Autophagic cell death is dependent on lysosomal membrane permeability through Bax and Bak
- Jason Karch
- Tobias G Schips
- Bryan D Maliken
- Matthew J Brody
- Michelle A Sargent
- Onur Kanisciak
- Jeffery D Molkentin
- University of Cincinnati, United States
- Howard Hughes Medical Institute, United States
Figures
Figure 1 with 1 supplement
The expression of Bax or Bak are required for autophagic cell death.
(A) Wild type (WT) mouse embryonic fibroblasts (MEFs), Bax/Bak1 double knockout (DKO) MEFs, and DKO MEFs stably expressing Bax or Bak1 were subjected to serum starvation for the indicated time …
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Figure 1—source data 1
Raw western gel images for Figure 1C.
- https://doi.org/10.7554/eLife.30543.004
Figure 1—figure supplement 1
The Expression of Bax or Bak are required for autophagic cell death induced by amino acid deprivation.
(A) WT and DKO MEFs were subjected to amino acid-free media for 4 days and cell death was measured by loss of membrane integrity. (B) Confocal images of LysoTracker red fluorescence in amino …
Figure 2 with 1 supplement
Autophagy can culminate in a unique form of cell death.
(A) WT MEFs treated with or without a pan caspase inhibitor (zVad) and subjected to serum starvation for 1 or 2 days. Cell death was measured by plasma membrane rupture. (B) WT and Ppif null MEFs …
Figure 2—figure supplement 1
Bax/Bak activation, oligomerization, and translocation does not occur during autophagic cell death.
(A) Western blots for Bax and α-tubulin on cell lysates from WT, DKO and DKO MEFs stably reconstituted with WT and the indicated mutant versions of GFP-Bax. (B) Confocal images of WT MEFs labeled …
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Figure 2—figure supplement 1—source data 1
Raw western gel images for Figure 2—figure supplement 1A and C.
- https://doi.org/10.7554/eLife.30543.007
Figure 3
Loss of Bax and Bak1 maintains lysosomal integrity.
(A) Transmission EM images (30,000x) of WT and DKO MEFs subjected to serum starvation for the specified time points. Autolysosomes and translucent vesicles are noticeable in the serum-starved WT …
Figure 4
Bax and Bak localize to lysosomes and targeting Bax to lysosomes restores autophagic cell death in DKO MEFs.
(A) Western blot analysis of the indicated protein fractions from starved and control WT MEFs for Bax, Bak, Lamp1 and the oxidative phosphorylation mitochondrial proteins UQCRC2 (upper band) and …
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Figure 4—source data 1
Raw western gel images for Figure 4A.
- https://doi.org/10.7554/eLife.30543.010
Figure 5
The lysosomotropic detergent MSDH restores autophagic death in Bax/Bak1 DKO MEFs.
(A) A time course of confocal images of DKO MEFs that were serum starved for 24 hr and then loaded with LysoTracker red, treated with or without 30 µM MSDH for the indicated times. Scale bars = 10 …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| gene (Mus Musculus) | Bak1 | N/A | ID: 12018 | N/A |
| gene (Mus Musculus) | Bax | N/A | ID: 12028 | N/A |
| cell line (Mus Musculus) | Wild Type SV40 Mouse Embryonic Fibroblasts; WT MEFs | PMID: 11326099 | N/A | N/A |
| cell line (Mus Musculus) | Bax Bak1 double knockout SV40Mouse Embryonic Fibroblasts; DKO MEFs | PMID: 11326099 | N/A | N/A |
| cell line (Mus Musculus) | DKO MEFs stably reconstitutied with wild type Bax; DKO-WT-Bax | PMID: 21255726 | N/A | N/A |
| cell line (Mus Musculus) | DKO MEFs stably reconstitutied with 92-94A mutant Bax; DKO-92-94A-Bax | PMID: 21255726 | N/A | N/A |
| cell line (Mus Musculus) | DKO MEFs stably reconstitutied with 63-65A mutant Bax; DKO-63-65A-Bax | PMID: 21255726 | N/A | N/A |
| antibody | Bax6A7 | Thermo Fisher Scinentific | MA5-14003 | 1 to 100 |
| antibody | TOM20 | Santa Cruz Biotechnology | sc-11415 | 1 to 100 |
| antibody | Lamp-1 | DSHB-University of Iowa | 1D4B | 1 to 200 and 1 to 1000 |
| antibody | Bax | Santa Cruz Biotechnologies | sc-493 | 1 to 300 |
| antibody | Bak | Millipore | 06–536 | 1 to 1000 |
| antibody | LC3I/II | Abcam | ab58610 | 1 to 500 |
| antibody | Bcl-2 | Santa Cruz Biotechnologies | sc-7382 | 1 to 300 |
| antibody | Lamp1 | Abcam | ab24170 | 1 to 500 |
| antibody | Cathepsin D | Novus | AF1029 | 1 to 800 |
| antibody | GAPDH | Fitzgerald | 10R-G109A | 1 to 25,000 |
| antibody | Total OXPHOS Rodent WB Antibody Cocktail | Abcam | ab110413 | 1 to 10,000 |
| antibody | αTubulin | Santa Cruz Biotechnologies | sc-8035 | 1 to 250 |
| recombinant DNA reagent | Lysosomal targeted Bax; Lyso-Bax | Bio Basic | N/A | special order |
| recombinant DNA reagent | MiniSOG-Bax | Bio Basic | N/A | special order |
| sequence-based reagent | N/A | N/A | N/A | N/A |
| peptide, recombinant protein | N/A | N/A | N/A | N/A |
| commercial assay or kit | Lysosome Isolation Kit | Sigma | LYSISO1 Sigma | N/A |
| commercial assay or kit | intracellular pH detection kit | Invitrogen | P35379 | N/A |
| commercial assay or kit | Muse Count and Viability assay | EMD Millipore | MCH100102 | N/A |
| chemical compound, drug | ABT-737 | Selleck Chemicals | S1002 | 20 μM |
| chemical compound, drug | Gossypol | Tocris | 1964 | 10 μM |
| chemical compound, drug | caspase inhibitor zVAD-fmk | Promega | G7232 | 20 μM |
| chemical compound, drug | (S)-O-methyl-serine dodecylamide hydrochloride; MSDH | Avanti Polar Lipids | 850546 | 30 μM |
| chemical compound, drug | staurosporine | Sigma-Aldrich | S6942 | 200 nM |
| chemical compound, drug | LysoTracker Red DND-99 | ThermoFisher Scientific | L7528 | 50 nM |
| chemical compound, drug | Rhodamine-123 | ThermoFisher Scientific | R-302 | 25 μM |
| chemical compound, drug | pHrodo Red AM Intracellular pH Indicator | ThermoFisher Scientific | P35372 | N/A |
| chemical compound, drug | Protease inhibitor cocktail | ThermoFisher Scientific | 88666 | N/A |
| software, algorithm | Excel 2013 | Microsoft Office | N/A | N/A |
| software, algorithm | Power Point 2013 | Microsoft Office | N/A | N/A |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.30543.012
