Identifying and characterizing genes required for the meiosis-to-mitosis transition

在受精期间，精子传递一个信号，指示卵子完成雌性减数分裂，并过渡到有丝分裂细胞分裂，从而构建新的有机体。然而，目前还不清楚哪些分子成分参与其中，以及它们如何指定正确的细胞分裂。这一建议集中在我们发现的一条分子途径上，这是线虫从减数分裂到有丝分裂转变所必需的。我们的方法确定了卵母细胞和精子的特定成分，现在我们准备利用我们的系统来进一步了解这一关键途径。我们可以在线虫身上进行实验，这是不可能在人类或其他哺乳动物身上做的。受精、减数分裂和第一次有丝分裂都发生在一小时内，所有过程都可以在子宫内用高分辨率活细胞显微镜观察到。每个阶段的具体参数都可以精确地量化。此外，通过突变或RNA干扰(RNAi)可以有效地改变单个基因的活性，以测试发育中的有机体内的影响。 利用NSERC-DG Funds，我们发现了3个高度相似的基因，Memi-1，Memi-2和Memi-3，它们编码减数分裂II(MII)所需的卵母细胞因子。Memi蛋白在减数分裂早期存在，但在MII开始后不久就被降解，在有丝分裂胚胎中检测不到。所有这三个基因的缺失会导致受精卵终止减数分裂I并提前进入有丝分裂(即它们跳过了MII)。这与静止的精子无法使卵母细胞受精时发生的情况非常相似。因此，我们假设MEMI“感觉”到精子进入。对我们有利的是，我们有一个多态突变，Memi-1(Sb41)，它会导致MEMI活性“过剩”。SB41干扰MEMI-1的降解，导致胚胎无法从MII正确过渡到有丝分裂。利用基于RNAi的Memi-1(Sb41)抑制子筛选，我们发现了精子PP1磷酸酶，它是Memi的遗传激活剂，是触发Memi的难以捉摸的精子信号的理想候选者。 在最近使用EMS突变的Memi-1(Sb41)抑制子的中试筛选中，我们恢复了相同的PP1磷酸酶，以及许多新的基因。为了进一步了解MEMI途径，我们将： 1)鉴定和鉴定EMS诱导的新的Memi-1(Sb41)抑制子。 2)确定MEMI蛋白的翻译后修饰，假设其发生在减数分裂到有丝分裂的转变过程中。 3)用邻近连接和酵母双杂交分析MEMI的物理相互作用。 尽管对协调受精胚胎复杂细胞分裂的机制知之甚少，但我们提出的目标有可能揭示核心成分及其分子作用。发现的每一个新基因都将为研究生和本科生的独立研究项目提供理想的培训机会。

During fertilization, sperm deliver a signal that instructs the egg to complete female meiosis and transition to mitotic cell divisions that build the new organism. However, it is unclear which molecular components are involved and how they specify the correct cell divisions. This proposal focuses on a molecular pathway that we discovered, which is required for the meiosis-to-mitosis transition in the nematode C. elegans. Our approach identified oocyte- and sperm-specific components, and we are now poised to exploit our system to further understand this critical pathway. We can perform experiments on C. elegans that would be impossible to do in humans or other mammals. Fertilization, the meiotic divisions, and the first mitotic division all occur within an hour, and all processes can be viewed in utero with high-resolution live-cell microscopy. Specific parameters of each stage can be quantified with precision. Furthermore, the activity of individual genes can be efficiently altered by mutation or RNA interference (RNAi) to assay the effects within a developing organism. Using NSERC-DG funds, we discovered 3 highly-similar genes, memi-1, memi-2, and memi-3, which encode oocyte factors required for meiosis II (MII). MEMI proteins are present during early meiosis, but they are degraded shortly after MII begins, and are undetected in mitotic embryos. Loss of all three genes results in fertilized embryos that abort meiosis I and enter mitosis prematurely (i.e., they skip MII). This is very similar to what happens when immotile sperm cannot fertilize the oocyte. Thus, we hypothesize that MEMI “senses” sperm entry. To our advantage, we have a hypermorphic mutation, memi-1(sb41), which results in “excess” MEMI activity. sb41 interferes with MEMI-1 degradation, resulting in embryos that fail to transition from MII to mitosis properly. Using an RNAi-based suppressor screen of memi-1(sb41), we discovered a sperm PP1 phosphatase, which is a genetic activator of memi and ideal candidate for the elusive sperm signal that triggers MII. In a recent pilot screen for memi-1(sb41) suppressors using EMS-mutagenesis, we recovered the same PP1 phosphatase, as well as many new genes. To further understand the MEMI pathway, we will: 1) identify and characterize the new EMS-induced suppressors of memi-1(sb41). 2) identify post-translational modifications in MEMI proteins that are hypothesized to occur during the meiosis-to-mitosis transition. 3) analyze MEMI physical interactions using proximity ligation and yeast 2-hybrid assays. Although there is limited knowledge of the mechanisms that coordinate the complex cell divisions characteristic of fertilized embryos, our proposed aims have a potential to reveal core components and their molecular roles. Each of the new genes identified will provide ideal training opportunities for independent research projects, at both the graduate and undergraduate level.