Mechanisms of mitochondrial inheritance

线粒体遗传机制

• 批准号: 10365938
• 负责人: Melissa Pamula
• 金额: $ 6.98万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10365938
• 关键词: Actin-Binding Protein; Actins; Address; Affinity; Alleles; Animal Model; Binding; Binding Proteins; Biochemical; Biochemistry; Biological; Cell Lineage; Cell Nucleus; Cells; Complex; Cytoskeleton; DNA sequencing; Defect; Development; Disease; Drosophila genus; Drosophila melanogaster; Embryo; Embryonic Development; Ensure; Event; F-Actin; Fluorescence Microscopy; Gene Frequency; Genes; Genetic; Genome; Germ; Germ Cells; Goals; Human; In Vitro; Individual; Inherited; Knock-out; Knowledge; Label; Light; Maps; Mass Spectrum Analysis; Measures; Membrane; Membrane Proteins; Methods; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Mitochondrial Inheritance; Mitochondrial Proteins; Molecular; Mothers; Mus; Mutation; Myopathy; Neurodegenerative Disorders; Neuromuscular Diseases; Nuclear; Oocytes; Organelles; Outer Mitochondrial Membrane; Ploidies; Point Mutation; Population; Process; Proliferating; Protein Isoforms; Proteins; Proteomics; Quantitative Microscopy; RNA Interference; RNA interference screen; Resolution; Respiration; Rest; Risk; Sampling; Siblings; Site; Structure of primordial sex cell; System; Temperature; Testing; Tropomyosin; base; crosslink; cytochrome c oxidase; experimental study; fly; high resolution imaging; insight; interdisciplinary approach; knock-down; mitochondrial DNA mutation; mitochondrial genome; mitochondrial membrane; molecular imaging; mutant; nervous system disorder; offspring; precursor cell; preimplantation; recruit; segregation; single molecule; tandem mass spectrometry; transgenerational epigenetic inheritance; transmission process

Project Summary The survival of eukaryotic species depends on the faithful transmission of both nuclear and mitochondrial genomes. Mutations in mitochondrial DNA (mtDNA) cause neurodegenerative and neuromuscular diseases in humans. Strikingly, though mitochondria are inherited exclusively through the maternal lineage, rapid changes in mtDNA allele frequency can occur, resulting in severe mitochondrial disease in a subset of offspring due to an increased mutational load. The long-term goal of this project is to decipher the molecular mechanisms regulating mitochondrial segregation in the germline. To achieve this goal, I will take a multidisciplinary approach combining genetics, proteomics, biochemistry, and high-resolution quantitative microscopy using the model organism, Drosophila melanogaster. The following aims will be pursued: (1) Analyze mtDNA allele frequency in gamete precursor cells termed primordial germ cells (PGCs). During embryogenesis, a small subset of mitochondria is permanently separated from the rest of the oocyte into PGCs, resulting in an ~1000-fold reduction in mtDNA content. To examine the consequence of this mitochondrial population bottleneck on the segregation of mtDNA alleles, I will use a heteroplasmic fly strain harboring both wild-type and mutant mitochondrial genomes. I will determine mtDNA allele frequency in individual PGCs using high-resolution imaging of single mtDNA molecules and quantitative PCR and will examine how these ratios change when the size of the bottleneck is genetically constricted. (2) Determine the network of Long Oskar interacting proteins. Long Oskar is the master regulator of mitochondrial inheritance. To recruit mitochondria to the site of PGC formation, Long Oskar stimulates F-actin reorganization, but it does not contact mitochondria directly. To identify proteins downstream of Long Oskar, I will use proximity labelling and tandem mass spectrometry. I will then map Long Oskar-binding regions on direct binding partners. (3) Identify nuclear-encoded mitochondrial proteins required for mitochondrial inheritance. Currently, our understanding of how mitochondria are targeted to sites of PGC formation is limited by an incomplete parts list of the mitochondrial segregation machinery. I will perform a comprehensive RNAi screen of mitochondrial membrane-associated proteins to identify those required for mitochondrial localization. Together, these aims will reveal how the mitochondrial bottleneck impacts the segregation of mtDNA alleles and will likely inform on the population risk of mitochondrial associated diseases. In addition, these experiments will identify molecular components of the mtDNA segregation machinery that is used to transmit mitochondria to germline cells during early Drosophila embryogenesis. Together, these results have the potential to shed light on how similar events may occur in pre-implantation human embryos.

项目摘要 真核物种的生存依赖于核和线粒体的忠实传递。 基因组。线粒体DNA(MtDNA)突变可导致神经退行性疾病和神经肌肉疾病 人类。引人注目的是，尽管线粒体完全通过母系遗传，但变化很快 在线粒体DNA等位基因频率可以发生，导致严重的线粒体疾病的子代由于 突变负荷增加。这个项目的长期目标是破译分子机制。 调节生殖系中的线粒体分离。为了实现这一目标，我将采取多学科的方法 使用该模型将遗传学、蛋白质组学、生物化学和高分辨率定量显微镜结合在一起 有机体，黑腹果蝇。本研究的主要目的如下：(1)分析中国人线粒体DNA等位基因频率。 配子前体细胞称为原始生殖细胞(PGCs)。在胚胎发育过程中，一小部分 线粒体从卵母细胞的其余部分永久分离成原生殖细胞，导致大约1000倍的减少 线粒体DNA含量。来检验线粒体种群瓶颈对分离的影响 在mtdna等位基因中，我将使用一种同时含有野生型和突变型线粒体的异质型苍蝇品系。 基因组。我将使用单个PGC的高分辨率成像来确定单个PGC的mtDNA等位基因频率 MtDNA分子和定量聚合酶链式反应，并将检查这些比率如何变化时，大小 瓶颈是由基因决定的。(2)确定长Oskar相互作用蛋白网络。长奥斯卡 是线粒体遗传的主要调节者。将线粒体招募到PGC形成的部位，Long Oskar刺激F-肌动蛋白重组，但它不直接接触线粒体。鉴定蛋白质 在Long Oskar的下游，我将使用邻近标记和串联质谱仪。然后我会把地图做长 直接结合伙伴上的Oskar结合区。(3)确定所需的核编码线粒体蛋白 线粒体遗传。目前，我们对线粒体如何靶向PGC部位的理解 形成受到线粒体分离机制部件清单不完整的限制。我将表演一场 线粒体膜相关蛋白的全面RNAi筛选 线粒体定位。总而言之，这些目标将揭示线粒体瓶颈如何影响 线粒体DNA等位基因的分离，可能会对线粒体相关疾病的人群风险提供信息。 此外，这些实验将确定线粒体DNA分离机制的分子组成，即 用于在果蝇早期胚胎发育过程中将线粒体传递给生殖细胞。总而言之，这些结果 有可能阐明类似的事件如何发生在植入前的人类胚胎中。