Molecular mechanisms of mtDNA inheritance

项目摘要 线粒体DNA（mtDNA）编码对细胞功能至关重要的RNA和蛋白质。然而， 控制哺乳动物细胞中mtDNA分离和拷贝数的机制还不清楚。这个的目标 我们的工作是鉴定和描述分离复制mtDNA的未知蛋白质机制， 在ER-线粒体接触位点的离散类核结构，并描述机制和途径 调节线粒体DNA拷贝数。拟议的实验将提供基本的洞察力， mtDNA分离和拷贝数控制的机制，以及这些过程是如何调节的， 可能导致发现和表征新的途径，调节遗传 mtDNA疾病单倍型。在目标1中，尖端的活细胞显微镜、蛋白质组学和高通量 测序技术将被用来剖析线粒体分离体候选者的分子功能 在初步实验中发现的蛋白质。目标2将讨论哪些细胞通路对 人类细胞中mtDNA拷贝数的调节。这将通过系统的全基因组 筛选线粒体DNA耗竭恢复的调节剂，单细胞RNA测序，通过其 线粒体DNA丢失的转录反应，以及候选线粒体DNA拷贝数效应子的表征 线粒体DNA缺失的人类细胞中的蛋白质这些实验将提供基本的洞察力， mtDNA分离和拷贝数控制的机制，以及这些过程是如何调节的， 可能导致发现和表征新的途径，调节遗传 mtDNA疾病单倍型。

Project Summary Mitochondrial DNA (mtDNA) encodes RNAs and proteins critical for cell function. However, pathways that control mtDNA segregation and copynumber in mammalian cells are not well understood. The goals of this work are to identify and characterize the unknown protein machinery that segregates replicating mtDNAs into discrete nucleoid structures at ER-mitochondria contact sites and to describe the mechanisms and pathways that regulate mtDNA copy number. The proposed experiments will provide fundamental insight into the mechanism of mtDNA segregation and copynumber control, and how those processes are regulated, potentially leading to the discovery and characterization of novel pathways that regulate the inheritance of mtDNA disease haplotypes. In Aim 1, cutting-edge live-cell microscopy, proteomics, and high-throughput sequencing technologies will be employed to dissect the molecular functions of mtDNA segrosome candidate proteins identified in preliminary experiments. Aim 2 will address which cellular pathways are critical to regulation of mtDNA copy number in human cells. This will be accomplished by systematic genome-wide screens for modulators of mtDNA depletion recovery, single-cell RNA sequencing to cluster genes by their transcriptional responses to mtDNA loss, and the characterization of candidate mtDNA copynumber effector proteins in human cells depleted of mtDNA. These experiments will provide fundamental insight into the mechanism of mtDNA segregation and copynumber control, and how those processes are regulated, potentially leading to the discovery and characterization of novel pathways that regulate the inheritance of mtDNA disease haplotypes.