OPA1-dependent cristae modulation is essential for cellular adaptation to metabolic demand

Abstract

Cristae, the organized invaginations of the mitochondrial inner membrane, respond structurally to the energetic demands of the cell. The mechanism by which these dynamic changes are regulated and the consequences thereof are largely unknown. Optic atrophy 1 (OPA1) is the mitochondrial GTPase responsible for inner membrane fusion and maintenance of cristae structure. Here, we report that OPA1 responds dynamically to changes in energetic conditions to regulate cristae structure. This cristae regulation is independent of OPA1's role in mitochondrial fusion, since an OPA1 mutant that can still oligomerize but has no fusion activity was able to maintain cristae structure. Importantly, OPA1 was required for resistance to starvation-induced cell death, for mitochondrial respiration, for growth in galactose media and for maintenance of ATP synthase assembly, independently of its fusion activity. We identified mitochondrial solute carriers (SLC25A) as OPA1 interactors and show that their pharmacological and genetic blockade inhibited OPA1 oligomerization and function. Thus, we propose a novel way in which OPA1 senses energy substrate availability, which modulates its function in the regulation of mitochondrial architecture in a SLC25A protein-dependent manner.

Keywords: ATP synthase; OPA1; SLC25A; cristae; mitochondria.

Figure 1. Cristae condense, mitochondria elongate and OPA1 oligomerizes rapidly and reversibly during cell starvation

1. MEFs were starved (STV) or not (Ctrl) for 2 h, fixed with 2% PFA and 1.6% glutaraldehyde and analysed by EM. Cristae and mitochondrial width were quantified from mitochondria and cristae within 10 cells from two independent cultures (n = 20). Scale bars: 100 nm.

2. Cells were starved or not for 2 h and crosslinked with BMH (1 mM), and OPA1 oligomerization was analysed by gradient gel Western blot.

3. MEFs were starved for 4 h and fixed, and mitochondrial length was measured by immunofluorescence using Tom20 antibodies (averages ± SEM of three independent experiments).

4. Time-course experiment of EBSS starvation on OPA1 oligomerization status as performed in (B) (averages ± SEM of four independent experiments). Whole-cell lysates of a parallel experiment were analysed by Western blot.

5. After 2 h of EBSS starvation, MEFs were recovered (REC) in regular growth media for 30 min or not and OPA1 oligomers were then analysed as above. A non-crosslinked control (NC) is also included.

Data information: Student's t-tests were performed relative to control, *P < 0.05, ***P < 0.005. Source data are available online for this figure.

Figure 2. OPA1 responds to energy substrate availability and responds by oligomerizing and maintaining cytochrome c in isolated mitochondria

1. OPA1 oligomerization was analysed in isolated mouse liver mitochondria incubated with or without the indicated ETC substrates (NC: non-crosslinked control; NS: no substrate control; CI: complex I, malate and glutamate; CII: complex II, succinate; G: glucose control; all at 5 mM each) for 1 h at 37°C and subsequently crosslinked with EDC for 30 min at room temperature. OPA1 oligomers were then analysed by gradient gel Western blot.

2. Mitochondria were incubated with or without complex I substrates, spun down and resuspended in the indicated buffer for 5 and 10 min and analysed as above.

3. Mitochondrial ultrastructure was analysed as the distribution of intercristal cytochrome c from liver mitochondria and solubilized with low digitonin concentrations (1 μg/μg mitochondria, 0.1%). Mobilized cytochrome c was separated by centrifugation and visualized by Western blot analysis where released cytochrome c was redistributed from the pellet (P) to the supernatant (SN) fraction.

4. Cytochrome c mobilization was analysed on mitochondria isolated from WT and OPA1 KO MEFs treated as indicated.

5. Liver mitochondria were incubated with or without indicated substrates as above with or without rotenone (2 μM), an ETC complex I poison.

6. Liver mitochondria were incubated with or without indicated substrates as above with or without CCCP (10 μM), a mitochondrial uncoupler.

Source data are available online for this figure.

Figure 3. OPA1 is required for resistance to starvation-induced cell death independently of its fusion activity

1. Cell death of WT and OPA1 KO cells starved or not for 6 h was analysed with propidium iodide (PI) and Hoechst (averages ± SEM of four independent experiments).

2. Representative Western blot of OPA1 expression in the transient transfection experiments performed in (C–E).

3. OPA1 KO MEFs were transiently transfected for 48 h with the indicated plasmids, and mitochondrial length was binned according to the top panels by immunofluorescence where cells that had any long mitochondria were binned as intermediate (averages ± SEM of three independent experiments).

4. Representative EM of mitochondria from cells transfected as indicated. Scale bars: 100 nm.

5. OPA1 KO MEFs were transfected as indicated for 48 h and starved or not for 6 h, and cell death was analysed as in (A) (averages ± SEM of four independent experiments).

Data information: Student's t-tests were performed as indicated, *P < 0.05, **P < 0.01 and ***P < 0.005. Source data are available online for this figure.

