Article Figures & Data
Figures
- Figure 1. MIC26 and MIC27 cooperatively determine cristae morphology and are required for formation of crista junctions (CJs).
(A) Western blots from total cell lysates from HAP1 WT, MIC26 KO, MIC27 KO or double knockout (DKO) cells show loss of respective protein and increased level of the respective other protein (reciprocal regulation). DKO cells lacking MIC26 and MIC27 show virtually a complete loss of both full-length proteins. (B) Quantification from qRT-PCR using HAP1 WT, MIC26 KO or MIC27 KO cells and probed for the mRNA levels of MIC26 or MIC27 using specific primers. The house-keeping genes HPRT1 and GAPDH were used as controls. Data from three independent experiments represented as mean ± SEM. P-values calculated using t test show no significant differences (ns). (C) Representative images from electron microscopy in HAP1 WT, MIC26 KO, MIC27 KO, or DKO cells show accumulation of concentric cristae in DKO cells and loss of CJs in single knockouts (SKOs) and DKO cells. Scale bar 0.5 μm. (D) Bar graph show the percentage of mitochondria that have abnormal cristae in respective cell lines. Data from total of 60–90 mitochondrial sections of two independent experiments are represented. (E) Box plot showing the number of concentric onion-like cristae per mitochondrial section. DKO cells show high accumulation of concentric cristae. Data from total of 60–90 mitochondrial section of two independent experiments. ****P-value ≤ 0.0001 indicated in the plot shows comparison between WT and DKO. t test was used for statistical analysis. (F) Box plot showing the number of CJs per mitochondrial section in respective cell lines. SKOs and DKO cells have significant reduction in CJs per mitochondrial section. Data from total of 60–90 mitochondrial section of two independent experiments. ****P-value ≤ 0.0001. P-value indicated in the graph show comparison between WT and respective cell lines. Comparisons between MIC26 KO and MIC27 KO as well as DKO with MIC26 KO were not significantly different (P-value > 0.5). Comparison between MIC27 KO and DKO show slightly significant difference (P-value = 0.04). t test was used for statistical analysis. (G) Box plot showing the number of cristae per mitochondrial section in respective cell lines. SKOs and DKO cells have significant reduction in cristae per mitochondrial section. Data from total of 60–90 mitochondrial sections of two independent experiments. ****P-value ≤ 0.0001. P-value indicated in the graph show comparison between WT and respective cell lines. Comparisons between MIC26 KO and MIC27 KO as well as DKO with MIC26 KO or MIC27 KO were not significantly different (P-value > 0.5). t test was used for statistical analysis.
- Figure 2. Mitochondrial respiration is impaired and mitochondria show fragmentation in double knockout (DKO) cells lacking MIC26 and MIC27.
(A) Oxygen consumption rates (pmol O2/s, normalized for cell numbers by Hoechst staining), including basal respiration (Basal), proton leak, maximal respiration (Maximal) after uncoupling by FCCP, spare respiratory capacity (Spare capacity), non-mitochondrial respiration (Non-mitochondrial), and ATP production is shown for HAP1 WT, MIC26 KO, MIC27 KO, or DKO cells. Data are normalized to basal respiration from HAP1 WT and the mean ± SEM from four independent experiments is shown. DKO cells lacking MIC26 and MIC27 show reduced respiration, whereas MIC27 KO show slight but significant increase compared with HAP1 WT. *P-value ≤ 0.05, **P-value ≤ 0.01, ***P-value ≤ 0.001 (t test). For comparison of basal respiration, one sample t test was performed. (B) Representative confocal images of mitochondria from HAP1 WT, MIC26 KO, MIC27 KO, or DKO cells show mitochondrial fragmentation in MIC26 KO and DKO cells. (C) Quantification of percentage of cells having tubular, intermediate, or fragmented mitochondrial morphology in HAP1 WT, MIC26 KO, MIC27 KO, or DKO cells. Data show mean ± SEM from three independent experiments. t test was used for comparison of percentage of cells having tubular mitochondria in MIC26 KO, MIC27 KO, or DKO cells with HAP1 WT. ***P-value ≤ 0.001.
- Figure S1. Stable expression of MIC26 or/and MIC27 in double knockout (DKO) cell lines rescue respiration.
