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The functional organization of mitochondrial genomes in human cells.

Iborra FJ, Kimura H, Cook PR - BMC Biol. (2004)

Bottom Line: This mitochondrial RNA colocalizes with components of the cytoplasmic machinery that makes and imports nuclear-encoded proteins - that is, a ribosomal protein (S6), a nascent peptide associated protein (NAC), and the translocase in the outer membrane (Tom22).The results suggest that clusters of mitochondrial genomes organize the translation machineries on both sides of the mitochondrial membranes.Then, proteins encoded by the nuclear genome and destined for the mitochondria will be made close to mitochondrial-encoded proteins so that they can be assembled efficiently into mitochondrial complexes.

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Affiliation: MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, UK. francisco.iborra@imm.ox.ac.uk <francisco.iborra@imm.ox.ac.uk>

ABSTRACT

Background: We analyzed the organization and function of mitochondrial DNA in a stable human cell line (ECV304, which is also known as T-24) containing mitochondria tagged with the yellow fluorescent protein.

Results: Mitochondrial DNA is organized in approximately 475 discrete foci containing 6-10 genomes. These foci (nucleoids) are tethered directly or indirectly through mitochondrial membranes to kinesin, marked by KIF5B, and microtubules in the surrounding cytoplasm. In living cells, foci have an apparent diffusion constant of 1.1 x 10(-3) microm2/s, and mitochondria always split next to a focus to distribute all DNA to one daughter. The kinetics of replication and transcription (monitored by immunolabelling after incorporating bromodeoxyuridine or bromouridine) reveal that each genome replicates independently of others in a focus, and that newly-made RNA remains in a focus (residence half-time approximately 43 min) long after it has been made. This mitochondrial RNA colocalizes with components of the cytoplasmic machinery that makes and imports nuclear-encoded proteins - that is, a ribosomal protein (S6), a nascent peptide associated protein (NAC), and the translocase in the outer membrane (Tom22).

Conclusions: The results suggest that clusters of mitochondrial genomes organize the translation machineries on both sides of the mitochondrial membranes. Then, proteins encoded by the nuclear genome and destined for the mitochondria will be made close to mitochondrial-encoded proteins so that they can be assembled efficiently into mitochondrial complexes.

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Fusion and fission of mitochondria and mtDNA foci. (A-C) Four pairs of frames (collected at 10 s intervals) from three movies of living cells (made as in Figure 4A,4B) showing YFP and ethidium fluorescence (marking mitochondria and mtDNA, respectively) are illustrated. Arrows mark a mtDNA focus that splits (A), two foci that fuse (B), and the point where a mitochondrion splits next to one mtDNA focus to give two separate tips (C). Bar: 1 μm. (D-F) Cells with YFP-tagged mitochondria were fixed, DNA and Drp1 immunolabelled with Cy5 and Cy3, and imaged; two views of one cell are shown in (D) and (E). In the merge (E), mtDNA often lies next to Drp1, which is also found in the cytoplasm [34]. Bar: 1 μm. A cross-correlation analysis of the distribution of mtDNA and Drp1 (data from 15 images like those in (D,E)); Pearson's coefficient (rP) indicates that Drp1 is excluded from ~0.3 μm to each side of mtDNA foci (F).
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Figure 5: Fusion and fission of mitochondria and mtDNA foci. (A-C) Four pairs of frames (collected at 10 s intervals) from three movies of living cells (made as in Figure 4A,4B) showing YFP and ethidium fluorescence (marking mitochondria and mtDNA, respectively) are illustrated. Arrows mark a mtDNA focus that splits (A), two foci that fuse (B), and the point where a mitochondrion splits next to one mtDNA focus to give two separate tips (C). Bar: 1 μm. (D-F) Cells with YFP-tagged mitochondria were fixed, DNA and Drp1 immunolabelled with Cy5 and Cy3, and imaged; two views of one cell are shown in (D) and (E). In the merge (E), mtDNA often lies next to Drp1, which is also found in the cytoplasm [34]. Bar: 1 μm. A cross-correlation analysis of the distribution of mtDNA and Drp1 (data from 15 images like those in (D,E)); Pearson's coefficient (rP) indicates that Drp1 is excluded from ~0.3 μm to each side of mtDNA foci (F).

Mentions: In 25 films of 20 min duration, ~6% mtDNA foci were seen to split (Figure 5A). If fission were coupled to replication, and as mtDNA is replicated throughout the cell cycle [32], we would expect only ~1.5% foci to split (since these cells have a cycle of ~22 h or ~1,320 min, and 20/1320 × 100 = 1.5); therefore, fissions seem uncoupled to replication. While some foci split, others fuse (Figure 5B); some fuse and remain together for 30–600 sec before splitting (not shown). In 49 out of 50 fusion-fission events the emerging foci retained the intensities of fusing foci.


