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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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mtDNA dynamics in living cells with YFP-tagged mitochondria. Cells were grown (30 min) in 0.1 μg/ml ethidium, the ethidium washed away, the cells regrown for 10 min, and single confocal sections collected every 10 sec for ≤500 sec; mitochondria and mtDNA are marked by YFP and ethidium respectively. (A,B) Two views of mitochondria in one cell at one time. Ethidium is locally concentrated in (mtDNA) foci, against a background (bound to RNA). S1: distance between two foci. S2: distance between one focus and mitochondrial tip. (C,D) Frames showing ethidium fluorescence (marking mtDNA) from two movies. In each case, the first (0 sec; left) and last (500 sec; middle) frames show the two mtDNA foci move little relative to each other; this is confirmed by superimposing all frames in each movie (right). Bar: 2 μm. (E) Changes in separations S1 and S2 seen in (B) over time; movement is erratic. (F) Mean square displacement (MSD; n = 20) of one mt DNA focus relative to another (DNA), or one tip relative to another (tip); movement of foci is more restrained than that of tips. The MSD of one terminal focus relative to the tip is similar to that of one focus relative to another and is not shown. (G) Autocorrelation analysis. The autocorrelation (ac) between the displacement distances (Δd) for time points separated by different time intervals (Δt) of 10 sec (dashed lines show limits of 5% significance). The pattern is typical of a random walk; there is no periodicity, and the first value (the only significant one) is negative. (H) The distribution of step lengths (Δd) is typical of a random walk. Mean = 0.01 μm (SD ± 0.4). The maximum value was 2.4 μm, and is not shown.
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Figure 4: mtDNA dynamics in living cells with YFP-tagged mitochondria. Cells were grown (30 min) in 0.1 μg/ml ethidium, the ethidium washed away, the cells regrown for 10 min, and single confocal sections collected every 10 sec for ≤500 sec; mitochondria and mtDNA are marked by YFP and ethidium respectively. (A,B) Two views of mitochondria in one cell at one time. Ethidium is locally concentrated in (mtDNA) foci, against a background (bound to RNA). S1: distance between two foci. S2: distance between one focus and mitochondrial tip. (C,D) Frames showing ethidium fluorescence (marking mtDNA) from two movies. In each case, the first (0 sec; left) and last (500 sec; middle) frames show the two mtDNA foci move little relative to each other; this is confirmed by superimposing all frames in each movie (right). Bar: 2 μm. (E) Changes in separations S1 and S2 seen in (B) over time; movement is erratic. (F) Mean square displacement (MSD; n = 20) of one mt DNA focus relative to another (DNA), or one tip relative to another (tip); movement of foci is more restrained than that of tips. The MSD of one terminal focus relative to the tip is similar to that of one focus relative to another and is not shown. (G) Autocorrelation analysis. The autocorrelation (ac) between the displacement distances (Δd) for time points separated by different time intervals (Δt) of 10 sec (dashed lines show limits of 5% significance). The pattern is typical of a random walk; there is no periodicity, and the first value (the only significant one) is negative. (H) The distribution of step lengths (Δd) is typical of a random walk. Mean = 0.01 μm (SD ± 0.4). The maximum value was 2.4 μm, and is not shown.

Mentions: The dynamics of mtDNA in living cells can be monitored after staining with fluorescent dyes like 4',6'-diamidino-2-phenylindole (DAPI) [27], SYTO13 [28], or ethidium [29]; here, we use ethidium. Cells were grown briefly in 0.1 μg/ml ethidium, washed, and regrown in its absence; some dye is then seen in nuclei and mitochondria, with little in the rest of the cytoplasm. (This brief exposure has no long-term effects on cell doubling, but transcription is temporarily inhibited as bromouridine (BrU) incorporation into mitochondrial RNA falls to 60% of controls (measured as in Figure 7H (see later) during the 20 min following removal of ethidium; 1 μg/ml ethidium completely inhibits incorporation; not shown).) Immediately after exposure to 0.1 μg/ml, ethidium fluorescence in mitochondria is concentrated in discrete foci against a diffuse background (Figure 4B). On regrowth in the absence of ethidium, foci faded much less rapidly than the background; as a result, foci could still be seen after 8 h against a now non-fluorescent background. This suggests that foci reflected tight binding to mtDNA, and the initial background an unstable binding to RNA. Marking RNA in fixed cells using a tagged RNA-binding protein (that is, RNase tagged with Cy3) confirms that this background contains RNA (see Methods).


