Abstract
The regulation of both mitochondrial dynamics and apoptosis is key for maintaining the health of a cell. Bcl-2 family proteins, central in apoptosis regulation, also have roles in the maintenance of the mitochondrial network. Here we report that Bax and Bak participate in the regulation of mitochondrial fusion in mouse embryonic fibroblasts, primary mouse neurons and human colon carcinoma cells. To assess how Bcl-2 family members may regulate mitochondrial morphogenesis, we determined the binding of a series of chimeras between Bcl-xL and Bax to the mitofusins, mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2). One chimera (containing helix 5 (H5) of Bax replacing H5 of Bcl-xL (Bcl-xL/Bax H5)) co-immunoprecipitated with Mfn1 and Mfn2 significantly better than either wild-type Bax or Bcl-xL. Expression of Bcl-xL/Bax H5 in cells reduced the mobility of Mfn1 and Mfn2 and colocalized with ectopic Mfn1 and Mfn2, as well as endogenous Mfn2 to a greater extent than wild-type Bax. Ultimately, Bcl-xL/Bax H5 induced substantial mitochondrial fragmentation in healthy cells. Therefore, we propose that Bcl-xL/Bax H5 disturbs mitochondrial morphology by binding and inhibiting Mfn1 and Mfn2 activity, supporting the hypothesis that Bcl-2 family members have the capacity to regulate mitochondrial morphology through binding to the mitofusins in healthy cells.
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Abbreviations
- Mfn1:
-
mitofusin 1
- Mfn2:
-
mitofusin 2
- OMM:
-
outer mitochondrial membrane
- IMM:
-
inner mitochondrial membrane
- Opa1:
-
optic atrophy 1
- Drp1:
-
dynamin-related protein 1
- H5:
-
helix 5
- WT:
-
wild type
- MEF:
-
mouse embryonic fibroblast
- FRAP:
-
fluorescence recovery after photobleaching
- ROI:
-
region of interest
- PAGFP:
-
photoactivatable GFP
- DIV:
-
days in vitro
- EndoB1:
-
endophilin B1
- EndoB2:
-
endophilin B2
- STS:
-
staurosporine
- DMSO:
-
dimethyl sulfoxide
- IP:
-
immunoprecipitate
- WCL:
-
whole cell lysate
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Acknowledgements
We would like to thank Drs. David Huang and Kym Lowes for the C57BL/6 WT and Bax−/−Bak−/− cells and their comments on the manuscript; Dr. Kuan Hong Wang for his thoughtful reading of the manuscript; Susan Smith for her tissue culture assistance; and the NINDS DNA sequencing facility, the NINDS imaging facility and Sungyoung Auh for her assistance with statistical analysis. We would also like to thank Dr. Fred Bunz for the pSEPT vector and advice on gene targeting strategy; Dr. Seung-Wook Ryu for cloning FLAG-Mfn2; Dr. Liqiang Zhang for cloning GFP-Bax/Bcl-xL H5; and Dr. Benoit Pierrat for the gift of anti-endophilin B2. MK and XL thankfully acknowledge financial support from National Institute of General Medical Science (RO1-GM083131 to MK and RO1-GM76237 to XL). This work was supported in part by the Intramural Research Program, NINDS, NIH.
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Cleland, M., Norris, K., Karbowski, M. et al. Bcl-2 family interaction with the mitochondrial morphogenesis machinery. Cell Death Differ 18, 235–247 (2011). https://doi.org/10.1038/cdd.2010.89
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DOI: https://doi.org/10.1038/cdd.2010.89
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