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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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Electron microscopy reveals mitochondrial DNA in discrete foci. Bars: 200 nm. (A) Cytoplasmic section after immunogold labelling with anti-DNA; gold particles marking mtDNA are found near the mitochondrial membrane. (B) Whole mount view of cytoplasm after extraction with CSK buffer and immunogold labelling with anti-DNA; mtDNA (marked by gold particles) resists extraction.
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Figure 2: Electron microscopy reveals mitochondrial DNA in discrete foci. Bars: 200 nm. (A) Cytoplasmic section after immunogold labelling with anti-DNA; gold particles marking mtDNA are found near the mitochondrial membrane. (B) Whole mount view of cytoplasm after extraction with CSK buffer and immunogold labelling with anti-DNA; mtDNA (marked by gold particles) resists extraction.

Mentions: Immunogold labelling and electron microscopy confirmed that mtDNA was found in discrete sites; moreover, the sites often abutted the inner mitochondrial membrane as in Figure 2A. DNA also remained associated with cytoskeletal elements when mitochondrial membranes and most cytoplasmic proteins were extracted (Figure 2B), consistent with earlier results [22-24]. These sites had an average diameter of ~68 nm (Table 1, line 4). As expected [2,7,25], mitochondria also associated with microtubules (Figure 3A,3B,3C,3D). Moreover, the kinesin motor implicated in moving mitochondria along microtubules – KIF5B [25] – often colocalized with individual mtDNA foci (Figure 3E,3F,3G), and a close association was confirmed by the peak in Pearson's coefficient at a Δx of zero (Figure 3H). These results are consistent with mtDNA foci being tethered through mitochondrial membranes to kinesin and the microtubule network.


The functional organization of mitochondrial genomes in human cells.

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

Electron microscopy reveals mitochondrial DNA in discrete foci. Bars: 200 nm. (A) Cytoplasmic section after immunogold labelling with anti-DNA; gold particles marking mtDNA are found near the mitochondrial membrane. (B) Whole mount view of cytoplasm after extraction with CSK buffer and immunogold labelling with anti-DNA; mtDNA (marked by gold particles) resists extraction.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC425603&req=5

Figure 2: Electron microscopy reveals mitochondrial DNA in discrete foci. Bars: 200 nm. (A) Cytoplasmic section after immunogold labelling with anti-DNA; gold particles marking mtDNA are found near the mitochondrial membrane. (B) Whole mount view of cytoplasm after extraction with CSK buffer and immunogold labelling with anti-DNA; mtDNA (marked by gold particles) resists extraction.
Mentions: Immunogold labelling and electron microscopy confirmed that mtDNA was found in discrete sites; moreover, the sites often abutted the inner mitochondrial membrane as in Figure 2A. DNA also remained associated with cytoskeletal elements when mitochondrial membranes and most cytoplasmic proteins were extracted (Figure 2B), consistent with earlier results [22-24]. These sites had an average diameter of ~68 nm (Table 1, line 4). As expected [2,7,25], mitochondria also associated with microtubules (Figure 3A,3B,3C,3D). Moreover, the kinesin motor implicated in moving mitochondria along microtubules – KIF5B [25] – often colocalized with individual mtDNA foci (Figure 3E,3F,3G), and a close association was confirmed by the peak in Pearson's coefficient at a Δx of zero (Figure 3H). These results are consistent with mtDNA foci being tethered through mitochondrial membranes to kinesin and the microtubule network.

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