Molecular mechanisms of cell fate determinant assembly

项目摘要 在胚胎发育过程中，细胞必须被正确地分配给生物体发育所需的不同命运。 生长和繁殖。生殖细胞产生卵子和精子，必须详细说明，以避免疾病的发生。 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 由其他蛋白质成核，这些蛋白质在动物中的序列可能不同，但它们具有相似的 进化历史和生物物理特性。这些成核剂的分子机制 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子的组装和功能的分子机制。在这 建议，我们将阐明机制，果蝇核，由oskar基因编码， 聚集生殖质。这一建议意义重大，因为它有可能揭示可推广的 种质组装的原则，这可能是广泛适用于形成和功能的 在许多不同情况下调节细胞命运的无膜、凝胶样细胞质聚集体。我们 生物信息学发现了数百个新的奥斯卡序列，结合X射线晶体学， 生化分析，提出了以前未探索的假设的分子机制奥斯卡 功能，我们将测试如下：在目标1中，我们将阐明保守的LOTUS结构域的作用 在种质装配中，采用生物学的体外生物化学测定和体内转基因测定， 功能和生物物理特性的种质，测试假设的重要性 二聚化、高阶聚集体形成、相分离和Vasa与种质结合 组装件.在目标2中，我们将首次确定 长Oskar结构域使长Oskar同种型能够在胚芽中募集和锚线粒体 血浆在目标3中，我们将测试保守的OSK结构域与特异性的 类脂质在卵母细胞后，以帮助锚奥斯卡的后极。由于细菌的缺陷 细胞发育可导致生殖系统病变，影响高达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， 对人类健康的影响。更一般地说，种质是一种核糖核蛋白多聚体（RNP）， 在生殖细胞和体细胞中调节翻译的无膜、凝胶样细胞器。 因此，了解胚质组装可能有助于了解胚质组装的一般原理。 多种关键细胞类型的正常功能所需的细胞质RNP。

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.