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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.

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.

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Mitochondrial transcription. Cells with YFP-tagged mitochondria were grown in 2.5 mM BrU for different times, fixed, Br-RNA immunolabelled with Cy3, and confocal images collected. In some cases, cells were grown for 30 min, washed, and regrown in 5 mM U for up to 2 h (I,J); in others, DNA was immunolabelled with Cy5 (E-G,J) or stained with Hoechst 33342 (K). (A,B) Two views of one field; after growth in BrU for 20 min, Br-RNA is seen mainly in mitochondria. (C,D) Two views of one field; after growth in BrU for 60 min, Br-RNA is seen mainly in nuclei. Bar: 10 μm. (E-G) Three views of mitochondria of one cell, after growth in BrU for 30 min and immunolabelling DNA with Cy5; the merge reveals Br-RNA (red) in some, but not all, mtDNA foci (green). Bar: 2 μm. (H) On growth in BrU, the intensity of Br-RNA labelling (arbitrary units/μm2) in mitochondria increases progressively. (I) After growth for 30 min in BrU and regrowth in U, the intensity of mitochondrial Br-RNA labelling falls exponentially (red line; t1/2 ~ 45 min). (J) The effects of incubation time on distance between a Br-RNA focus and the nearest mtDNA focus. Distances (μm) are binned (that is, 0–0.2, 0.2–0.4, etc). After 20 or 30 min in BrU, most Br-RNA foci lie between 0–0.2 μm of the nearest mtDNA foci (labelled with Cy5); however, after 1 h (or a 30 min pulse followed by a 2 h chase), Br-RNA foci appear to be spread randomly. The decline in Br-RNA colocalizing with mtDNA (that is, lying within 0.2 μm) between 20 and 60 min is consistent with a residence half-life of 43 min. (K) After growth in BrU for 30 min, the intensity of labelling in a Br-RNA focus (au, arbitrary units) does not correlate with the DNA concentration seen in the associated mtDNA focus. The line (slope = 0.0042) is that of best fit. In an analogous experiment in which mtDNA was marked with the antibody, the line of best fit had a similar slope of 0.0068.
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Figure 7: Mitochondrial transcription. Cells with YFP-tagged mitochondria were grown in 2.5 mM BrU for different times, fixed, Br-RNA immunolabelled with Cy3, and confocal images collected. In some cases, cells were grown for 30 min, washed, and regrown in 5 mM U for up to 2 h (I,J); in others, DNA was immunolabelled with Cy5 (E-G,J) or stained with Hoechst 33342 (K). (A,B) Two views of one field; after growth in BrU for 20 min, Br-RNA is seen mainly in mitochondria. (C,D) Two views of one field; after growth in BrU for 60 min, Br-RNA is seen mainly in nuclei. Bar: 10 μm. (E-G) Three views of mitochondria of one cell, after growth in BrU for 30 min and immunolabelling DNA with Cy5; the merge reveals Br-RNA (red) in some, but not all, mtDNA foci (green). Bar: 2 μm. (H) On growth in BrU, the intensity of Br-RNA labelling (arbitrary units/μm2) in mitochondria increases progressively. (I) After growth for 30 min in BrU and regrowth in U, the intensity of mitochondrial Br-RNA labelling falls exponentially (red line; t1/2 ~ 45 min). (J) The effects of incubation time on distance between a Br-RNA focus and the nearest mtDNA focus. Distances (μm) are binned (that is, 0–0.2, 0.2–0.4, etc). After 20 or 30 min in BrU, most Br-RNA foci lie between 0–0.2 μm of the nearest mtDNA foci (labelled with Cy5); however, after 1 h (or a 30 min pulse followed by a 2 h chase), Br-RNA foci appear to be spread randomly. The decline in Br-RNA colocalizing with mtDNA (that is, lying within 0.2 μm) between 20 and 60 min is consistent with a residence half-life of 43 min. (K) After growth in BrU for 30 min, the intensity of labelling in a Br-RNA focus (au, arbitrary units) does not correlate with the DNA concentration seen in the associated mtDNA focus. The line (slope = 0.0042) is that of best fit. In an analogous experiment in which mtDNA was marked with the antibody, the line of best fit had a similar slope of 0.0068.

