In situ architecture of Opa1-dependent mitochondrial cristae remodeling

Abstract

Cristae membrane state plays a central role in regulating mitochondrial function and cellular metabolism. The protein Optic atrophy 1 (Opa1) is an important crista remodeler that exists as two forms in the mitochondrion, a membrane-anchored long form (l-Opa1) and a processed short form (s-Opa1). The mechanisms for how Opa1 influences cristae shape have remained unclear due to lack of native three-dimensional views of cristae. We perform in situ cryo-electron tomography of cryo-focused ion beam milled mouse embryonic fibroblasts with defined Opa1 states to understand how each form of Opa1 influences cristae architecture. In our tomograms, we observe a variety of cristae shapes with distinct trends dependent on s-Opa1:l-Opa1 balance. Increased l-Opa1 levels promote cristae stacking and elongated mitochondria, while increased s-Opa1 levels correlated with irregular cristae packing and round mitochondria shape. Functional assays indicate a role for l-Opa1 in wild-type apoptotic and calcium handling responses, and show a compromised respiratory function under Opa1 imbalance. In summary, we provide three-dimensional visualization of cristae architecture to reveal relationships between mitochondrial ultrastructure and cellular function dependent on Opa1-mediated membrane remodeling.

Keywords: Cristae Remodeling; Cryo-Electron Tomography; Cryo-Focused Ion Beam Milling; Mitochondrial Biology.

Figure 1. In situ mitochondrial membrane morphology is influenced by Opa1 processing.

Mitochondria with distinguishable inner mitochondrial membrane (IMM) and outer mitochondrial membrane (OMM) visualized by cryo-ET. (A) (Right) Summed, projected central slices of cryo-electron tomograms visualizing representative mitochondria in indicated MEF cell lines. (Left) Three-dimensional (3D) rendering of segmented membranes with mitochondria shown across Z slices. Green and yellow surfaces indicate OMM and IMM, respectively. (Bottom right) Schematic of Opa1 forms present in respective cell lines. Scale bar = 200 nm. (B) Graph bar representing the relative proportion of different mitochondrial shapes observed. N refers to number of mitochondria: wild-type = 57, Opa1-OE = 17, l-Opa1* = 39, s-Opa1* = 55, Opa1-KO = 12. (C) Plot of mitochondria size (µm²) observed in cryo-electron tomograms in MEF lines. N refers to number of mitochondria: wild-type = 57, Opa1-OE = 17, l-Opa1* = 39, s-Opa1* = 55, Opa1-KO = 12. Data Information: Scatter plots show data distribution, the mean is marked by a bold black line. Significance of difference is tested relative to wild type using Mann–Whitney test; **p < 0.005, ****p < 0.0001. Source data are available online for this figure.

Figure 2. In situ crista ultrastructure.

(A) Graph bars showing the proportions of straight, tilted, and no attachment observed (NAO) crista observed in indicated MEF lines. N refers to the number of cristae analyzed: WT = 131, Opa1-OE = 166, l-Opa1* = 380, s-Opa1* = 495, Opa1-KO = 112. (B) Graph bars showing the proportions of lamellar, tubular, globular and unusual crista observed in indicated MEF lines. N refers to the number of cristae analyzed: WT = 131, Opa1-OE = 166, l-Opa1* = 380, s-Opa1* = 495, Opa1-KO = 112. (C) Measured cristae length across cell lines. N = 50 for all cell lines. (D) Measured cristae width across cell lines. N refers to the number of cristae subvolumes: WT = 222, Opa1-OE = 430, l-Opa1* = 323, s-Opa1* = 653, Opa1-KO = 243. (E) (Top rows) Computational slices of straight, tilted, disconnected, globular and tubular crista across cell lines and the corresponding 3D renderings (bottom rows) from cryo-electron tomograms (n.a. = not applicable). Scale bar = 50 nm. (F) Histograms of crista widths across cell conditions (see Methods). N refers to the number of cristae subvolumes: WT = 222, Opa1-OE = 430, l-Opa1* = 323, s-Opa1* = 653, Opa1-KO = 243. (G) Subtomogram averages of mitochondrial cristae membranes with the average width indicated. Data information: Scatter plots show data distribution, the mean is marked by a bold black line. Removal of outliers did not change statistical test results (see source data). Significance of difference is tested relative to WT using Mann–Whitney test; *p < 0.05, ***p < 0.001, ****p < 0.0001. For (D), mean ± SD in WT = 14.5 ± 5.8; Opa1-OE = 15.2 ± 10.5; l-Opa1* = 17.6 ± 15.4; s-Opa1* = 12.9 ± 7.9; Opa1-KO = 20 ± 15.8. Source data are available online for this figure.

