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Role of Bax and Bak in mitochondrial morphogenesis

Nature volume 443, pages 658–662 (2006)Cite this article

Abstract

Bcl-2 family proteins are potent regulators of programmed cell death. Although their intracellular localization to mitochondria and the endoplasmic reticulum has focused research on these organelles, how they function remains unknown. Two members of the Bcl-2 family, Bax and Bak, change intracellular location early in the promotion of apoptosis to concentrate in focal clusters at sites of mitochondrial division. Here we report that in healthy cells Bax or Bak is required for normal fusion of mitochondria into elongated tubules. Bax seems to induce mitochondrial fusion by activating assembly of the large GTPase Mfn2 and changing its submitochondrial distribution and membrane mobility—properties that correlate with different GTP-bound states of Mfn2. Our results show that Bax and Bak regulate mitochondrial dynamics in healthy cells and indicate that Bcl-2 family members may also regulate apoptosis through organelle morphogenesis machineries.

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Figure 1: Mitochondrial morphology defects in Bax/Bak DKO cells.
Figure 2: vMIA-induced mitochondrial fragmentation is reversed by Bax overexpression.
Figure 3: Ectopic Mfn2 but not Mfn2-G12V reverses fragmentation owing to loss of Bax/Bak.
Figure 4: Mfn2 complex formation, subcellular distribution and membrane mobility are altered by Bax expression.
Figure 5: Mitochondrial fusion is inhibited in the absence of Bax and Bak.

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Acknowledgements

We thank N. Swarup for help in the early stages of this work; C. Thompson, J. Lum and T. Lindsten for Bax/Bak DKO bone marrow cells, Bak-transfected DKO cells and DKO primary MEFs; E. White, S. Korsmeyer and D. Chan for Bax/Bak DKO BMK cells, Bax/Bak DKO MEFs and Mfn KO MEFs, respectively; B. Vogelstein for HCT116 Bax-/- cells, J. Norris for DU145 cells, V. Goldmacher for vMIA HeLa cells, H. McBride for antibodies to Mfn2 and mutant Mfn2 constructs; P. Hajek and G. Attardi for antibodies to Mfn2; A. Antignani, A. Neutzner, B. Fell, S.-W. Ryu and S. Smith for technical assistance; and C. Smith for assistance with confocal microscopy and data analysis and for reading the manuscript. This work was supported by the Intramural Research Program of the NIH, National Institute of Neurological Disorders and Stroke. Author Contributions M.K., K.N. and R.Y. contributed to the conception, interpretation, execution and presentation of the experiments; S-Y.J. contributed to the RNAi experiments; M.C. participated in FRAP experiments and data analysis.

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Author notes

  1. Mariusz Karbowski

    Present address: University of Maryland Biotechnology Institute, Medical Biotechnology Center, UMBI Building, 725 West Lombard Street, Baltimore, Maryland, 21201, USA

  2. Seon-Yong Jeong

    Present address: Department of Medical Genetics, School of Medicine, Ajou University, Suwon, 443-721, Korea

  3. Mariusz Karbowski and Kristi L. Norris: *These authors contributed equally to this work

Authors and Affiliations

  1. Biochemistry Section, SNB, NINDS, NIH, Bethesda, Maryland, 20892, USA

    Mariusz Karbowski, Kristi L. Norris, Megan M. Cleland, Seon-Yong Jeong & Richard J. Youle

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Correspondence to Mariusz Karbowski or Richard J. Youle.

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Supplementary information

Supplementary Notes

This file contains Supplementary Methods and Supplementary Figure Legends (DOC 107 kb)

Supplementary Figure 1

Quantification of mitochondrial morphology with and without Bax/Bak expression in baby mouse kidney cells (BMK) and primary mouse embryonic fibroblasts (MEFs). (JPG 16 kb)

Supplementary Figure 2

Development and mitochondrial phenotype of Bax and Bak double RNAi cells (JPG 22 kb)

Supplementary Figure 3

Viral mitochondria-associated inhibitor of apoptosis (vMIA) interacts with both Bax and Bak and inhibits apoptosis induced by Bak-overexpression (JPG 17 kb)

Supplementary Figure 4

Certain Bcl-2 family members other than Bax do not reverse vMIA-induced mitochondrial fragmentation (JPG 14 kb)

Supplementary Figure 5

Blocking fission does not reverse fragmentation induced by vMIA expression or loss of Bax/Bak (JPG 63 kb)

Supplementary Figure 6

vMIA blocks mitochondrial fusion (JPG 26 kb)

Supplementary Figure 7

Drp1 RNAi effect on mitochondria in Bax/Bak DKO MEFs (JPG 12 kb)

Supplementary Figure 8

Expression and sub-cellular distribution of mitochondrial fusion and fission proteins in 129/CD1 WT and 129/CD1 Bax/Bak DKO MEFs (JPG 10 kb)

Supplementary Figure 9

Quantification of Mfn2-YFP sub-mitochondrial localization in WT and Bax/Bak DKO MEFs (JPG 10 kb)

Supplementary Figure 10

FRAP of mitochondrial outer membrane-associated proteins in Bax/Bak DKO MEFs (JPG 17 kb)

Supplementary Figure 11a-d

Sub-mitochondrial distribution of YFP chimeras of several Mfn2 mutants. (JPG 27 kb)

Supplementary Figure 11e-h

Sub-mitochondrial distribution of YFP chimeras of several Mfn2 mutants. (JPG 54 kb)

Supplementary Figure 12

Opa1 complex formation is not altered in WT and Bax/Bak DKO MEFs (JPG 9 kb)

Supplementary Figure 13

Gel Filtration analysis of Mfn2 complex formation (JPG 10 kb)

Supplementary Figure 14

Mitochondrial membrane potential (Δψm) and ATP levels in WT and Bax/Bak DKO MEFs (JPG 15 kb)

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Karbowski, M., Norris, K., Cleland, M. et al. Role of Bax and Bak in mitochondrial morphogenesis. Nature 443, 658–662 (2006). https://doi.org/10.1038/nature05111

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