Novel reverse genetics approach to probe cytoskeletal functions in mammalian oocytes

探测哺乳动物卵母细胞细胞骨架功能的新型反向遗传学方法

• 批准号: 10018066
• 负责人: JANICE P EVANS
• 金额: $ 7.75万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-13 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10018066
• 关键词: Actins; Address; Auxins; Biology; Breathing; Cell Division Process; Cell division; Cells; Chronic; Communities; Complex; Cullin Proteins; Cytoskeleton; Data; Dominant-Negative Mutation; Embryonic Development; Event; Experimental Designs; F-Box Proteins; Family member; Female; Female infertility; Future; Germ Cells; Goals; Growth; Haploidy; Impairment; Investigation; Knock-out; Knowledge; Ligase; Mediating; Meiosis; Membrane; Metaphase; Methods; Mitosis; Molecular; Mus; Nuclear Envelope; Oocytes; Paper; Pathway interactions; Phenotype; Plant Growth Regulators; Plants; Play; Positioning Attribute; Preparation; Process; Protein Family; Proteins; Publishing; Research; Role; Running; Saccharomycetales; Specificity; System; Testing; Time; Ubiquitin; Variant; Work; actin depolymerizing factor; actin depolymerizing proteins; base; cofilin; egg; experimental study; ezrin; genetic approach; in vivo Model; innovation; insight; interest; knock-down; knockout gene; moesin; multicatalytic endopeptidase complex; mutant; novel; novel strategies; null mutation; oocyte maturation; p19(SKP1) Protein; protein degradation; protein function; public health relevance; radixin protein; reproductive success; reverse genetics; sperm cell; tool; ubiquitin ligase; zygote

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 The hypotheses to be pursued here are based on our published data and on new unpublished data presented here.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

项目摘要 基于肌动蛋白细胞骨架的过程在减数分裂中在哺乳动物卵母细胞中起关键作用，包括 减数分裂主轴和不对称细胞分裂的定位。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导致女性不育症。总体 这里提出的研究的目标是分析假设调节功能的关键蛋白的作用 女性减数分裂过程中肌动蛋白细胞骨架的。这里要提出的假设是基于我们的 发布的数据以及此处介绍的新的未发表数据。该项目的第二个目标是技术 一个 - 提供有价值的原则证明，以证明新型系统的实用性 翻译蛋白耗竭，作为卵母细胞生物学研究界的资产。标准 卵母细胞中使用的蛋白质耗竭方法（即敲除，敲除）的方法相对较少 部署目标目标的临时精度。相比之下，请考虑以下示例 研究萌芽的酵母。通过无效突变对蛋白质的慢性消耗仅产生缓慢的生长 表型，而在减数分裂的特定阶段的耗竭为大量的见解提供了很多 更具体而有趣的表型。这是这里提出的项目的灵感。 研究将使用相同的创新方法 - 生长素诱导的降解（AID）系统 - 获得 通过减数分裂的哺乳动物卵母细胞进展的新见解。我们在这里表明我们有 辅助系统在鼠标卵母细胞中启动和运行。这些研究的总体概念是表达功能 - 改变感兴趣的蛋白质的变体（在野生型中显性阴性[DN]或组成型活性[Ca]） 卵母细胞到特定途径的扰动功能。辅助系统允许降解此功能 - 在减数分裂成熟的不同时间改变蛋白质（例如，M相进入[核包膜分解]， 早期的M期，晚期M相等）。本质上，这将卵母细胞从突变状态转化为野生型 在我们选择的时候，允许评估蛋白质功能的临时特异性。我们将使用 分析卵母细胞减数分裂的不同阶段的援助系统，从进入减数分裂I到 减数分裂II的结论。那个项目不仅对于它将提供的发现很有价值，而且将是有价值的 还用于在减数分裂的多个阶段展示援助系统实用程序。 AIM 1专注于主轴 在减数分裂I中定位，并将确定肌动蛋白 - 脱聚合蛋白Cofilin通过 对纺锤体定位需要何时何地需要的何时何地进行研究。 AIM 2考试减数分裂I，中期 II逮捕和减数分裂II的逮捕和完成，并将检验以下假设：肌动蛋白到肌动蛋白的活性不适当 被称为ERMS的膜接头蛋白家族会损害主轴功能和极性体发射。拍摄 这些研究将共同​​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，包括导致未来的体内模型工作。