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Comparative Study
. 2003 Apr;14(4):1583-96.
doi: 10.1091/mbc.e02-07-0399.

Composition and dynamics of human mitochondrial nucleoids

Nuria Garrido  1 , Lorena Griparic, Eija Jokitalo, Jorma Wartiovaara, Alexander M van der Bliek, Johannes N Spelbrink
Affiliations
Comparative Study

Composition and dynamics of human mitochondrial nucleoids

Nuria Garrido et al. Mol Biol Cell. 2003 Apr.

Abstract

The organization of multiple mitochondrial DNA (mtDNA) molecules in discrete protein-DNA complexes called nucleoids is well studied in Saccharomyces cerevisiae. Similar structures have recently been observed in human cells by the colocalization of a Twinkle-GFP fusion protein with mtDNA. However, nucleoids in mammalian cells are poorly characterized and are often thought of as relatively simple structures, despite the yeast paradigm. In this article we have used immunocytochemistry and biochemical isolation procedures to characterize the composition of human mitochondrial nucleoids. The results show that both the mitochondrial transcription factor TFAM and mitochondrial single-stranded DNA-binding protein colocalize with Twinkle in intramitochondrial foci defined as nucleoids by the specific incorporation of bromodeoxyuridine. Furthermore, mtDNA polymerase POLG and various other as yet unidentified proteins copurify with mtDNA nucleoids using a biochemical isolation procedure, as does TFAM. The results demonstrated that mtDNA in mammalian cells is organized in discrete protein-rich structures within the mitochondrial network. In vivo time-lapse imaging of nucleoids show they are dynamic structures able to divide and redistribute in the mitochondrial network and suggest that nucleoids are the mitochondrial units of inheritance. Nucleoids did not colocalize with dynamin-related protein 1, Drp1, a protein of the mitochondrial fission machinery.

