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Abstract:
Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs,rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial networkthat undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward techniqueto characterize a nucleoid's position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their proteincomponents, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct,which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages.However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking intoaccount the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling ofmitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantificationof mitochondrial nucleoid motion using time series of super-resolution structured illumination images.