(A) Western blot showing the expression level of MIC26 or/and MIC27 in the stable cell lines of HAP1 WT expressing empty vector (ev) and DKO that contain either of the following constructs, ev (pMSCVpuro), pMSCVpuro-MIC26, pMSCVpuro-MIC27, and both pMSCVpuro-MIC26 and pMSCVpuro-MIC27 together. (B) Oxygen consumption rates (pmol O2/s), including basal respiration (Basal), proton leak, maximal respiration (Maximum) after uncoupling by FCCP, spare respiratory capacity (Spare capacity), non-mitochondrial respiration (Non-mitochondrial), and ATP production are shown for HAP1 WT+EV (empty vector) and DKO+EV, DKO+MIC26, DKO+MIC27, DKO+MIC26+MC27, and DKO+CRLS1. Data are normalized to basal respiration from HAP1 WT+EV and the mean ± SEM from three independent experiments is shown. Overexpression of both MIC26 and MIC27 in DKO cells significantly rescued respiration of DKO cells. *P-value ≤ 0.05, **P-value ≤ 0.01 (t test).
- Figure 3. MIC26 and MIC27 are not required for the stability of other MICOS subunits.
(A) Representative STED super-resolution images of MIC26 or MIC27 in control cells show the punctae rail-like arrangement within mitochondria that resemble the staining from MIC60 or MIC10 (see also Fig 4A). Scale bar 0.5 μm. (B) Western blots of total cell lysates from HAP1 WT, MIC26 KO, MIC27 KO, or double knockout (DKO) cells probed for various subunits of the MICOS complex. (C) Densitometric quantification of Western blots from four independent experiments (mean ± SEM) in HAP1 WT, MIC26 KO, MIC27 KO, or DKO cells that are normalized to levels of each MICOS subunits to the HAP1 WT. Except MIC26 or MIC27 levels (showing reciprocal change), steady-state levels of other MICOS subunits were not drastically reduced in single knockouts or DKO cells of MIC26 and MIC27. One sample t test was used for comparison. Marginal increase in MIC25 was found in DKO cells. *P-value ≤ 0.05. **P-value ≤ 0.01.
- Figure 4. MIC26 and MIC27 are dispensable for the spatial arrangement of MIC10 and MIC60 in mitochondria and the incorporation of other MICOS subunits into the complex.
(A) Representative images of endogenous staining of MIC60 or MIC10 in HAP1 WT, MIC26 KO, MIC27 KO, or double knockout (DKO) cells using STED super-resolution nanoscopy show that the rail-like punctae arrangement of MIC60 or MIC10 remain unaltered in single knockouts or DKO cells. (B) Complexome profiling data representing the heat map of abundance of occurrence of MICOS subunits in isolated mitochondria from HAP1 WT, MIC26 KO, MIC27 KO, or DKO cells show that cluster of MICOS complex shifts to a lower molecular weight in DKO mitochondria but the remaining subunits remain associated to this complex. (C) Blue-native gel electrophoresis blotted for anti-MIC60 show MICOS complex in single knockouts or DKO cells lacking MIC26 and/or MIC27. BHM, bovine heart mitochondria.
- Figure S2. Reference complexome profile for MICOS complex in HAP1 WT, MIC26 KO, MIC27 KO, and double knockout cells.
Averaged MICOS subunit quantification values were used for complex reference profiles. Reference profiles were normalized to maximum appearance between the samples.
- Figure 5. MIC26 and MIC27 are cooperatively required for the stability and assembly of the F1Fo–ATP synthase complex.
(A) Blot showing the in-gel activity of F1Fo–ATP synthase using isolated mitochondria of HAP1 WT, MIC26 KO, MIC27 KO, or double knockout (DKO) cells that were solubilized with increasing concentration of digitonin (g/g). The blot show oligomers, dimers, and monomers forms of F1Fo–ATP synthase. The intensity (or activity) was reduced in DKO cells. The same mitochondrial lysate was blotted on SDS–PAGE to probe for equal loading among the samples. (B) The quantification of ratio of oligomers or dimers or monomers of F1Fo–ATP synthase to the total intensity in the lane specific for 0.75 g/g digitonin was calculated from three independent experiments (mean ± SEM) show no significant difference among them in single knockouts or DKO cells lacking MIC26 and/or MIC27 compared with HAP1 WT. ns = P-value > 0.05 (nonsignificant). t test was used for statistical analysis. (C) Blue-native gel electrophoresis of isolated mitochondria from HAP1 WT, MIC26 KO, MIC27 KO, or DKO cells that were solubilized with increasing concentration of digitonin (g/g) is blotted and probed for F1Fo–ATP synthase subunit, ATP5D show reduced staining in DKO cells lacking MIC26 and MIC27 with concomitant appearance of lower molecular weight complex (F1). (D) Western blot from the lysate of HAP1 WT, MIC26 KO, MIC27 KO, or DKO cells were probed with antibodies specific to various subunits of F1Fo–ATP synthase complex, do not show any consistent change in either of them in single knockouts or DKO cells. (E) Complexome profiling of isolated mitochondria from HAP1 WT, MIC26 KO, MIC27 KO, or DKO cells for the F1Fo–ATP synthase complex showing the heat map of occurrence of subunits of F1Fo–ATP synthase. F1Fo–ATP synthase complex is reduced and subunits of the F1 part are partially dissociated from the complex in DKO cells lacking MIC26 and MIC27.
