SIMH is associated with ultrastructural changes. Ultrastructural analysis of WT, Mfn1^−/−, hMFN1-HA:Mfn1^−/−, OPA1^−/−, mOPA1-V1:OPA1^−/−, shLuc, and shSLP-2 MEFs. Cells were untreated (left part) or exposed to 10 μM CHX (right part) and fixed after 6 h for electron microscopy analysis. Scale bar, 1 μm.

Stress-induced mitochondrial hyperfusion protects against apoptosis. (A) Death of Mfn1^−/− and hMFN1-HA:Mfn1^−/− cells exposed to 3 μg/ml Act D or 60 mJ/cm² UV-C. (B) Death of HeLa and MEF cells transiently transfected with shSLP-2 or shLuc after exposure to 60 mJ/cm² UV-C. (C) Death of Mfn1^−/− and hMFN1-HA:Mfn1^−/− MEF cells exposed to 1 μM staurosporine. In all cases cells were harvested at the indicated time points and stained with annexin V FITC for flow-cytometric analysis. Data represent the mean±s.e.m of at least three independent experiments (^*P<0.05; ^**P<0.01).

SIMH promotes mitochondrial ATP production. (A, B) Measurement of total ATP levels in WT MEFs treated with Act D (3 μM), CHX (10 μM), UV (60 mJ/cm²), and STS (0.25 μM) (A), in SIMH competent (WT, Mfn2^−/−, shLuc) and incompetent (Mfn1^−/−, OPA1^−/−, shSLP-2) cells (B) exposed to 10 μM CHX treatment for 6 h. (C–E) Measurement of mitochondrial ATP production. Cells were pretreated with 10 μM CHX for 6 h, followed by 10 mM methylpyruvate (MeP). Cells were harvested after 5, 10 or 15 min and total ATP levels assayed. In (C) ATP levels were also measured 15 min after co-addition of methylpyruvate+oligomycin (10 μM). Data represent the mean±s.e.m of at least three independent experiments. ^*P<0.05; ^**P<0.01; ^***P<0.005; students t-test, unpaired, two-tailed.

Mitochondrial tubulation in response to stress stimuli requires Opa1 and Mfn1. (A, B) Wild-type MEFs after exposure to 60 mJ/cm² UV-C, 3 μg/ml Act D or 10 μM CHX. (A) MEFs stained with antibodies to cytochrome c were analyzed in untreated conditions and 9 h after stress exposure by fluorescence microscopy. Scale bar, 25 μm. (B) Quantification of cells with a majority of connected, very long (>5 μm) tubular mitochondria the extremities of which are difficult to visualize (defined as hyperfused mitochondria) at the indicated time points after stress exposure. Data represent the mean±s.d. of three independent experiments, each with >300 cells counted per condition. (C, D) Wild type, Mfn1^−/−, Mfn2^−/−, Mfn1/2^−/−, Opa1^−/−, and Bax/Bak^−/− MEFs after exposure to UV-C irradiation. (C) Quantification of cells with hyperfused mitochondria 9 h after exposure with 60 mJ/cm² UV-C, 3 μg/ml Act D, or 10 μM CHX. Data represent the mean±s.d. of three independent experiments, each with >300 cells counted per condition. Wild-type MEFs shown here are controls for Mfn1^−/− cells and are representative of all WT MEFs used as controls for mutant MEFs. (D) MEFs stained with antibodies to cytochrome c were analysed in untreated conditions and 9 h after UV-C irradiation by fluorescence microscopy. Scale bar, 25 μm.

