Mol Cell. 2015 Jan 8;57(1):69-82. doi: 10.1016/j.molcel.2014.10.028. Epub 2014 Dec 4.

Mitochondrial shape governs BAX-induced membrane permeabilization and apoptosis.

Renault TT¹, Floros KV², Elkholi R³, Corrigan KA⁴, Kushnareva Y⁵, Wieder SY⁶, Lindtner C⁷, Serasinghe MN⁸, Asciolla JJ⁹, Buettner C¹⁰, Newmeyer DD⁵, Chipuk JE¹¹.

Author information

1: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA; The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA; The Diabetes, Obesity, and Metabolism Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.
2: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA; The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.
3: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA; The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA; The Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.
4: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.
5: Division of Immune Regulation, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037, USA.
6: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA; Department of Dermatology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.
7: Department of Medicine, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.
8: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA; The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA; The Diabetes, Obesity, and Metabolism Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA; Department of Dermatology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.
9: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA; The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA; Department of Dermatology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.
10: The Diabetes, Obesity, and Metabolism Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA; Department of Medicine, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA; Department of Neuroscience, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.
11: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA; The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA; The Diabetes, Obesity, and Metabolism Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA; The Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA; Department of Dermatology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA. Electronic address: jerry.chipuk@mssm.edu.

Abstract

Proapoptotic BCL-2 proteins converge upon the outer mitochondrial membrane (OMM) to promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Here we investigated the mechanistic relationship between mitochondrial shape and MOMP and provide evidence that BAX requires a distinct mitochondrial size to induce MOMP. We utilized the terminal unfolded protein response pathway to systematically define proapoptotic BCL-2 protein composition after stress and then directly interrogated their requirement for a productive mitochondrial size. Complementary biochemical, cellular, in vivo, and ex vivo studies reveal that Mfn1, a GTPase involved in mitochondrial fusion, establishes a mitochondrial size that is permissive for proapoptotic BCL-2 family function. Cells with hyperfragmented mitochondria, along with size-restricted OMM model systems, fail to support BAX-dependent membrane association and permeabilization due to an inability to stabilize BAXα9·membrane interactions. This work identifies a mechanistic contribution of mitochondrial size in dictating BAX activation, MOMP, and apoptosis.

PMID:: 25482509
PMCID:: PMC4289414
DOI:: 10.1016/j.molcel.2014.10.028

[Indexed for MEDLINE]

Free PMC Article

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Figure 7

BAX α9 displays requirements for membrane shape

(A) LUVs were combined with BAX or BAX^OG (0.25 μM) for 15 min at 37°C. (B) BAX (100 ng) was incubated in the presence of BIM BH3 (2.5 μM) and LUVs for 30 min prior to 6A7 IP and western blot. (C) LUVs were combined with BAX^WT or BAX^ΔC (0.25 μM) for 1 h at 37°C. The required incubation time is longer for BAX^ΔC compared to BAX^WT, which increases BAX^WT activity. (D) LUVs were combined with BAX^WT or BAX^S184A for 30 min at 37°C. (E) Same as D, but with BIM BH3 (2.5 μM). (F) LUVs were combined with BAX^WT or BAX^S184A (100 nM) for 30 min at 37°C prior to centrifugation, solubilization, and western blot for associated BAX. (G) NBD-BAX^WT or NBD-BAX^S184A was incubated with 1 μm LUVs for 5 min, ± BIM BH3 (2.5 μM). An increase in NBD fluorescence indicates BAX·LUV interactions, and is reported as fold increase compared to NBD-BAX^WT + LUVs. (H) LUVs (1 μm) were combined with BAX^WT or BAX^S184A (50, 75, 100 nM) with BIM BH3 (2.5 μM) for 30 min at 37°C. (I) LUVs were combined with BAX^WT or BAX^S184A (50 nM) with BIM BH3 (2.5 μM) for 30 min at 37°C. (J) OMVs were combined with BAX^WT or BAX^S184A (50 nM) and BIM BH3 (2.5 μM) for 30 min at 37°C. (K) NBD-BAX^WT or NBD-BAX^S184A ± BIM BH3 (2.5 μM) was incubated with OMVs for 30 min at 37°C. The interaction between NBD-BAX^WT + BIM BH3 with 1 μm OMVs is reported as 100%. (L) Digitonin-permeabilized, JC-1 loaded Mfn1^−/− MEFs were incubated with BIM BH3 (0.1 μM), BAX^WT (50 nM), and BAX^S184A (50 nM), and ΔΔψ_M was determined. (M) Mfn1^−/− MEFs expressing shBax were reconstituted with human BAX^WT or BAX^S184A, treated with DTT (1.5 mM), and the kinetics of tUPR was evaluated by IncuCyte. (N) A schematic summarizing the relationship between BAX, mitochondrial shape, and apoptosis. All data are representative of at least triplicate experiments, and reported as ± S.D., as required. See also .

Outer mitochondrial membrane shape engages BAX α9 to initiate mitochondrial outer membrane permeabilization and apoptosis

Mol Cell. ;57(1):69-82.