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)

Mitochondrial transcription. Cells with YFP-tagged mitochondria were grown in 2.5 mM BrU for different times, fixed, Br-RNA immunolabelled with Cy3, and confocal images collected. In some cases, cells were grown for 30 min, washed, and regrown in 5 mM U for up to 2 h (I,J); in others, DNA was immunolabelled with Cy5 (E-G,J) or stained with Hoechst 33342 (K). (A,B) Two views of one field; after growth in BrU for 20 min, Br-RNA is seen mainly in mitochondria. (C,D) Two views of one field; after growth in BrU for 60 min, Br-RNA is seen mainly in nuclei. Bar: 10 μm. (E-G) Three views of mitochondria of one cell, after growth in BrU for 30 min and immunolabelling DNA with Cy5; the merge reveals Br-RNA (red) in some, but not all, mtDNA foci (green). Bar: 2 μm. (H) On growth in BrU, the intensity of Br-RNA labelling (arbitrary units/μm2) in mitochondria increases progressively. (I) After growth for 30 min in BrU and regrowth in U, the intensity of mitochondrial Br-RNA labelling falls exponentially (red line; t1/2 ~ 45 min). (J) The effects of incubation time on distance between a Br-RNA focus and the nearest mtDNA focus. Distances (μm) are binned (that is, 0–0.2, 0.2–0.4, etc). After 20 or 30 min in BrU, most Br-RNA foci lie between 0–0.2 μm of the nearest mtDNA foci (labelled with Cy5); however, after 1 h (or a 30 min pulse followed by a 2 h chase), Br-RNA foci appear to be spread randomly. The decline in Br-RNA colocalizing with mtDNA (that is, lying within 0.2 μm) between 20 and 60 min is consistent with a residence half-life of 43 min. (K) After growth in BrU for 30 min, the intensity of labelling in a Br-RNA focus (au, arbitrary units) does not correlate with the DNA concentration seen in the associated mtDNA focus. The line (slope = 0.0042) is that of best fit. In an analogous experiment in which mtDNA was marked with the antibody, the line of best fit had a similar slope of 0.0068.
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Related In: Results  -  Collection

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Figure 7: Mitochondrial transcription. Cells with YFP-tagged mitochondria were grown in 2.5 mM BrU for different times, fixed, Br-RNA immunolabelled with Cy3, and confocal images collected. In some cases, cells were grown for 30 min, washed, and regrown in 5 mM U for up to 2 h (I,J); in others, DNA was immunolabelled with Cy5 (E-G,J) or stained with Hoechst 33342 (K). (A,B) Two views of one field; after growth in BrU for 20 min, Br-RNA is seen mainly in mitochondria. (C,D) Two views of one field; after growth in BrU for 60 min, Br-RNA is seen mainly in nuclei. Bar: 10 μm. (E-G) Three views of mitochondria of one cell, after growth in BrU for 30 min and immunolabelling DNA with Cy5; the merge reveals Br-RNA (red) in some, but not all, mtDNA foci (green). Bar: 2 μm. (H) On growth in BrU, the intensity of Br-RNA labelling (arbitrary units/μm2) in mitochondria increases progressively. (I) After growth for 30 min in BrU and regrowth in U, the intensity of mitochondrial Br-RNA labelling falls exponentially (red line; t1/2 ~ 45 min). (J) The effects of incubation time on distance between a Br-RNA focus and the nearest mtDNA focus. Distances (μm) are binned (that is, 0–0.2, 0.2–0.4, etc). After 20 or 30 min in BrU, most Br-RNA foci lie between 0–0.2 μm of the nearest mtDNA foci (labelled with Cy5); however, after 1 h (or a 30 min pulse followed by a 2 h chase), Br-RNA foci appear to be spread randomly. The decline in Br-RNA colocalizing with mtDNA (that is, lying within 0.2 μm) between 20 and 60 min is consistent with a residence half-life of 43 min. (K) After growth in BrU for 30 min, the intensity of labelling in a Br-RNA focus (au, arbitrary units) does not correlate with the DNA concentration seen in the associated mtDNA focus. The line (slope = 0.0042) is that of best fit. In an analogous experiment in which mtDNA was marked with the antibody, the line of best fit had a similar slope of 0.0068.
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