The functional organization of mitochondrial genomes in human cells.

Iborra FJ, Kimura H, Cook PR - BMC Biol. (2004)

Fusion and fission of mitochondria and mtDNA foci. (A-C) Four pairs of frames (collected at 10 s intervals) from three movies of living cells (made as in Figure 4A,4B) showing YFP and ethidium fluorescence (marking mitochondria and mtDNA, respectively) are illustrated. Arrows mark a mtDNA focus that splits (A), two foci that fuse (B), and the point where a mitochondrion splits next to one mtDNA focus to give two separate tips (C). Bar: 1 μm. (D-F) Cells with YFP-tagged mitochondria were fixed, DNA and Drp1 immunolabelled with Cy5 and Cy3, and imaged; two views of one cell are shown in (D) and (E). In the merge (E), mtDNA often lies next to Drp1, which is also found in the cytoplasm [34]. Bar: 1 μm. A cross-correlation analysis of the distribution of mtDNA and Drp1 (data from 15 images like those in (D,E)); Pearson's coefficient (rP) indicates that Drp1 is excluded from ~0.3 μm to each side of mtDNA foci (F).
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Figure 5: Fusion and fission of mitochondria and mtDNA foci. (A-C) Four pairs of frames (collected at 10 s intervals) from three movies of living cells (made as in Figure 4A,4B) showing YFP and ethidium fluorescence (marking mitochondria and mtDNA, respectively) are illustrated. Arrows mark a mtDNA focus that splits (A), two foci that fuse (B), and the point where a mitochondrion splits next to one mtDNA focus to give two separate tips (C). Bar: 1 μm. (D-F) Cells with YFP-tagged mitochondria were fixed, DNA and Drp1 immunolabelled with Cy5 and Cy3, and imaged; two views of one cell are shown in (D) and (E). In the merge (E), mtDNA often lies next to Drp1, which is also found in the cytoplasm [34]. Bar: 1 μm. A cross-correlation analysis of the distribution of mtDNA and Drp1 (data from 15 images like those in (D,E)); Pearson's coefficient (rP) indicates that Drp1 is excluded from ~0.3 μm to each side of mtDNA foci (F).
Mentions: In 25 films of 20 min duration, ~6% mtDNA foci were seen to split (Figure 5A). If fission were coupled to replication, and as mtDNA is replicated throughout the cell cycle [32], we would expect only ~1.5% foci to split (since these cells have a cycle of ~22 h or ~1,320 min, and 20/1320 × 100 = 1.5); therefore, fissions seem uncoupled to replication. While some foci split, others fuse (Figure 5B); some fuse and remain together for 30–600 sec before splitting (not shown). In 49 out of 50 fusion-fission events the emerging foci retained the intensities of fusing foci.

Bottom Line: This mitochondrial RNA colocalizes with components of the cytoplasmic machinery that makes and imports nuclear-encoded proteins - that is, a ribosomal protein (S6), a nascent peptide associated protein (NAC), and the translocase in the outer membrane (Tom22).The results suggest that clusters of mitochondrial genomes organize the translation machineries on both sides of the mitochondrial membranes.Then, proteins encoded by the nuclear genome and destined for the mitochondria will be made close to mitochondrial-encoded proteins so that they can be assembled efficiently into mitochondrial complexes.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, UK. francisco.iborra@imm.ox.ac.uk <francisco.iborra@imm.ox.ac.uk>

ABSTRACT

Background: We analyzed the organization and function of mitochondrial DNA in a stable human cell line (ECV304, which is also known as T-24) containing mitochondria tagged with the yellow fluorescent protein.

Results: Mitochondrial DNA is organized in approximately 475 discrete foci containing 6-10 genomes. These foci (nucleoids) are tethered directly or indirectly through mitochondrial membranes to kinesin, marked by KIF5B, and microtubules in the surrounding cytoplasm. In living cells, foci have an apparent diffusion constant of 1.1 x 10(-3) microm2/s, and mitochondria always split next to a focus to distribute all DNA to one daughter. The kinetics of replication and transcription (monitored by immunolabelling after incorporating bromodeoxyuridine or bromouridine) reveal that each genome replicates independently of others in a focus, and that newly-made RNA remains in a focus (residence half-time approximately 43 min) long after it has been made. This mitochondrial RNA colocalizes with components of the cytoplasmic machinery that makes and imports nuclear-encoded proteins - that is, a ribosomal protein (S6), a nascent peptide associated protein (NAC), and the translocase in the outer membrane (Tom22).

Conclusions: The results suggest that clusters of mitochondrial genomes organize the translation machineries on both sides of the mitochondrial membranes. Then, proteins encoded by the nuclear genome and destined for the mitochondria will be made close to mitochondrial-encoded proteins so that they can be assembled efficiently into mitochondrial complexes.

Show MeSH
Related in: MedlinePlus