Christian Kuka, Karen M. Davies, Christian A. Wurm, Henrik Spåhr, Nina A. Bonekamp, Inge Kühl, Friederike Joos, Paola Loguercio Polosa, Chan Bae Park, Viktor Posse, Maria Falkenberg, Stefan Jakobs, Werner Kühlbrandt and Nils-Göran Larsson
Mitochondrial DNA is found to exist in protein-DNA complexes called nucleoids. The number of mtDNA molecules per nucleoid is an important quantity to know, as it has consequences for how mtDNA is distributed amongst successive generations. The compactness of mtDNA also determines its ability to be transcribed and replicated, so the way it is packaged is also important to understand.
In their first experiment, the authors use electron microscopy in vitro, to observe how mtDNA is compacted, with increasing concentrations of the molecule TFAM (the only protein known to package mtDNA). They find that upon binding to mtDNA, TFAM causes the DNA to bend by 180°. At intermediate concentrations 1 TFAM/30 bp, dense protein-DNA spots were dispersed amongst regions of naked DNA (perhaps being a concentration appropriate for translation or replication of mtDNA). At concentrations of 1 TFAM/6 bp, mtDNA was completely compacted, and a further increase in TFAM concentration had no further effect. This may be the density of TFAM required for storage of mtDNA.
They then continued their analysis in vivo, by studying TFAM-overexpressing mouse embryonic fibroblasts (OE MEFS), which had ~2.5-fold higher mtDNA copy number. Nucleoids can cluster in cells, and confocal microscopy cannot always resolve individual nucleoids. The number of nucleoids detected in wild-type cells using superresolution STED microscopy / confocal was 1.36+/-0.76. By counting nucleoids using STED microscopy, and mtDNA copy number with rt-qPCR, they found that wild-type cells had ~1.1 mtDNA molecules / nucleoid and OE MEFS had ~1.5 mtDNA molecules / nucleoid. Thus, overexpressing TFAM has only minor changes in the mean amount of mtDNA per nucleoid. Instead, the number of nucleoids was observed to increase, relative to wild-type cells.