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First published online 11 May 2004
doi: 10.1242/jcs.01134

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Organization and dynamics of human mitochondrial DNA

INSERM U582 (IFR 14, UPMC) Institut de Myologie, Groupe Hospitalier Pitié-Salpêtrière, 47, Boulevard de l'Hôpital, 75651 Paris CEDEX 13, France

View larger version (114K): [in a new window]   Fig. 1. Mitochondrial DNA localizes to punctate structures that are distributed throughout the mitochondrial compartment. Primary skin fibroblasts were labeled with antibodies against DNA, COX2 and/or BrdU under normal conditions (A) or after a 20 hour pulse with BrdU (B). Insets 1 and 2 depict enlargements of the boxed areas. (A) Monoclonal antibodies against DNA label punctate structures that are distributed throughout mitochondria labeled with antibodies against the inner membrane protein COX2. Only a few mitochondria appear devoid of DNA-labeling (arrowheads). (B) Antibodies against the thymidine analogue BrdU label the nucleus as well as punctate mitochondrial structures labeled with DNA-antibodies. Some of the mitochondrial DNA-positive structures are devoid of BrdU (arrowheads) and very few BrdU-positive structures escape detection with DNA-antibodies (arrows).

View larger version (151K): [in a new window]   Fig. 3. Mitochondrial transcription factor A (mtTFA) is enriched in punctate DNA-positive structures. Primary skin fibroblasts, HeLa cells and 143B cells were decorated with antibodies against DNA and mitochondrial transcription factor A (mtTFA) and analyzed by conventional (Fibroblasts) or confocal (HeLa, 143B) fluorescence microscopy. The insets are enlargments of the boxed image regions. In fibroblasts, mtTFA is restricted to punctate mtDNA-positive structures. In Hela and 143B cells, mtTFA has a more homogeneous intramitochondrial distribution and is partially enriched in some mtDNA-positive structures. Bars, 10 µm.

View larger version (63K): [in a new window]   Fig. 4. The majority of mitochondria remain DNA-positive after fragmentation of mitochondrial filaments in vivo. HeLa cells expressing mtRFP (A-C) and primary skin fibroblasts (D,E) were fixed and decorated with COX2 and/or DNA-specific antibodies under control conditions (A) or after a 4-hour treatment with cccp (B-E). C and E are enlargements of the areas boxed in B and D, respectively. (A) In HeLa cells expressing mtRFP, DNA-specific antibodies label the cell nucleus and punctate intramitochondrial structures. Numerous DNA-positive structures colocalize within elongated mitochondrial filaments. (B-E) Inhibition of mitochondrial fusion with cccp leads to mitochondrial fragmentation in HeLa cells (B,C) and fibroblasts (D,E). The majority of punctate mitochondria are DNA-positive. Arrowheads point to mitochondria devoid of mtDNA. Bars, 10 µm.

View larger version (107K): [in a new window]   Fig. 6. Mitochondrial nucleoids are mobile and diffuse into 0-mitochondria. Stably transfected 143B-0 cells expressing mtRFP and untransfected 143B-+ cells were co-plated and analyzed without further treatment (A) or 12 hours after PEG-mediated cell fusion (B,C). (A) 143B-0 cells expressing mtRFP have punctate mitochondria that are devoid of mtDNA and of mtTFA. The mitochondria of 143B-+ cells contain mtDNA and mtTFA. (B) Twelve hours after PEG-mediated fusion, the polykaryons derived from the fusion of + and 0 cells depict filamentous mitochondria that contain mtRFP, mtDNA and mtTFA. Nucleoids and mtRFP are distributed throughout the entire mitochondrial compartment. (C) An enlargement of the area boxed in B shows that mtDNA nucleoids are enriched in mtTFA. The position of some mtDNA nucleoids is indicated with arrowheads. Bars, 20 µm.

View larger version (72K): [in a new window]   Fig. 2. Antibody characterization by subcellular fractionation and western-blot analysis. HeLa and 143B cells (normal + and mtDNA-less 0) were subjected to subcellular fractionation. Equal protein amounts of total homogenate (TH), mitochondrial pellet (MP) and post-mitochondrial supernatant (PMS) were separated by SDS-PAGE and transferred to membranes. Arrowheads point to the positions of marker proteins with the indicated molecular mass (kDa). The distributions of outer membrane Mfn2 and inner membrane COX2 demonstrate mitochondrial enrichment in both cell types. The 0 cells do not have any mtDNA-encoded COX2. The mtTFA-protein has an apparent molecular mass of 25 kDa and is strongly downregulated in 0 cells. The mtSSB-protein has an apparent molecular mass of 16 kDa and is present at similar levels in fractions of + and 0 cells.

View larger version (113K): [in a new window]   Fig. 5. Cytochrome c oxidase (COX) complexes diffuse into 0 mitochondria. Stably transfected 143B-0 cells expressing mtRFP and 143B-+ cells expressing mtGFP were co-plated and analyzed without further treatment (A), or 8 hours (B) or 12 hours (C) after PEG-mediated cell fusion. To inhibit mitochondrial (B) and/or cytosolic (B,C) protein synthesis, fused cells were treated with chloramphenicol (B) and/or cycloheximide (B,C). (A) 143B-0 cells expressing mtRFP display a punctate morphology and are devoid of mitochondrially encoded COX2. 143B-+ cells expressing mtGFP are filamentous and contain mitochondrially encoded COX2. Eight (B) and twelve (C) hours after PEG-mediated cell fusion, polykaryons derived from the fusion of + and 0 cells depict filamentous mitochondria that are positive for mtRFP, mtGFP and COX2. In some polykaryons, the amount of COX2 is lower than that of mtGFP in certain regions of the mitochondrial network (arrowheads), revealing a lower mobility of COX2.

View larger version (108K): [in a new window]   Fig. 7. Mitochondrial DNA nucleoids are mobile within + mitochondria. Stably transfected human 143B cells expressing GFPOM were co-plated with human HeLa cells that had been pre-incubated with BrdU. Co-plated cells were analyzed without further treatment (A), or 12 hours (B,C) or 24 hours (D) after PEG-mediated cell fusion. (A) The nuclear and mitochondrial DNA of untransfected HeLa cells is labeled with BrdU, and GFPOM-transfected cells are devoid of BrdU-labeling. (B-D) The polykaryons derived from the fusion of HeLa and 143B cells are positive for GFPOM and BrdU. The GFPOM-protein is distributed throughout the entire mitochondrial network (B-D). Twelve hours after fusion, BrdU-labeled nucleoids are absent from some regions of the mitochondrial network (B) or distribute to the entire mitochondrial compartment (C). Twenty-four hours after fusion, BrdU-labeled nucleoids are seen throughout the entire mitochondrial compartment. The outlines of the polykaryons are depicted with a white line.