Journal: The Journal of Biological Chemistry

Article Title: The short variant of the mitochondrial dynamin OPA1 maintains mitochondrial energetics and cristae structure

doi: 10.1074/jbc.M116.762567

Figure Lengend Snippet: OPA1 variants and MEFs differentially expressing these variants. A, OPA1 proteolytic cleavages occur at S1 and S2 in exons 5 and 5b. CC, coiled-coil domain; MID, middle domain; GED, GTPase effector domain; MPP, mitochondrial processing peptidase. The variant 1 (OPA1-v1) contains the S1 site where partial cleavage occurs, producing both L- and S-OPA1 (L/S-OPA1). OPA1-v1ΔS1 only produces L-OPA1 due to the deletion of the S1 site in OPA1-v1. OPA1-v5 generates S-OPA1 only because of full cleavage at the S1 site. Amino acid numbers are indicated for each construct. B, three OPA1 variants were expressed in OPA1-KO MEFs, and stable clones were isolated. Immunoblots of cell lysates from these cells show exclusive expression of L-OPA1 and S-OPA1 in MEFs expressing OPA1-v1ΔS1 and OPA1-v5, respectively, and the presence of both L- and S-OPA1 in cells expressing OPA1-v1.

Article Snippet: OPA1- KO MEFs were from American Type Culture Collection (ATCC CRL2995) ( 6 ).

Techniques: Expressing, Variant Assay, Construct, Clone Assay, Isolation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The short variant of the mitochondrial dynamin OPA1 maintains mitochondrial energetics and cristae structure

doi: 10.1074/jbc.M116.762567

Figure Lengend Snippet: L-OPA1 alone has the capacity to elongate mitochondria in pro-fusion conditions. Mitochondrial tubule formation was assessed in normal conditions (A) and conditions of fission inhibition (B) and SIMH (C and D) in cells expressing different OPA1 variants. Mitochondrial lengths were categorized into long tubules, intermediate, and fragmented. In both fission inhibition and SIMH conditions, OPA1-v1 and v1ΔS1 significantly increased the formation of tubular mitochondria. Mitochondrial tubule formation by OPA1-v5 was minimal. n = 4. Error bars are S.E.

Article Snippet: OPA1- KO MEFs were from American Type Culture Collection (ATCC CRL2995) ( 6 ).

Techniques: Inhibition, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The short variant of the mitochondrial dynamin OPA1 maintains mitochondrial energetics and cristae structure

doi: 10.1074/jbc.M116.762567

Figure Lengend Snippet: Assessments of fusion competency by PEG cell hybrid assays. Mixed cultures of OPA1-KO cells with DsRed-labeled mitochondria and OPA1 variant cells with GFP-labeled mitochondria were treated with PEG, and mixing of red and green fluorescence was evaluated in hybrid cells. A, examples of mitochondrial images from the fusion assay representing no fusion, partial fusion, and complete fusion. Scale bar, 20 μm. B, OPA1-v1 and OPA1-v1ΔS1 are fusion-competent, whereas OPA1-v5 has very little fusion capacity. n = 3. Error bars are S.E.

Article Snippet: OPA1- KO MEFs were from American Type Culture Collection (ATCC CRL2995) ( 6 ).

Techniques: Labeling, Variant Assay, Fluorescence, Single Vesicle Fusion Assay

Journal: The Journal of Biological Chemistry

Article Title: The short variant of the mitochondrial dynamin OPA1 maintains mitochondrial energetics and cristae structure

