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标题：Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid
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作者：Christian Kukat ; Karen M. Davies ; Christian A. Wurm 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：36
页码：11288-11293
DOI：10.1073/pnas.1512131112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceAltered expression of mitochondrial DNA (mtDNA) is heavily implicated in human disease and aging, but the basic organizational unit of mtDNA, the mitochondrial nucleoid, is poorly understood. Here, we have used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mammalian mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Furthermore, we show that the nucleoid ultrastructure is independent of cellular mtDNA copy number and that the core nucleoid structure is formed by cross-strand binding of mitochondrial transcription factor A (TFAM) to a single copy of mtDNA. The clarification of the ultrastructure of the mammalian mitochondrial nucleoid provides the fundamental basis for the understanding of regulation of mtDNA maintenance and expression in mammals. Mammalian mitochondrial DNA (mtDNA) is packaged by mitochondrial transcription factor A (TFAM) into mitochondrial nucleoids that are of key importance in controlling the transmission and expression of mtDNA. Nucleoid ultrastructure is poorly defined, and therefore we used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Rotary shadowing electron microscopy revealed that nucleoid formation in vitro is a multistep process initiated by TFAM aggregation and cross-strand binding. Superresolution microscopy of cultivated cells showed that increased mtDNA copy number increases nucleoid numbers without altering their sizes. Electron cryo-tomography visualized nucleoids at high resolution in isolated mammalian mitochondria and confirmed the sizes observed by superresolution microscopy of cell lines. We conclude that the fundamental organizational unit of the mitochondrial nucleoid is a single copy of mtDNA compacted by TFAM, and we suggest a packaging mechanism.
关键词：nucleoids ; mitochondria ; cryo-ET ; STED ; nanoscopy