Figure 3. Quantification of cristae junction (CJ) properties.

(A) Plot of measured cristae junction width across cell lines. (see Appendix Fig. S3C,D and “Methods” section for measurement methods). N refers to number of cristae analyzed: WT = 103, Opa1-OE = 33, l-Opa1* = 107, s-Opa1* = 92, Opa1-KO = 34. (B) Plot of measured cristae junction angle across cell lines (see Appendix Fig. S3C,D and “Methods” section for measurement methods). N refers to number of cristae analyzed: WT = 103, Opa1-OE = 33, l-Opa1* = 107, s-Opa1* = 92, Opa1-KO = 34. (C) (Top) Summed, projected central slices of cryo-electron tomograms of representative mitochondria analyzed in (A, B) with magnified cristae junction (bottom insets). WT, Opa1-OE, l-Opa1* and Opa1-KO are the same tomograms from Fig. 1A. Scale bar = 100 nm. Inset scale bar = 25 nm. Data information: Scatter plots show data distribution, the mean is marked by a bold black line. Significance of difference is tested relative to wild type using Mann–Whitney test; *p < 0.05, **p < 0.01, ***p < 0.001. Source data are available online for this figure.

Figure 4. l-Opa1* cells show WT apoptotic priming.

(A) BH3 profiling of MEF lines with BIM BH3 peptide at indicated concentrations compared with positive control DFNA5 and negative control PUMA2A. Error bars represent range of N = 3–4 biological replicates (see methods for description). (B) MEF lines were treated with indicated agents for 24 h and apoptosis was detected via flow cytometry after staining with Annexin V. Error bars represent range of N = minimum 4 biological replicates. (C) Representative traces of mitochondrial calcium retention capacity assays done in indicated MEF lines. (D) Quantification of CaCl₂ concentration required to induce mPTP opening in (C). Error bars represent SD from N = 3 biological replicates. Data information: Significance of difference is tested relative to wild-type using the Holm–Sidak’s multiple comparison test; *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001. Source data are available online for this figure.

Figure 5. Defects to mitochondrial functions are observed in cells with altered Opa1 levels.

(A) OCR plotted against time with the addition of each compound indicated by an arrow for oligomycin (2 µM), FCCP (1 µM), and rotenone/antimycin A (0.5 µM). Error bars represent SD from N = 3 biological replicates. (B) Aspects of mitochondrial respiration; basal respiration rates, the amount of respiration used for ATP production, maximum respiration and spare capacity, are extracted by the data plotted in (A). Error bars represent range for N = 3 biological replicates. Data Information: Significance of difference is tested relative to WT using Welch’s t test; *p < 0.05, **p < 0.01. Source data are available online for this figure.

Figure 6. Summary of cryo-ET cristae observations.

Cartoon schematic summarizing mitochondrial morphological observations dependent on Opa1 state. Mitochondria in l-Opa1* cells displayed WT-like CJ widths and perpendicularity. The majority of mitochondria displayed a stacking phenotype and multijunction cristae. Tubular and vesicular cristae were found in s-Opa1* cells. Summaries of functional dysfunctions corresponding to observed cristae morphological changes. (A) Respiratory defects correlate with a large number of unstructured cristae. (B) Defects in cytochrome c (CytC) release properties (as evaluated by BH3 profiling) and calcium handling correlate with wider CJ and vesicular cristae. (C) Cartoon scheme with hypothesized modes of action for l-Opa1 in the crista stacking. (D) Cartoon schematic with hypothesized modes of action for l-Opa1 in BH3 and calcium handling process.

Figure EV1. Mitochondrial subcompartment volumes.