Figure 4. OPA1 regulates mitochondrial metabolism independently of mitochondrial fusion

A WT and OPA1 knockout MEFs were infected with viruses encoding GFP, OPA1 or OPA1(Q297V) with dual promoters expressing GFP to allow for puromycin selection and FACS sorting cells for GFP expression. The resulting cultures were lysed, analysed by Western blot analysis and used for all subsequent experiments. B OPA1 oligomers were analysed from isolated mitochondria incubated with no substrate or complex I substrates. C, D To assess the impact of the fusion-incompetent OPA1 mutant on mitochondrial bioenergetics, we studied cells in the Seahorse XF-24 analyzer. Cells were plated on Seahorse TC plates 24 h prior to analysis, washed and incubated for 15 min in modified KRB and analysed. Cells in (D) were pre-starved for 2 h before analysis. At the indicated times, oligomycin (O), FCCP (F) and antimycin A (AA) with rotenone (R) were injected (averages ± SEM of three independent experiments). E Quantification of ATP-linked OCR (resting OCR minus oligomycin-insensitive OCR) and reserve OCR (maximal minus resting) in (C) and (D). F Long-term cell cultures were grown in glucose (top panel) or galactose media (bottom panel). Cells were passaged as required, medium was changed every 3 days if required, and cell number was determined once per week (averages ± SEM of four independent experiments). Data information: Student's t-tests were performed relative to control, *P < 0.05 **P < 0.01. Source data are available online for this figure.

Figure 5. The dual role of OPA1 in regulating the ATP synthase

A Isolated mitochondria incubated with complex I substrates were analysed by BN-PAGE and blotted for ATP5A of the ATP synthase. The oligomers [mostly dimers (D)] and monomer (M) of the ATP synthase were quantified as relative to complex II monomer (averages ± SEM of five independent experiments). B Isolated liver mitochondria incubated with or without complex I in parallel with (A), lysed and analysed for ATP5A by straight Western blot. C, D Long-term cultures of control cells (C) or cells grown for 1–2 weeks in galactose (D) were extracted for BN-PAGE directly from cells and blotted for ATP5A and complex II. E DNA was extracted from long-term cultures, and mtDNA was analysed by qPCR relative to nDNA (averages ± SEM of three independent experiments). F RNA was extracted from long-term cultures, and nuclear and mtDNA-encoded ATP synthase transcripts were analysed by RT–PCR (averages ± SEM of four independent experiments). G Schematic diagram demonstrates the dual roles of OPA1 on the F₁F_o ATP synthase where OPA1 regulates the stabilization of the ATP synthase independently of its fusion activity and regulates mtDNA stability which is required for ATP6 (F_o subunit) expression and thus full F₁F_o ATP synthase assembly. Data information: Student's t-tests were performed relative to control, *P < 0.05, **P < 0.01 and ***P < 0.005. Source data are available online for this figure.

Figure 6. OPA1 interacts with SLC25 proteins that regulate OPA1's response to starvation

A Mito-YFP-3×flag, DIC-3×flag, OGC-3×flag, AGC1-3×flag and AGC2-3×flag constructs were transiently transfected into MEFs. Twenty-four hours post-transfection, cells were lysed, immunoprecipitated with anti-flag antibodies and analysed by Western blot. B Endogenous OPA1 was immunoprecipitated from MEF mitochondrial lysates with or without 15 mM phenylsuccinate (PhS), and the eluted samples were analysed by Western blot. Complex II and cytochrome c were used as negative controls. C Representative OGC knockdown for experiments in (D-H) (see also Supplementary Figs S8B and S9A–E). MEFs were transfected twice with siOGC for 120 h total, and mitochondrial lysates were analysed by Western blot. D Isolated mitochondria from siOGC-treated cells were incubated with the indicated ETC substrates (NC: non-crosslinked control; NS: no substrate control; CI: complex I, malate and glutamate at 5 mM each) for 30 min at 37°C and crosslinked with EDC, and OPA1 oligomers were then analysed by gradient gel Western blot. E Isolated mitochondria from siOGC and siCtrl cells were incubated in no substrate or complex I buffers for 30 min and analysed for cytochrome c retention. F, G SiOGC and siCtrl cells were plated onto Seahorse TC plates 24 h prior to analysis, washed and incubated for 15 min in modified KRB and analysed by the XF analyzer. Cells in (G) were pre-starved for 2 h before analyses. At the indicated times, oligomycin (O), FCCP (F) and antimycin A (AA) with Rotenone (R) were injected. H Quantification of ATP-linked (resting OCR minus oxygen leak) and reserve OCR (maximal minus resting OCR) in (F) and (G) (averages ± SEM of four independent experiments). I MEFs were grown for 1 week in galactose media, to revert to a more oxidative phenotype, or in regular glucose media, and these cells (1 × 10⁶) were transfected with either siCtrl, siOGC or siOPA1 and maintained in their respective media. Cells were then left to grow, passaged when required, and cell number in regular growth medium (left panel) or galactose media (right panel) was counted 6 days later (averages ± SEM of 4 independent experiments). Data information: Student's t-tests were performed as indicated: *P < 0.05, **P < 0.01, ***P < 0.005. ▪ indicates a background band in (B) and (C). Source data are available online for this figure.

Figure 7. Pharmacological blockade of SLC25A proteins drastically inhibit OPA1 oligomerization and function

1. Isolated liver mitochondria were incubated with phenylsuccinate (PhS) or butylmalonate (BM), and OPA1 oligomerization was assessed as previously.

2. Mouse liver mitochondria were incubated with 50 mM PhS or BM, and then, cytochrome c retention was analysed where released CytC was mobilized from the pellet (P) to the supernatant (SN) fraction.

3. Mouse liver mitochondria were incubated with 50 mM PhS or BM, and lysates were analysed by BN-PAGE. ATP synthase monomers were quantified (right panel) relative to complex II monomer (averages ± SEM of 6 independent experiments).

4. Mouse liver mitochondria were prepared and incubated in parallel with 7C and analysed by regular Western blot for ATP5A.

Data information: Student's t-tests were performed relative to control, ***P < 0.005. Source data are available online for this figure.