Journal: Molecular Biology of the Cell

Article Title: Rapid degradation of mutant SLC25A46 by the ubiquitin-proteasome system results in MFN1/2-mediated hyperfusion of mitochondria

doi: 10.1091/mbc.E16-07-0545

Figure Lengend Snippet: SLC25A46 comigrates with mitochondrial dynamics and MICOS proteins. (A) Glycerol gradient density centrifugation of mitochondrial extracts generated from a stable cell line expressing 2xHA-SLC25A46 WT. Mitochondria were solubilized in 1% digitonin, and lysates were loaded on a linear 10–50% glycerol gradient and centrifuged overnight. Fractions 1–23 were analyzed with the indicated antibodies. A similar sample was included, and the HA antibody was used to determine SLC25A46 L341P assembly. SM, starting material. (B) Immunoprecipitation (IP) with the HA antibody of 2xHA-SLC25A46 WT from the indicated fractions (1, 9, 11,13) of the glycerol gradient density centrifugation in A. Input samples correspond to 10% of each fraction. Antibodies against HA, MFN1, and MFN2 were used. (C) Mitochondria isolated form HEK293T cells stably expressing 2xHA-SLC25A46 WT or L341P were solubilized with digitonin, and mitochondrial extracts were resolved by two-dimensional BN-PAGE. Antibodies against MFN1, TOMM40, PreP, and HA were used for immunoblotting. For MFN1, TOMM40, and PreP, the analysis from WT mitochondria is shown, and the complexes migrated identically in mitochondria from the SLC25A46 L341P cell line. (▪) The MFN1-SLC25A46 complex. (D, E) Mitochondria were isolated from LAN5 cells and then lysed in 1% digitonin. Mitochondrial extracts were resolved by one- (D) or two- (E) dimensional BN-PAGE; antibodies for endogenous SLC25A46, MIC19, MIC60, MFN2, and MTCH2 were included. In D, shorter and longer exposures for SLC25A46 are shown to highlight the larger complex that comigrates with the MICOS complex. (▪) The MFN1-SLC25A46 complex; (▴) the MICOS-SLC25A46 complex; (*) the MTCH2-SLC25A46 complex.

Article Snippet: Antibodies used in this study were SLC25A46 (Proteintech); HA (HA.11; Biolegend); MFN1 (ab57602) and histidine (HIS.H8; Abcam); LC3A/B (Cell Signaling); MFN2 (D1E9; Cell Signaling); MTCH2 (Abgent); MIC60/IMMT (Abgent and Proteintech); MID51 (Proteintech); Ubiquitin (FK2, Enzo Biochem); Mortalin (University of California, Davis/National Institutes of Health NeuroMab Facility); MIC19/CHCHD3 (Everest Biotech); MIC27/Apool (Assay BioTech); OPA1, P97/VCP, and TIMM23 (BD Biosciences); TOMM20 (FL-145), LRP130 (H-300), and glyceraldehyde-3-phosphate dehydrogenase (FL-335; Santa Cruz Biotechnology); MULAN (HPA017681) and Flag (M2) (Sigma-Aldrich); MARCH5 (provided by Richard Youle); myc (9E10; Developmental Studies Hybridoma Bank, University of Iowa); and Cullin 4a (PA5-29857; Thermo Fisher Scientific).

Techniques: Centrifugation, Generated, Stable Transfection, Expressing, Immunoprecipitation, Isolation, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Rapid degradation of mutant SLC25A46 by the ubiquitin-proteasome system results in MFN1/2-mediated hyperfusion of mitochondria

