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. 2010 Jun;11(6):459-65.
doi: 10.1038/embor.2010.50. Epub 2010 Apr 30.

The BH3-only Bnip3 binds to the dynamin Opa1 to promote mitochondrial fragmentation and apoptosis by distinct mechanisms

Thomas Landes  1 , Laurent J Emorine, Delphine Courilleau, Manuel Rojo, Pascale Belenguer, Laetitia Arnauné-Pelloquin
Affiliations

The BH3-only Bnip3 binds to the dynamin Opa1 to promote mitochondrial fragmentation and apoptosis by distinct mechanisms

Thomas Landes et al. EMBO Rep. 2010 Jun.

Abstract

Opa1 modulates mitochondrial fusion, cristae structure and apoptosis. The relationships between these functions and autosomal dominant optic atrophy, caused by mutations in Opa1, are poorly defined. We show that Bnip3 interacts with Opa1, leading to mitochondrial fragmentation and apoptosis. Fission is due to inhibition of Opa1-mediated fusion and is counteracted by Opa1 in an Mfn1-dependent manner. Bnip3-Opa1 interaction is necessary to trigger Opa1 complex disruption in a Bax- and/or Bak-dependent manner, ultimately leading to apoptosis. Our results uncover a direct link between Opa1 on the inner mitochondrial membrane and the apoptotic machinery on the outer membrane that modulates fusion and cristae structure by separate mechanisms. These findings might help to unravel optic atrophy aetiology as retinal ganglion cells are particularly prone to hypoxia, an inductor of Bnip3 expression.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Bnip3 interacts with Opa1. (A) HeLa cells were co-transfected with plasmids for Opa1 and HA–Bnip3, or (B) HA–Bnip3 mutants, as indicated. (C) Endogenous interactions were observed in HeLa cells exposed (+) or not (−) to hypoxia to induce Bnip3 expression. Immunoprecipitations (IPs) were performed on crosslinked mitochondria (mito input, 1/20 of IP) using anti-Opa1, anti-HA or no antibodies (−), and analysed by western blots using the indicated antibodies. Migration of molecular weight markers is shown on the right. The batch of affinity media used in (C) released a major contaminant (indicated by the asterisk), probably immunoglobulin light chains (MW∼25 kDa). Note in (C) that low basal levels of endogenous Bnip3 expressed under normoxia (IP Opa1, hypoxia−) are associated with Opa1. HA, haemagglutinin; HA–Bnip3, haemagglutinin-tagged Bnip3.
Figure 2
Figure 2
Bnip3 induces mitochondrial fragmentation by inhibiting fusion. HeLa cells were transfected with GFP and either empty (control), HA–Bnip3 or Drp1K38A vectors during 24 h. Mitochondria were observed after MitoTracker staining. Typical morphologies are exemplified (A) and were quantified (B). Data represent mean±s.d. of three independent experiments; n⩾200 GFP-expressing cells per condition. (C) HeLa cells co-transfected separately with mtDsRed or mtGFP, and HA–Bnip3 or empty (control) vectors were fused with polyethylene glycol and observed by fluorescence microscopy. (D) HeLa cells were transfected with mito-Dendra and either HA–Bnip3 or empty vectors (control). Images were acquired in GFP and TRITC channels before photoconversion, and in TRITC channel immediately after (t=0 min) and every 10 min subsequently. The graph depicts the time course of fluorescence. Data represent mean±s.d. of four separate experiments; n⩾20 single time lapse per condition. (A,C,D) Scale bars, 10 μm. GFP, green fluorescent protein; HA–Bnip3, haemagglutinin-tagged Bnip3; TRITC, tetramethyl rhodamine isothiocyanate.
Figure 3
Figure 3
Bnip3 induces apoptotic cell death. HeLa cells were transfected with CFP–Bnip3 for 24 h with or without z-VAD-fmk. The cells were observed by immunofluorescence using antibodies against cytochrome-c (cyt. c) or subunit IV of the cytochrome-c oxidase (Cox IV) and stained by Hoescht 3325. (A) The data represent the percentage of CFP–Bnip3-transfected cells with apoptotic nuclei, cytosolic cytochrome-c or fragmented mitochondria and are the mean±s.d. of three independent experiments; n⩾200 cells per condition. (B) Representative images are shown. Scale bar, 10 μm. CFP–Bnip3, cerulean fluorescent protein-tagged Bnip3.
Figure 4
Figure 4
Interactions of Bnip3 and Opa1 induce mitochondrial fragmentation and cell death by distinct mechanisms. (A) Established HeLa Tet-Off cells were induced (+) or not (−) for Opa1 expression during 48 h. For the last 24 h, cells were co-transfected with GFP and either HA–Bnip3 (+) or empty (−) vectors. (B) HeLa cells were transfected for 24 h with GFP and either empty (−) or the indicated wild-type or mutant HA–Bnip3 vectors. (C) Cells were as in (A) and cultured with siRNA to Mfn1 (Mfn1) or the off-target luciferase (luc) during the Opa1 induction period. (D) HeLa cells were cultured under hypoxia (+) or normoxia (−) in the presence of siRNA to Bnip3 or luciferase. Fragmentation occurred rapidly (24 h) and was later (72 h) followed by apoptosis. For each panel, data (mean±s.d. of ⩾3 independent experiments; n⩾200 cells per condition) show quantification of mitochondrial fragmentation (left axis) and of apoptosis (right axis) on observation by fluorescence microscopy after MitoTracker and Hoescht 33258 staining. GFP, green fluorescent protein; HA–Bnip3, haemagglutinin-tagged Bnip3; Mfn, mitofusin; siRNA, short interfering RNA.
Figure 5
Figure 5
Bnip3 promotes Opa1 oligomer disassembly. (A) Mitochondria isolated from HeLa cells were incubated without (−) or with t-Bid, or with thrombin cleavage products from GST (mock), GST-Bnip3, GST-Bnip3ΔC, GST-Bnip3TMBcl2, GST-Bnip3TMOmp25 or GST-Bnip3mutBH3 beads. (B) HeLa cells were treated with siRNA to luciferase (luc) or Bnip3 for 24 h and cultured (+) or not (−) under hypoxia for an additional 24 h. For (A) and (B), mitochondria were then crosslinked and analysed by western blots using the indicated antibodies. Quantification of Opa1 complex-to-monomer ratios is shown for each figure. Data represent the mean±s.e.m. of ⩾3 independent experiments; *P<0.05. GST, glutathione-S-transferase; siRNA, short interfering RNA.
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