Quality matters: how does mitochondrial network dynamics and quality control impact on mtDNA integrity?

Abstract

Mammalian mtDNA encodes for 13 core proteins of oxidative phosphorylation. Mitochondrial DNA mutations and deletions cause severe myopathies and neuromuscular diseases. Thus, the integrity of mtDNA is pivotal for cell survival and health of the organism. We here discuss the possible impact of mitochondrial fusion and fission on mtDNA maintenance as well as positive and negative selection processes. Our focus is centred on the important question of how the quality of mtDNA nucleoids can be assured when selection and mitochondrial quality control works on functional and physiological phenotypes constituted by oxidative phosphorylation proteins. The organelle control theory suggests a link between phenotype and nucleoid genotype. This is discussed in the light of new results presented here showing that mitochondrial transcription factor A/nucleoids are restricted in their intramitochondrial mobility and probably have a limited sphere of influence. Together with recent published work on mitochondrial and mtDNA heteroplasmy dynamics, these data suggest first, that single mitochondria might well be internally heterogeneous and second, that nucleoid genotypes might be linked to local phenotypes (although the link might often be leaky). We discuss how random or site-specific mitochondrial fission can isolate dysfunctional parts and enable their elimination by mitophagy, stressing the importance of fission in the process of mtDNA quality control. The role of fusion is more multifaceted and less understood in this context, but the mixing and equilibration of matrix content might be one of its important functions.

Keywords: OXPHOS; TFAM; mitochondrial dynamics; mitophagy; mtDNA; nucleoids.

Figure 1.

Assays to determine the exchange rates of compounds between mitochondria. (a) Polyethylene glycol- (PEG-)-mediated fusion of cells with differently fluorescent-tagged compounds, here OXPHOS complexes in red (RFP-tagged) and green (GFP-tagged). (b) Use of photo-activatable fluorescent proteins, here OXPHOS complex CI-paGFP to monitor spreading in MitoTracker stained mitochondria. (c) Scheme of photo-activation assay: paGFP is photo-activated in single mitochondria that express paGFP-tagged proteins. Owing to ongoing fusion and fission and mixing of compounds, the fluorescent signal spreads throughout the mitochondrial reticulum. Asterisks indicate nuclei. Scale bars, 10 µm. Images from K.B. (Online version in colour.)

Figure 2.

Repetitive fusion and fission cycles are required for good mixing of inner membrane proteins. (a) Dual-colour super-resolution imaging of fused mitochondria with differently labelled OXPHOS complexes (CI-EGFP + CII-mRFP) after few (early state) and frequent (intermediate and late states) fusion and fission events. In short, two stable cell lines expressing OXPHOS complex I fused to monomeric EGFP (CI-EGFP) and complex II fused to monomeric RFP (CII-mRFP), respectively, were co-plated and cell fusion was induced by PEG treatment. Owing to ongoing mitochondrial dynamics, mitochondria in the syncytium fused and divided frequently and mitochondria with a hybrid composition were generated. The yellow arrowheads and colour, respectively, show co-localization of OXPHOS complexes of different origin. (b) Schematic model of the sequence of events eventually generating well-mixed mitochondria. Scale bars, 300 nm (a), 100 nm (b). Adapted with permission from Wilkens et al. [39]. (Online version in colour.)

Figure 3.

Mitochondrial nucleoids marked by TFAM display remote spreading throughout the chondriom. (a) mtDNA and TFAM localize in similar foci patterns: mtDNA (stained with picogreen) and TFAM-GFP in mitochondria (stained with MitoTracker Red). (b) TFAM/nucleoids from a photo-activated (pa) area show very little dynamics within a single cell during 20 min. (c) Spreading of OXPHOS complex I and TFAM from photo-activated areas in comparison. TFAM*: control measurement in fragmented mitochondria (fragmentation was induced by bacterial contamination). Scale bar, 3 µm (a), 10 µm (b). (Online version in colour.)

Figure 4.

Putative spatio-temporal organization of nucleoids in microcompartments. (a) TEM micrograph of mammalian HeLa mitochondria. (b) Trajectory map of TFAM-Halo/TMR (see electronic supplementary material, Materials and methods) in a single mitochondrion of the same type. The detailed view at the bottom (b′) shows a putative microcompartment explored by three TFAM molecules (magenta, cyan, brown trajectories). (c) Comparison of ultra-structural and dynamics defined microcompartments, respectively, resolved by EM (matrix) and tracking and localization microscopy [38] (TFAM). (d) Apparent diffusion coefficients of the mobile fractions of TFAM, OXPHOS complex IV, mitofilin (IMMT) and mitochondrial matrix processing peptidase (MPP). Mean values from at least two independent measurements, including 12 cells, 50 mitochondria and more than a thousand individual trajectories are depicted (ANOVA significance test; letters indicate significance levels p ≤ 0.05 for a,a′–b; p ≤ 0.001 for a–d). Scale bar, 300 nm (a,b). TEM micrograph courtesy of Verena Wilkens.

Figure 5.

The organelle control theory predicts a ‘leaky link’ between genotype and phenotype [35]. The tight spatial organization of cristae combined with their microcompartment character leads automatically to such a connection, because the diffusion of mRNAs and proteins synthesized by different nucleoids, N, is impeded by these structures.

Figure 6.

Genotype–phenotype linkage as the critical parameter to eliminate dominant negative mtDNA. (a) Mitochondria with wild-type mtDNA can undergo (spontaneous or induced) depolarization, but recovery followed by fusion brings them back into the dynamic mitochondrial network. (b) In a very leaky link, the presence of mutant next to wild-type mtDNA in the same mitochondrion would allow intramitochondrial functional complementation. As long as the decrease of proton motive force (pmf) and membrane potential, respectively, is not too harsh, elimination by mitophagy will not work and mutant mtDNA can escape the quality control. This spares biomaterial on cost on stringency. (c) In a less leaky link, with closer genotype-phenotype coupling, fission can generate fragments with low pmf, unable to fuse again, prone to be eliminated by mitophagosomes.