doi: 10.1074/jbc.M116.762567

Figure Lengend Snippet: L- or S-OPA1 alone is sufficient to support mitochondrial respiratory function. A, OXPHOS assessment by cell growth in galactose media. All cell lines including OPA1-KO cells grew well in the glycolytic media. In galactose media, OPA1-KO did not grow, whereas WT and OPA1 variant cells grew with no discernible difference in their growth rates. Experiments were done in quadruplicate and repeated three times. Representative data are presented. B and C, oxygen consumption rates (OCR) of OPA1 variant cells show no difference from that of WT, whereas OPA1-KO cells are respiration-deficient. The respiration control ratios of uncoupled maximum respiration (state 3u) to leak respiration under oligomycin (state 4o) were indistinguishable in OPA1 variants and WT. n = 6. Error bars are S.E. ***, p < 0.0001; #, p = 0.0002; **, p = 0.0089 (one-way ANOVA with Tukey's post hoc test). D and E, immunoblotting of WT and OPA1 variant cells in galactose and SIMH conditions. Cells were incubated in 10 mm glucose (Glc) or galactose (Gal) for 24 h (D). For SIMH, cells were incubated in HBSS for 3 h (Strv) or in 10 μm cycloheximide (Chx) for 6 h (E). There are no changes in L- and S-OPA1 in galactose and HBSS incubations. Cycloheximide incubation appears to increase L-OPA1 cleavage in WT and OPA1-v1 cells. Olm, oligomycin; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; Con, control.

Article Snippet: OPA1- KO MEFs were from American Type Culture Collection (ATCC CRL2995) ( 6 ).

Techniques: Variant Assay, Control, Western Blot, Incubation

Journal: The Journal of Biological Chemistry

Article Title: The short variant of the mitochondrial dynamin OPA1 maintains mitochondrial energetics and cristae structure

doi: 10.1074/jbc.M116.762567

Figure Lengend Snippet: L- and S-OPA1 are indistinguishable in restoring mitochondrial respiratory complexes. BNGE was used to examine respiratory complexes and supercomplexes. A, OPA1-KO cells have significantly decreased levels of respiratory complexes except complex I. Note that supercomplex assembly was not affected by OPA1-KO. OPA1-KO cells accumulate F1 domain of complex V (shown in the long exposure). OPA1-v1, -v1ΔS1, and -v5 restored the levels of complexes III, IV, and V with a minimal recovery of complex II. There is no distinction between OPA1-v1ΔS1 and OPA1-v5 in restoring respiratory complexes. Numbers in parentheses indicate the size of the complexes in kDa. B, quantification of total band density for individual complexes in each cell line. n = 5. Error bars are S.E. (one-way ANOVA with Tukey's post hoc test). IB, immunoblotting.

Article Snippet: OPA1- KO MEFs were from American Type Culture Collection (ATCC CRL2995) ( 6 ).

Techniques: Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The short variant of the mitochondrial dynamin OPA1 maintains mitochondrial energetics and cristae structure

doi: 10.1074/jbc.M116.762567

Figure Lengend Snippet: No functional distinction between L- and S-OPA1 in maintaining respiratory complexes and mtDNA. A, respiratory complexes after starvation assessed by BNGE. Starvation does not significantly affect the complex levels except for complex I. Increased complex I levels were seen after starvation across the cell lines. * in the complex IV blot indicates a potential dimer. B, expression of OPA1 variants in OPA1-KO cells restores mtDNA levels. mtDNA and nuclear DNA were quantified using PCR with primer sets for ATP6 and GAPDH, respectively. OPA1-KO cells contain ∼30% of mtDNA copy number compared with WT cells. All three OPA1 variant cells showed ∼60% of the WT level, indicating partial recovery of mtDNA copy numbers. n = 4. Error bars are S.E. (Student's t test (two-tailed)).

Article Snippet: OPA1- KO MEFs were from American Type Culture Collection (ATCC CRL2995) ( 6 ).

Techniques: Functional Assay, Expressing, Variant Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: The short variant of the mitochondrial dynamin OPA1 maintains mitochondrial energetics and cristae structure

doi: 10.1074/jbc.M116.762567

Figure Lengend Snippet: EM images of mitochondria in WT, OPA1-KO, and OPA1 variant cells. No or a few cristae are in OPA1-KO mitochondria. OPA1 variant cells restored cristae structure, showing increased numbers of cristae and tight lamellae similar to those in WT cells. Scale bar, 1 μm.

Article Snippet: OPA1- KO MEFs were from American Type Culture Collection (ATCC CRL2995) ( 6 ).

