Mitochondrial cristae shape determines respiratory chain supercomplexes assembly and respiratory efficiency

Abstract

Respiratory chain complexes assemble into functional quaternary structures called supercomplexes (RCS) within the folds of the inner mitochondrial membrane, or cristae. Here, we investigate the relationship between respiratory function and mitochondrial ultrastructure and provide evidence that cristae shape determines the assembly and stability of RCS and hence mitochondrial respiratory efficiency. Genetic and apoptotic manipulations of cristae structure affect assembly and activity of RCS in vitro and in vivo, independently of changes to mitochondrial protein synthesis or apoptotic outer mitochondrial membrane permeabilization. We demonstrate that, accordingly, the efficiency of mitochondria-dependent cell growth depends on cristae shape. Thus, RCS assembly emerges as a link between membrane morphology and function.

Graphical abstract

Figure 1

Two Conserved Lys in BID α6 Helix Are Required for Cristae Remodeling (A) Mitochondria were treated for the indicated times with the indicated mutants of cBID, and cytochrome c release was measured by ELISA. Data represent average ± SEM of five independent experiments. (B) Mitochondria treated with the indicated mutants of cBID for the indicated min were crosslinked with 1 mM BMH for 30 min, spun, and the pellets were separated by SDS-PAGE and immunoblotted using anti-BAK antibody. The asterisks denote BAK oligomers. (C) Mitochondria were treated as indicated (Ca²⁺, 200 μM), and cytochrome b₅-dependent NADH fluorescence changes were recorded. a.u., arbitrary units. (D) Mitochondria were treated for 15 min with the indicated BID mutants, transferred into the chamber of a Clark’s type O₂ electrode, and the ascorbate/TMPD-driven respiration ratio was determined. Data represent average ± SEM of four independent experiments. (E) Representative electron microscopy fields of mitochondria treated for 15 min with the indicated cBID mutants. Arrows indicate class II and III mitochondria. The scale bar represents 450 nm. (F) Blind morphometric analysis of randomly selected EM of mitochondria treated with the indicated cBID mutants (as in [E]). Mitochondria were assigned to morphological classes I–III as in Scorrano et al. (2002). Data represent average ± SEM of three independent experiments. The asterisk denotes p < 0.05 in a paired sample Student’s t test versus untreated. (G and H) BNGE analysis of OPA1 oligomers in MLM treated for 30 min (G) or for the indicated minutes (H), as indicated. The boxed area indicates the HMW complexes of OPA1. (I) Densitometric analysis of OPA1 HMW complexes. Experiments were as in (H). Data represent average ± SEM of four independent experiments. (J) MEFs were transfected with the pMIG plasmid containing the indicated insert and after 48 hr cell death was determined cytofluorimetrically as the percentage of Annexin-V⁺, GFP⁺ cells. Data represent average ± SEM of four independent experiments. The asterisk denotes p < 0.05 in a paired sample Student’s t test versus tBID. See also Figure S1.