doi: 10.1091/mbc.E16-07-0545

Figure Lengend Snippet: SLC25A46 interacts with proteins important for mitochondrial biogenesis and morphology. (A) IP with the HA antibody from whole-cell extracts of HEK293T or stable HEK293T cells expressing 2xHA-SLC25A46 WT or 2xHA-SLC25 L341P. Cells were cross-linked with dithiobis(succinimidyl propionate) before lysis. Samples were analyzed with the indicated antibodies. Input samples represent 2% of the material used for the IP. Short (S) and long (L) forms of OPA1 are marked. (B) Steady-state level of SLC25A46 interaction partners in HCT116 WT or SLC25A46 knockout ( SLC25A46 − / − ) cells was determined. Mitochondria from each cell line were isolated, solubilized, separated by SDS–PAGE, and analyzed with immunoblotting. Three different single-cell knockout clones designated 1.7, 4.1, and 4.3, using two different gRNAs. The asterisk marks a nonspecific band of the antibody. (C) Steady-state levels of MFN1 and MFN2 in HEK293 T-REx Flp-In WT, and SLC25A46 -/- cells were measured in three independent experiments. Mitochondria from each cell line were isolated, solubilized, separated by SDS–PAGE, and analyzed with immunoblotting. TOMM40 was included as a loading control. (D) Densitometric analysis of C. Results are shown as mean ± SEM of three independent experiments. t test, p < 0.05. (E) Analysis of MFN1 and MFN2 expression in HEK293 T-REx Flp-In WT and SLC25A46 –/– cells by qPCR (mean ± SEM, n = 3). (F) Knockout of SLC25A46 does not alter the phospholipid composition of mitochondria. Phospholipids were extracted from 0.75 mg of mitochondria in the cell lines as in B, separated by thin-layer chromatography, and visualized using molybdenum blue staining. Scanned images were analyzed using Quantity 1 software, and the relative abundance of each phospholipid was calculated as percentage of the total phospholipid in each sample (mean ± SEM, n = 3). CL, cardiolipin; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PG, phosphatidylglycerol; PS, phosphatidylserine.

Article Snippet: Antibodies used in this study were SLC25A46 (Proteintech); HA (HA.11; Biolegend); MFN1 (ab57602) and histidine (HIS.H8; Abcam); LC3A/B (Cell Signaling); MFN2 (D1E9; Cell Signaling); MTCH2 (Abgent); MIC60/IMMT (Abgent and Proteintech); MID51 (Proteintech); Ubiquitin (FK2, Enzo Biochem); Mortalin (University of California, Davis/National Institutes of Health NeuroMab Facility); MIC19/CHCHD3 (Everest Biotech); MIC27/Apool (Assay BioTech); OPA1, P97/VCP, and TIMM23 (BD Biosciences); TOMM20 (FL-145), LRP130 (H-300), and glyceraldehyde-3-phosphate dehydrogenase (FL-335; Santa Cruz Biotechnology); MULAN (HPA017681) and Flag (M2) (Sigma-Aldrich); MARCH5 (provided by Richard Youle); myc (9E10; Developmental Studies Hybridoma Bank, University of Iowa); and Cullin 4a (PA5-29857; Thermo Fisher Scientific).

Techniques: Expressing, Lysis, Knock-Out, Isolation, SDS Page, Western Blot, Clone Assay, Control, Thin Layer Chromatography, Staining, Software

Journal: Molecular Biology of the Cell

Article Title: Rapid degradation of mutant SLC25A46 by the ubiquitin-proteasome system results in MFN1/2-mediated hyperfusion of mitochondria

doi: 10.1091/mbc.E16-07-0545

Figure Lengend Snippet: SLC25A46 levels affect the steady-state levels and assembly of MFN1 and MFN2. (A) The oligomerization of MFN1 and MFN2 was determined in isolated mitochondria from LAN5 cell lines in which SLC25A46 was knocked down with different shRNA constructs after doxycycline induction using BN-PAGE and immunoblotting. (B) The steady-state levels of the indicated proteins were determined using the same mitochondrial extracts loaded on BN-PAGE in A. (C) Analysis of MFN1 and MFN2 expression in LAN5 cells in which SLC25A46 was knocked down with a specific shRNA construct by qPCR (mean ± SEM, n = 3). (D) The mitochondrial network in LAN5 cell lines in which SLC25A46 was knocked down after doxycycline induction was investigated by immunofluorescence staining using an antibody against the matrix protein Mortalin or OM protein TOMM20. 4′,6-Diamidino-2-phenylindole was used to stain the nucleus. Scale bar, 10 μm. (E) Quantification of the mitochondrial network from D. For each experiment, 100 cells were counted (mean ± SEM, n = 3). (F) Live cells were quantitated using a trypan blue assay in the LAN5 and HEK294 T-REx Flp-In lines. An equal number of cells from each line was plated, and then live cells were counted after 4 d. The expression of SLC25A46 in LAN5 was knocked down for 2 wk before the experiment. Data represent mean ± SEM. ( n = 3). (G) As in A, but the oligomerization of MFN1 and MFN2 was analyzed in HEK293 T-REx Flp-In cells lacking SLC25A46. SLC25A46 knockout was rescued by expressing 2xHA-SLC25A46 WT or L341P for 48 h after induction with 10 ng/ml doxycycline. (H) Lysates from G were separated by SDS–PAGE and the steady-state levels of indicated proteins investigated by immunoblot. 2xHA-SLC25A46 and endogenous SLC25A46 were detected with anti-SLC25A46; a longer exposure to detect HA-SLC25A46 L341P of the panel was included. A panel with anti-HA was also included to detect expressed HA-SLC25A46 WT and L341P.

