Molecular mechanisms of cell fate determinant assembly

细胞命运决定簇组装的分子机制

• 批准号: 10626885
• 负责人: Cassandra G Extavour
• 金额: $ 48.61万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10626885
• 关键词: Adopted; Affect; Affinity; Animals; Binding; Biochemical; Bioinformatics; Biological Assay; Biological Process; Cells; Cytoplasm; Data; Defect; Development; Developmental Biology; Dimerization; Drosophila genus; Embryo; Embryonic Development; Ensure; Failure; Gel; Genes; Genetic; Germ; Germ Cells; Germ Lines; Germ cell tumor; Goals; Growth; Health; Human; In Vitro; Individual; Infertility; Inherited; Light; Lipid Binding; Lipids; Liposomes; Malignant neoplasm of ovary; Mediating; Membrane; Messenger RNA; Mitochondria; Modification; Molecular; Molecular Biology; Mutagenesis; Mutation; N-terminal; Oocytes; Oogenesis; Organelles; Organism; Pathology; Phase; Population; Property; Protein Isoforms; Proteins; RNA; Recording of previous events; Reproduction; Reproductive system; Ribonucleoproteins; Role; Series; Site; Somatic Cell; Specific qualifier value; Structure; Teratoma; Testing; Time; Transgenic Organisms; Translations; Variant; X-Ray Crystallography; biophysical properties; cell fate specification; cell type; egg; experimental study; gonadal cancer; human disease; in vivo; innovation; novel; novel strategies; protein aggregation; protein protein interaction; recruit; reproductive; reproductive system disorder; sperm cell; stoichiometry

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.

项目概要 在胚胎发育过程中，细胞必须正确分配到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，必须对其进行指定以避免疾病 生殖系统，包括性腺癌和卵巢癌、畸胎瘤和其他生殖细胞肿瘤，以及 最终导致不孕。生殖细胞通常通过继承细胞质成分来获得其命运 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，或者 由其他蛋白质成核，这些蛋白质在动物之间的序列可能不同，但具有相似的 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因素的组装和功能的分子机制。在这个 提案中，我们将阐明由 oskar 基因编码的果蝇成核剂的机制， 组装种质。这个提议很重要，因为它有可能发现可推广的 种质组装原理，可广泛应用于 无膜、凝胶状细胞质聚集体，可在许多不同的情况下调节细胞命运。我们的 数百个新的奥斯卡序列的生物信息发现，结合X射线晶体学和 生化检测，提出了先前未探索的关于 oskar 分子机制的假设 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 通过体外生化测定和生物体内转基因测定进行种质组装 种质的功能和生物物理特性，检验关于种质重要性的假设 二聚化、高阶聚集体形成、相分离以及 Vasa 与种质的结合 集会。在目标2中，我们将首次确定其具体序列和结构特性 长奥斯卡结构域，使长奥斯卡亚型能够招募并锚定胚芽中的线粒体 血浆。在目标 3 中，我们将测试保守的 OSK 结构域与特定的相互作用的新假设。 卵母细胞后部的脂质类别，有助于将奥斯卡锚定在后极。由于胚芽缺陷 细胞发育可能导致生殖系统病变，影响多达 12% 的美国人口， 阐明确保种系决定因素的组装和功能的机制具有高度相关性 为了人类的健康。更一般地说，种质是一种核糖核蛋白多聚体（RNP），它们是 调节生殖细胞和体细胞翻译的无膜凝胶状细胞器。 因此，了解种质组装可能有助于揭示种质组装的一般原理。 多种关键细胞类型正常发挥功能所需的细胞质 RNP。