Fig 2: Crista junctions (CJs) regulate the difference in ??m between cristae and IBM The role of cristae structure as well as CJ formation and sealing on the generation of the difference in ??m between cristae and IBM were studied by disrupting cristae using Mic10-, Mic13-, and Mic60-deficient cells. Mic13 and Mic60 support cristae formation. Mic10 is essential for CJ formation, and, in its absence, cristae tend to remain as vesicles detached from IBM. Live-cell Airyscan imaging of TMRE was used in all figure panels and models. Representative images of mitochondria in control (top row) vs. Mic13-KO (bottom row) HeLa cells. Note that, TMRE FI in Mic13-KO mitochondria is distributed more evenly along the IMM, so that ??Cr-IBM is decreased (arrowheads). Scale bar = 500 nm. N = 3 independent experiments.Quantification of (A), showing deletion of Mic13 in HeLa cells results in significant decrease in ??Cr-IBM. While Mic13-KO cells show a substantial decrease in cristae number, the loss of cristae does not appear absolute, making it feasible to compare TMRE FI at cristae relative to IBM. N = 3 independent experiments.Representative images of Hap1 control (top row) vs. Mic60 KO (middle row) and Mic10 KO (bottom row); Mic60-KO mitochondria show very few cristae structures. TMRE staining along the IMM is relatively homogeneous, indicating decreased ??Cr-IBM. In general, Mic10 KO in Hap1 cells results in decreased TMRE signal intensity at cristae relative to IBM (arrowheads) as compared to control cells. Deletion of Mic10 induces detachment of cristae from IBM (arrows; see Fig 8). Except for the detached cristae vesicles, the TMRE staining is homogeneously distributed in Mic10-KO cells, indicating decreased ??Cr-IBM. Scale bar = 500 nm. N = 3 independent experiments.Quantification of (C), showing Mic60 KO in Hap1 cells results in significant decrease in ??Cr-IBM. While Mic60-KO cells show a marked loss of normal cristae structures, depletion of cristae does not appear absolute, making it possible to compare TMRE FI at cristae relative to IBM. N = 3 independent experiments.Quantification of (C), showing Mic10 KO in Hap1 cells results in a significant decrease in ??Cr-IBM. Note: quantification of ??m at cristae relative to IBM was performed only on cristae structures that appeared to maintain attachment to IBM. N = 3 independent experiments.Data information: Data were analyzed with 2-tailed Student's t-tests, and P values < 0.05 were considered statistically significant. Specific P values are indicated in the figure. Error bars indicate SEM.

Fig 3: Mitochondrial Mic60 in COS7 cells.A larger view, showing the entire image, for the sample displayed in Fig. 6c. As described, the cell is stained with antibodies against Mic60, and secondary antibodies labeled with Alexa Fluor 647. The localizations were color-coded according to their z coordinate to show the depth information. A three-dimensional rendering of this dataset is shown in Supplementary Video 6. Scale bar: 1 µm.

Fig 4: TRAP1 protected cardiac function and mitochondrial cristae of rats’ heart by increasing MIC60 protein levels in extracellular acidosis.A Transmission electron microscopy was used to detect the ultrastructure of mitochondria of rats’ heart tissue. Twenty-five images were obtained from five rats for each group. The average cristae number in one mitochondrion (n = 30 mitochondria for each group randomly selected from 25 pictures) of each group was quantified. B HE staining was used to detect tissue injury of rats’ heart tissue. C Echocardiography was used to detect cardiac functions of rats including LVEF. D–E Elisa assay was used to detect serum BNP (D) and cTnI (E). Data were the means ± SD from six rats. Group comparisons were performed by one-way analysis of variance followed by Tukey’s post hoc test. &P < 0.05 vs. Normal group. *P < 0.05 vs. sh-Con group. #P < 0.05 vs. ov-Con group. ?P < 0.05 vs. ov-TRAP1 group or ov-MIC60 group.

Fig 5: Unwrapped view of Mic60 in a COS-7 cell.A longer section of the mitochrondrion shown in Fig. 6c, digitally unwrapped to show the organization of Mic60 at the inner boundary membrane. (a) Overview image, showing the region which was unwrapped (white box). The white arrow at one end of the box indicates the origin (coordinate 0) of the analysis and the direction of propagation. Scale bar: 1 µm. (b) The organization of Mic60 relative to the plane of the mitochondrial membrane. Mic60 density is plotted as a function of the normalized distance around the circumference of the membrane (x-axis) and the distance along the length of the mitochondrion (y-axis). The measured circumference is plotted to the right of the Mic60 density. (c) Zoomed view of the boxed region in (b). Scale bar: 100 nm.