Heteroplasmy at the single mitochondrion level

描述(由申请者提供)：申请者的直接目标是在K02奖项的五年支持期内，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假期将使他能够更专注于基于单个细胞器分析的新策略的开发，然后这些策略将被用来更好地了解疾病和衰老。这些策略是他实验室正在进行的两个R01项目的一部分，这两个项目调查(I)线粒体DNA(MtDNA)突变在衰老和老年相关疾病中的作用，以及(Ii)亚细胞药物代谢。这一奖项还将允许候选人将更多的时间用于(I)将机器人、赛博德技术和蛋白质组学的专业知识整合到他的实验室中；(Ii)培训科学家；(Iii)建立坚实的合作网络；以及(Iv)为一个多学科的研究团队提供凝聚力的领导。在这项应用的研究计划中，候选人建议根据单个线粒体的测量来研究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。这一应用的假设是，单个线粒体同时包含野生型和突变的DNA，这种情况被称为异质性，它决定了突变的分布和传播方式。将使用两个模型来验证这一假设：存在大量mtDNA缺失的Cybrid细胞系，以及预计会随着年龄增长积累类似缺失的老年Fisher 344大鼠的骨骼肌组织。这项研究的三个目标是：(I)确定单个线粒体内异质性的存在，(Ii)监测环体融合后异质性的变化，以及(Iii)测量骨骼肌纤维的异质性。由于不存在直接检验这一假说的技术，这一应用将需要申请人进一步开发最初基于毛细管电泳和激光诱导荧光检测的策略，以表征单个细胞器中的线粒体DNA和多肽表达。在完成这项K02奖项后，预计申请者将为科学界提供新技术，并指导一项得到广泛认可的研究计划。

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.