。

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 The hypotheses to be pursued here are based on our published data and on new unpublished data presented here.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