Mechanisms of mitochondrial inheritance - Pamula Admin Childcare Supplement

线粒体遗传机制 - Pamula Admin Childcare Supplement

• 批准号: 10747188
• 负责人: Melissa Pamula
• 金额: $ 0.25万
• 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/10747188
• 关键词: Actin-Binding Protein; Actins; Address; Affinity; Alleles; Apical; Binding; Binding Proteins; Biochemical; Biochemistry; Biological; Cell Lineage; Cell Nucleus; Cells; Child Care; Complex; Cytoskeleton; 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; 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; 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; constriction; crosslink; cytochrome c oxidase; experimental study; fly; high resolution imaging; insight; interdisciplinary approach; knock-down; mitochondrial DNA mutation; mitochondrial genome; mitochondrial membrane; model organism; 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 真核生物的生存依赖于细胞核和线粒体的忠实传递 基因组线粒体DNA（mtDNA）突变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， 在线粒体DNA等位基因频率可能发生，导致严重的线粒体疾病的后代的子集，由于 增加的突变负荷。这个项目的长期目标是破译 调节生殖系中的线粒体分离。为了实现这一目标，我将采取多学科方法 结合遗传学，蛋白质组学，生物化学，和高分辨率定量显微镜使用的模型 黑腹果蝇Drosophila melanogaster本研究的主要目的是：（1）分析中国汉族人群mtDNA等位基因频率， 配子前体细胞称为原始生殖细胞（PGCs）。在胚胎发生过程中，一小部分 线粒体与卵母细胞的其余部分永久分离成PGCs，导致约1000倍的减少 线粒体DNA含量。为了研究这种线粒体群体瓶颈对分离的影响， 为了确定线粒体DNA等位基因的数量，我将使用一种异质性果蝇品系，该品系同时具有野生型和突变型线粒体DNA， 基因组我将使用高分辨率成像技术确定单个PGC中mtDNA等位基因的频率。 线粒体DNA分子和定量PCR，并将研究如何改变这些比例时，大小的 瓶颈是基因限制的。(2)确定Long Oskar相互作用蛋白的网络。朗·奥斯卡 是线粒体遗传的主要调节者。为了将线粒体募集到PGC形成的位点，Long Oskar刺激F-肌动蛋白重组，但它不直接接触线粒体。来鉴定蛋白质 在朗奥斯卡下游，我将使用近距离标记和串联质谱法。然后我会绘制Long地图 直接结合伴侣上的奥斯卡结合区。(3)识别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位点的理解， 形成受到线粒体分离机制的不完整部分列表的限制。我会行一个 线粒体膜相关蛋白的全面RNAi筛选，以确定 线粒体定位总之，这些目标将揭示线粒体瓶颈如何影响细胞的生长。 线粒体DNA等位基因的分离，并可能告知线粒体相关疾病的人群风险。 此外，这些实验将确定线粒体DNA分离机制的分子组成， 用于在果蝇胚胎发育早期将线粒体传递到生殖细胞。这些结果一起 有可能揭示类似事件如何发生在植入前的人类胚胎中。