The cristae modulator Optic atrophy 1 requires mitochondrial ATP synthase oligomers to safeguard mitochondrial function

Abstract

It is unclear how the mitochondrial fusion protein Optic atrophy 1 (OPA1), which inhibits cristae remodeling, protects from mitochondrial dysfunction. Here we identify the mitochondrial F₁F_o-ATP synthase as the effector of OPA1 in mitochondrial protection. In OPA1 overexpressing cells, the loss of proton electrochemical gradient caused by respiratory chain complex III inhibition is blunted and this protection is abolished by the ATP synthase inhibitor oligomycin. Mechanistically, OPA1 and ATP synthase can interact, but recombinant OPA1 fails to promote oligomerization of purified ATP synthase reconstituted in liposomes, suggesting that OPA1 favors ATP synthase oligomerization and reversal activity by modulating cristae shape. When ATP synthase oligomers are genetically destabilized by silencing the key dimerization subunit e, OPA1 is no longer able to preserve mitochondrial function and cell viability upon complex III inhibition. Thus, OPA1 protects mitochondria from respiratory chain inhibition by stabilizing cristae shape and favoring ATP synthase oligomerization.

Fig. 1

OPA1 prevents mitochondrial electrochemical gradient loss caused by CIII inhibition. a Representative color-coded frames from real time imaging of TMRM fluorescence in MAFs of the indicated genotype. Where indicated, cells were treated for 30 min with 10 µM antimycin A (AA). Scale bar, 20 µm. b Quantitative analysis of TMRM fluorescence over mitochondrial regions in real time imaging experiments as in a. Where indicated, cells were treated with AA (10 µM) and with FCCP (2 µM). Data are average ± SEM. (n = 5 for each group). c Representative color-coded frames from real time imaging of mtSypHer fluorescence ratio in MAFs of the indicated genotype. Where indicated, cells were treated for 30 min with 10 µM AA. Rainbow color bar: pseudocolor scale of mtSypHer fluorescence ratio. Scale bar, 20 µm. d Quantitative analysis of mitochondrial mtSypHer fluorescence ratio in real time imaging experiments as in c. Where indicated, cells were treated with AA (10 µM). Data are mean ± SEM of at least four independent experiments. e Representative color-coded images of mitochondrial mtSypHer fluorescence in Opa1^flx/flx cells transfected with empty (EV) or CRE-encoding vectors and treated where indicated for 30 min with AA (10 µM). Rainbow color bar: pseudocolor scale of SypHer fluorescence ratio. Scale bar, 20 µm. f Quantitative analysis of mitochondrial mtSypHer fluorescence ratio in real time imaging experiments as in a. Data are mean ± SEM of at least four different experiments

Fig. 2

OPA1 requires ATP synthase activity to prevent mitochondrial ΔpH loss caused by CIII inhibition. a Representative color-coded frames from real time imaging of mtSypHer ratio fluorescence in MAFs of the indicated genotype. Where indicated, cells were incubated with oligomycin (oligo, 1 µM for 30 min) and antimycin A (AA) (10 µM added 5 min after oligo, for 25 min). Rainbow color bar: pseudocolor scale of SypHer fluorescence ratio. Scale bar, 20 µm. b Quantitative analysis of mitochondrial mtSypHer fluorescence ratio in real time imaging experiments as in a. Data are mean ± SEM of at least four independent experiments. c Representative pseudocolor-coded frames from real time imaging of TMRM fluorescence in MAFs of the indicated genotype. Where indicated, cells were incubated with oligomycin (1 µM for 5 min, before AA additions) and AA (2 µM for 25 min). Scale bar, 20 µm. d Quantitative analysis of mitochondrial TMRM fluorescence ratio in real time imaging experiments as in c. Where indicated, cells were treated with 2 µM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP). Data are mean ± SEM of at least four independent experiments. e Representative color-coded images of mitochondrial mtSypHer fluorescence in Opa1^flx/flx cells transfected with empty (EV) or Cre-encoding vectors and treated for 30 min with AA (10 µM) and oligomycin (1 µM, added 5 min before AA) where indicated. Rainbow color bar: pseudocolor scale of mtSypHer fluorescence ratio. Scale bar, 20 µm. f Quantitative analysis of mitochondrial mtSypHer fluorescence ratio in real time imaging experiments as in e. Data are mean ± SEM of at least 4 independent experiments. g Pseudocolor-coded frames of mtATeam FRET fluorescence ratio in cells of the indicated genotype. Where indicated, cells were treated for 10 min with AA (10 µM). Rainbow color bar: pseudocolor scale of ATeam fluorescence ratio. Scale bar, 20 µm. h Quantitative analysis of mitochondrial mtATeam fluorescence ratio in real time imaging experiments as in g. Data are mean ± SEM of at least three independent experiments. i Mean ± SEM of mtATeam fluorescence ratio slopes, calculated from experiments as in g. Where indicated, oligomycin (1 µM) was added 5 min before AA. *p < 0.05 in an unpaired two-sample Student’s t test