Techniques: Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: The short variant of the mitochondrial dynamin OPA1 maintains mitochondrial energetics and cristae structure

doi: 10.1074/jbc.M116.762567

Figure Lengend Snippet: Analyses of cristae structure in OPA1 variant cells. A, quantification of cristae density. The number of cristae was counted for each mitochondrion and divided by the mitochondrial area in megapixels. Six to 16 mitochondria were analyzed in each cell for 11 cells per cell line. n = 111, 82, 83, 91, and 79 for WT, OPA1-KO, -v1, -v1ΔS1, and -v5, respectively. Error bars are S.E. (one-way ANOVA with Tukey's post hoc test). B, matrix electron density is presented by matrix gray value (GV[mat]) after background subtraction (GV[cyt]) in individual mitochondria. Distribution of matrix electron density was plotted with the average gray value (horizontal line) for each cell line along with low magnification EM images. n = 139, 130, 135, 150, and 139 for WT, OPA1-KO, -v1, -v1ΔS1, and -v5, respectively. #, p < 0.0001 (one-way ANOVA with Tukey's post hoc test). Scale bar, 0.5 μm. C, septa (arrowheads) in mitochondria of OPA1-KO cells. Scale bars, 0.5 μm. D, STJ (arrows) in OPA1-KO and OPA1-v5 cells. Scale bars, 0.5 μm. E, quantification of CCJ and STJ. The numbers of CCJ and STJ were separately counted and divided by the mitochondrial area in megapixels. 11 cells were analyzed for each cell line. n = 101, 79, 68, 83, and 69 for WT, OPA1-KO, -v1, -v1ΔS1, and -v5, respectively. Error bars are S.E. (one-way ANOVA with Tukey's post hoc test). F, quantification of cristae width for assessing cristae tightness. Distribution of cristae width in nm was plotted with the median width (horizontal line) for each cell line. n = 146, 171, 177, 125, and 159 for WT, OPA1-KO, -v1, -v1ΔS1, and -v5, respectively. #, p < 0.001 (one-way ANOVA with Tukey's post hoc test).

Article Snippet: OPA1- KO MEFs were from American Type Culture Collection (ATCC CRL2995) ( 6 ).

Techniques: Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: The short variant of the mitochondrial dynamin OPA1 maintains mitochondrial energetics and cristae structure

doi: 10.1074/jbc.M116.762567

Figure Lengend Snippet: OPA1 GTPase activity is required for mitochondrial fusion and OXPHOS. A, mitochondrial tubule formation was assessed by SIMH using cycloheximide in cells expressing Lys-to-Ala mutant OPA1 variants. In both normal and SIMH conditions, mitochondria in mutant OPA1-expressing cells remained fragmented. n = 4. Error bars are S.E. B, OXPHOS assessment of Lys-to-Ala mutant OPA1 variant cells by cell growth in galactose media. Although all cells grew well in the glycolytic media, Lys-to-Ala mutant-expressing cells grew for 3 days and abruptly died on day 4. Experiments were done in quadruplicate and repeated three times. Representative data are presented.

Article Snippet: OPA1- KO MEFs were from American Type Culture Collection (ATCC CRL2995) ( 6 ).

Techniques: Activity Assay, Expressing, Mutagenesis, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: The short variant of the mitochondrial dynamin OPA1 maintains mitochondrial energetics and cristae structure

doi: 10.1074/jbc.M116.762567

Figure Lengend Snippet: OPA1 GTPase activity is critical for maintaining cristae tightness. A–C, EM images of OPA1 GTPase-defective mutant cells. Cristae are swollen and round. Scale bar, 0.5 μm. D, quantification of cristae width. Distribution of cristae width in nm was plotted with the mean width (horizontal line) for each cell line. n = 103, 94, and 124 for OPA1-v1-K301A, -v1ΔS1-K291A, and -v5-K319A, respectively. E, quantification of cristae density. The number of cristae was counted for each mitochondrion and divided by the mitochondrial area in μm2. Three to 10 mitochondria were analyzed in each cell for 10 cells per cell line. n = 44, 54, and 56 for OPA1-v1-K301A, -v1ΔS1-K291A, and -v5-K319A, respectively. Error bars are S.E. F, immunoblotting of BNGE for complex V. Lys-to-Ala mutant OPA1 variant cells show disrupted complex V assembly, accumulating F1 domain of complex V, identical to OPA1-KO cells. Numbers in parentheses indicate the size in kDa.

