Molecular and topological reorganizations in mitochondrial architecture interplay during Bax-mediated steps of apoptosis
- MRC Laboratory of Molecular Biology, United Kingdom
- National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States
Figures
Figure 1 with 1 supplement
Live confocal fluorescence microscopy of HeLa cells overexpressing GFP-Bax.
HeLa cells transfected with GFP-Bax (green) in the presence of Q-VD-OPh were imaged every 30 min for 24 hr after transfection. Cells were stained with MitoTracker Deep Red (magenta) prior to …
Figure 1—figure supplement 1
Immunofluorescence of cytochrome c release at different stages of GFP-Bax recruitment to mitochondria.
Confocal FM of fixed HeLa cells, 16 hr post-transfection with GFP-Bax (yellow) in the presence of Q-VD-OPh. Cells were stained with antibodies for the translocase of outer membrane 20 (TOM20) …
Figure 2 with 2 supplements
Correlative microscopy of resin-embedded HeLa cells overexpressing GFP-Bax.
Gallery of GFP-Bax locations and the associated mitochondrial morphologies, 16 hr post-transfection with GFP-Bax, in the presence of Q-VD-OPh. (A–D) FM images of sections of resin-embedded cells. …
Figure 2—figure supplement 1
Representation of mitochondrial morphology in different views.
(A) Virtual slice from electron tomogram of HeLa cells (16 hr post-transfection with GFP-Bax). (B) 3D segmentation model of outer membrane in A. (C–F’) The thin samples used in our experiments are …
Figure 2—figure supplement 2
Drug-induced GFP-Bax recruitment to mitochondria in HCT116 cells causes outer membrane ruptures and inner membrane rearrangement similar to those induced in HeLa cells upon GFP-Bax overexpression.
Live confocal FM of Bax/Bak DKO HCT116 cells stably expressing GFP-Bax (green), treated with ABT-737 and Q-VD-OPh for 3 hr. Cells were stained with MitoTracker Deep Red (magenta) prior to treatment. …
Figure 3 with 2 supplements
Ultrastructure of GFP-Bax clusters in HeLa cells visualized by correlative cryo-microscopy of vitreous sections, and by cryo-ET of FIB-milled cells.
(A, E) Cryo-FM of vitreous sections of HeLa cells (high-pressure frozen 16 hr post-transfection with GFP-Bax). GFP-Bax signal in green. White squares indicate areas shown in B and F, respectively. (B…
Figure 3—figure supplement 1
Analysis of GFP-Bax clusters versus control regions in vitreous sections.
(A) Cryo-FM (GFP channel) of vitreous section of HeLa cells (high-pressure frozen 16 hr post-transfection with GFP-Bax). White square indicates area shown in B. (B) Cryo-EM overview image of area …
Figure 3—figure supplement 2
Cryo-FM targeted cryo-FIB milling of human apoptotic cells.
Shown are all apoptotic cells that were used to acquire the cryo-ET data set presented in this study. A-E and G-K are HeLa cells that were grown on EM grids and plunge-frozen 16 hr after …
Figure 4
Mitochondrial outer membrane ruptures are accompanied by rearrangements of the inner membrane.
(A-C) Virtual slices through electron cryo-tomograms of HeLa cells (16 hr post-transfection with GFP-Bax), thinned by cryo-FIB milling. Black squares indicate areas magnified in D-I, respectively. (D…
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Figure 4—source data 1
Numerical data presented in the graph shown in Figure 4M.
- https://doi.org/10.7554/eLife.40712.013
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Figure 4—source data 2
Numerical data presented in the graph shown in Figure 4N.
- https://doi.org/10.7554/eLife.40712.014
Figure 5
Dilution of the mitochondrial matrix and organization of ATP synthases visualized by cryo-ET of cryo-FIB milled HeLa cells.
(A) Virtual slice through an electron cryo-tomogram of a HeLa cell (16 hr post-transfection with GFP-Bax) thinned by cryo-FIB milling. (B) Virtual slice through an electron cryo-tomogram of a …
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Figure 5—source data 1
Numerical data presented in the graph shown in Figure 5C.
- https://doi.org/10.7554/eLife.40712.018
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Figure 5—source data 2
Numerical data presented in the graph shown in Figure 5J.
- https://doi.org/10.7554/eLife.40712.019
Figure 6
HCT116 cells treated with the apoptotic drug ABT-737 contain inner membrane compartments that are lacking the enclosing outer membranes.
(A) FM of a section of resin-embedded Bax/Bak DKO HCT116 cells stably expressing GFP-Bax, treated with ABT-737 for 3 hr. GFP-Bax (green), MitoTracker Deep Red (magenta). Yellow square indicates the …
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Figure 6—source data 1
Numerical data presented in the graph shown in Figure 6J.
- https://doi.org/10.7554/eLife.40712.021
Figure 7
Model for the interplay of inner and outer membrane reorganization during Bax-mediated steps of apoptosis.
Bax clusters form at outer membrane ruptures. Clusters display a higher order organization of their components. Ruptures allow influx of ribosomes and thus mixing of cytosolic and intermembrane …
Videos
Video 1
Electron cryo-tomogram of GFP-Bax clusters obtained by correlative microscopy of vitreous sections, corresponding to Figure 3C–D’.
Movie through virtual slices of electron cryo-tomogram. Movie pauses at the virtual slice shown in Figure 3D and D’ to indicate structural features highlighted in red. Scale bar: 50 nm.
