Slc25a3-dependent copper transport controls flickering-induced Opa1 processing for mitochondrial safeguard

Abstract

Following the Goldilocks principle, mitochondria size must be "just right." Mitochondria balance division and fusion to avoid becoming too big or too small. Defects in this balance produce dysfunctional mitochondria in human diseases. Mitochondrial safeguard (MitoSafe) is a defense mechanism that protects mitochondria against extreme enlarging by suppressing fusion in mammalian cells. In MitoSafe, hyperfused mitochondria elicit flickering-short pulses of mitochondrial depolarization. Flickering activates an inner membrane protease, Oma1, which in turn proteolytically inactivates a mitochondrial fusion protein, Opa1. The mechanisms underlying flickering are unknown. Using a live-imaging screen, we identified Slc25a3 (a mitochondrial carrier transporting phosphate and copper) as necessary for flickering and Opa1 cleavage. Remarkably, copper, but not phosphate, is critical for flickering. Furthermore, we found that two copper-containing mitochondrial enzymes, superoxide dismutase 1 and cytochrome c oxidase, regulate flickering. Our data identify an unforeseen mechanism linking copper, redox homeostasis, and membrane flickering in mitochondrial defense against deleterious fusion.

Keywords: division; fusion; mitochondria; stress response; transporter.

Figure 1.. A knockdown screen identifies that Slc25a3 is critical for flickering and Opa1 cleavage in Drp1-KO cells.

(A) Drp1-KO MEFs expressing matrix-targeted Su9-GFP were viewed by laser scanning confocal microscopy for 30 min with 10-s intervals in the presence of TMRE. The arrows indicate mitochondria that showed flickering. Three frames from the time-lapse analysis are shown. Scale bar, 10 μm. (B) A list of genes tested. (C) 30 genes were individually knocked down in Drp1-KO cells using esiRNA and analyzed for flickering by TMRE staining and live-cell imaging. Knockdown of four genes highlighted in yellow in (B) decreased the frequency of flickering. (D) Drp1-KO MEFs were transduced by lentiviruses carrying shRNAs for each of the four candidates for 1-2 weeks. Cells were stained with TMRE and viewed by laser scanning confocal microscopy for 30 min with 10-s intervals. The percentage of cells that showed flickering are presented (average ± SD, n = 3). (E) Slc25a3 was knocked out using CRISPR in Drp1-KO MEFs. Flickering frequency is quantified (average ± SD, n = 3). (F) The mitochondrial membrane potential was measured in Drp1-KO and Drp1Slc25a3-KO MEFs using MitoLite and flow cytometry. As a negative control, Drp1-KO MEFs were treated with 10 μM FCCP. Significance was calculated using ANOVA with post-hoc Tukey in (D) and Student’s t-test in (E): **p<0.01. See also Figures S1 and S2.

Figure 2.. OPA1 cleavage.

(A) Western blotting of WT, Drp1-KO, Slc25a3-KO, and Drp1Slc25a3-KO MEFs with the indicated antibodies. (B) Quantification of band intensity (average ± SD, n = 3). (C) Drp1-KO and Drp1Slc25a3-KO MEFs were transduced with lentiviruses carrying Opa1 L2- HA. The expression of L2-HA was induced for 4 h (0.1 μg/ml doxycycline). Western blotting was performed with the indicated antibodies. (D) Quantification of band intensity (average ± SD, n = 3). Significance was calculated using ANOVA with post-hoc Tukey in (B) and Student’s t-test in (D): *p<0.05, **p<0.01, ***p<0.001. See also Figures S3, S4, and S5.

Figure 3.. Mitochondrial morphology and cristae structure.

(A) WT, Drp1-KO, Slc25a3-KO, and Drp1Slc25a3-KO MEFs were subjected to laser confocal immunofluorescence microscopy with antibodies to PDH and Tom20. The boxed regions are magnified. Scale bar, 10 μm. (B) Quantification of mitochondrial morphology (average ± SD, n = 3 experiments). In each experiment, 10 cells were analyzed. (C) The size of spherical mitochondria (average ± SD, n = 7 for WT, 79 for Drp1-KO, 3 for Slc25a3-KO, and 211 for Drp1Slc25a3-KO). (D) Mitochondria in the same set of MEFs were analyzed by transmission electron microscopy. Scale bar, 1 μm. Significance was calculated using ANOVA with post-hoc Tukey in (C): ***p<0.001. See also Figure S6.

Figure 4.. mtDNA distribution.

(A) MEFs were stained with SYBR Green I and MitoTracker and viewed using live-cell imaging. Scale bar, 10 μm. (B) The size of mtDNA signal (average ± SD, n = 30). (C) Drp1Slc25a3-KO MEFs were analyzed by laser confocal immunofluorescence microscopy with antibodies to DNA and Hsp60. (D-J) WT, Drp1-KO, and Drp1Slc25a3-KO MEFs were transfected with a plasmid carrying mUNG (Y147A). (D) Seven days after transfection, cells were subjected to laser confocal immunofluorescence microscopy. Scale bar, 10 μm. (E, G, I) The percentage of cells with spherical mitochondria (average ± SD, n = 30). (F, H, J) The size of spherical mitochondria (average ± SD, n = ~20 [F], ~30 [H], and ~130 [J]). (K) Flickering frequency is quantified in the indicated cells (average ± SD, n = 3). Significance was calculated using ANOVA with post-hoc Tukey in (B and K) and Student’s t-test in (F, H, J): *p<0.05, ***p<0.001.

