Heteroplasmy at the single mitochondrion level

单线粒体水平的异质性

• 批准号: 6877114
• 负责人: EDGAR A ARRIAGA
• 金额: $ 10.81万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6877114
• 关键词: aging; cell line; confocal scanning microscopy; drug metabolism; gene mutation; immunocytochemistry; mathematical model; matrix assisted laser desorption ionization; mitochondrial DNA; molecular genetics; polymerase chain reaction; tissue /cell culture

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.

描述（由申请人提供）：申请人的近期目标是在 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，然后将其用于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，这些项目研究 (i) 线粒体 DNA (mtDNA) 突变在衰老和年龄相关疾病中的作用，以及 (ii) 亚细胞药物代谢。该奖项还将使候选人能够投入更多时间：（i）将机器人技术、细胞混合技术和蛋白质组学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，候选人提出根据个体线粒体测量来研究线粒体DNA突变的分布。虽然这些突变的积累与衰老过程和与年龄相关的疾病有关，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。该应用的假设是，单个线粒体同时含有野生型和突变型 DNA，这种情况称为异质性，它决定了突变如何分布和传播。将使用两个模型来检验这一假设：带有大量 mtDNA 缺失的 cybrid 细胞系，以及来自老年 Fisher 344 大鼠的骨骼肌组织，预计随着年龄的增长，该组织也会积累类似的缺失。该研究的三个目标是：（i）确定个体线粒体内异质性的存在，（ii）监测细胞融合后异质性的变化，以及（iii）测量沿骨骼肌纤维的异质性。由于不存在直接检验这一假设的技术，因此本申请将需要进一步开发申请人的策略，该策略最初基于毛细管电泳和激光诱导荧光检测，用于表征单个细胞器中的 mtDNA 和肽表达。获得 K02 奖项后，申请人预计将为科学界提供新技术并指导一项得到广泛认可的研究项目。