Mechanisms of mitochondrial inheritance

线粒体遗传机制

• 批准号: 10155867
• 负责人: Melissa Pamula
• 金额: $ 6.64万
• 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/10155867
• 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.

项目总结