OPA1 requires mitofusin 1 to promote mitochondrial fusion - PubMed (original) (raw)

OPA1 requires mitofusin 1 to promote mitochondrial fusion

Sara Cipolat et al. Proc Natl Acad Sci U S A. 2004.

Abstract

The regulated equilibrium between mitochondrial fusion and fission is essential to maintain integrity of the organelle. Mechanisms of mitochondrial fusion are largely uncharacterized in mammalian cells. It is unclear whether OPA1, a dynamin-related protein of the inner membrane mutated in autosomal dominant optic atrophy, participates in fusion or fission. OPA1 promoted the formation of a branched network of elongated mitochondria, requiring the integrity of both its GTPase and C-terminal coiled-coil domain. Stable reduction of OPA1 levels by RNA interference resulted in small, fragmented, and scattered mitochondria. Levels of OPA1 did not affect mitochondrial docking, but they correlated with the extent of fusion as measured by polyethylene glycol mitochondrial fusion assays. A genetic analysis proved that OPA1 was unable to tubulate and fuse mitochondria lacking the outer membrane mitofusin 1 but not mitofusin 2. Our data show that OPA1 functionally requires mitofusin 1 to regulate mitochondrial fusion and reveal a specific functional difference between mitofusin 1 and 2.

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Figures

Fig. 1.

Fig. 1.

Overexpression of OPA1 promotes mitochondrial elongation. (A_–_H) Effect of mutations in the GTPase and coiled-coil domains. Mitochondrial shape in MEFs transfected with mitochondria-shaping proteins is shown. WT MEFs grown on coverslips were cotransfected with mtCFP and empty vector (A), WT OPA1 (B), K301A OPA1 (C), R905stop OPA1 (D), Mfn1 (E), Mfn2 (F), WT DRP1 (G), or K38A DRP1 (H). After 24 h, images of mtCFP fluorescence from randomly selected cells were acquired, deconvoluted, and stored. (Scale bar, 15 μm.) (I) Morphometric analysis of mitochondrial elongation. WT MEFs grown on coverslips were cotransfected with mtCFP and empty vector or the indicated mitochondria-shaping protein. After 24 h, 20 randomly selected images of mtCFP fluorescence were acquired, deconvoluted, stored, and subsequently classified as described in Supporting Materials and Methods. Data represent mean ± SE of 14 different experiments. (J_–_M) Volume-rendered 3D reconstructions of mitochondrial network in MEFs. MEFs grown on coverslips were cotransfected with mtYFP and empty vector (J) or OPA1 (L), and, after 24 h, randomly selected confocal _z_-axis stacks of mtYFP fluorescence were acquired, stored, reconstructed, and volume-rendered as described in Supporting Materials and Methods. K and M represent 3× magnification of the boxed area in J and L, respectively. The depth of stacks was 20 μm. (Scale bar, 15 μm.)

Fig. 2.

Fig. 2.

Mitochondrial shape, mitochondrial fusion, and response to mitofusins after RNAi against OPA1. (A) Expression levels of OPA1 in MEF clones. Cells (5 × 106) from clone H4 carrying pSilencer-control and clone A7 carrying pSilencer-OPA1 were lysed, and equal amounts of protein were separated and immunoblotted with anti-OPA1 (1:2,000) antiserum and an anti-actin monoclonal antibody (1:2,000, Chemicon). (B) Mitochondrial shape and response to mitofusins after OPA1 ablation. MEFs from clones H4 and A7 grown on coverslips were transfected with mtCFP or cotransfected with the indicated mitofusins. After 24 h, images of mtCFP fluorescence were acquired exactly as in Fig. 1 A_–_H. (Scale bar, 15 μm.) (C) Morphometric analysis. H4 and A7 MEFs grown on coverslips were transfected as indicated. Experiments were performed exactly as in Fig. 1_I_. Data represent mean ± SE of four different experiments. (D) Representative heteropolykaryons from H4 and A7 clones. MEFs from the indicated clones were transfected with mtYFP or mtRFP, coplated on glass coverslips, fused as described in Experimental Procedures, and fixed after 4 h. Confocal images of representative polykaryons are shown. (Scale bar, 20 μm.) (E) Quantitation of the effects of ablation of OPA1 on a mitochondrial PEG fusion assay. Experiments were carried out as described in D, except that cells were fixed at the indicated times. Mitochondrial fusion was evaluated as described in Experimental Procedures from 30 randomly selected polykaryons. Data represent mean ± SE of three different experiments.

Fig. 3.

Fig. 3.

OPA1 changes the shape of the mitochondrial reticulum by promoting MFN1-dependent mitochondrial fusion. (A) Effect of OPA1 on the shape of the mitochondrial network in WT, _Mfn1_–/–, and _Mfn2_–/– MEFs. MEFs of the indicated genotype grown on coverslips were cotransfected with mtCFP and empty vector (Left) or OPA1 (Right). When indicated, MFN1 or MFN2 were cotransfected with mtCFP in _Mfn1_–/– MEFs. After 24 h, images of mtCFP fluorescence were acquired exactly as in Fig. 1 A_–_H.(B) Morphometric analysis of WT, _Mfn1_–/–, and _Mfn2_–/– mitochondria. MEFs of the indicated genotype grown on coverslips were cotransfected with mtCFP and empty vectors (black bars) or with OPA1 (gray bars). Where indicated, Mfn1_–/– cells were complemented by cotransfection with MFN1 or MFN2. Experiments were performed exactly as in Fig. 1_I. Data represent mean ± SE of nine different experiments. (C) MEFs of the indicated genotype were transfected with mtYFP or mtRFP (Left) or cotransfected with mtYFP or mtRFP plus OPA1 (Right), coplated on glass coverslips, fused with PEG as described in Experimental Procedures, and fixed after 4 h. Confocal images of representative polykaryons are shown. (Scale bar, 20 μm.) (D) Quantification of the effect of OPA1 on fusion of WT, _Mfn1_–/–, and Mfn2_–/– mitochondria. The experiments were conducted as described in C, except that heteropolykaryons were fixed at the indicated times. In the experiments depicted with diamonds, Mfn1_–/– cells were cotransfected with mtYFP or mtRFP plus MFN1 (⋄) and with mtYFP or mtRFP plus MFN1 and OPA1 (♦). Mitochondrial fusion was evaluated as described in Experimental Procedures from 30 randomly selected polykaryons. Data represent mean ± SE of four different experiments.

Fig. 4.

Fig. 4.

The lack of MFN1 does not affect mitochondrial juxtaposition but blocks OPA1-induced mitochondrial tubulation. WT (A), _Mfn1_–/– (B), and _Mfn2_–/– (C) MEFs were cotransfected with mtYFP and empty vector (Upper) or with mtYFP and OPA1 (Lower), and, after 24 h, confocal _z_-stacks were acquired as described in Supporting Materials and Methods. Stacks were reconstructed and volume-rendered, and movements of individual mitochondria were tracked. Five-fold magnified portions of single _z_-planes corresponding to the indicated frames are shown. For the sake of clarity, mitochondria were individually labeled in different colors. Arrowheads indicate sites of mitochondrial contact. Colors of tubular mitochondria resulted from the merging of the colors of the individual mitochondria from which they originated. (Scale bar, 4 μm.)

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