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Figures

Figure 1
Figure 1
Localization of endogenous TFAM and mtSSB in intramitochondrial foci. 143B osteosarcoma cells grown on coverslips for 1–2 d were stained with Mitotracker Red (B and E). Detection of TFAM (A) and mtSSB (D) by ICC used polyclonal rabbit antibodies and a secondary fluorescein labeled antibody. (C; TFAM, F; SSB) Merged fluorescent images. (G) Immunolabeling for TFAM in a HEK293 cell mitochondrion. Seven gold particles were concentrated along a stretch of 300 nm of the mitochondrion. Very little background labeling was observed outside mitochondria (circle). (H) Blow-up of the boxed area in G, showing TFAM label (arrow) in the mitochondrial matrix near cross-sectioned cristae (C).
Figure 2
Figure 2
Colocalization of Twinkle-EGFP with endogenous TFAM, mtSSB, and POLG2. 143B osteosarcoma cells (or A549 lung-carcinoma cells) were grown on coverslips and transfected with a Twinkle-EGFP constructs. One to 2 d after transfection ICC for the detection of TFAM, mtSSB, or POLG2 was performed. Secondary antibodies had rhodamine or Texas Red fluorescent groups. (A, D, and G) ICC for TFAM, mtSSB, and POLG2, respectively. (B, E, and H) Same cells as for A, D, and G showing the expression of Twinkle-EGFP. (C, F, and I) Merged fluorescent images showing colocalization of Twinkle-EGFP with TFAM, SSB, and POLG2, respectively. Some but not all foci containing the apparent highest POLG2 concentrations also contained Twinkle-EGFP as indicated by arrows in G and H, but overall POLG2 fluorescence was more uniform than TFAM or mtSSB fluorescence.
Figure 3
Figure 3
Intramitochondrial foci that are positive for mtSSB or Twinkle are sites of BrdU incorporation. MtDNA was labeled with BrdU for 24 h (A–C) or 2 h (D–F) in 143B(TK−) cells seeded at low density 24–48 h before labeling. BrdU incorporation was detected by a BrdU-specific mAb (A and D). The cells shown in A were subsequently processed for mtSSB-ICC (B), whereas the cells shown in D, transfected with a Twinkle-MycHis construct 24 h before BrdU labeling, were processed for Twinkle-MycHis-ICC (E). (C and F) The merged images for A+B and D+E, respectively. Results show an abundant and specific BrdU labeling in most foci that also specifically stain for either mtSSB or Twinkle, demonstrating that these foci are nucleoids.
Figure 4
Figure 4
Distribution and expression levels of endogenous TFAM and mtSSB in rho-zero cells. (A) ICC for endogenous TFAM in A549 rho+ and rho-zero (C) cells. (B) ICC for endogenous mtSSB in A549 rho+ and rho-zero (D) cells. The results show a significant reduction in detectable TFAM protein levels. Note that the exposure and image enhancement for C is different from that in A in order to obtain the most informative image in each case.
Figure 5
Figure 5
Biochemical isolation of nucleoids from cultured human cells. HEK 293 Cells were grown to ∼80% confluence and isolated by low-speed centrifugation. The mitochondrial isolation procedure and subsequent steps of the nucleoid isolation procedure is depicted in A. Abbreviations for the various fractions as used in the other panels are also indicated. (B) Analysis of sucrose gradients from the various purification steps as indicated in A. (C) A more thorough analysis of fractions from each step of the isolation procedure, using various specific antibodies against mitochondrial proteins. The S1 fraction used in this case was from the top of the sucrose gradient. Please note that a major cross-reacting non-POLG protein (indicated by asterisk) was observed with slightly higher mobility than POLG itself (indicated by open arrow). The cross-reacting species is prominent in fractions mt, S0, P0, and S1, whereas it is only faintly visible in P1 and no longer visible in P2. POLG was very faintly visible in P0 while becoming more prominent in P1 and P2, indicating its enrichment. Two asterisks indicate a TFAM breakdown product (see main text for further explanations). (D) The same samples as used for C, but the gel was Coomassie and subsequently silver-stained to reveal all proteins. Protein markers are indicated on the right in kDa. We tentatively assigned the TFAM protein by comparison of the gel with the immunoblots shown in C and the adenine nucleotide translocator (ANT) on the basis of its molecular mass and abundance. Note also that because of gel loading limitations total protein concentration in the final P2 fraction is significantly less than, for example, that in P1, Also note that bands in P2 are slightly upshifted possibly because of incomplete dialysis to remove excess sucrose.
Figure 6
Figure 6
Nucleoids and mitochondrial dynamics. Nucleoid dynamics were studied in MitoTracker Red–stained and Twinkle-GFP–transfected COS7 cells. Mitotracker shows as uniform mitochondrial staining, whereas Twinkle-GFP appears as brighter intramitochondrial spots. (A) A section of a cell with several nucleoid divisions, indicated by solid arrows. In addition it shows an apparent redistribution of a nucleoid moving into what appears to be a daughter mitochondrion about to be split of (indicated by an open arrow). Both in A and B, frames of 15-s intervals are shown going from left to right and top to bottom. (B) Two mitochondrial division events (solid arrows) in which nucleoids are positioned so that each daughter mitochondrion has at least one new nucleoid element. Although nucleoids appear close to the tips after division, they do not appear to actively participate in the division event itself.
Figure 7
Figure 7
Drp1 does not colocalize with mitochondrial nucleoids. To further demonstrate that nucleoids were excluded from sites of mitochondrial fission we looked at colocalization, in human osteosarcoma cells, of nucleoids stained by mtSSB ICC (A), with the mitochondrial fission protein Drp1, used here as a GFP fusion protein (B). The merged image (C) shows a general lack of colocalization of Drp1-GFP with mtSSB foci. Indicated in an enlarged section of the cell shown in C is a mitochondrial constriction site containing Drp1-GFP with mtSSB foci on either site of this putative future fission site (solid arrow). Indicated by open arrows are two faint Drp1-GFP foci at mitochondrial tips, suggestive of a recent fission event.
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