Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA

Abstract

Mammalian mtDNA is packaged in DNA-protein complexes denoted mitochondrial nucleoids. The organization of the nucleoid is a very fundamental question in mitochondrial biology and will determine tissue segregation and transmission of mtDNA. We have used a combination of stimulated emission depletion microscopy, enabling a resolution well below the diffraction barrier, and molecular biology to study nucleoids in a panel of mammalian tissue culture cells. We report that the nucleoids labeled with antibodies against DNA, mitochondrial transcription factor A (TFAM), or incorporated BrdU, have a defined, uniform mean size of approximately 100 nm in mammals. Interestingly, the nucleoid frequently contains only a single copy of mtDNA (average approximately 1.4 mtDNA molecules per nucleoid). Furthermore, we show by molecular modeling and volume calculations that TFAM is a main constituent of the nucleoid, besides mtDNA. These fundamental insights into the organization of mtDNA have broad implications for understanding mitochondrial dysfunction in disease and aging

Quality matters: how does mitochondrial network dynamics and quality control impact on mtDNA integrity?

“…Mitochondrial transcription factor A (TFAM) is an appropriate marker protein because it is not only a mitochondrial transcription factor, but also a packaging factor of mtDNA. It has been shown on several occasions, both for the endogenous protein and overexpressed tagged variants that it constitutively labels all mtDNA molecules [37,65,66]. Indeed, here also the distribution pattern of TFAM (figure 3b) resembles that of mtDNA (figure 3a).…”

“…The size of mtDNA nucleoids roughly ranges from 50 to 300 nm based on EM and super-resolution microscopy [37,65]. This implies that in mitochondria with a regular arrangement of cristae discs with a mean distance of 53 nm, such as observed in HeLa cells [38], most nucleoids would not fit.…”

“…This also forms the basis for the suggestion that each nucleoid has a limited sphere of influence that could result in a focal mitochondrial OXPHOS deficiency if it contains mutant mtDNA [35]. It has been suggested that nucleoids contain multiple copies of mtDNA [36], but recent work using superresolution microscopy indicates that each nucleoid contains on average only a single mtDNA copy in various cell types [37]. Furthermore, the confinement of OXPHOS complexes to single cristae owing to their restricted diffusion adds to the focal perspective [38,39].…”

TFAM forces mtDNA to make a U-turn

“…Current estimates from mammalian cell lines and mouse tissues indicate that TFAM is present at 1 copy per 15-20bp of mtDNA and it is thus likely to fully coat mtDNA 10,14 (Fig. 1).…”

“…1). Super-resolution microscopy has revealed that the apparent mean size of nucleoids is ~100 nm in mammals 14 . By combining imaging and quantitative molecular approaches, it has been demonstrated that most nucleoids in human fibroblasts contain one single copy of mtDNA 14 .…”