Parker Danitra J, Moran Aida, Mitra Kasturi
Department of Genetics, School of Medicine, University of Alabama at Birmingham.
Department of Genetics, School of Medicine, University of Alabama at Birmingham;
J Vis Exp. 2017 Jan 4(119):54989. doi: 10.3791/54989.
Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.
为了全面了解线粒体功能的调控机制，需要对线粒体的结构 - 功能关系进行分析。荧光显微镜是直接评估活细胞中线粒体结构和功能以及研究线粒体结构 - 功能关系不可或缺的工具，线粒体结构 - 功能关系主要由控制线粒体之间裂变和融合事件的分子调节。本文描述并展示了在模式生物黑腹果蝇的活体以及固定组织中研究线粒体结构和功能的具体方法。这里选择的组织是果蝇卵巢，它可以被分离并适用于体外活体共聚焦显微镜检查。此外，本文还描述了如何在果蝇卵巢中对线粒体裂变蛋白Drp1进行基因操作，以研究由Drp1驱动的线粒体裂变在调节线粒体结构 - 功能关系中的作用。已发表的数据以及新数据都证明了这些方法的广泛应用。所描述的方法可以进一步扩展，以了解营养物质和/或生长因子对体外线粒体特性的直接影响。鉴于线粒体功能失调是各种疾病病因的基础，预计在遗传上易于处理的模式生物果蝇中开发的这些创新方法将对理解线粒体结构 - 功能关系的机制细节以及线粒体定向治疗策略的发展做出重大贡献。