(A) Three-dimensional renderings of segmented inter-membrane space (IMS, pink surface), cristae lumen (CL, magenta surface), and matrix (translucent gray surface) volumes. Scale bar = 200 nm. (B) Total mitochondrial volume across indicated cell lines. N = 5 cells for all cell lines. (C) Quantification of IMS volume relative to total volume of each mitochondrion indicated in (B). N = 5 cells for all cell lines. (D) Quantification of CL volume relative to total volume of each mitochondrion indicated in (B). N = 5 cells for all cell lines. (E) Quantification of matrix volume relative to total volume of each mitochondrion indicated in (B). N = 5 cells for all cell lines. (F) CL to matrix ratio across cell lines. N = 5 cells for all cell lines. (G) Normalized gray scale mitochondrial matrix value across cell lines. N = 5 cells for all cell lines. (H) Graph bar representing percentage of cells with detected calcium deposits in cryo-electron tomograms. N refers to the number of mitochondria: WT = 57, Opa1-OE = 17, l-Opa1* = 39, s-Opa1* = 55, Opa1-KO = 12. Data information: Scatter plots show data distribution, the mean is shown by a bold black line. Significance of difference is tested relative to wild type using Mann–Whitney test in (B, D, E, G); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; and unpaired t test in (C): **p < 0.01.

Figure EV2. Cristae analysis.

(A) Cristae density (cristae per µm²) represented as a scatter plot. N refers to number of cells; WT = 33, Opa1-OE = 7, l-Opa1* = 21, s-Opa1* = 28, Opa1-KO = 11. (B) Number of cristae per mitochondria represented as a scatter plot. N refers to number of cells; WT = 51, Opa1-OE = 17, l-Opa1* = 39, s-Opa1* = 55, Opa1-KO = 12. (C) (Top) Summed, projected central slices of cryo-electron tomograms visualizing mitochondria with stacking crista characteristics, supported by 3D representations consisting of their sub compartments (bottom) in indicated MEF lines. Scale bar = 200 nm. N refers to number of cells; WT = 57, Opa1-OE = 17, l-Opa1* = 39, s-Opa1* = 55, Opa1-KO = 12. The representative tomograms for WT, Opa1-OE, l-Opa1*, and Opa1-KO are the same as in Fig. 1A. The representative s-Opa1* tomogram is the same as the second from the left in Appendix Fig. S2A. (D) Graph bar representing percentage of mitochondria with stacking crista formation in each MEF line. N refers to number of cells; WT = 57, Opa1-OE = 17, l-Opa1* = 39, s-Opa1* = 55, Opa1-KO = 12. Data information: Significance of difference is tested relative to WT using Mann–Whitney test; ****p < 0.0001.

Figure EV3. Mitochondrial network morphology in MEF lines by fluorescence microscopy.

(A) Representative images of mitochondrial morphology in indicated MEF lines labeled with MitoTracker^TM Deep Red FM. Insets show magnified view of regions indicated with dashed boxes. Scale bar = 10 µm. Inset scale bar = 5 µm. (B) Graph bar representing mitochondrial network morphology scored in indicated MEF lines. N = 100 cells analyzed per cell line.

Figure EV4. Unusual cristae morphology.

(A) Graph bar representing the relative proportion of unusual cristae morphology observed in indicated MEF lines. Unusual cristae were categorized into vesicular, zipped, ring, split, amorphous, straight-across, pinched and loop. N refers to number of cristae analyzed, N: wild-type = 222, Opa1-OE = 430, l-Opa1* = 323, s-Opa1* = 653, Opa1-KO = 243. (B) Summed, projected central slices of cryo-electron tomograms showing examples of unusual cristae in mitochondria across cell lines in 2D (top) and 3D (bottom). Loop (from Fig. 1A, s-Opa1*), ring, straight-across (from Fig. 1A, l-Opa1*), pinched (from Appendix Fig. S2A, Opa1-KO second from the left), vesicular (from Fig. 1A, Opa1-KO), and amorphous cristae are shown. Scale bar = 200 nm.

Figure EV5. Multijunction cristae.

(A) Scatter plot showing the percentage of multijunction cristae per mitochondrion in indicated MEF lines. N refers to number of cristae; WT = 18, Opa1-OE = 5, l-Opa1* = 30, s-Opa1* = 16, Opa1-KO = 3. (B) Graph bar representing percentage of multijunction cristae categorized into straight-across and loop morphology in each MEF line. N refers to number of cristae; WT = 26, Opa1-OE = 9, l-Opa1* = 79, s-Opa1* = 29, Opa1-KO = 4. Data information: Scatter plot shows data distribution, the mean is marked by a bold black line. Significance of difference is tested relative to wild type using Mann–Whitney; ****p < 0.0001.