The modified mitochondrial outer membrane carrier MTCH2 links mitochondrial fusion to lipogenesis

Abstract

Mitochondrial function is integrated with cellular status through the regulation of opposing mitochondrial fusion and division events. Here we uncover a link between mitochondrial dynamics and lipid metabolism by examining the cellular role of mitochondrial carrier homologue 2 (MTCH2). MTCH2 is a modified outer mitochondrial membrane carrier protein implicated in intrinsic cell death and in the in vivo regulation of fatty acid metabolism. Our data indicate that MTCH2 is a selective effector of starvation-induced mitochondrial hyperfusion, a cytoprotective response to nutrient deprivation. We find that MTCH2 stimulates mitochondrial fusion in a manner dependent on the bioactive lipogenesis intermediate lysophosphatidic acid. We propose that MTCH2 monitors flux through the lipogenesis pathway and transmits this information to the mitochondrial fusion machinery to promote mitochondrial elongation, enhanced energy production, and cellular survival under homeostatic and starvation conditions. These findings will help resolve the roles of MTCH2 and mitochondria in tissue-specific lipid metabolism in animals.

Figure 1.

MTCH2 regulates mitochondrial fusion. (A) COS7 cells transiently transfected with matrix-targeted mito-mCherry and a low (25 ng) or high (250 ng) amount of MTCH2-GFP. Scale bars = 5 µm. (B) MitoTracker red staining of WT HCT116 cells, two independently generated MTCH2^−/− CRISPR cell lines, and a MTCH2^−/− line that stably expresses MTCH2-GFP (MTCH2^−/− rescue). Scale bars = 5 µm. (C and D) Immunoblot for MTCH2 expression in lysates from indicated cell lines. Fold expression was determined by relative density analysis. *, Nonspecific bands. (E) Quantification of mitochondrial morphology in cell lines described in B. Error bars show mean + SEM of at least three independent experiments. Statistical significance was evaluated between fragmented values by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. Source data are available for this figure: SourceData F1.

Figure S1.

MTCH2 regulates mitochondrial fusion. (A) Individual channels and GFP pixel intensity of fluorescent images shown in Fig. 1 A. Scale bars = 5 µm. (B) Pixel intensity of a line scan drawn across mitochondrial tubules of cell expressing MTCH2-GFP and matrix-targeted mito-mCherry. (C) DNA sequence of CRISPR-generated mutations in HCT116 MTCH2^−/− clonal cell lines. (D) MTCH2^−/− rescue line stably expressing MTCH2-GFP stained with MitoTracker red. Scale bars = 5 µm. (E) Immunofluorescence for endogenous DRP1 and TOM20 in WT and MTCH2^−/− cells. Yellow line demarcates the cell periphery. Scale bars = 5 µm. (F) Quantification of the percent of total cellular DRP1 signal localized to mitochondria in images described in A. Error bars show mean + SEM of 30 cells. Statistical significance was evaluated by two-sided unpaired t test. (G) Immunoblot for total, phospho-S637, or phospho-S616 DRP1 in lysates from WT and MTCH2^−/− cells. (H) Quantification of relative density of phospho/total normalized to WT. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated by one-way ANOVA. Source data are available for this figure: SourceData FS1.

Figure 2.

The mitochondrial dynamics machinery is functional in MTCH2^−/− cells. (A) Immunoblot for expression of the indicated mitochondrial dynamics proteins in lysates of WT and MTCH2^−/− cells. (B) Quantification of the relative density normalized to GAPDH. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (C) WT and MTCH2^−/− cells transfected with dominant-negative mCherry-DRP1^K38A or control empty mCherry vector and stained with MitoTracker green. Scale bars = 5 µm. (D) Quantification of mitochondrial morphology in cells described in C. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated between fragmented values by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (E) Top: Schematic representation of content mixing in vitro mitochondrial fusion assay. Bottom: In vitro fusion efficiency of mitochondria isolated from WT or MTCH2^−/− cells. Data are presented as fusion efficiency normalized to the WT reaction. Error bars show mean + SEM of seven independent experiments. Statistical significance was evaluated between fragmented values by two-sided nonparametric t test. Source data are available for this figure: SourceData F2.

Figure 3.

