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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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DNA in the mitochondria of fixed ECV-304 cells expressing subunit VIII of cytochrome oxidase tagged with YFP. (A,B) Two views taken with a confocal microscope of fixed cells after immunolabelling DNA. Mitochondria marked with YFP (A) contain discrete foci of mtDNA (B); nuclear DNA appears weakly labelled, presumably because most of it is inaccessible to the anti-DNA antibody, an IgM. Pretreatment (60 min; 20°C) with 25 μg/ml DNase removed >96% foci like those in (B). Insets: high-power views (bar: 2 μm). (C,D) Two similar views after growth (24 h) in BrdU, and immunolabelling the resulting Br-DNA; Br-DNA is found in nuclei and mitochondrial foci. Bar: 4 μm. (E) The distribution seen experimentally (exp) of distances between consecutive DNA foci within mitochondria (determined using images like (B); n > 500) differs significantly from a random one (p = 0.0013); foci tend to be closer together than expected. Distances (μm) are binned (that is, 0–0.4, 0.4–0.8, etc). (F) Experimental distribution (determined using 15 images like (D)) of intensities of mtDNA foci. Intensities (arbitrary units, au) are binned (that is, 0–0.5, 0.5–1, etc), the first bin contained no examples, and the arrow marks the average intensity. If the weakest focus (in bin 2) contains one genome, then the average contains ~9.2. (G) Cross-correlation analysis of mtDNA foci and the YFP-tagged subunit (using images like those in the insets in (A,B)); the low Pearson's coefficient (rP) at Δx values close to zero indicates that mtDNA is excluded from sites containing YFP.
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Figure 1: DNA in the mitochondria of fixed ECV-304 cells expressing subunit VIII of cytochrome oxidase tagged with YFP. (A,B) Two views taken with a confocal microscope of fixed cells after immunolabelling DNA. Mitochondria marked with YFP (A) contain discrete foci of mtDNA (B); nuclear DNA appears weakly labelled, presumably because most of it is inaccessible to the anti-DNA antibody, an IgM. Pretreatment (60 min; 20°C) with 25 μg/ml DNase removed >96% foci like those in (B). Insets: high-power views (bar: 2 μm). (C,D) Two similar views after growth (24 h) in BrdU, and immunolabelling the resulting Br-DNA; Br-DNA is found in nuclei and mitochondrial foci. Bar: 4 μm. (E) The distribution seen experimentally (exp) of distances between consecutive DNA foci within mitochondria (determined using images like (B); n > 500) differs significantly from a random one (p = 0.0013); foci tend to be closer together than expected. Distances (μm) are binned (that is, 0–0.4, 0.4–0.8, etc). (F) Experimental distribution (determined using 15 images like (D)) of intensities of mtDNA foci. Intensities (arbitrary units, au) are binned (that is, 0–0.5, 0.5–1, etc), the first bin contained no examples, and the arrow marks the average intensity. If the weakest focus (in bin 2) contains one genome, then the average contains ~9.2. (G) Cross-correlation analysis of mtDNA foci and the YFP-tagged subunit (using images like those in the insets in (A,B)); the low Pearson's coefficient (rP) at Δx values close to zero indicates that mtDNA is excluded from sites containing YFP.

Mentions: We first derived a stable cell line containing mitochondria tagged with the yellow fluorescent protein (YFP). A plasmid encoding subunit VIII of cytochrome c oxidase fused with YFP was transfected into an epithelial-like human line derived from a bladder carcinoma (ECV304), and a clone (cox18) expressing YFP was established. Expression of the plasmid in other cells has no effect on viability [17]. The tagged subunit is incorporated into mitochondria, making them autofluorescent (Figure 1A). However, photobleaching experiments show it diffuses rapidly throughout the interior of the mitochondrion ([17]; our unpublished results); this makes it unlikely that it is incorporated into the membrane to become functional. This clone is used for all experiments described here.


The functional organization of mitochondrial genomes in human cells.