Article Snippet: Antibodies used in this study were SLC25A46 (Proteintech); HA (HA.11; Biolegend); MFN1 (ab57602) and histidine (HIS.H8; Abcam); LC3A/B (Cell Signaling); MFN2 (D1E9; Cell Signaling); MTCH2 (Abgent); MIC60/IMMT (Abgent and Proteintech); MID51 (Proteintech); Ubiquitin (FK2, Enzo Biochem); Mortalin (University of California, Davis/National Institutes of Health NeuroMab Facility); MIC19/CHCHD3 (Everest Biotech); MIC27/Apool (Assay BioTech); OPA1, P97/VCP, and TIMM23 (BD Biosciences); TOMM20 (FL-145), LRP130 (H-300), and glyceraldehyde-3-phosphate dehydrogenase (FL-335; Santa Cruz Biotechnology); MULAN (HPA017681) and Flag (M2) (Sigma-Aldrich); MARCH5 (provided by Richard Youle); myc (9E10; Developmental Studies Hybridoma Bank, University of Iowa); and Cullin 4a (PA5-29857; Thermo Fisher Scientific).

Techniques: Isolation, shRNA, Construct, Western Blot, Expressing, Immunofluorescence, Staining, Knock-Out, SDS Page

Journal: Molecular Biology of the Cell

Article Title: Rapid degradation of mutant SLC25A46 by the ubiquitin-proteasome system results in MFN1/2-mediated hyperfusion of mitochondria

doi: 10.1091/mbc.E16-07-0545

Figure Lengend Snippet: MARCH5 and MULAN mediate SLC25A46 L341P ubiquitylation and degradation. (A) Increased half-life of SLC25A46 L341P by overexpressing the RING-finger mutant MULAN H319A. A stable cell line expressing 2xHA-SLC25A46 L341P was transiently transfected with an empty vector, WT MULAN, or MULAN H319A and treated with CHX for indicated time points. Mitochondria were isolated, and MULAN was detected with an anti-myc antibody. The asterisk marks monoubiquitylated SLC25A46. (B) As in A, with the exception that the cells were transiently transfected with WT MARCH5 or the RING-mutant MARCH5 H43W. (C) HEK293T cells stably expressing Doxy-inducible control shRNA or a shRNA targeting MULAN were transfected with constructs for control shRNA or shRNA targeting MARCH5 and 2xHA-SLC25A46 L341P. Doxycycline (1 μg/ml) was added during transfection. After 48 h, CHX was added, and cells were harvested just before CHX addition (0) and at 15 and 30 min. Mitochondria were isolated and analyzed by SDS–PAGE and immunoblotting. (D) As in C, but cells were transfected with 3xFlag-SLC25A46 L341P and HA-Ubiquitin-GFP. After treatment of the cells with 25 μM MG132 for 3 h, HA-tagged ubiquitylated proteins were immunoprecipitated under denaturing conditions with anti-HA. Ubiquitylated 3xFlag-SLC25A46 L341P was detected with an anti-Flag antibody. The asterisk marks polyubiquitylated species. TOMM40 is a loading control. (E) Determination of SLC25A46 L341P abundance in HEK293 T-REx Flp-In WT, MARCH5, MULAN, or MARCH5/MULAN knockout cells. Cells were transfected with a 2xHA-SLC25A46 L341P construct; mitochondria from the cell lines were isolated, solubilized, separated by SDS–PAGE, and analyzed by immunoblotting. PreP was included as a loading control. (F) As in E, with CHX treatment for the indicated times. Cells were harvested at each time point, and mitochondria were isolated. SLC25A46 L341P and endogenous SLC25A46 were detected by immunoblotting, and TOMM40 was included as a loading control. (G) Under physiological expression of WT SLC25A46, the levels of MFN1, MNF2, and dimers are kept constant so as not to disturb the fine balance between fission and fusion, which is important for development and survival (left). However, when the gene SLC25A46 is disrupted by deletions or the gene product destabilized by point mutations like L341P, which is quickly degraded by OM–associated degradation, MFN1 and MFN2 dimers accumulate on the OM and shift the balance towards fusion. This leads to pontocerebellar hypoplasia and early death (right).

