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.

项目摘要 在胚胎发育过程中，必须正确分配给生物所需的不同命运 生长和繁殖。生殖细胞产生卵和精子，必须指定以避免 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。生殖细胞经常通过遗传细胞质成分而获得命运 蛋白质和RNA的母体合成，无膜，凝胶状聚集体 血浆。聚集了高度保守的蛋白质，RNA和细胞器，或者组装 由其他蛋白质成核的，这些蛋白质可能会在动物之间序列不同，但共享 进化史和生物物理特性。这些成核器的分子机制 确保种植血浆的组装和功能尚不清楚。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，我们将阐明由Oskar基因编码的果蝇核定剂的机制， 组装种植血浆。该提议很重要，因为它有可能揭示可概括的 细菌等离子组装原理，可能广泛适用于形成和功能 在许多不同情况下调节细胞命运的无膜的凝胶样细胞质聚集体。我们的 生物信息学发现数百个新的Oskar序列，并结合X射线晶体学和 生化测定，提出了先前未开发的关于Oskar分子机制的假设 功能，我们将测试如下：在AIM 1中，我们将阐明保守的莲花域的作用 在细菌等离子体组装中，带有体外生化测定和生物学的体内转基因测定 种植血浆的功能和生物物理特性，检验了关于重要性的假设 二聚化，高阶聚集体形成，相位分离和与种植浆的VASA结合 集会。在AIM 2中，我们将首次确定特定序列和结构特性 长长的Oskar域，使长Oskar同工型可以在细菌中募集和锚定线粒体 血浆。在AIM 3中，我们将测试保守的OSK域与特定相互作用的新假设 卵母细胞后部的脂质类，以帮助将Oskar锚定在后极。由于细菌缺陷 细胞开发可能导致生殖系统病理，影响多达12％的美国人群， 阐明确保生殖系决定因素组装和功能的机制高度相关 对人类健康。更一般地，种菌等血浆是一种核糖核蛋白多聚蛋白（RNP），它是 无膜的凝胶状细胞器，可调节生殖系和体细胞中的翻译。 因此，了解细菌等离子组件可能会阐明 多种关键细胞类型的适当功能所需的细胞质RNP。