The role of SPE-11 in C. elegans egg activation

为了确定spe-11突变胚胎的主要缺陷，我们进行了详细的表型分析。鉴于SPE-11的末端表型是一个圆形的、脆弱的1-细胞胚胎，我们已经检查了两种蛋壳完整性的诊断方法。首先，我们已经证明了spe-11(Hc90)突变胚胎是渗透敏感的，这表明蛋壳内层发生了破坏，从而提供了渗透屏障。其次，在这些突变体中，蛋壳的几丁质层是有缺陷的。在SPE-11(Hc90)缺失型胚胎中，甲壳素仅在胚胎表面的限制性新月体中观察到，这与野生型胚胎不同，在野生型胚胎中，甲壳素存在于胚胎周围。作为早期胚胎发生的另一个标志，我们研究了被称为皮质颗粒的细胞内小泡的运输，这些小泡在卵子激活过程中经历了一种特征性的易位。SPE-11突变体在依赖细胞周期的皮质颗粒运动过程中不受影响，因为皮质颗粒的标志物CAV-1：：GFP在SPE-11突变体中表现出正常的运输。我们还询问了在没有SPE-11的情况下，其他卵子激活基因的定位是否正常。在spe-11(Hc90)突变体中受精后，Egg-3：：GFP通常被定位。因此，我们在spe-11突变体中检测到的最早缺陷是蛋壳形成。我们正在继续使用与GFP或mCherry融合的额外皮质和蛋壳标记进行这种类型的分析。这一分析应该有助于确定早期发育的哪些方面受到SPE-11缺乏的干扰，以及哪些功能独立于SPE-11。 在过去，我们进行了非互补筛选，以恢复Spe-11的一个强温度敏感等位基因，该等位基因可用于遗传抑制筛选。到目前为止，我们已经恢复了Spe-11的两个新等位基因，Spe-11(av33和av34)。两者都被发现是产生截短蛋白产物的非条件等位基因，与大多数现有的spe-11等位基因一样。这些新等位基因的行为类似于参考等位基因，因此我们认为它们也是零等位基因。我们正在继续进行这一筛选，希望识别出可以用于抑制子筛选的Spe-11的温度敏感等位基因。或者，我们希望至少识别一个错义等位基因，这也是一个很好的抑制者筛查候选。由于这一筛选表明存在非等位非互补等位基因，我们计划在未来分离这些增强子等位基因，因为它们可能对spe-11发挥作用的遗传途径提供相当多的信息。 我们还使用spe-11、spe-11(Bn65)的截断等位基因启动了遗传抑制基因的筛选。Spe-11的这个等位基因编码80%的全长蛋白质。我们预计，这一筛选将分离SPE-11的显性和隐性旁路抑制因子，这可能会使卵母细胞对激活更加敏感，或者促进SPE-11的定位或功能。 我们还获得了一个在内源spe-11启动子和3UTR的作用下表达GFP：spe-11的转基因株系。这条线在成熟精子中非常明亮地表达GFP：SPE-11，并将被用于进行显微镜研究，以确定受精后SPE-11蛋白的命运。此外，我们正在使用CBD-1和CPG-1的翻译融合构建在体内表达蛋壳荧光(GFP)标记的转基因动物。这些转基因将为评估蛋壳的细微变化提供重要的工具，这将对我们研究SPE-11在蛋壳形成中的作用非常有用。 除了上述策略外，我们还在进行酵母双杂交筛选以寻找SPE-11相互作用子，并通过询问哪些变体能够挽救空来对SPE-11变体进行结构功能分析。

To identify the primary defect in the spe-11 mutant embryos, we have undertaken a detailed phenotypic analysis. Given that the spe-11 terminal phenotype is a round, fragile 1-cell embryo, we have examined two diagnostics of eggshell integrity. First, we have shown that spe-11(hc90) mutant embryos are osmotically sensitive, indicating a disruption in the inner layer of the eggshell, which confers the osmotic barrier. Second, the chitin layer of the eggshell is defective in these mutants. Chitin is observed only in a restricted crescent at the surface of the embryo in the null spe-11(hc90) embryos, in contrast to wild type embryos where chitin is present around the periphery of the embryo. As an additional marker of early embryogenesis, we have investigated the trafficking of intracellular vesicles called cortical granules, which undergo a characteristic translocation during egg activation. The spe-11 mutants are not compromised in the cell cycle dependent process of cortical granule movement because CAV-1::GFP, a marker of cortical granules, exhibits normal trafficking in spe-11 mutants. We also asked if the localization of other egg activation genes was normal in the absence of SPE-11. EGG-3::GFP is normally localized following fertilization in spe-11(hc90) mutants. Thus, the earliest defects we have detected in spe-11 mutants are in eggshell formation. We are continuing this type of analysis using additional cortical and eggshell markers fused to GFP or mCherry. This analysis should help determine which aspects of early development are perturbed by the lack of SPE-11 and which function independently of SPE-11. In the past, we performed a non-complementation screen in order to recover a strong temperature-sensitive allele of spe-11 that could be used for a genetic suppressor screen. Thus far, we have recovered two new alleles of spe-11, spe-11(av33 and av34). Both were found to be a non-conditional alleles that produce truncated protein products, like most of the existing spe-11 alleles. These new alleles behave like the reference allele and thus we believe they too are null alleles. We are continuing this screen in hopes of identifying a temperature-sensitive allele of spe-11 that can be used in a suppressor screen. Alternatively, we hope to at least identify a missense allele, which would also be a good candidate for a suppressor screen. Because this screen has suggested the existence of non-allelic non-complementing alleles, we are planning to isolate these enhancer alleles in the future as they may be quite informative as to the genetic pathways in which spe-11 functions. We have also initiated a genetic suppressor screen using a truncation allele of spe-11, spe-11(bn65). This allele of spe-11 encodes 80% of the full protein length. We anticipate that this screen will isolate dominant and recessive bypass suppressors of spe-11, perhaps that will make the oocyte more sensitive to activation or that facilitate SPE-11 localization or function. We have also generated a transgenic line that expresses GFP:SPE-11 under the endogenous spe-11 promoter and 3UTR. This line expresses GFP:SPE-11 very brightly in the mature sperm and will be used to perform microscopic studies to determine the fate of the SPE-11 protein following fertilization. In addition, we are constructing transgenic animals expressing fluorescent (GFP) in vivo markers of the eggshell using cbd-1 and cpg-1 translational fusions. These transgenics will provide important tools for evaluating the subtle alterations of the eggshell, which will be of great utility in our investigations of the role of SPE-11 in eggshell formation. In addition to the above strategies, we are also performing yeast two hybrid screens to find SPE-11 interactors and are performing structure-function assays on spe-11 variants by asking which variants are capable of rescuing a null.