Molecular mechanisms of cell fate determinant assembly

项目总结 在胚胎发育过程中，细胞必须正确地分配给有机体所需的不同命运 生长和繁殖。生殖细胞产生卵子和精子，必须指定以避免疾病 生殖系统，包括性腺和卵巢癌、畸胎瘤和其他生殖细胞肿瘤，以及 最终导致不孕不育。生殖细胞通常通过遗传细胞质成分来获得它们的命运 母体合成的、无膜的、凝胶状的蛋白质和RNA聚集体，统称为胚芽 血浆。种质内高度保守的蛋白质、RNA和细胞器被组装，或 核化，由其他蛋白质组成，这些蛋白质在不同动物中的序列可能不同，但具有相似的特征 进化史和生物物理特性。这些成核子的分子机制 确保种质的组装和功能仍不清楚。我们的长期目标是了解 驱动细胞质命运决定因素的组装和功能的分子机制。在这 建议，我们将阐明由奥斯卡基因编码的果蝇核仁的机制， 组装种质。这一建议意义重大，因为它有可能揭示可推广的 种质组装原理，可广泛适用于细菌的形成和功能 无膜凝胶状细胞质聚集体，在许多不同的环境中调节细胞的命运。我们的 生物信息学发现数百个新的Oskar序列，结合X射线结晶学和 生化分析，对奥斯卡的分子机制提出了以前未曾探索过的假说 功能，我们将测试如下：在目标1中，我们将阐明保守的莲花结构域的作用 在种质组装中采用体外生化检测和体内转基因生物检测 种质的功能和生物物理特性，测试关于 二聚、高阶聚集体形成、相分离和VASA与种质结合 集合。在目标2中，我们将首次确定其特定的序列和结构特性 长Oskar结构域，使长Oskar亚型能够在胚胎中招募和固定线粒体 血浆。在目标3中，我们将测试保守的OSK结构域与特定的 卵母细胞后部的类脂，帮助将奥斯卡锚定在后极。因为胚芽中的缺陷 细胞发育可能导致生殖系统疾病，影响多达12%的美国人口。 阐明确保生殖系决定因素的组装和功能的机制是高度相关的 为人类健康干杯。更广泛地说，种质是一种核糖核蛋白多聚体(RNP)，它是 无膜凝胶状细胞器，调节生殖系和体细胞中的翻译。 因此，了解种质组装可能有助于阐明潜在的组装的一般原理 多种关键细胞类型正常运作所需的细胞质RNPs。

PROJECT SUMMARY During embryonic development cells must be correctly allocated to distinct fates needed for organismal growth and reproduction. Germ cells generate eggs and sperm and must be specified to avoid disorders of the reproductive system, including gonadal and ovarian cancers, teratomas and other germ cell tumors, and ultimately infertility. Germ cells often acquire their fate by inheriting cytoplasmic components that are maternally synthesized, membrane-free, gel-like aggregates of proteins and RNAs collectively called germ plasm. The highly conserved proteins, RNAs and organelles within germ plasm are assembled, or nucleated, by other proteins that can be different in sequence across animals, but that share similar evolutionary histories and biophysical properties. The molecular mechanisms by which these nucleators ensure assembly and function of germ plasm remain unclear. Our long-term goal is to understand the molecular mechanisms that drive the assembly and function of cytoplasmic fate determinants. In this proposal, we will elucidate the mechanisms by which the Drosophila nucleator, encoded by the oskar gene, assembles germ plasm. This proposal is significant because it has the potential to uncover generalizable principles of germ plasm assembly, which may be broadly applicable to the formation and function of membrane-less, gel-like cytoplasmic aggregates that regulate cell fates in many different contexts. Our bioinformatic discovery of hundreds of new oskar sequences, combined with X-ray crystallography and biochemical assays, suggested previously unexplored hypotheses for the molecular mechanism of oskar function, which we will test as follows: In Aim 1, we will elucidate the role of the conserved LOTUS domain in germ plasm assembly with in vitro biochemical assays and in vivo transgenic assays of the biological function and biophysical properties of germ plasm, testing hypotheses regarding the importance of dimerization, higher-order aggregate formation, phase separation, and Vasa binding to germ plasm assembly. In Aim 2, we will determine for the first time the specific sequences and structural properties of the Long Oskar Domain that enable the Long Oskar isoform to recruit and anchor mitochondria in the germ plasm. In Aim 3, we will test the novel hypothesis that the conserved OSK domain interacts with specific classes of lipids in the oocyte posterior, to help anchor Oskar to the posterior pole. Since defects in germ cell development can lead to reproductive system pathologies affecting up to 12% of the US population, elucidating the mechanisms that ensure assembly and function of germ line determinants is highly relevant to human health. More generally, germ plasm is a type of ribonucleoprotein multimer (RNP), which are membrane-free, gel-like organelles that regulate translation in both germ line and somatic cells. Understanding germ plasm assembly may thus shed light on the general principles underlying assembly of cytoplasmic RNPs required for the proper function of multiple critical cell types.