Mechanisms of mitochondrial inheritance

线粒体遗传机制

基本信息

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
项目类别：
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

项目摘要

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.

项目概要真核物种的生存取决于核和线粒体的忠实传递基因组。线粒体 DNA (mtDNA) 突变会导致神经退行性疾病和神经肌肉疾病人类。引人注目的是，尽管线粒体仅通过母系遗传，但快速变化 mtDNA 等位基因频率中的突变可能会发生，导致部分后代出现严重的线粒体疾病，原因是突变负荷增加。该项目的长期目标是破译分子机制调节种系中的线粒体分离。为了实现这个目标，我将采取多学科的方法使用该模型结合遗传学、蛋白质组学、生物化学和高分辨率定量显微镜有机体，果蝇。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，一小部分线粒体与卵母细胞的其余部分永久分离成 PGC，从而导致约 1000 倍的减少线粒体DNA含量。检查线粒体群体瓶颈对分离的影响 mtDNA 等位基因，我将使用同时具有野生型和突变型线粒体的异质蝇菌株基因组。我将使用单个 PGC 的高分辨率成像来确定单个 PGC 中的 mtDNA 等位基因频率。 mtDNA 分子和定量 PCR 并将检查当 mtDNA 分子大小变化时这些比率如何变化瓶颈是基因限制的。 (2)确定Long Oskar相互作用蛋白的网络。长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 形成位点，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法。然后我将绘制长直接结合伙伴上的奥斯卡结合区域。 (3) 鉴定所需的核编码线粒体蛋白用于线粒体遗传。目前，我们对线粒体如何靶向 PGC 位点的了解线粒体分离机制的零件清单不完整，限制了其形成。我将执行一个对线粒体膜相关蛋白进行全面的 RNAi 筛选，以确定所需的蛋白线粒体定位。这些目标将共同揭示线粒体瓶颈如何影响线粒体DNA等位基因的分离，可能会提示线粒体相关疾病的人群风险。此外，这些实验将鉴定 mtDNA 分离机制的分子成分，即用于在早期果蝇胚胎发生过程中将线粒体传递给生殖细胞。综合起来，这些结果有可能揭示类似事件如何在植入前的人类胚胎中发生。