Oligomer aggregation of green-to-red photoconvertible fluorescent protein Eos (EosFP) is a natural feature of the wild‑type variant. The aim of the present study was to follow up mitochondrial nucleoid behavior under natural conditions of living cells transfected with mitochondrial single‑strand DNA‑binding protein (mtSSB) conjugated with EosFP. HEPG2 and SH‑SY5Y cells were subjected to lentiviral transfection and subsequently immunostained with anti‑DNA, anti‑transcription factor A, mitochondrial (TFAM) or anti‑translocase of the inner membrane 23 antibodies. Fluorescent microscopy, conventional confocal microscopy, superresolution biplane fluorescence photo-activation localization microscopy and direct stochastic optical reconstruction microscopy were used for imaging. In the two cell types, apparent couples of equally‑sized mtSSB‑EosFP‑visualized dots were observed. During the time course of the ongoing transfection procedure, however, a small limited number of large aggregates of mtSSB‑EosFP‑tagged protein started to form in the cells, which exhibited a great co‑localization with the noted coupled positions. Antibody staining and 3D immunocytochemistry confirmed that nucleoid components such as TFAM and DNA were co‑localized with these aggregates. Furthermore, the observed reduction of the mtDNA copy number in mtSSB‑EosFP‑transfected cells suggested a possible impairment of nucleoid function. In conclusion, the present study demonstrated that coupled nucleoids are synchronized by mtSSB‑EosFP overexpression and visualized through their equal binding capacity to mtSSB‑EosFP‑tagged protein. This observation suggested parallel replication and transcription activity of nucleoid couples native from a parental one. Preserved coupling in late stages of artificial EosFP‑mediated aggregation of tagged proteins suggested a rational manner of mitochondrial branching that may be cell-type specifically dependent on hierarchical nucleoid replication.

Department of Membrane Transport Biophysics Institute of Physiology The Czech Academy of Sciences Prague 14220 Czech Republic

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