Mechanisms of Nucleolar Stress in Drosophila

核糖体是我们细胞内的纳米机器，它读取由mRNAs携带的遗传密码，合成所有维持细胞存活和功能的蛋白质。细胞中核糖体产生的失败(称为核仁应激)对细胞和生物体的健康有深远的影响。重要的是，并不是所有的细胞都是平等的，不同的干细胞和祖细胞对核仁压力表现出不同的敏感性：虽然一些干细胞和祖细胞对核仁压力表现出强烈的敏感性，并因细胞程序性死亡而死亡，但许多其他干细胞和祖细胞似乎对核仁压力具有弹性。问题是，为什么一些干细胞和祖细胞对核仁压力敏感，而另一些则不敏感？这个项目将使用果蝇，黑腹果蝇。该项目测试了一种假设，即发育中的幼虫大脑中不同的果蝇神经母细胞(干细胞)对核仁压力表现出不同的敏感性。初步数据显示，“蘑菇体”神经母细胞比大多数其他神经母细胞对核仁压力更具弹性。蘑菇体是昆虫大脑的一部分，对昆虫的嗅觉和学习至关重要。研究人员通过耗尽核糖体生物生成因子，一种名为Nopp140的蛋白质，在果蝇体内诱导核仁应激。这项研究确定了核仁应激的机制，因为它干扰了细胞的活性和功能，特别是在神经发生期间。这一点至关重要，因为最近的证据表明，核糖体生物发生失败是导致神经疾病衰弱的一个因素。更广泛的影响将向地区高中代表不足的学生介绍遗传学和细胞生物学的基本概念，为代表不足的本科生提供实践研究培训的机会，并培训研究生高级遗传学和细胞生物学。不同的干细胞和祖细胞对核仁压力表现出不同的敏感性。哺乳动物糖浆是一种核仁核糖体生物发生因子(RBF)，它的单倍体缺失突变会耗尽选定的胚胎神经脊细胞中的核糖体，这些细胞会因细胞凋亡而死亡，从而导致头面部出生缺陷。胚胎内的大多数其他胚胎干细胞和祖细胞似乎对这种核仁压力具有弹性。问题是，为什么对RBF中的系统性突变会有不同的反应？该项目将使用果蝇作为模型系统来回答这个问题。核仁应激将通过耗尽Nopp140来诱导，Nopp140是一种在结构和功能上与糖蜜相关的核仁RBF。目的1验证果蝇幼虫脑内的蘑菇体(MB)神经母细胞对核仁应激具有弹性的假说，这是因为它们在持续增殖期间通过翻译母体转录来储存RBF，而大多数其他神经母细胞通常进入静止期。目的1在野生型和Nopp140-幼虫脑中，使用镶嵌分析来确定RBF对MB神经母细胞的母体贡献与其他神经母细胞相比。目的1B利用相对光镜和电子显微镜(CLEM)检测野生型和Nopp140缺失背景下MB神经母细胞与其他神经母细胞相比的RBF和核糖体丰度。目的2验证JNK是后生动物核仁应激效应的中枢机制。果蝇JNK对各种形式的应激做出反应，初步数据表明，果蝇的JNK在核仁应激时被激活。然而，JNK的上游激活物对核仁应激的作用尚不清楚。该项目通过使用RNAi在Nopp140空(核仁应激)背景中耗尽已知的上游JNK激活子，然后在失去JNK激活时对挽救的核仁应激表型进行评分来识别这些激活子。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Ribosomes are nano-machines within our cells that read the genetic code, carried by mRNAs, to synthesize all proteins that keep our cells alive and functional. Failures in ribosome production in cells (referred to as nucleolar stress) have far-reaching effects on the well-being of cells and the organism. Importantly, not all cells are equal and various stem cells and progenitor cells show differential sensitivities to nucleolar stress: while some stem cells and progenitor cells display acute sensitivities to nucleolar stress and die by programed cell death, many other stem cells and progenitor cells seem to be resilient to nucleolar stress. The question is, why are some stem cells and progenitor cells sensitive to nucleolar stress, while others are not? This project will use the fruit fly, Drosophila melanogaster. The project tests the hypothesis that different Drosophila neuroblasts (stem cells) in the developing larval brain display differential sensitivities to nucleolar stress. Preliminary data show that “mushroom body” neuroblasts are more resilient to nucleolar stress than are most other neuroblasts. Mushroom bodies are portions of the insect brain essential for olfaction and learning in insects. The investigators induce nucleolar stress in Drosophila by depleting the ribosome biogenesis factor, a protein called Nopp140. The research determines the mechanisms of nucleolar stress as it interferes with cell viability and function, especially during neurogenesis. This is critical, as recent evidence suggests that failure in ribosome biogenesis is a contributing factor in debilitating neurological diseases. Broader impacts will introduce basic concepts in genetics and cell biology to underrepresented students in area high schools, provide an opportunity for hands-on research training for underrepresented undergraduates, and train graduate students in advanced genetics and cell biology. Various stem cells and progenitor cells show differential sensitivities toward nucleolar stress. Haplo-insufficiency mutations in mammalian treacle, a nucleolar ribosome biogenesis factor (RBF), deplete ribosomes in select embryonic neural crest cells that die by apoptosis leading to craniofacial birth defects. Most other embryonic stem cells and progenitor cells within the embryo appear resilient to this nucleolar stress. The question is, why are there differential responses to a systemic mutation in an RBF? The project will use Drosophila as a model system to answer this question. Nucleolar stress will be induced by depleting Nopp140, a nucleolar RBF structurally and functionally related to treacle. Aim 1 tests the hypothesis that mushroom body (MB) neuroblasts within the Drosophila larval brain are resilient to nucleolar stress because they stockpile RBFs by translation of maternal transcripts during a period of continued proliferation, while most other neuroblasts normally enter a period of quiescence. Aim 1A uses mosaic analyses to determine maternal contributions of RBFs to MB neuroblasts versus other neuroblasts in wild type and Nopp140-/- larval brains. Aim 1B uses co-relative light and electron microscopy (CLEM) to assess RBF and ribosome abundances in MB neuroblasts versus other neuroblasts in wild type and null Nopp140 genetic backgrounds. Aim 2 tests the mechanism that JNK is the central nucleolar stress effector in metazoans. Drosophila JNK responds to various forms of stress, and preliminary data shows that JNK is activated upon nucleolar stress in Drosophila. However, the upstream activators of JNK upon nucleolar stress remain unknown. This project identifies these activators by using RNAi to deplete known upstream JNK activators in the Nopp140 null (nucleolar stress) background, and then scores for rescued nucleolar stress phenotypes upon loss of JNK activation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.