Article Snippet: OPA1- KO MEFs were from American Type Culture Collection (ATCC CRL2995) ( 6 ).

Techniques: Activity Assay, Mutagenesis, Western Blot, Variant Assay

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: ( A ) Western blot determining the expression levels of MFN1 and MFN2 in wt, Mfn2-null, and Mfn1-null MEFs. ( B, C ) Spread area ( B ) and circularity ( C ) of wt, Mfn1-null, and Mfn2-null MEFs after overnight culture. The individual points represent individual MEF cells. ( D–G ) representative images with individual tracks ( D ), Wind–Rose plots ( E ), quantification of velocity ( F ), and directionality ( G ) of wt, Mfn1-null, and Mfn2-null MEFs cells during random migration. ( H, I ) Quantification of cell circularity ( H ) and representative images ( I ) of indicated MEFs during cell spreading at indicated time points. Data are presented as mean ± SD in ( F ) and were pooled from a total of 18 cells in three independent experiments. Bars represent arithmetic means ± SD. One representative result of three biological repeats is shown in ( A, D, E, I ). Data are pooled from three independent experiments in ( B, C, F, G ). n = 50 cells are tracked and counted in ( B, C ). N = 30 cells are quantified in ( D ). ****p<0.0001 (one-way ANOVA). Scale bars: 50 µm. Figure 1—source data 1. Original blots and figures with the bands labeled for .

Article Snippet: Cell line ( M. musculus ) , Mfn1 -null MEF , ATCC , CRL-2992 , .

Techniques: Western Blot, Expressing, Migration, Labeling

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: ( A ) Immunofluorescence of F-actin (phalloidin), α-tubulin, and mitochondria (Mito Tracker) in wt , Mfn1 -null, and Mfn2 -null MEFs. ( B–D ) Cre-induced Mfn2 disruption in MEFs from Mfn2 flox/flox mice displays similar cell morphology as Mfn2 -null MEFs ( B ). ( E, F ) Cell spread area ( E ) and circularity ( F ) of indicated cells in . The individual points represent the circularity or spread area of individual MEF cells in ( C–F ). ( G ) Cells were transfected with mitochondria probes. Mfn2 -null MEFs and Mfn2 -null MEFs overexpressing MFN1 display large and fragmented mitochondria, while MFN2 re-expression in Mfn2 -null MEFs restored mitochondria tubules. One representative result of three biological repeats is shown in ( A, B ). n = 25 cells in each group are quantified in ( C–-F ). ***p≤0.001, ****p<0.0001 (one-way ANOVA). Scale bars: 10 µm in ( A, B ), 20 µm in ( G ).

Article Snippet: Cell line ( M. musculus ) , Mfn1 -null MEF , ATCC , CRL-2992 , .

