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Cell Culture and Transfection. SV40-transformed WT, Mfn1-/- and Mfn2-/- mouse embryonic fibroblasts (MEFs) were cultured as described in ref. 1. Transfection was performed by using FuGENE6 (Roche Molecular Biochemicals, Indianapolis) according to the manufacturer’s instructions.

RNA Interference (RNAi) Generation. A 19-mer corresponding to the nucleotide region 1813-1831 from murine OPA1 was selected to synthesize a hairpin oligonucleotide sequence (5'-GATCCCGTCATCAGTCTGAGCCAGGTTCAAGAGACCTGGCTCAGACTGATGACTTTTTGGAA-3') and a scrambled control that were cloned in the BamH1 and HindIII sites of pSilencer2.1-U6/hygro (Ambion, Austin, TX). The resulting pSilencer-OPA1 and pSilencer-control were confirmed by sequencing and used to transfect WT MEFs from mixed 129/CD1 background (2). Forty-eight hours after transfection, cells expressing pSilencer were selected by adding 0.45 mg/ml hygromycin to complete medium. Single-cell clones were generated by limiting dilution, and OPA1 levels were measured by immunoblotting.

Imaging. For epifluorescence imaging, cells seeded onto 25-mm round glass coverslips, incubated in Hanks’ balanced salt solution (HBSS), and supplemented with 10 mM Hepes were placed on the stage of an Olympus IX inverted microscope (Olympus, Melville, NY) equipped with a Xe-illumination system and a 12-bit charge-coupled device camera (TILL Photonics, Planegg, Germany). Cells were excited at 425 ± 2.5 nm for cyan fluorescent protein or at 460 ± 2.5 nm for yellow fluorescent protein with a Polychrome IV monochromator (TILL Photonics), and emitted light was collected by using a 480 ± 20 band-pass and a 520-nm long-pass filter, respectively. Images were acquired with exposure times of 800 msec with a 60 × 1.3 NA Plan Apo objective (Olympus). 2D Blind deconvolution was carried out by using AutoDeblur 2D (Autoquant Imaging), with deconvolution parameters kept constant. For confocal z-axis stacks of mitochondrial network, coverslips were placed on the stage of a Nikon Eclipse TE300 inverted microscope equipped with a spinning-disk PerkinElmer Ultraview LCI confocal system, a piezoelectric z-axis motorized stage (Pifoc, Physik Instrumente, Karlsruhe Germany), and a Orca ER 12-bit charge-coupled device camera (Hamamatsu Photonics, Hamamatsu City, Japan). Cells expressing mitochondrially targeted yellow fluorescent protein (mtYFP) were excited using the 488-nm line of the HeNe laser (PerkinElmer) with exposure times of 50 msec by using a 60 × 1.4 NA Plan Apo objective (Nikon), and stacks of 20 and 5 images separated by 1 m m along the z-axis were acquired for steady-state and time-resolved 3D imaging, respectively. Total acquisition time for each stack was 1.1 sec for the 20 planes and 260 msec for the 5 planes stacks to minimize reconstruction artifacts caused by mitochondrial movement. 3D reconstruction and volume rendering of the stacks were performed with the appropriate plugins of imagej (National Institutes of Health, Bethesda).

For imaging of polykarions, fixed cells on 13-mm round coverslips were placed on the stage of a Nikon Eclipse E600FN upright microscope equipped with a Bio-Rad Radiance 2100 Confocal Laser Scanning system. Cells were excited with the 457-nm laser line for yellow fluorescent protein and the 543-nm line for dsRED, and emitted light was collected with 515/30 band-pass and 570 long-pass filters, respectively.

Morphometric and Contact Analysis. Morphometric analysis was performed with imagetool 3.0 (University of Texas Health Science Center, San Antonio). Images of cells expressing mitochondrially targeted yellow fluorescent protein (mtYFP) or mitochondrially targeted cyan fluorescent protein (mtCFP) were thresholded by using the automatic threshold function. For each identified object, the major axis length and the roundness index were calculated. Cells were scored with elongated mitochondria when >50% of the objects in the image (i.e., mitochondria) displayed a major axis longer than 5 m m and a roundness index below 0.5 (maximum value is 1).

For analysis of the intermitochondrial contacts, individual mitochondria were randomly chosen in the initial 3D reconstructed, volume rendered z-stack of a time stack performed as described above. These mitochondria were then individually followed in each stack of the time sequence, and total and productive contacts with other mitochondria were counted. Two mitochondria were classified as in contact when no black pixels could be identified between the two organelles. A contact was considered to be productive when it resulted in changes in the shape of both mitochondria involved, i.e., in the production of a structure that remained tubular for at least 40 s.

Subcellular Fractionation and Western Blotting. Twenty-four hours after transfection, cells (10⁹) were harvested and subcellular fractionation and mitochondria lysis were performed as described in ref. 3. Extracted proteins (45 m g) were separated by 4–12% SDS/PAGE (NuPAGE, Invitrogen), transferred onto polyvinylidene fluoride membranes (Millipore), and probed with the indicated antibodies. Isotype-matched, horseradish peroxidase-conjugated secondary antibodies (Sigma) were used, followed by detection by chemiluminescence (Pierce).

Anti-Mouse OPA1 Antibody. A peptide (LKKVREIQEKLDAFI) corresponding to amino acids 940-954 of murine OPA1 was coupled via N-terminal cysteine to keyhole limpet hemocyanin and used to immunize two rabbits by s.c. injection. Immunizations, bleeds, and affinity purification were preformed by Primm (Milan). Specificity of both antiserum and affinity purified was confirmed by Western blotting. To detect the R905stop mutant, a specific antibody generated against recombinant OPA1 (residues 261–904) (kindly provided by P. Belenguer, Université Paul Sabatier, Toulouse, France) was used (4).

Plasmids. mtCFP (pECFP-mito) and mtYFP (pEYFP-mito) were purchased from BD-Clontech. Mitochondrially targeted dsRED (mtRFP) was a gift from M. Zaccolo (Venetian Institute of Molecular Medicine, Padua, Italy). pCB6-MYC-Mfn1 and pCB6-MYC-Mfn2 were kindly provided by M. Rojo (Groupe Hospitalier Pitié-Salpêtrière, Paris) (5), and pcDNA3.1-HA-DRP1 and pcDNA3.1-HA-K38A-DRP1 (6) were a gift from A. Van der Bliek (University of California, Los Angeles).

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3. Scorrano, L., Oakes, S. A., Opferman, J. T., Cheng, E. H., Sorcinelli, M. D., Pozzan, T. & Korsmeyer, S. J. (2003) Science 300, 135-139.

4. Olichon, A., Emorine, L. J., Descoins, E., Pelloquin, L., Brichese, L., Gas, N., Guillou, E., Delettre, C., Valette, A., Hamel, C. P., et al. (2002) FEBS Lett. 523, 171-176.

5. Rojo, M., Legros, F., Chateau, D. & Lombes, A. (2002) J. Cell Sci. 115, 1663-1674.

6. Smirnova, E., Griparic, L., Shurland, D. L. & van der Bliek A. M. (2001) Mol. Biol. Cell 12, 2245-2256.