Figure 2

Respiratory Chain Supercomplexes Are Disassembled during Cristae Remodeling (A) RCR of mitochondria energized with 5 mM/2.5 mM glutamate/malate (GLU/MAL) or 10 mM succinate (SUCC) treated for the indicated times with cBID. Data represent average ± SEM of five independent experiments. Inset, RCR values normalized to t = 0. (B) Experiments were as in (A), except that mitochondria were incubated with the indicated mutants of cBID for 15 min. Data represent average ± SEM of four independent experiments. The asterisk denotes p < 0.05 in a paired sample Student’s t test versus untreated. (C) BNGE of mitochondrial OXPHOS protein from DKO MEFs transduced as indicated and, after 2 days, metabolically labeled for 2 hr and lysed after 24 hr. Equal amounts of protein (100 μg) were separated by BNGE, and radioactivity was detected in the fixed and dried gels for 1 week. RCC and RCS of the respiratory chain are indicated. (D) Densitometric analysis of the autoradiographic RCS/complex V signal ratio. Data represent average ± SEM of three independent experiments performed as in (E). The asterisk denotes p < 0.05 in a paired sample Student’s t test versus untreated. (E) 2D BN/SDS PAGE analysis of mitochondrial OXPHOS proteins from DKO MEFs transduced as indicated, metabolically labeled for 2 hr, and lysed after 24 hr. Equal amount (50 μg) of proteins were separated in native condition, and then the lanes were excised and proteins separated by a second dimension SDS-PAGE. The gels were dried and the signal was detected following 1 week of exposure. RCC and RCS of the respiratory chain as well as the single-labeled proteins are indicated. Boxes and circles indicate RCS and cytochrome b, respectively. (F) Densitometric analysis of the ratio of autoradiographic signal between supercomplex (boxed) and complex assembled cytochrome b (circled). Data represent average ± SEM of three independent experiments. The asterisk denotes p < 0.05 in a paired sample Student’s t test versus untreated. (G) BNGE analysis of OXPHOS proteins in mitochondria from DKO MEFs transduced as indicated. Equal amounts (100 μg) of proteins were separated in native conditions, transferred onto PVDF membranes, and probed with the indicated antibodies. RCC and RCS are indicated. (H) RCR of DKO mitochondria energized with 5 mM/2.5 mM GLU/MAL or 10 mM SUCC treated for 15 min with the indicated cBID mutants. Data represent average ± SEM of four independent experiments. The asterisk denotes p < 0.05 in a paired sample Student’s t test versus untreated. See also Figure S2.

Figure 3

Generation of Opa1^flx/flx and Opa1^tg Mice (A) Maps of the wild-type Opa1 allele, the targeting vector, the conditional floxed allele, and the inactivated Opa1 allele. The 5′ UTR, exons (black boxes), LoxP sites (white arrows), FRT recombination sites, and PGK-neomycin cassette (white box) are indicated. The locations of PCR primers (ck1 = primer check1 forward, ck2 = primer check2 reverse) are indicated. Dimensions are not in scale. (B) Prediction of the maximal possible aberrant OPA1 protein. (C) RT-PCR analysis of transcripts in heterozygous (flx/−), homozygous (flx/flx), and WT mice. (D) Maps of the targeting vector, the mutant Hprt locus of BPES cells, and the transgenic targeted and functional Hprt locus. The 5′ UTR, the human β-ACTIN promoter and Opa1 gene (light gray box), poly A (white box), human exon 1 of Hprt locus (h1, dark gray box), and Hprt locus exons (black boxes) are indicated. The locations of PCR primers (WTF, WT forward; WTR, WT reverse; tgF, transgenic forward; tgR, transgenic reverse) are indicated. Dimensions are not in scale. (E) RT-PCR screening of ESC clones. The positive clones are indicated. (F) RT-PCR analysis of transcripts in heterozygous (tg/−), homozygous (tg/tg), and WT mice. See also Figure S3.