Fig. 3

OPA1 promotes stabilization of ATP synthase oligomers. a Equal amounts (40 µg) of digitonin-solubilized mitochondrial extracts from MAFs of the indicated genotype were separated by BNGE and immunoblotted with anti-ATP synthase subunit α (ATPase) and Succinate Dehydrogenase (SDHA) antibodies. ATPase oligomers (Vo), dimers (Vd), monomers (Vm) and F₁ are indicated. b Densitometric analysis of experiments performed as in a. Data represent mean ± SEM of Vo + Vd (oligomeric)/total ATP synthase (Vo + Vd + Vm + F₁) from five different experiments. *p < 0.05 in a two-way ANOVA vs. control. c Equal amounts (40 µg) of digitonin-solubilized mitochondrial extracts from MAFs of the indicated genotype were separated by BNGE and immunoblotted with the indicated antibodies (left) or processed for ATPase activity (right). ATPase oligomers (Vo), dimers (Vd), monomers (Vm) and F₁ are indicated. d, e Quantitative densitometric analysis of total ATP synthase/CII (SDHA) (d) and of oligomeric/total ATP synthase conformations (e) in experiments as in c. Data show mean ± SEM of at least three independent experiments. *p < 0.05 in a two-way ANOVA versus EV. f Mitochondria from MAFs of the indicated genotypes were treated with recombinant BID as indicated for 30 min, lysed and equal amounts (40 µg) of digitonin-solubilized extracts were separated by BNGE and immunoblotted with the indicated antibodies. g, h Quantitative densitometric analysis of total/CII (SDHA) (g) and of oligomeric/total ATP synthase conformations (h) in experiments as in f. Data are normalized to untreated cells and represent mean ± SEM of at least three independent experiments. *p < 0.05 in an unpaired two-sample Student’s t test versus untreated

Fig. 4

ATP synthase oligomers are reduced by cristae remodeling. a, b Mouse heart mitochondria were incubated as indicated and after 30 min protein lysates (30 µg) were separated by BNGE and Coomassie stained (a) or immunoblotted with the indicated antibody (b). c Representative color-contour plots of spectral counts of OPA1 peptides from quantitative DiS-MS analysis of extracted mitochondrial complexes. Experiments were as in a. Numbers indicate the bands excised for MS analysis. The rainbow color scale indicates the number of spectral counts. d, e Color-contour plot of spectral counts of F₁ core components (α, β, γ, δ, OSCP, panel d) and of the F_O ATPase dimerization subunit e (ATP5k) from experiments as in c. f Experiments were as in b, except that membranes were decorated with the anti-ATPase subunit e antibody. Asterisks: cross-reactive unspecific bands. g Quantification of experiments as in d, e. The median values of the spectral counts of the indicated ATP synthase subunits were calculated in mitochondria pooled from three experiments performed as in a. The ratio of the median values in the dimer+oligomer region over the total forms is plotted