The functional organization of mitochondrial genomes in human cells.

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

mtDNA dynamics in living cells with YFP-tagged mitochondria. Cells were grown (30 min) in 0.1 μg/ml ethidium, the ethidium washed away, the cells regrown for 10 min, and single confocal sections collected every 10 sec for ≤500 sec; mitochondria and mtDNA are marked by YFP and ethidium respectively. (A,B) Two views of mitochondria in one cell at one time. Ethidium is locally concentrated in (mtDNA) foci, against a background (bound to RNA). S1: distance between two foci. S2: distance between one focus and mitochondrial tip. (C,D) Frames showing ethidium fluorescence (marking mtDNA) from two movies. In each case, the first (0 sec; left) and last (500 sec; middle) frames show the two mtDNA foci move little relative to each other; this is confirmed by superimposing all frames in each movie (right). Bar: 2 μm. (E) Changes in separations S1 and S2 seen in (B) over time; movement is erratic. (F) Mean square displacement (MSD; n = 20) of one mt DNA focus relative to another (DNA), or one tip relative to another (tip); movement of foci is more restrained than that of tips. The MSD of one terminal focus relative to the tip is similar to that of one focus relative to another and is not shown. (G) Autocorrelation analysis. The autocorrelation (ac) between the displacement distances (Δd) for time points separated by different time intervals (Δt) of 10 sec (dashed lines show limits of 5% significance). The pattern is typical of a random walk; there is no periodicity, and the first value (the only significant one) is negative. (H) The distribution of step lengths (Δd) is typical of a random walk. Mean = 0.01 μm (SD ± 0.4). The maximum value was 2.4 μm, and is not shown.
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Figure 4: mtDNA dynamics in living cells with YFP-tagged mitochondria. Cells were grown (30 min) in 0.1 μg/ml ethidium, the ethidium washed away, the cells regrown for 10 min, and single confocal sections collected every 10 sec for ≤500 sec; mitochondria and mtDNA are marked by YFP and ethidium respectively. (A,B) Two views of mitochondria in one cell at one time. Ethidium is locally concentrated in (mtDNA) foci, against a background (bound to RNA). S1: distance between two foci. S2: distance between one focus and mitochondrial tip. (C,D) Frames showing ethidium fluorescence (marking mtDNA) from two movies. In each case, the first (0 sec; left) and last (500 sec; middle) frames show the two mtDNA foci move little relative to each other; this is confirmed by superimposing all frames in each movie (right). Bar: 2 μm. (E) Changes in separations S1 and S2 seen in (B) over time; movement is erratic. (F) Mean square displacement (MSD; n = 20) of one mt DNA focus relative to another (DNA), or one tip relative to another (tip); movement of foci is more restrained than that of tips. The MSD of one terminal focus relative to the tip is similar to that of one focus relative to another and is not shown. (G) Autocorrelation analysis. The autocorrelation (ac) between the displacement distances (Δd) for time points separated by different time intervals (Δt) of 10 sec (dashed lines show limits of 5% significance). The pattern is typical of a random walk; there is no periodicity, and the first value (the only significant one) is negative. (H) The distribution of step lengths (Δd) is typical of a random walk. Mean = 0.01 μm (SD ± 0.4). The maximum value was 2.4 μm, and is not shown.
Mentions: The dynamics of mtDNA in living cells can be monitored after staining with fluorescent dyes like 4',6'-diamidino-2-phenylindole (DAPI) [27], SYTO13 [28], or ethidium [29]; here, we use ethidium. Cells were grown briefly in 0.1 μg/ml ethidium, washed, and regrown in its absence; some dye is then seen in nuclei and mitochondria, with little in the rest of the cytoplasm. (This brief exposure has no long-term effects on cell doubling, but transcription is temporarily inhibited as bromouridine (BrU) incorporation into mitochondrial RNA falls to 60% of controls (measured as in Figure 7H (see later) during the 20 min following removal of ethidium; 1 μg/ml ethidium completely inhibits incorporation; not shown).) Immediately after exposure to 0.1 μg/ml, ethidium fluorescence in mitochondria is concentrated in discrete foci against a diffuse background (Figure 4B). On regrowth in the absence of ethidium, foci faded much less rapidly than the background; as a result, foci could still be seen after 8 h against a now non-fluorescent background. This suggests that foci reflected tight binding to mtDNA, and the initial background an unstable binding to RNA. Marking RNA in fixed cells using a tagged RNA-binding protein (that is, RNase tagged with Cy3) confirms that this background contains RNA (see Methods).

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