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. 2008 Jun 30;181(7):1117-28.
doi: 10.1083/jcb.200712101. Epub 2008 Jun 23.

Mitochondrial nucleoids maintain genetic autonomy but allow for functional complementation

Robert W Gilkerson  1 , Eric A Schon, Evelyn Hernandez, Mercy M Davidson
Affiliations

Mitochondrial nucleoids maintain genetic autonomy but allow for functional complementation

Robert W Gilkerson et al. J Cell Biol. .

Abstract

Mitochondrial DNA (mtDNA) is packaged into DNA-protein assemblies called nucleoids, but the mode of mtDNA propagation via the nucleoid remains controversial. Two mechanisms have been proposed: nucleoids may consistently maintain their mtDNA content faithfully, or nucleoids may exchange mtDNAs dynamically. To test these models directly, two cell lines were fused, each homoplasmic for a partially deleted mtDNA in which the deletions were nonoverlapping and each deficient in mitochondrial protein synthesis, thus allowing the first unequivocal visualization of two mtDNAs at the nucleoid level. The two mtDNAs transcomplemented to restore mitochondrial protein synthesis but were consistently maintained in discrete nucleoids that did not intermix stably. These results indicate that mitochondrial nucleoids tightly regulate their genetic content rather than freely exchanging mtDNAs. This genetic autonomy provides a molecular mechanism to explain patterns of mitochondrial genetic inheritance, in addition to facilitating therapeutic methods to eliminate deleterious mtDNA mutations.

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Figures

Figure 1.
Figure 1.
mtDNAs and experimental design. (A) Linearized maps of mtDNAs discussed. The WT human mtDNA is shown at top. FLP and CW Δ-mtDNAs are shown below. White box denotes deleted region for each Δ-mtDNA. Red box denotes CW Δ-mtDNA FISH probe position and size. Green box denotes FLP Δ-mtDNA FISH probe position and size. (B) Schematic of cell fusion experiment. FLPΔ and CWΔ homoplasmic cell lines are fused in the presence of PEG, allowed to recover, and placed in uridine-minus medium to select for mitochondrial function. Homoplasmic FLPΔ (green) and CWΔ (red) nucleoids are shown with four to five copies of mtDNA per nucleoid. Two models for complementation are shown: faithful, in which each Δ-mtDNA (red only or green only) remains segregated from the other in homoplasmic nucleoids, and dynamic, in which the Δ-mtDNAs are exchanged among nucleoids, resulting in heteroplasmic nucleoids (red plus green).
Figure 2.
Figure 2.
mtDNA analysis of cybrid cell lines. (A) Long-distance PCR of mtDNAs. Lanes denote long-distance amplification from 1 ng of template from total cellular DNA of the indicated cell line. Primers amplify 14,320 bp from nt 3066 (forward primer) to nt 812 (reverse primer) of the WT mtDNA molecule, encompassing both deleted regions. (B) Conventional PCR of ND4 and CO2/3 from isolated total DNA of cybrid cell lines. Lanes denote PCR reactions from 100 ng of template. (C) Southern blot analysis of FC II day-90 cells, including schematic of CW and FLP Δ-mtDNA restriction sites, as well as that of a potential FLP/CW recombinant molecule. Both Δ-mtDNAs carry a PvuII site. FLP Δ-mtDNA carries a SnaBI site but not a Tth111I site, whereas CW Δ-mtDNA carries a Tth111I site but not a SnaBI. 2 μg of total cellular DNA was digested with the enzymes listed, transferred to PVDF, and probed with DNA corresponding to nt 3778–6051 of human mtDNA. Single-lane panel shows a Southern blot of FC II cells subjected to ketogenic selection, digested with SnaBI and Tth111I, and electrophoresed for several days to achieve better separation of the FLP and CW Δ-mtDNA–linearized molecules to examine for the presence of FC recombinant restriction digest products (13.7 and 11.8 kb) intermediate to the two parental Δ-mtDNA restriction products (FLP Δ-mtDNA, 14.7 kb; CW Δ-mtDNA, 10.8 kb).
Figure 3.
Figure 3.
Two-color FISH of cultured cells. Green (FLP Δ-mtDNA) and red (CW Δ-mtDNA) images were captured sequentially. Bar, 20 μm.
Figure 4.
Figure 4.
Immunofluorescence microscopy of cybrid cell lines. Cells were immunolabeled for fluorescence microscopy with an antibody against mitochondrially encoded MTCO2 (green). Mitochondria were visualized with MitoTracker (red). Bar, 20 μm. Outlined boxes in merge are enlarged in merge detail.
Figure 5.
Figure 5.
A novel combined immunofluorescence/FISH method. (A) Cells were first immunolabeled for MTCO2 protein (blue) and subsequently labeled for mtDNA via FISH with probes against FLP Δ-mtDNA (green) and CW Δ-mtDNA (red). Indicated controls lacking primary antibody and/or FISH probes demonstrate signal specificity. Cells were visualized by confocal microscopy. Bar, 10 μm. (B) High-resolution immunofluorescence/FISH images of WT and FC cells. Cells were visualized by conventional microscopy. Bar, 10 μm.
Figure 6.
Figure 6.
Time course of FC cell fusion. (A) Confocal microscopy images of FC I cells viewed by green/red in situ hybridization at days 22 and 32 of selection in medium lacking uridine. Bar, 20 μm. Outlined boxes in merge are enlarged in detail. Bar, 10 μm. (B) Long-term culture of FC II cells visualized by FISH using conventional microscopy. Arrowheads (day 120) denote minority genotype nucleoids in essentially homoplasmic cells (Table I). Bar, 20 μm. (C) Anti-MTCO2 immunofluorescence microscopy of FC I cells at days 4, 22, 32, 104, and 274 of selection. The boundaries of cells with little or no MTCO2 signal were added freehand in Photoshop. Days 22 and 32 were detected with an Alexa 350 (blue) goat anti–rabbit secondary as in Fig. 5, and images were pseudocolored green via ImageJ. Bar, 20 μm.
Figure 7.
Figure 7.
Nucleoid segregation in FC cells. Representative FISH images, visualized with conventional microscopy, used to generate the colocalization data in Table II. FLP Δ-mtDNA is labeled in green, whereas the CW Δ-mtDNA is labeled in red, with colocalization appearing as yellow in the merge. ImageJ colocalization is shown in white in the far right, illustrating the pixels having both significant red and green signal. WT and ρ0 controls are also shown. Bar, 0.5 μm.
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