Fig. 5

OPA1 does not directly stimulate ATP synthase oligomerization. a Purified mitochondria were treated where indicated with 10 mM EDC. After 30 min the reaction was quenched, mitochondria lysed and equal amounts (40 µg) of proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. Dashed box: OPA1 and ATPase cross-reactive adduct. Arrowheads: non-crosslinked OPA1 and ATPase. Bottom panels: short exposure blots of non-crosslinked OPA1 and ATPase. b, c Mitochondrial lysates (200 µg) were immunoprecipitated with the indicated antibodies coupled to Protein A agarose beads; bound proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. Input was diluted 1:10. d Representative EM image of a negatively stained proteoliposome containing rOPA1 in the lumen either in the absence (left panel) or presence (right panel) of recombinant ATP synthase in the membrane. Arrowheads: F₁ oriented towards the outside of the liposome. Scale bar, 50 nm. e Proteoliposomes harboring ATPase and either empty or containing rOpa1 were solubilized and proteins separated by BN-PAGE. f Densitometric quantification of ATPase composition in experiments as in e. Data are mean ± SEM of four independent experiments. g, h Representative BNGE (g) and densitometric quantification (h) of ATPase oligomeric forms upon incubation of the indicated ratios of rOPA1 with ATP synthase (3 µg). Data are mean ± SEM of four independent experiments. Vo: ATPase oligomers, Vd: dimers, Vm: monomers

Fig. 6

OPA1 requires ATP5k to stabilize ATP synthase oligomers. a Equal amounts (30 µg) of lysates from MAFs of the indicated genotype transfected for 48 h with the indicated shRNA were separated by SDS-PAGE and immunoblotted with the indicated antibodies (ATPase is ATP5A). b Equal amounts (40 µg) of digitonin-solubilized mitochondrial extracts from MAFs of the indicated genotype transfected with the indicated shRNAs were separated by BNGE and immunoblotted with the indicated antibodies. c Quantitative densitometric analysis of ATPase dimer vs. total ATPase. Data are mean ± SEM of three independent experiments performed as in b. d Representative EM micrographs of cells of the indicated genotype transfected with the indicated shRNA and GFP and after 48 h sorted for GFP⁺ and processed for EM. Boxed areas are magnified 12× in the bottom images. Scale bars: 500 nm; 20 nm in bottom magnifications. e–g Morphometric analysis of cristae maximal width (e), number of cristae per mitochondrion (f) and cristae junctions per cristae (g) in experiments as in d. At least 30 mitochondria per experimental condition were analyzed. Mean values ± SEM of three independent experiments are shown. *p < 0.05 in a two-way ANOVA versus control (e) or paired two-sample Student’s t test versus scramble shRNA (shScr) (f, g)

Fig. 7

OPA1 requires ATP5k to protect from mitochondrial dysfunction. a Representative pseudocolor-coded frames from real time imaging of TMRM fluorescence in MAFs of the indicated genotype transfected with the indicated shRNA. Where indicated, cells were incubated with antimycin A (AA) (2 µM for 25 min). Scale bar, 20 µm. b, c Quantitative analysis of mitochondrial TMRM fluorescence ratio in experiments as in a. Where indicated, cells were treated with 2 µM FCCP. Data are mean ± SEM of at least four independent experiments. d Pseudocolor-coded frames of mtATeam FRET fluorescence ratio in cells of the indicated genotype. Where indicated, cells were treated for 20 min with AA (10 µM). The rainbow indicates the pseudocolor scale. Scale bar, 20 µm. e Mean ± SEM of maximal mtATeam fluorescence ratio slopes in at least three independent experiments as in (d). *p < 0.05 in an unpaired two-sample Student’s t test. versus scramble shRNA (shScr). f MAFs of the indicated genotype were cotransfected with GFP and the indicated shRNA and after 72 h were treated with AA (5 µM, 6 h). Data are mean ± SEM of three independent experiments. *p < 0.05 in a two-way ANOVA test versus shScr