Figure 4

Acute Ablation of Opa1 Alters Mitochondrial Morphology, Cristae Shape, and RCS Assembly (A) Opa1^flx/flx MAFs were infected with bicistronic adenoviruses carrying the indicated insert (EV, empty vector; CRE, Cre recombinase) upstream of GFP and, after 24 hr, equal amounts of proteins (20 μg) were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (B) Opa1^flx/flx MAFs were infected with the indicated adenoviruses and, after 24 hr, fixed, immunostained using an anti-TOM20 antibody, and representative confocal images acquired. The green cytoplasmic staining identifies the coexpression of GFP. The scale bar represents 20 μm. (C) Average mitochondrial major axis length. Experiments were as in (B). Data represent average ± SEM of four independent experiments (five mitochondria per cell, at least 50 cells/experiment). The asterisk denotes p < 0.05 in a paired sample Student’s t test versus ad-EV. (D) Opa1^flx/flx MAFs were infected with the indicated adenoviruses and, after 24 hr, fixed and processed for electron microscopy. The scale bars represent 2 μm (top) and 200 nm (bottom). (E) Morphometric analysis of cristae width in 40 randomly selected mitochondria of Opa1^flx/flx MAFs infected with the indicated adenoviruses. Data represent average ± SEM of three independent experiments. The asterisk denotes p < 0.05 in a paired sample Student’s t test versus ad-EV. (F) Mitochondrial DNA copy number quantification. mtDNA was amplified by RT-PCR from total DNA of Opa1^flx/flx MAFs infected with the indicated adenoviruses. Data are normalized to MAFs infected with control adenovirus and represent average ± SEM of four independent experiments. (G) mtDNA translation assay. Opa1^flx/flx MAFs infected as indicated were metabolic labeled in presence of emetine and lysed after 30 min. Protein samples (40 μg) were separated by SDS-PAGE, and the radioactivity was detected in the fixed and dried gels for 3 days. The mtDNA encoded proteins are indicated. (H) Densitometric analysis of the mtDNA-encoded proteins. Experiments are as in (G). Data represent average ± SEM of four independent experiments. (I) RCS assembly assay. Opa1^flx/flx MAFs were infected as indicated and, after 24 hr, metabolically labeled for 2 hr and then chased for the indicated times. Equal amounts of protein (100 μg) were separated by BN PAGE, and radioactivity was detected in the fixed and dried gels for 1 week. RCC and RCS of the respiratory chain are indicated. (J) Densitometric analysis of the incorporation rate of radioactivity into supercomplexes. Values are normalized for the autoradiographic signal of complex V. Data represent average ± SEM from three independent experiments performed as in (H). (K) RCR of mitochondria isolated from livers of Opa1^flx/flx mice 3 days after tail-vein injection of the indicated adenoviruses, energized with 5 mM/2.5 mM GLU/MAL or 10 mM SUCC. Data represent average ± SEM of four independent experiments. The asterisk denotes p < 0.05 in a paired sample Student’s t test versus ad-EV. See also Figure S4.

Figure 5

Transgenic Overexpression of OPA1 Increases RCS Assembly (A) Equal amounts of proteins (20 μg) from MAFs of the indicated genotypes were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (B) Representative confocal micrographs of mitochondrial morphology in WT and Opa1^tg MAFs. Mitochondria were visualized by anti-TOM20 immunostaining. The scale bar represents 20 μM. (C) Average mitochondrial major axis length. Experiments were as in (B). Data represent average ± SEM of four independent experiments (five mitochondria per cell, at least 50 cells/experiment). The asterisk denotes p < 0.05 in a paired sample Student’s t test versus WT. (D) Electron micrographs of MAFs of the indicated genotype. The scale bars represent 2 μm (top) and 200 nm (bottom). (E) Morphometric analysis of cristae width in 40 randomly selected mitochondria of MAFs of the indicated genotype. Data represent average ± SEM of three independent experiments. The asterisk denotes p < 0.05 in a paired sample Student’s t test versus WT. (F) Mitochondrial DNA copy number quantification. mtDNA was amplified by RT-PCR from total DNA of MAFs of the indicated genotype. Data are normalized to WT MAFs and represent the average ± SEM of four independent experiments. (G) mtDNA translation assay. MAFs of the indicated genotype were metabolic labeled in presence of emetine and lysed after 30 min. Protein samples (40 μg) were separated by SDS-PAGE, and the radioactivity was detected in the fixed and dried gels for 3 days. The mtDNA-encoded proteins are indicated. (H) Densitometric analysis of the mtDNA-encoded proteins. Experiments are as in (G). Data represent average ± SEM of four independent experiments. (I) RCS assembly assay. MAFs of the indicated genotype were metabolically labeled for 2 hr and then chased for the indicated times. Equal amounts of protein (100 μg) were separated by BN PAGE, and radioactivity was detected in the fixed and dried gels for 1 week. Individual complexes and supercomplexes of the respiratory chain are indicated. (J) Densitometric analysis of the incorporation rate of radioactivity into RCS. Values are normalized for the autoradiographic signal of complex V. Data represent average ± SEM of three independent experiments performed as in (H). (K) RCR of mitochondria isolated from livers of mice of the indicated genotype energized with 5 mM/2.5 mM GLU/MAL or 10 mM SUCC. Data represent mean ± SEM of four independent experiments. The asterisk denotes p < 0.05 in a paired sample Student’s t test versus WT. See also Figure S5.