MTCH2 is essential for and specific to starvation stress–induced mitochondrial hyperfusion. (A) MitoTracker red staining of WT and MTCH2^−/− cells treated with cycloheximide (CHX; 10 µM) or starved in HBSS for 5 h. Scale bars = 5 µm. (B) Quantification of mitochondrial morphology in cells described in A. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated between hyperfused values by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (C–E) Top: Western blot for phospho- and total levels of the indicated proteins in lysates from WT and MTCH2^−/− cells starved with HBSS for the specified amount of time. Bottom: Quantification of relative density of phospho/total normalized to WT 0 h. Error bars show mean + SD of two independent experiments. (F) Left: Schematic representation of content mixing in vitro mitochondrial fusion assay. Addition of cytosol to the reaction enhances fusion efficiency in WT cells. Right: In vitro fusion efficiency of mitochondria isolated from WT or MTCH2^−/− cells with or without addition of cytosol from WT cells. Data are presented as fusion efficiency normalized to the WT no-cytosol reaction. Error bars show mean + SEM of five independent experiments. Statistical significance was evaluated by two-sided unpaired t test. *, P < 0.05. Source data are available for this figure: SourceData F3.

Figure S2.

MTCH2 is essential for and specific to starvation stress–induced mitochondrial hyperfusion. (A) Quantification of mitochondrial morphology in WT and MTCH2^−/− clone 2 cells treated with cycloheximide (CHX; 10 µM) or starved in HBSS for 5 h. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated between hyperfused values by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (B) Quantification of mitochondrial morphology in WT and MTCH2^−/− rescue line starved in HBSS for 5 h. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated between hyperfused values by two-tailed unpaired t test. *, P < 0.05. (C) MitoTracker red staining of WT cells incubated for 5 h in HBSS (left) or medium (right) with no supplements (n/a) or supplemented with Serum (FBS), glutamine (Q), or glucose (gluc), as indicated. Scale bars = 5 µm. (D and E) Traces of oxygen consumption rates (OCR; D) and extracellular acidification rate (ECAR; E) of WT, MTCH2^−/−, and rescue cell lines sequentially treated with glucose (gluc), oligomycin (oligo), FCCP, and rotenone with antimycin A (rot/AA) to assess respiratory states. Error bars show mean + SEM of 18 replicates. (F) Total, oxidative phosphorylation-derived (ox), and glycolysis-derived (glyc) ATP synthesis rates of WT, MTCH2^−/−, and MTCH2^−/− rescue cell lines as calculated from measurements shown in A and B at baseline, before addition of glucose (top) and after the addition of glucose (bottom). Error bars show mean + SEM of 18 replicates. Statistical significance was evaluated by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (G) Quantification of total mitochondrial pixel area in WT and MTCH2^−/− cells stained with MitoTracker red. Error bars show mean + SEM of 15 cells. Statistical significance was evaluated between hyperfused values by two-tailed unpaired t test. (H) Immunoblot for TOM20 and cytochrome C (CYT C) expression in lysates from indicated cell lines. (I) Quantification of the relative density normalized to GAPDH. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated by one-way ANOVA. (J and K) Relative growth rate (J) and doubling time (K) of WT and MTCH2^−/− cells grown in media containing glucose or galactose. Error bars show mean + SEM of seven time points measured over two independent experiments. Statistical significance was evaluated between hyperfused values by two-tailed unpaired t test. *, P < 0.05. (L) In vitro fusion efficiency of mitochondria isolated from WT cells with or without addition of cytosol from WT or MTCH2^−/− cells. Data are presented as fusion efficiency normalized to the no-cytosol reaction. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (M) In vitro fusion efficiency of mitochondria derived from WT cells, MTCH2^−/− cells, or a mix of both with or without addition of cytosol from WT cells. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated by one-way ANOVA. Source data are available for this figure: SourceData FS2.

Figure 4.

Disruption of de novo fatty acid synthesis phenocopies loss of MTCH2. (A) Volcano plot of significant interactors identified by mass spectrometry analysis of MTCH2-GFP immunoprecipitated from cross-linked MTCH2^−/− rescue cells. Significant interactors with a log2 fold-change (Log2FC) >1.0 are shown in black. Significant interactors involved in fatty acid/TAG de novo synthesis or catabolism are indicated in red and listed in B. (C) MitoTracker red staining of WT cells treated with vehicle or with the CPT1a inhibitor etomoxir (CPT1i; 100 nM) for 16 h. CPT1i-treated cells were then incubated with cycloheximide (CHX; 10 µM) or starved in HBSS for 5 h in the presence of inhibitor. Scale bars = 5 µm. (D) Quantification of mitochondrial morphology in cells described in C. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated between hyperfused values by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (E) MitoTracker red staining of WT cells treated with vehicle or with the ACC inhibitor TOFA (ACCi; 2 µg/ml) for 16 h. ACCi-treated cells were then incubated with CHX (10 µM) or starved in HBSS for 5 h in the presence of inhibitor. Scale bars = 5 µm. (F) Quantification of mitochondrial morphology in cells described in E. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated between hyperfused values by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05.

