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 真核物种的生存取决于核和线粒体的忠实传播 基因组。线粒体DNA（mtDNA）的突变引起神经退行性和神经肌肉疾病 人类。令人惊讶的是，尽管线粒体仅通过母体谱系遗传，但快速变化 在mtDNA等位基因频率中可能发生，导致严重的线粒体疾病在后代的一部分中 突变负荷增加。该项目的长期目标是破译分子机制 调节种系中的线粒体隔离。为了实现这一目标，我将采用多学科的方法 使用模型结合遗传学，蛋白质组学，生物化学和高分辨率定量显微镜 有机体，果蝇Melanogaster。将追求以下目标：（1）分析mtDNA等位基因频率 配子前体细胞称为原始生殖细胞（PGC）。在胚胎发生过程中，一小部分 线粒体从卵母细胞中永久分离为PGC，导致降低约1000倍 在mtDNA含量中。检查这种线粒体种群瓶颈对隔离的结果 MtDNA等位基因，我将使用携带野生型和突变的线粒体的异质蝇菌株 基因组。我将使用单个单个PGC中的MtDNA等位基因频率来确定单个PGC的频率 mtDNA分子和定量PCR，并将检查当这些比率的大小时如何变化 瓶颈在遗传上是限制的。 （2）确定长Osk​​ar相互作用蛋白的网络。长奥斯卡 是线粒体遗传的主调节器。要募集线粒体到PGC组的位置， Oskar刺激F-肌动蛋白的重组，但不会直接接触线粒体。鉴定蛋白质 长奥斯卡（Long Oskar）的下游，我将使用接近标记和串联质谱。然后我会绘制长时间 Oskar结合区域有关直接约束伙伴的区域。 （3）确定所需的核编码线粒体蛋白 用于线粒体遗传。目前，我们对线粒体如何针对PGC的位置的理解 形成受线粒体隔离机制的不完整零件列表的限制。我会表演 线粒体膜相关蛋白的全面RNAi屏幕，以识别所需的蛋白 线粒体定位。这些目标在一起将揭示线粒体瓶颈如何影响 mtDNA等位基因的分离，可能会告知人口相关疾病的人口风险。 此外，这些实验将确定mtDNA隔离机制的分子成分 用于在早期果蝇胚胎发生期间将线粒体传输到种系细胞。在一起，这些结果 有可能阐明在植入前的人类胚胎中如何发生类似事件。