Abstract
Mitochondrial DNA molecules coated with proteins form compact particles called mitochondrial nucleoids. They are redistributed within mitochondrial network undergoing morphological changes. The straightforward technique to characterize nucleoids’ motions is fluorescence microscopy. Mitochondrial nucleoids are commonly labelled with fluorescent protein tags, which is not always feasible and was reported to cause artifacts. Organic DNA-binding dyes are free of these drawbacks, but they lack specificity to mitochondrial DNA. Here, considering physico-chemical properties of such dyes, we achieved preferential live-cell labelling of mitochondrial nucleoids by a nucleic acid staining dye SYBR Gold. It enabled time-lapse imaging of mitochondrial nucleoids by structured illumination microscopy and quantification of their motions.
Highlights
Genetic material of mitochondria consists of a circular 16.5 kbp DNA molecule, which encodes tRNAs, 22 rRNAs, and 13 polypeptides needed for mitochondrial oxidative phosphorylation complexes
We observed that upon short (1 hr or less) incubation at 1:10000 dilution, SYBR Gold stained nuclei only weakly, while bright green dotted pattern appeared in mitochondria (Fig 1); the ratio of integrated cytoplasmic signal intensity to integrated nuclear signal intensity was 1.06 (Fig 2), which corresponds to highly preferential staining of mitochondrial nucleoids, considering their small volume and small total mtDNA content, in comparison to that of the nucleus
To get insights into the processes in which mitochondrial nucleoids are involved, one needs a specific, live-cell compatible and convenient labelling combined with a microscopy technique with resolution beyond the diffraction limit (“super-resolution”)
Summary
Genetic material of mitochondria consists of a circular 16.5 kbp DNA molecule, which encodes tRNAs, 22 rRNAs, and 13 polypeptides needed for mitochondrial oxidative phosphorylation complexes. Mitochondria communicate with the rest of the cell in both directions: mitochondrial dysfunction alters expression of nuclear signaling factors MtDNA in complex with mitochondrial transcription factor A (TFAM) and several other proteins form compact structures called nucleoids [4] [5,6,7,8]. Mitochondrial network undergoes morphological remodeling, fission/fusion etc, depending on cell cycle phase, stress and other factors (reviewed e.g. in [9]). These morphology rearrangements are accompanied by mitochondrial nucleoids redistributions and motions [10] [11,12,13]. Mitochondrial nucleoids motions may play a role in diseases: e.g. extrusion of mtDNA
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