Figure 6

Mitochondria-Dependent Cellular Growth Requires Assembled RCS (A) Growth curves of DKO MEFs transduced with the indicated retroviruses and grown in DMEM supplemented with the indicated monosaccharides. Data represent mean ± SEM of five independent experiments. (B–D) Growth curves of the indicated cell lines cultured in DMEM supplemented with the indicated monosaccharides. Data represent mean ± SEM of five independent experiments. See also Figure S6.

Figure S1

The Conserved BID α6 Helix K157, K158 Residues Are Required to Destabilize OPA1 HMW Complexes, Related to Figure 1 (A) ClustalW alignment between the amino acid sequence of the BID hydrophobic α-6helix of BID (red) and Mastoparan (green). Asterisk: identical aa.; colon: high homology; dot: homology. Two conserved Lysine residues (K157, 158) are highlighted in gray. (B) ClustalW alignment between the amino acidic sequence of the transmembrane domain of BimS and Bnip3 and the α6-helix of BID. (C) ClustalW alignment of the amino acidic sequence of BID of different species. Identical aa. are indicated in the same color. (D) Mouse liver mitochondria were treated with the indicated mutants of cBID for 30 min and peripheral proteins were extracted by incubation in 0.1 M Na₂CO₃ pH 11.3, for 30 min. Membrane and soluble fractions were recovered after centrifugation. Equal amount of proteins (10 μg) were separated by SDS-PAGE and immunoblotted with the indicated antibodies (T: total lysate, P: pellet, SN: supernatant). (E) 2D BN/SDS-PAGE analysis of the OPA1 oligomers and complex IV. Mitochondria were treated as indicated for 30 min. Equal amount (50 μg) of proteins were separated in native condition and then the lanes were excised and proteins separated by a second dimension SDS-PAGE. After immunoblotting, the proteins were immunodecorated with the indicated antibody. Asterisk indicates high molecular weight complexes of OPA1. (F) 2D BN/BN-PAGE analysis of OPA1 oligomers in mitochondria treated as indicated for 30 min. Equal amounts (50 μg) of proteins were separated in native condition and then the lane was cut and proteins were separated by a second dimension native page. The complexes were immunoblotted and probed with anti-OPA1 antibody. The circled areas were used for densitometry and the ratio between the HMW and the monomeric (mono) OPA1 forms are reported below each panel. For the sake of clarity, the ratio in untreated mitochondria has been set to 1 and the values in cBID treated mitochondria normalized to the untreated one. (G) Mitochondria treated with the indicated mutants of cBID for the indicated minutes were crosslinked with 1 mM EDC for 30 min, and equal amount (20 μg) of proteins in the pellet were separated by SDS-PAGE and immunoblotted with anti-OPA1 antibody. Asterisks indicate OPA1 oligomers. (H) Kinetics of OPA1 oligomers destabilization by cBID. Experiments were as in (C). Data represent average ± SEM of 5 independent experiments.

Figure S2

Cristae Remodeling Impairs Glutamate-Supported Uncoupled Respiration, Related to Figure 2 (A) Mitochondria isolated from mouse liver were treated with the indicated mutants of cBID for 10 min and 3uncoupled respiration rate was determined. Data represent average ± SEM of 3 independent experiments. ^∗p < 0.05 in a pair sample Student’s t test versus untreated. (B) DKO MEFs were transduced as indicated and after 36 hr fixed and processed for electron microscopy. Bars, 1 μm (left) and 200 nm (right).

