Multiple assay systems to analyze the dynamics of mitochondrial nucleoids in living mammalian cells
Multiple assay systems to analyze the dynamics of mitochondrial nucleoids in living mammalian cells
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DOI：
10.1016/j.bbagen.2021.129874
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发表时间：
2021-04-14
影响因子：
3
通讯作者：
Ishihara, Naotada
中科院分区：
文献类型：
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作者：
Ishihara, Takaya;Kanon, Hirotaka;Ishihara, Naotada
背景：线粒体在氧化呼吸产生能量中发挥着关键作用，是高度动态的细胞器，其双层膜经常发生融合和裂变事件。线粒体被认为源自变形菌的内共生，因此线粒体仍然含有自己的DNA，即线粒体DNA（mtDNA）。多个 mtDNA 拷贝与 DNA 结合蛋白形成线粒体核仁。最近，据报道线粒体膜和核的形态和分布在动态运动过程中受到协同调节。然而，其分子机制尚不清楚，因为对所涉及的分子知之甚少，并且尚未完全开发出合适的分析类核的技术。结果：为了解决这些问题，我们通过两种方法研究了类核动力学的分子机制。首先，我们构建了一种新的探针，使用线粒体转录因子 A (TFAM) 的 DNA 结合域和光转换荧光蛋白 Kikume Green-Red (KikGR) 进行核动力学实时成像。使用新探针可以长时间稳定地观察类核。其次，我们使用 HeLa 细胞通过小干扰 RNA 筛选来寻找类核调节因子，并鉴定了影响类核形态的 MARCH 家族泛素连接酶的子集。结论：本研究报道的因子和探针将有助于揭示线粒体调节的新机制。一般意义：线粒体 DNA 动力学应关注与线粒体膜动力学相关的线粒体活性的调节及其质量控制。
Background: 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.Results: 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.Conclusion: The factors and probe, reported in this study, would be useful to reveal novel mechanisms of mitochondrial regulation.General significance: The mtDNA dynamics should be concerned in the regulation of mitochondrial activity and its quality control, associated with mitochondrial membrane dynamics.