Possible frequent multiple mitochondrial DNA copies in a single nucleoid in HeLa cells

0574023 - FGÚ 2024 RIV US eng J - Journal Article
Pavluch, Vojtěch - Špaček, Tomáš - Engstová, Hana - Dlasková, Andrea - Ježek, Petr
Possible frequent multiple mitochondrial DNA copies in a single nucleoid in HeLa cells.
Scientific Reports. Roč. 13, č. 1 (2023), č. článku 5788. ISSN 2045-2322. E-ISSN 2045-2322
R&D Projects: GA ČR(CZ) GA21-01205S; GA ČR(CZ) GA22-02203S; GA ČR(CZ) GA23-05798S
Institutional support: RVO:67985823
Keywords : HeLa cells * mitochondrial proteins * fluorescence in situ hybridization
OECD category: Physiology (including cytology)
Impact factor: 4.6, year: 2022
Method of publishing: Open access
https://doi.org/10.1038/s41598-023-33012-6

Previously, a number of ~ 1.4 of mitochondrial DNA (mtDNA) molecules in a single nucleoid was reported, which would reflect a minimum nucleoid division. We applied 3D-double-color direct stochastic optical reconstruction microscopy (dSTORM), i.e. nanoscopy with ~ 25–40 nm x,y-resolution, together with our novel method of Delaunay segmentation of 3D data to identify unbiased 3D-overlaps. Noncoding D-loops were recognized in HeLa cells by mtDNA fluorescence in situ hybridization (mtFISH) 7S-DNA 250-bp probe, containing biotin, visualized by anti-biotin/Cy3B-conjugated antibodies. Other mtFISH probes with biotin or Alexa Fluor 647 (A647) against ATP6-COX3 gene overlaps (1,100 bp) were also used. Nucleoids were imaged by anti-DNA/(A647-)-Cy3B-conjugated antibodies. Resulting histograms counting mtFISH-loci/nucleoid overlaps demonstrated that 45% to 70% of visualized nucleoids contained two or more D-loops or ATP6-COX3-loci, indicating two or more mtDNA molecules per nucleoid. With increasing number of mtDNA per nucleoid, diameters were larger and their distribution histograms peaked at ~ 300 nm. A wide nucleoid diameter distribution was obtained also using 2D-STED for their imaging by anti-DNA/A647. At unchanged mtDNA copy number in osteosarcoma 143B cells, TFAM expression increased nucleoid spatial density 1.67-fold, indicating expansion of existing mtDNA and its redistribution into more nucleoids upon the higher TFAM/mtDNA stoichiometry. Validation of nucleoid imaging was also done with two TFAM mutants unable to bend or dimerize, respectively, which reduced both copy number and nucleoid spatial density by 80%. We conclude that frequently more than one mtDNA molecule exists within a single nucleoid in HeLa cells and that mitochondrial nucleoids do exist in a non-uniform size range.
Permanent Link: https://hdl.handle.net/11104/0344385