Heteroplasmy at the single mitochondrion level

描述（由申请人提供）：申请人的近期目标是在K02奖的五年支持期内，将75%的时间用于探索亚细胞生物学的新方法。该奖项提供的教学和服务的休息时间将使他能够更加专注于基于个体细胞器分析的新策略的开发，这些新策略将用于更好地了解疾病和衰老。这些策略是他的实验室正在进行的两个R01项目的一部分，研究(i)线粒体DNA （mtDNA）突变在衰老和与年龄相关的疾病中的作用，以及（ii）亚细胞药物代谢。该奖项还将允许候选人投入更多时间(i)将机器人技术，混合技术和蛋白质组学专业知识整合到他的实验室；（ii）培训科学家，（iii）建立坚实的合作网络，以及（iv）为多学科研究团队提供有凝聚力的领导。在本申请的研究计划中，候选人提出基于个体线粒体测量来研究mtDNA突变的分布。虽然这些突变的积累与衰老过程和年龄相关疾病有关，但突变水平与年龄相关表型或疾病症状之间的联系尚不清楚。这种应用的假设是，单个线粒体既包含野生型DNA，也包含突变DNA，这种情况被称为异质性，它决定了突变如何分布和繁殖。将使用两种模型来验证这一假设：一种是含有大量mtDNA缺失的杂交细胞系，另一种是来自老年Fisher 344大鼠的骨骼肌组织，该组织预计会随着年龄的增长而积累类似的缺失。该研究的三个目标是：(i)确定单个线粒体内异质性的存在，（ii）监测杂交融合后异质性的变化，以及（iii）测量骨骼肌纤维的异质性。由于没有直接测试这一假设的技术存在，该应用将需要进一步发展申请人的策略，最初是基于毛细管电泳和激光诱导荧光检测，以表征单个细胞器中的mtDNA和肽表达。在完成该K02奖项后，申请人有望为科学界提供新技术，并指导一个广泛认可的研究项目。

DESCRIPTION (provided by applicant): The immediate goal of the applicant is to devote 75% of his time during the five-year support period of this K02 award to the exploration of novel approaches to subcellular biology. The time off from teaching and service provided by the award would allow him to focus more intensely on the development of new strategies based on individual organelle analysis that would then be used to better understand disease and aging. These strategies are being developed as part of two ongoing R01 projects in his laboratory that investigate (i) the role of mitochondrial DNA (mtDNA) mutations in aging and age-related diseases, and (ii) subcellular drug metabolism. This award would also allow the candidate to devote more time to (i) integrating robotics, cybrid technology, and proteomics expertise into his laboratory; (ii) training scientists, (iii) establishing a solid network of collaborations, and (iv) providing cohesive leadership to a multi-disciplinary research team. In the research plan of this application, the candidate proposes to study the distributions of mtDNA mutations based on individual mitochondrion measurements. While the accumulation of these mutations has been implicated in the aging process and age-related diseases, the link between mutation levels and age-related phenotypes or disease symptoms is not known. The hypothesis of this application is that individual mitochondria contain both wild-type and mutated DNA, a condition known as heteroplasmy, which determines how mutations are distributed and propagated. Two models will be used to test this hypothesis: cybrid cell lines harboring large mtDNA deletions, and skeletal muscle tissue from aged Fisher 344 rats that is expected to have accumulated similar deletions with age. The three goals of the study are: (i) establish the existence of heteroplasmy within individual mitochondria, (ii) monitor changes in heteroplasmy following cybrid fusion, and (iii) measure heteroplasmy along skeletal muscle fibers. Since no technology exists to directly test this hypothesis, this application will require the further development of the applicant's strategies initially on based capillary electrophoresis with laser-induced fluorescence detection for characterizing mtDNA and peptide expression in individual organelles. Upon the completion of this K02 award, the applicant is expected to have provided the scientific community with new technologies and to be directing a widely recognized research program.