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

DNA in the mitochondria of fixed ECV-304 cells expressing subunit VIII of cytochrome oxidase tagged with YFP. (A,B) Two views taken with a confocal microscope of fixed cells after immunolabelling DNA. Mitochondria marked with YFP (A) contain discrete foci of mtDNA (B); nuclear DNA appears weakly labelled, presumably because most of it is inaccessible to the anti-DNA antibody, an IgM. Pretreatment (60 min; 20°C) with 25 μg/ml DNase removed >96% foci like those in (B). Insets: high-power views (bar: 2 μm). (C,D) Two similar views after growth (24 h) in BrdU, and immunolabelling the resulting Br-DNA; Br-DNA is found in nuclei and mitochondrial foci. Bar: 4 μm. (E) The distribution seen experimentally (exp) of distances between consecutive DNA foci within mitochondria (determined using images like (B); n > 500) differs significantly from a random one (p = 0.0013); foci tend to be closer together than expected. Distances (μm) are binned (that is, 0–0.4, 0.4–0.8, etc). (F) Experimental distribution (determined using 15 images like (D)) of intensities of mtDNA foci. Intensities (arbitrary units, au) are binned (that is, 0–0.5, 0.5–1, etc), the first bin contained no examples, and the arrow marks the average intensity. If the weakest focus (in bin 2) contains one genome, then the average contains ~9.2. (G) Cross-correlation analysis of mtDNA foci and the YFP-tagged subunit (using images like those in the insets in (A,B)); the low Pearson's coefficient (rP) at Δx values close to zero indicates that mtDNA is excluded from sites containing YFP.
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Related In: Results  -  Collection

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Figure 1: DNA in the mitochondria of fixed ECV-304 cells expressing subunit VIII of cytochrome oxidase tagged with YFP. (A,B) Two views taken with a confocal microscope of fixed cells after immunolabelling DNA. Mitochondria marked with YFP (A) contain discrete foci of mtDNA (B); nuclear DNA appears weakly labelled, presumably because most of it is inaccessible to the anti-DNA antibody, an IgM. Pretreatment (60 min; 20°C) with 25 μg/ml DNase removed >96% foci like those in (B). Insets: high-power views (bar: 2 μm). (C,D) Two similar views after growth (24 h) in BrdU, and immunolabelling the resulting Br-DNA; Br-DNA is found in nuclei and mitochondrial foci. Bar: 4 μm. (E) The distribution seen experimentally (exp) of distances between consecutive DNA foci within mitochondria (determined using images like (B); n > 500) differs significantly from a random one (p = 0.0013); foci tend to be closer together than expected. Distances (μm) are binned (that is, 0–0.4, 0.4–0.8, etc). (F) Experimental distribution (determined using 15 images like (D)) of intensities of mtDNA foci. Intensities (arbitrary units, au) are binned (that is, 0–0.5, 0.5–1, etc), the first bin contained no examples, and the arrow marks the average intensity. If the weakest focus (in bin 2) contains one genome, then the average contains ~9.2. (G) Cross-correlation analysis of mtDNA foci and the YFP-tagged subunit (using images like those in the insets in (A,B)); the low Pearson's coefficient (rP) at Δx values close to zero indicates that mtDNA is excluded from sites containing YFP.
Mentions: We first derived a stable cell line containing mitochondria tagged with the yellow fluorescent protein (YFP). A plasmid encoding subunit VIII of cytochrome c oxidase fused with YFP was transfected into an epithelial-like human line derived from a bladder carcinoma (ECV304), and a clone (cox18) expressing YFP was established. Expression of the plasmid in other cells has no effect on viability [17]. The tagged subunit is incorporated into mitochondria, making them autofluorescent (Figure 1A). However, photobleaching experiments show it diffuses rapidly throughout the interior of the mitochondrion ([17]; our unpublished results); this makes it unlikely that it is incorporated into the membrane to become functional. This clone is used for all experiments described here.

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