Mechanisms of Nucleolar Stress in Drosophila

核糖体是我们细胞内的纳米机器，它读取由mRNA携带的遗传密码，以合成保持细胞存活和功能的所有蛋白质。细胞中核糖体生产的失败（称为核仁应激）对细胞和生物体的健康具有深远的影响。重要的是，并非所有细胞都是平等的，各种干细胞和祖细胞对核仁应激表现出不同的敏感性：虽然一些干细胞和祖细胞对核仁应激表现出急性敏感性并通过程序性细胞死亡而死亡，但许多其他干细胞和祖细胞似乎对核仁应激有弹性。问题是，为什么有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。该项目测试的假设，不同的果蝇神经母细胞（干细胞）在发育中的幼虫大脑显示不同的敏感性核仁的压力。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更能适应核仁压力。蘑菇体是昆虫大脑中对嗅觉和学习至关重要的部分。研究人员通过消耗核糖体生物合成因子（一种称为Nopp 140的蛋白质）来诱导果蝇的核仁应激。这项研究确定了核仁应激的机制，因为它干扰了细胞的活力和功能，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，核糖体生物合成的失败是导致神经系统疾病的一个因素。更广泛的影响将介绍遗传学和细胞生物学的基本概念，在地区高中代表性不足的学生，为代表性不足的本科生提供动手研究培训的机会，并培养研究生在先进的遗传学和细胞生物学。不同的干细胞和祖细胞对核仁应激表现出不同的敏感性。哺乳动物糖蜜单倍不足突变，核仁核糖体生物发生因子（RBF），耗尽核糖体在选择胚胎神经嵴细胞，导致颅面出生缺陷的细胞凋亡死亡。大多数其他胚胎干细胞和胚胎内的祖细胞似乎对这种核仁压力有弹性。问题是，为什么在RBF中对系统性突变有不同的反应？该项目将使用果蝇作为模型系统来回答这个问题。 核仁压力将通过耗尽Nopp 140而诱导，Nopp 140是结构和功能上与糖蜜相关的核仁RBF。目的1测试的假设，蘑菇体（MB）的神经母细胞在果蝇幼虫的大脑是有弹性的核仁压力，因为他们储存RBFs的翻译的母体转录在一段时间内的持续增殖，而大多数其他神经母细胞通常进入一段时间的静止。目的1A使用镶嵌分析来确定野生型和Nopp140-/-幼虫脑中RBFs对MB神经母细胞与其他神经母细胞的贡献。目的1B使用相关光学和电子显微镜（CLEM）来评估野生型和空Nopp 140遗传背景中MB神经母细胞与其他神经母细胞的RBF和核糖体丰度。目的2探讨JNK作为后生动物核仁应激效应子的机制。果蝇JNK对各种形式的应激有反应，初步数据显示果蝇中JNK在核仁应激时被激活。然而，JNK在核仁应激后的上游激活剂仍然未知。该项目通过使用RNAi在Nopp140 null（核仁应激）背景下耗尽已知的上游JNK激活剂来识别这些激活剂，然后在JNK激活丧失后对拯救的核仁应激表型进行评分。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估而被认为值得支持。

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.