Techniques: Immunofluorescence, Disruption, Transfection, Expressing

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: ( A ) Western blot determining the expression level of MFN1 and MFN2 in indicated MEF cells. Percentages of knockdown or re-expression were calculated by normalizing the intensity to vinculin first, then normalizing to the wt group. ( B–D ) Representative images with individual tracks ( B ), quantification of velocity ( C ), and Wind–Rose plots ( D ) of indicated MEF cells during random migration. ( E ) Quantification of cell circularity of wt and Mfn2-null MEFs with vec, MFN1, or MFN2 re-expressed during spreading at indicated time points. Data are presented as mean ± SD in ( E ) (n = 5). ( F, G ) Cell circularity ( F ) and cell spreading area ( G ) of indicated MEFs measured after overnight culture. ( H, I ) Percentage of Actin abundance in the cell border region ( H ) and peripheral actin band (PAB) cell percentage in each view was quantified using our custom algorism (see ). ( J ) Representative images of wt, Mfn2-null with doxycycline-induced MEF2 (DIn-MFN2) MEF cells treated with or without doxycycline for 48 hr. The cells are immunostained with phalloidin and MFN2. One representative result of three biological repeats is shown in ( A, B, D, H ). Data are pooled from three independent experiments in ( C, F–I ). n = 30 cells are tracked and counted in ( C ); n = 35 cells are quantified in ( F–H ). Five different views from three biological repeats are quantified in ( I ). *p≤0.05, **p≤0.01, ***p≤0.001, ****p<0.0001 (one-way ANOVA in C, E, F , unpaired t -test in H, I ). Scale bars: 50 µm in ( B ), 10 µm in ( J ). Figure 2—source data 1. Original blots and figures with the bands labeled for .

Article Snippet: Cell line ( M. musculus ) , Mfn1 -null MEF , ATCC , CRL-2992 , .

Techniques: Western Blot, Expressing, Knockdown, Migration, Labeling

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: ( A, B ) Western blot ( A ) and quantification ( B ) of the amount of pMLCII and total MLCII in wt , Mfn1 -null, and Mfn2 -null MEFs. ( C, D ) Increased pMLCII in Mfn2 -null MEFs can be corrected by re-expressing MFN2 or inducing a mitochondria-endoplasmic reticulum (ER) tether. ( C ) Western blot and ( D ) quantification determining the amount of pMLCII and total MLCII protein in wt , Mfn2 -null MEFs, Mfn2 -null MEFs with MFN2 re-expressed, or with an artificial ER-mitochondria tether. ( E ) Representative images of wt and Mfn2 -null MEFs immunostained for F-actin (phalloidin), pMLCII, and DAPI. ( F ) Western blot determining the expression levels of MLCK or ROCK in Mfn2 -null MEFs with shMLCK or shROCK . ( G ) Western blot of pMLCII and total MLCII Mfn2 -null MEFs with shMLCK or shROCK . ( H ) Representative images of Mfn2 -null MEFs with shMLCK or shROCK immunostained for F-actin (green) and paxillin (red). ( I, J ) Cellular spread area and circularity of wt , Mfn2 -null MEFs with vec, shMLCK, or shROCK were measured after overnight culture. ( K ) Percentage of actin abundance in the cell border region in wt , Mfn2 -null MEFs, Mfn2 -null MEFs with shMLCK or shROCK . ( L ) Percentage of PAB cells identified by a custom algorithm in wt , Mfn2 -null MEFs, Mfn2 -null MEFs with shMLCK or shROCK . The individual points stand for the size or circularity of individual MEF cells. One representative result of three biological repeats is shown in ( A, B, F, G ). Four biological repeats were done in ( C, D ). Data are pooled from three independent experiments in ( I, J ). n = 30 cells are quantified in ( I, K ). Five different views from three biological repeats are quantified in ( L ). *p≤0.05, **p≤0.01, ***p≤0.001, ****p<0.0001 (one-way ANOVA, comparing each group to the average of Mfn2 -/- vec group in I, K ). Scale bars: 20 µm in ( H ), 10 µm in ( E ). Figure 7—source data 1. Original blots and figures with the bands labeled for . Figure 7—source data 2. Original blots and figures with the bands labeled for . Figure 7—source data 3. Original blots and figures with the bands labeled for . Figure 7—source data 4. Original blots and figures with the bands labeled for .

Article Snippet: Cell line ( M. musculus ) , Mfn1 -null MEF , ATCC , CRL-2992 , .

Techniques: Western Blot, Expressing, Labeling

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet:

Article Snippet: Cell line ( M. musculus ) , Mfn1 -null MEF , ATCC , CRL-2992 , .

Techniques: Transfection, Construct, Over Expression, Expressing, Dominant Negative Mutation, Recombinant, Plasmid Preparation, Control, Knockdown, Sequencing, Staining, Activation Assay, Imaging, Cloning, Software, Microscopy