- Figure S3. Reference complexome profile for whole F1Fo–ATP synthase, F1 or Fo complexes in HAP1 WT, MIC26 KO, MIC27 KO, and double knockout cells.
Averaged F1, Fo, and F1Fo–ATP synthase subunit quantification values were used for complex reference profiles for indicated (sub-)complexes. Reference profiles were normalized to maximum appearance between the samples.
- Figure 6. MIC26 and MIC27 are required for the stability of respiratory chain (super) complexes.
(A) Blue-native gel electrophoresis for isolated mitochondria from HAP1 WT, MIC26 KO, MIC27 KO, or double knockout (DKO) cells that were solubilized and blotted for antibodies specific for complex I (NDUFB4), complex III (UQCRC2), or complex IV (COX1V) show reduced staining of respiratory chain complexes (RCs) and their higher assemblies (supercomplexes). The same mitochondrial lysate was blotted on SDS–PAGE to probe for equal loading among the samples. (B) Complexome profiling of isolated mitochondria from HAP1 WT, MIC26 KO, MIC27 KO, or DKO cells for respiratory chain complexes (RCs)/supercomplexes (SCs) showing the heat map of occurrence of subunits of respiratory chain complexes which were reduced in DKO cells lacking MIC26 and MIC27.
- Figure S4. 2D Reference complexome profile for respiratory chain complexes in HAP1 WT, MIC26 KO, MIC27 KO, and double knockout cells.
Averaged electron transport chain subunits quantification values were used for complex reference profiles for indicated complexes. Reference profiles were normalized to maximum appearance between the samples.
- Figure S5. Steady-state levels of certain subunits of RCs remain unchanged in double knockout (DKO) cells lacking MIC26 and MIC27.
(A) Western blots from the lysate of HAP1 WT, MIC26 KO, MIC27 KO, or DKO cells were probed with antibodies specific to subunits of respiratory chain complex, do not show any consistent change in either of them in single knockouts or DKO cells except for SDHB that show significant increase in MIC26 KO. (B) Bar graph obtained using densitometry of Western blots show normalized (to HAP1 WT) levels of respective antibody staining specific to subunits of respiratory chain complexes from three to six independent experiments. ns = P-value > 0.05 (non-significant), **P-value ≤ 0.01. One sample t test was performed for statistical analysis.
- Figure S6. OPA1 forms are unaltered in double knockout cells lacking MIC26 and MIC27.
(A) Western blot showing the distribution and levels of OPA1 forms long or short in HAP1 WT, MIC26 KO, MIC27 KO, or double knockout cells show no change in distribution of OPA1 forms. (B) Bar graph represent percentage of OPA1 in either L-OPA1 (long form) or S-OPA1 (Short form) in respective cell lines from six independent experiments. ns = P-value > 0.05 (non-significant). t test was used for all comparisons.
- Figure 7. MIC26 and MIC27 maintain cardiolipin levels that are required for stability of respiratory chain (super) complexes.
(A) Graph representing the levels of cardiolipin shown as arbitrary units normalized to mg of protein in each cell types show significant reduction in MIC26 KO and double knockout (DKO) cells. *P-value ≤ 0.05, **P-value ≤ 0.01. t test was used for statistical analysis. (B) Graph showing the distribution of various cardiolipin species (arbitrary units normalized to mg of protein) in HAP1 WT, MIC26 KO, MIC27 KO, and DKO cells. (C) Blue-native gel electrophoresis for isolated mitochondria from HAP1 WT expressing empty vector (ev) and DKO cell lines that are stably expressing ev or MIC26 or MIC27 or MIC26 and MIC27 together or CRLS1 (cardiolipin synthase) were solubilized and blotted for antibodies specific for complex I (NDUFB4), complex III (UQCRC2), or complex IV (COX1V). The restoration of staining of respiratory chain (super) complexes compared with DKO (with ev) was found upon expression of MIC26 and MIC27 as well as CRLS1 in DKO cell lines. The part of the BN–PAGE stained with Coomassie is shown to represent the loading among the cell lines. (D) The scheme summarizing the phenotype occurring due to loss of MIC26 and MIC27 that show MIC26 and MIC27 are cooperatively required for the formation of crista junctions, maintenance of cardiolipin levels, and stability of respiratory chain (super) complexes and F1FO–ATP synthase. In addition, MIC26 and MIC27 are required for the assembly of F1Fo–ATP synthase by facilitating the association of F1 and Fo part. Loss of MIC26 and MIC27 leads to impaired respiration.
