Multiple assay systems to analyze the dynamics of mitochondrial nucleoids in living mammalian cells

Mitochondria, which play a critical role in energy production by oxidative respiration, are highly dynamic organelles and their double membranes undergo frequent events of fusion and fission. Mitochondria are believed to be derived from the endosymbiosis of proteobacteria, and thus mitochondria still contain their own DNA, mitochondrial DNA (mtDNA). Several copies of mtDNA form mitochondrial nucleoid with DNA-binding proteins. Recently, the morphology and distribution of the mitochondrial membrane and nucleoid were reported to be cooperatively regulated during their dynamic movement. However, the molecular mechanism is unclear, because the involved molecules are poorly understood, and suitable techniques to analyze nucleoid have not been fully developed.

To solve these issues, we examined the molecular mechanism of nucleoid dynamics by two approaches. First, we constructed a new probe to perform live imaging of nucleoid dynamics using the DNA-binding domain of mitochondrial transcriptional factor A (TFAM) and the photo-convertible fluorescent protein Kikume Green-Red (KikGR). Nucleoids were visualized stably for a long period using the new probe. Second, we searched for nucleoid regulatory factors by small interfering RNA screening using HeLa cells and identified a subset of MARCH family ubiquitin ligases that affect nucleoid morphology.

The factors and probe, reported in this study, would be useful to reveal novel mechanisms of mitochondrial regulation.

The mtDNA dynamics should be concerned in the regulation of mitochondrial activity and its quality control, associated with mitochondrial membrane dynamics.