Figure 5.. The role of copper transport by Slc25a3 for flickering and Opa1 processing.

(A) Mitochondrial respiration was analyzed by measuring OCRs in WT, Drp1-KO, Slc25a3-KO, and Drp1Slc25a3-KO MEFs. (B) The basal and maximal OCRs (average ± SD, n = 3). (C) Glycolysis was examined by measuring ECARs in the same set of MEFs. (D) The basal glycolysis, glycolytic capacity, and glycolytic reserve (average ± SD, n = 3). (E) Drp1-KO MEFs and Drp1Slc25a3-KO MEFs expressing WT and mutant forms of the mouse (m) and human (h) Slc25a3 were analyzed for flickering (average ± SD, n = 3 experiments). In each experiment, about 15 cells were analyzed. (F) Drp1Slc25a3-KO MEFs were incubated with 0.1 μM Cu-ATSM for 3 days and analyzed for flickering (average ± SD, n = 3 experiments). (G) Copper levels in mitochondria isolated from Drp1Slc25a3-KO MEFs treated with Cu-ATSM were measured using atomic absorption spectrometry. (average ± SD, n = 3 experiments). (H) Western blotting of Drp1-KO MEFs and Drp1Slc25a3-KO MEFs expressing the indicated WT and mutant mouse Slc25a3. (I) Quantification of band intensity (average ± SD, n = 3). Significance was calculated using ANOVA with post-hoc Tukey in (B, D, E, and I) and Student’s t-test in (F and G): *p<0.05, **p<0.01, ***p<0.001.

Figure 6.. The role of copper transport by Slc25a3 for mitochondrial morphology, cristae structure, and respiration.

(A) Drp1-KO MEFs and Drp1Slc25a3-KO MEFs expressing the mouse (m) and human (h) Slc25a3 were subjected to laser confocal immunofluorescence microscopy with antibodies to PDH and Tom20. The boxed regions are magnified. Scale bar, 10 μm. (B) Quantification of cells with spherical mitochondria (average ± SD, n = 3 experiments). In each experiment, 10 cells were analyzed. (C) The size of spherical mitochondria (average ± SD, n >70). (D) Mitochondria in the indicated MEFs were analyzed by transmission electron microscopy. Scale bar, 500 nm. (E) Mitochondrial respiration was analyzed by measuring OCRs in Slc25a3-KO MEFs expressing the indicated constructs. (F and G) The basal OCRs (F) and maximal OCRs (G) (average ± SD, n = 3). Significance was calculated using ANOVA with post-hoc Tukey in (C, F, and G): *p<0.05, ***p<0.001.

Figure 7.. The effects of SOD1 and cytochrome c oxidase on flickering, and the impact of disease-associated Slc25a3 mutants on Opa1 processing and copper transport.

(A and B) Drp1-KO MEFs (A) and WT MEFs (B) were treated with 0.1 mM LCS-1 for 1 h and analyzed for flickering (average ± SD, n = 3 experiments). (C) Drp1-KO MEFs carrying Opa1 L2-HA were treated with LCS-1. The expression of L2-HA was induced for 4 h (0.1 μg/ml doxycycline). Western blotting was performed with the indicated antibodies. (D) Quantification of band intensity (average ± SD, n = 3). (E) Drp1-KO MEFs were treated with LCS-1 and then analyzed by laser confocal immunofluorescence microscopy with antibodies to DNA and Hsp60. Scale bar, 10 μm. (F) The percentage of cells with spherical mitochondria (average ± SD, n = 30). (G) The size of spherical mitochondria (average ± SD, n = 66 for DMSO and 98 for LCS-1). (H) Drp1-KO MEFs were transduced with lentiviruses carrying SOD1-FLAG and analyzed for flickering (average ± SD, n = 3 experiments). (I) Drp1-KO MEFs were treated with 1 mM NaN₃ for 1 h and analyzed for flickering (average ± SD, n = 3 experiments). (J) Western blotting of Drp1-KO MEFs and Drp1Slc25a3-KO MEFs expressing the indicated human Slc25a3 constructs. (K) Quantification of band intensity (average ± SD, n = 3). (L) Copper levels in mitochondria isolated from WT MEFs and Drp1Slc25a3-KO MEFs expressing either WT Slc25a3 or the G72E mutant were measured using atomic absorption spectrometry (average ± SD, n = 3 experiments). Significance was calculated using Student’s t-test in (A, B, D, G, H, and I) and ANOVA with post-hoc Tukey in (K and L): *p<0.05, **p<0.01, ***p<0.001. See also Figure S7.