Video 2
Electron cryo-tomogram of GFP-Bax clusters obtained by correlative microscopy of vitreous sections, corresponding to Figure 3G–H’.
Movie through virtual slices of electron cryo-tomogram. Movie pauses at the virtual slice shown in Figure 3H and H’ to indicate structural features highlighted in red. Scale bar: 50 nm.
Video 3
Electron cryo-tomogram of GFP-Bax cluster obtained from cryo-FIB milled cells, corresponding to Figure 3I–K’.
Movie through virtual slices of electron cryo-tomogram. 3D segmentation model of mitochondrion is shown as an overlay. Outer membranes in dark blue, inner membranes in light blue, and ribosomes in …
Video 4
Electron cryo-tomogram of GFP-Bax clusters and inner membrane rupture obtained from cryo-FIB milled cells, corresponding to Figure 3L–Q’.
Movie through virtual slices of electron cryo-tomogram. 3D segmentation model of mitochondrion is shown as an overlay. Outer membranes are in dark blue, inner membranes in light blue. White borders …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | Bax/Bak DKO HCT116 GFP-Bax | this paper | Cell line generated by transfection of GFP-Bax and selection for stable expression in Bax/Bak DKO HCT116 line generated in PMID:22056880 | |
| Cell line (Homo sapiens) | HeLa/wtOTC, TetOn promoter | PMID:24149988 | Cell line maintained in Richard Youle lab. | |
| Cell line (Homo sapiens) | HeLa control/Fsp27-EGFP | other | Cell line stably expressing Fsp27-EGFP from tet-inducible promoter. Obtained from Koini Lim (David Savage lab). | |
| Antibody | Mouse anti-cytochome c (monoclonal) | BD Pharmingen | BD Pharmingen:556432 | (1:250) |
| Antibody | Rabbit anti-TOM20 (polyclonal) | Santa Cruz Biotechnology | Santa Cruz:sc-11415 | (1:250) |
| Antibody | Goat anti-rabbit Alexa Fluor 405 nm | Invitrogen | Invitrogen:A31556 | (1:200) |
| Antibody | Donkey anti-mouse Alexa Fluor 647 nm | Invitrogen | Invitrogen:A31571 | (1:200) |
| Recombinant DNA reagent | hBax-C3-EGFP | Addgene | Addgene:19741 | |
| Chemical compound, drug | ABT-737 | Cayman | Cayman:11501 | assay concentration = 10 µM |
| Chemical compound, drug | Doxycycline | Takara | Takara:631311 | assay concentration = 1 µg/mL |
| Chemical compound, drug | Hygromycin B | Invitrogen | Invitrogen:10687010 | assay concentration = 0.2 µg/mL |
| Chemical compound, drug | Oleic acid | Sigma | Sigma:O3008 | assay concentration = 0.4 mM |
| Chemical compound, drug | Q-VD-Oph | APExBIO | APExBIO:A1901 | assay concentration = 10 µM |
| Commercial assay or kit | Lowicryl HM20 | Polysciences, Inc. | Polysciences, Inc.:15924–1 | |
| Software, algorithm | Correlative Microscopy, MATLAB-based scripts | PMID:22863005; PMID:24275379; Mathworks | https://www.embl.de/download/briggs/cryoCLEM/index.htm | |
| Software, algorithm | IMOD | PMID:8742726 | http://bio3d.colorado.edu/imod/ | |
| Software, algorithm | SerialEM | PMID:16182563 | http://bio3d.colorado.edu/SerialEM/ | |
| Software, algorithm | Ridge Detection 1.4.0 | DOI:10.5281/zenodo.845874 | https://imagej.net/Ridge_Detection | |
| Other | Specimen carrier, aluminum, B | Engineering Office M. Wohlwend | Engineering Office M. Wohlwend: Art. 1314 | |
| Other | Specimen carrier, copper gold-coated | Engineering Office M. Wohlwend | Engineering Office M. Wohlwend: Art. 1322 | |
| Other | EM grids, copper, 200 mesh carbon support | Agar Scientific Ltd. | Agar Scientific Ltd:S160 | |
| Other | EM grids, copper, 200 mesh, R 3.5/1 holey carbon | Quantifoil | ||
| Other | EM grids, gold, 200 mesh, R 2/2 holey carbon | Quantifoil | ||
| Other | MitoTracker Deep Red | Thermo | Thermo:22426 | assay concentration = 20 nM |
| Other | Sapphire disks, 3 mm | Engineering Office M. Wohlwend | Engineering Office M. Wohlwend: Art. 405 | |
| Other | TetraSpeck microspheres,50 nm | Invitrogen | custom order, (diluted 1:100 in PBS) | |
| Other | TetraSpeck microspheres, 100 nm | Invitrogen | T7279 | (diluted 1:50 in PBS) |
| Other | X-tremeGENE 9 | Roche | Roche:06365787001 | 3 µL reagent: 1 µg DNA |
Additional files
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Supplementary file 1
Sample sizes from which the analyzed electron tomography data sets were generated.
Counts include only samples that have contributed to the final data presented in this study. Additional samples and data have been excluded based on either one or more of the following criteria: poor vitrification/sample quality, poor tilt series acquisition quality, poor tomographic reconstruction, no structure of interest contained in the tomographic volume.
- https://doi.org/10.7554/eLife.40712.023
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Transparent reporting form
- https://doi.org/10.7554/eLife.40712.024