SLP-2 prevents OPA1 proteolysis during SIMH. (A) Immunoblot analysis of Opa1^−/− MEFs stably expressing shSLP-2 or shLuc after transient transfection of FLAG-tagged rat OPA1 variant 1 (rOPA1-V1) and its non-cleavable deletion mutant (rOPA1-V1-ΔS1) at the indicated time points after CHX treatment. Whole-cell lysates were analysed by SDS–PAGE using antibodies against OPA1. (B) Quantification of mitochondrial morphology in Opa1^−/− MEFs stably expressing shLuc or shSLP-2 after transient transfection of mitochondrial targeted YFP alone (not shown) or together with rOPA1-V1 and rOPA1-V1-ΔS1. Cells were exposed to 10 μM CHX for 8 h and YFP-positive cells were analysed by fluorescence microscopy (C). (D) Wild-type MEFs stably expressing shRNA against Luciferase (shLuc) or SLP-2 (shSLP-2) were treated with indicated concentrations of (1,10)-o-phenanthroline or 1 mM PMSF alone or in combination with 10 μM CHX. After 3 h, cells were harvested and whole-cell lysates were used for SDS–PAGE. (E) Mitochondrial morphology in Pbh2^fl/fl MEFs (WT), Prohibitin 2 knockout (Phb2^−/−) and Phb2^−/− re-expressing prohibitin 2 (PHB2∷Phb2^−/−) 6 h after exposure to 10 μM CHX. (F) Quantification of cells shown in (E). HSP70 served as a loading control (A, D). Data (B) represent the mean±s.d. of three independent experiments, each with >300 cells counted per condition.

Stress-induced mitochondrial hyperfusion requires SLP-2. (A) Quantification of mitochondrial morphology in wild-type MEFs stably expressing control shRNA against Luciferase (shLuc) or shRNA against SLP-2 (shSLP-2) 8 h after exposure to 60 mJ/cm² UV-C or 10 μM CHX. Tubular mitochondria are defined as isolated mitochondria with visible free ends; hyperfused mitochondria form a mesh of interconnected organelles, the extremities of which were difficult to visualize. (B) Immunoblot analysis of cells described in (A) at the indicated time points after CHX treatment. Whole-cell lysates were analysed by SDS–PAGE using antibodies against OPA1 and SLP-2. (C) Quantification of mitochondrial morphology in wild-type MEFs stably expressing shSLP-2 after transient transfection of FLAG-tagged rat OPA1 variant 1 (rOPA1-V1) and its non-cleavable deletion mutant (rOPA1-V1-ΔS1). Cells exposed to 10 μM CHX for 8 h were stained with MitoTracker and antibodies to FLAG, and mitochondrial morphology of transfected cells was analysed by fluorescence microscopy. (D) Immunoblot analysis of cells described in (C). Whole-cell lysates were analysed by SDS–PAGE using antibodies against OPA1 and SLP-2. HSP70 served as a loading control (B, D). Data (A, C) represent the mean±s.d. of three independent experiments, each with >300 cells counted per condition. The blot (B, D) is representative of three independent experiments.

SIMH is a result of increased mitochondrial fusion activity. (A) Visualization and (B) quantification of mitochondrial fusion using mito-PAGFP in DMSO-treated Mfn2^−/− MEFs (UT cells) (n=27), as well as Mfn2^−/− MEFs 3 h (n=30) and 7 h (n=30) after CHX treatment. Regions of interest within mito-PAGFP-expressing cells were photoactivated using 405-nm light, followed by whole-cell imaging with 488-nm light. Pre-activation images were taken with higher detector gain, and after the activation the gain was adjusted to avoid oversaturation of activated mitochondria. Post-acquisition processing was performed using MetaMorph software. The data represent mean±s.e.m. P-values (versus untreated Mfn2^−/−), t-test, two-tailed, unpaired. (C) Morphology of mitochondria in wild-type, Mfn1^−/−, Mfn2^−/−, Mfn1/2^−/−, and Opa1^−/− MEFs transiently expressing the dominant-negative DRP1 mutant HA-DRP1-K38. Mitochondria were stained using MitoTracker and Drp1 K38A revealed using an antibody to HA tag. (D) Quantification of cells described in (C) with elongated mitochondrial morphology. As a control, cells were transfected with GFP. Data represent the mean±s.d. of three independent experiments, each with >300 cells counted per condition. (E) Subcellular fractionation of untreated WT MEFs and after exposure to 60 mJ/cm² UV-C, 3 μg/ml ActD or 10 μM CHX. Cells were harvested at the indicated time points and mitochondria were isolated. Cytoplasmic and mitochondrial fractions were analysed by SDS–PAGE using antibodies against DRP1 and FIS1, respectively. Actin served as a loading control for the cytoplasmic fraction. The data are representative of three independent experiments. ^**P<0.01; ^***P<0.005; students t-test, unpaired, two-tailed.