Figure S3.

LPA promotes mitochondrial fusion via MTCH2. (A) Schematic of the de novo fatty acid and TAG synthesis pathway. Enzymes identified as interactors in the mass-spectrometry analysis of MTCH2-GFP immunoisolation are shown in red. (B) MitoTracker red staining of MTCH2^−/− cells treated with vehicle, CPT1i (100nM), ACCi (2 µg/ml), DGATi (20 µM), or GPATi (75 µM) for 16 h. Scale bars = 5 µm. (C) Quantification of mitochondrial morphology in cells described in B. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated between fragmented values by one-way ANOVA. (D) MitoTracker red staining of WT and MTCH2^−/− cells stably expressing scramble shRNA or one of two different shRNAs targeting CPT1a. Scale bars = 5 µm. (E) Immunoblot for CPT1a expression in lysates from indicated cell lines. The percentage knockdown efficiency (% KD) was determined by relative density analysis. (F) MitoTracker red and BODIPY 493/503 staining of WT cells treated with vehicle, ACCi (2 µg/ml), DGATi (20 µM), or GPATi (75 µM) for 16 h. Scale bars = 5 µm. (G) Quantification of mitochondrial morphology in WT and MTCH2^−/− clone 1 cells treated with vehicle or GPATi (75 µM) for 16 h and starved in HBSS for 5 h with or without BSA (0.1% wt/vol) in the presence of vehicle or inhibitor. Error bars show mean + SEM of three independent experiments. (H) Immunoblot for GPAT1 expression in lysates from indicated cell lines. The % KD was determined by relative density analysis. (I) Quantification of mitochondrial morphology in WT cells stably expressing scramble shRNA or one of three different shRNAs targeting mitochondrial GPAT1. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated between fragmented values by one-way ANOVA. *, P < 0.05. (J) In vitro fusion efficiency of mitochondria isolated from WT or MTCH2^−/− cells with or without addition of LPA 18:0 (400 µM), LPA 18:1 (400 µM), DAG (400 µM), or LPC (400 µM). Data are presented as fusion efficiency normalized to the WT control reaction. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated between fragmented values by one-way ANOVA. *, P < 0.05. Source data are available for this figure: SourceData FS3.

Figure 5.

LPA promotes mitochondrial fusion via MTCH2. (A) MitoTracker red staining of WT cells treated with vehicle or the DGAT1 inhibitor T863 (20 µM) and DGAT2 inhibitor PF-06424439 (20 µM; together, DGATi) for 16 h. DGATi-treated cells were then incubated with cycloheximide (CHX; 10 µM) or starved in HBSS for 5 h in the presence of inhibitor. Scale bars = 5 µm. (B) Quantification of mitochondrial morphology in cells described in A. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated between hyperfused values by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (C) MitoTracker red staining of WT cells treated with vehicle or the GPAT inhibitor FSG67 (GPATi; 75 µM) for 16 h. GPATi-treated cells were incubated with CHX (10 µM) or starved in HBSS for 5 h with or without BSA (0.1% wt/vol) in the presence of inhibitor. Scale bars = 5 µm. (D) Quantification of mitochondrial morphology in cells described in C. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated between hyperfused values by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (E) Top: Schematic representation of content-mixing in vitro mitochondrial fusion assay with addition of LPA. Bottom: In vitro fusion efficiency of mitochondria isolated from WT cells with or without addition of cytosol (cyto) or the indicated amount of LPA 16:0. Data are presented as fusion efficiency normalized to the control reaction. Error bars show mean + SEM of three or four independent experiments. Statistical significance was evaluated by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (F) In vitro fusion efficiency of mitochondria isolated from WT or MTCH2^−/− cells with or without addition of LPA 16:0 (400 µM) or PA (400 µM). Data are presented as fusion efficiency normalized to the WT control reaction. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (G and H) In vitro fusion efficiency of mitochondria isolated from WT cells with or without addition of cytosol from cells treated with vehicle, ACCi (2 µg/ml; G), or GPATi (75 µM; H) for 16 h. Data are presented as fusion efficiency normalized to the no-cytosol reaction. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (I) Schematic representation of the TAG synthesis pathway and the generation of LPA stimulating MTCH2-dependent mitochondrial fusion.