Figure S3

Chronic Opa1 Ablation Causes Loss of mtDNA and Impairs mtDNA Translation, Related to Figure 3 (A) Mitochondrial DNA copy number quantification. mtDNA was amplified by RT-PCR from total DNA of the indicated cell lines using specific primers. The mtDNA copy number was normalized to the mtDNA copy number of the correspondent WT cell line. Data are average ± SEM of 5 dependent experiments. ^∗p < 0.05 in a pair sample Student’s t test versus WT. (B) mtDNA translation assay. The indicated cell lines were metabolic labeled in presence of emetine and lysed after 30 min. Protein samples (40 μg) were separated by SDS-PAGE and the radioactivity was detected in the fixed and dried gels for 3 days. The mtDNA encoded proteins are indicated. (C) RCS assembly assay. MEFs of the indicated genotype were metabolically labeled and chased for the indicated times. Equal amounts of protein (100 μg) were separated by BN PAGE and radioactivity was detected in the fixed and dried gels for 1 week. Individual complexes and supercomplexes of the respiratory chain are indicated.

Figure S4

Acute Ablation of Opa1 Affects Cristae Shape and RCS In Vivo, Related to Figure 4 (A) Levels of OPA1 in mitochondria from livers of Opa1^flx/flx mice were isolated 3 days after tail-vein injection with the indicated adenoviruses. Protein samples (20 μg) were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (B) Representative electron micrographs of mitochondria isolated from livers of Opa1^flx/flx mice 3 days after tail-vein injection with the indicated adenoviruses. Bars, 2 μm (top), 200 nm (bottom). (C) BNGE analysis of RCS of mitochondria isolated from livers of Opa1^flx/flx mice 3 days after tail-vein injection with the indicated adenoviruses. Equal amounts (100 μg) of proteins were separated in native conditions, transferred onto PVDF membranes and probed with the indicated antibodies. Individual complexes and supercomplexes of the respiratory chain are indicated.

Figure S5

Overexpression of Opa1 Decreases Cristae Width and Increases RCS In Vivo, Related to Figure 5 (A) Levels of OPA1 in mitochondria isolated from liver of Opa1^tg mice. Protein samples (20 μg) were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (B) Representative electron micrographs of mitochondria isolated from liver of Opa1^tg mice. Bars, 0.2 μm (top), 60 μm (bottom). (C) BNGE analysis of RCS of mitochondria isolated from liver of Opa1^tg mice. Equal amounts (100 μg) of proteins were separated in native conditions, transferred onto PVDF membranes and probed with the indicated antibodies. Individual complexes and supercomplexes of the respiratory chain are indicated.

Figure S6

RCS Are Not Affected by Chronic Loss of Mfn 1 and 2, Related to Figure 6 (A) mtDNA copy number quantification. mtDNA was amplified by RT-PCR from total DNA of the indicated cell lines using specific primers. The mtDNA copy number was normalized to the mtDNA copy number of the correspondent WT cell line. Data are average ± SEM of 5 dependent experiments. ^∗p < 0.05 in a pair sample Student’s t test versus WT. (B) Representative electron micrographs of cells of the indicated genotype. Bar, 500 μM (C) Morphometric analysis of cristae width in mitochondria of the indicated genotype. Cristae width was measured in 40 randomly selected mitochondria. Data represent avarage ± SEM of 3 independent experiments. (D) mtDNA translation assay. The indicated cell lines were metabolic labeled in presence of emetine and lysed after 30 min. Protein samples (40 μg) were separated by SDS-PAGE and the radioactivity was detected in the fixed and dried gels for 3 days. The mtDNA encoded proteins are indicated. (E) BN PAGE analysis of RCS stability. MEFs of the indicated genotype were metabolically labeled. Equal amounts of protein (100 μg) were separated by BN PAGE and radioactivity was detected in the fixed and dried gels for 3 days. Individual complexes and supercomplexes of the respiratory chain are indicated. (F) RCS assembly assay. MEFs of the indicated genotype were metabolically labeled and chased for the indicated times. Equal amounts of protein (100 μg) were separated by BN PAGE and radioactivity was detected in the fixed and dried gels for 1 week. Individual complexes and supercomplexes of the respiratory chain are indicated.