Article Snippet: Antibodies used in this study were SLC25A46 (Proteintech); HA (HA.11; Biolegend); MFN1 (ab57602) and histidine (HIS.H8; Abcam); LC3A/B (Cell Signaling); MFN2 (D1E9; Cell Signaling); MTCH2 (Abgent); MIC60/IMMT (Abgent and Proteintech); MID51 (Proteintech); Ubiquitin (FK2, Enzo Biochem); Mortalin (University of California, Davis/National Institutes of Health NeuroMab Facility); MIC19/CHCHD3 (Everest Biotech); MIC27/Apool (Assay BioTech); OPA1, P97/VCP, and TIMM23 (BD Biosciences); TOMM20 (FL-145), LRP130 (H-300), and glyceraldehyde-3-phosphate dehydrogenase (FL-335; Santa Cruz Biotechnology); MULAN (HPA017681) and Flag (M2) (Sigma-Aldrich); MARCH5 (provided by Richard Youle); myc (9E10; Developmental Studies Hybridoma Bank, University of Iowa); and Cullin 4a (PA5-29857; Thermo Fisher Scientific).

Techniques: Mutagenesis, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Isolation, Control, shRNA, Construct, SDS Page, Western Blot, Ubiquitin Proteomics, Immunoprecipitation, Knock-Out

Journal: Frontiers in Endocrinology

Article Title: The Causal Role of Mitochondrial Dynamics in Regulating Innate Immunity in Diabetes

doi: 10.3389/fendo.2020.00445

Figure Lengend Snippet: Role of MFN1/MFN2 overexpression on innate immune response in cybrid B4 under nutrient excess. Cybrid B4 cells were overexpressed with GFP, GFP-MFN1, or GFP-MFN2 for 18 h, followed by a 6-h exposure with or without 0.25 mM FA. (A) Representative blot image of RIG-I, MDA5, MAVS, NLRP3, and caspase-1. β-Actin as loading control. (B) Histograms of densitometric results. Quantification value of β-actin was used for normalization. * p < 0.05 compared to vector control in the absence of FA. † p < 0.05 comparison between indicated groups. FA, fatty acid.

Article Snippet: Gene overexpression of Mfn1, Mfn2, Drp1, and Fis1 (RG207184; RG202218; RG202046; RG202560; Origene Technologies, Inc., Rockville, MD, USA) were performed using Lipofectamine® 2000 (1367620, Invitrogen) in the presence of Opti-MEM (31985-070, Gibco) for 18 h. A DNA (μg) to Lipofectamine® 2000 (μl) ratio of 1:1 was used for transfection.

Techniques: Over Expression, Plasmid Preparation

Journal: Frontiers in Endocrinology

Article Title: The Causal Role of Mitochondrial Dynamics in Regulating Innate Immunity in Diabetes

doi: 10.3389/fendo.2020.00445

Figure Lengend Snippet: Role of MFN1/MFN2 knockdown on innate immune response in cybrid B4 under nutrient excess. (A) Representative blot image of RIG-I, MDA5, MAVS, NLRP3, and caspase-1. β-Actin as loading control. (B) Histograms of densitometric results. Quantification value of β-actin was used for normalization. * p < 0.05 compared to siRNA control in the absence of FA or between indicated groups. † p < 0.05 comparison between indicated groups. CON, control; FA, fatty acid.

Article Snippet: Gene overexpression of Mfn1, Mfn2, Drp1, and Fis1 (RG207184; RG202218; RG202046; RG202560; Origene Technologies, Inc., Rockville, MD, USA) were performed using Lipofectamine® 2000 (1367620, Invitrogen) in the presence of Opti-MEM (31985-070, Gibco) for 18 h. A DNA (μg) to Lipofectamine® 2000 (μl) ratio of 1:1 was used for transfection.

Techniques: