Abstract
Mitochondrial fission occurs in many cellular processes, but the regulation of fission is poorly understood. We show that long-chain acyl-coenzyme A (LCACA) activates two related mitochondrial fission proteins, MiD49 and MiD51, by inducing their oligomerization, which activates their ability to stimulate the DRP1 GTPase. The 1:1 stoichiometry of LCACA:MiD in the oligomer suggests interaction in the previously identified nucleotide-binding pocket, and a point mutation in this pocket reduces LCACA binding and LCACA-induced oligomerization for MiD51. In cells, this LCACA binding mutant does not assemble into puncta on mitochondria or rescue MiD49/51 knockdown effects on mitochondrial length and DRP1 recruitment. Furthermore, cellular treatment with BSA-bound oleic acid, which causes increased LCACA, promotes mitochondrial fission in an MiD49/51-dependent manner. These results suggest that LCACA is an endogenous ligand for MiDs, inducing mitochondrial fission and providing a potential mechanism for fatty-acid-induced mitochondrial division. Finally, MiD49 or MiD51 oligomers synergize with Mff, but not with actin filaments, in DRP1 activation, suggesting distinct pathways for DRP1 activation.
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Data availability
Raw numerical data for all quantifications have been provided in Statistical Source Data. Unprocessed blots and gels for all relevant figures have been provided in Image Source Data. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank J. Delgado, S. Liu, Z. Svindrych and A. I. Ferreira Verissimo for much appreciated help during these investigations. We greatly value the efforts of M. Ryan and A. Sharpe for providing MiD KO cells made on the other side of the world. We also thank A. Divakaruni and A. Jones for advice on acyl-CoA extraction methods from cells, and Z. Comeny for working so well in transferring materials. This work was supported by the National Institutes of Health (NIH) R35 GM122545 and R01 DK088826 to H.N.H., NIH R35 GM150942 and a Boettcher Foundation Webb-Waring Biomedical Research Award to H.A., NIH P20 GM113132 to the Dartmouth BioMT and by the Deutsche Forschungsgemeinschaft fellowship KA5106/1-1 (418076373) to F.K.
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A.L. was the main experimentalist for the biochemical experiments (Figs. 1–4 and associated Extended Data figures). F.K. was the main experimentalist for the cellular experiments (Figs. 5–8 and associated Extended Data figures.). H.N.H. conducted acyl-CoA:MiD ratio experiments, RT–PCR analysis and CMC determination. G.S. and H.A. conducted negative-stain EM experiments. A.F.A. assisted with acyl-CoA binding preference experiments. M.P.H.A. assisted with mutant MiD51 biochemical analysis. A.L., F.K. and H.N.H. discussed the experimental approach and results and wrote/edited the paper (including figure preparation).
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Extended data
Extended Data Fig. 1 Proteins used in this study, and effect of palmitoyl-CoA on MiD49 oligomerization.
a, Bar diagrams of MiD49 and MiD51, showing the regions used in our biochemical studies: 125–454 for murine MiD49 (38.7 kDa), and 134-463 for human MiD51 (38.3 kDa). TM = transmembrane sequence, DR = disordered region. Green region = segment used for biochemical studies here, and for crystallization studies by others. b, Coomassie-stained SDS–PAGE of proteins used in this study. 2 µg protein loaded. Mass markers in kDa shown. Protein preparations were conducted independently with similar outcomes 3 times for MiD49 and MiD51-WT, and 2 times for MiD51-R342A and MiD51-Y185A. c, Superose 6 size-exclusion chromatography of MiD49 cytoplasmic region (100 µM) mixed with varying concentrations of palmitoyl-CoA. d, Velocity analytical ultracentrifugation of peak fractions from Superose 6 size-exclusion chromatography of MiD49 cytoplasmic region (100 µM) incubated without or with palmitoyl-CoA (100 µM). Molecular masses calculated by these vAUC data: 36.4 kDa (without palmitoyl-CoA), and 4815 kDa (with palmitoyl-CoA). Unprocessed gels are available in source data.
Extended Data Fig. 2 Negative-stain EM of MiD49 and MiD51 oligomers.
Peak oligomer fractions from Superose 6 chromatography after mixing MiD49 (100 µM) or MiD51 (50 µM) with palmitoyl-CoA (50 or 500 µM, respectively). a, Full-field examples for MiD49 and MiD51. Scale bars, 100 nm. b, Graph of long axis and short axis lengths for 18 particles of MiD49 or MiD51 oligomers. c, Montage of MiD49 (left) or MiD51 (right) oligomer particles. Scale bars, 50 nm. d, Palmitoyl-CoA alone (1 mM) in same buffer as MiDs (10 mM Hepes pH 7.4, 65 mM KCl, 1 mM MgCl2, 1 mM EGTA, 1 mM DTT). Scale bars: 100 nm (main panel), 50 nm (inset). e, Graph of particles/field (800 ×800 nm field) versus palmitoyl-CoA concentration. 3–5 fields quantified for each concentration. X intercept for linear fit of the 125, 250, 500, and 1000 μM data points (after accounting for background particles, 8.2/field) represents critical micelle concentration (cmc, 47 µM). Inset shows data points closer to the cmc (red line indicates mean of 0 μM palmitoyl-CoA particles).
Extended Data Fig. 3 Palmitoyl-CoA quantification in MiD49 oligomer fraction.
a, Reversed-phase HPLC profile of 1.5 nmole palmitoyl-CoA (red) or solvent alone (blue). Dashed grey line represents acetonitrile concentration. b, Palmitoyl-CoA peaks from four quantities of loaded palmitoyl-CoA (0, 0.5, 1, 1.5 and 2 nmole). c, Peak area as a function of palmitoyl-CoA loaded. d, Palmitoyl-CoA HPLC peak obtained from 200 mL of MiD49 oligomer fraction (Superose 6 oligomer fraction after incubating 100 µM MiD49 with 500 µM palmitoyl-CoA). Peak area, 3.42 A.U.-min, corresponding to 1.1 nmole (5.5 µM) palmitoyl-CoA. MiD49 concentration in this fraction (by Bradford assay), 5.3 µM. e, Standard curve of phosphate using the Fiske-Subbarow assay. f, Palmitoyl-CoA concentrations calculated from Fiske-Subbarow assay in which 400 µL oligomer fraction from Superose 6 was assayed. ‘Condition’ refers to the µM amount of palmitoyl-CoA that was incubated with 100 µM MiD49 (37˚C, 1 hr) prior to separation of oligomers from monomers by Superose 6. MiD49 concentration determined by Bradford assay.
Extended Data Fig. 4 HPLC analysis of acyl-CoAs bound to MiD49 oligomer.
a, Individual HPLC traces of each of the acyl-CoAs (4 nmole loaded). b, HPLC trace of the MiD49 oligomer fraction from the following experiment. MiD49 cytoplasmic construct (100 µM) was mixed simultaneously with 83.3 µM of six acyl-CoAs (stearoyl, oleoyl, palmitoyl, myristoyl, lauroyl, octanoyl) for 1 hr at 37˚C, then MiD49 oligomer was isolated by Superose 6 chromatography. Acyl-CoA distribution was then analysed in the oligomer fraction by reversed-phase HPLC. The Y axis label of A.U. in all graphs in this figure represents mA.U.
Extended Data Fig. 5 Effects of LCACA on MiD51 biochemical properties.
a-c, Effect of MiD49 and actin filaments on DRP1 activity. a, DRP1 GTPase assays (0.75 µM DRP1) alone (black points) or in the presence of 0.5 µM actin filaments (red). b, DRP1 GTPase assays (0.75 µM DRP1) alone (black points) or in the presence of 10 µM Mff alone (grey) or with 250 nM MiD49 oligomers (red) or monomers (blue) added. c, Effect of varying concentrations of Mff on DRP1 GTPase activity (0.75 µM DRP1) in the absence or presence of 250 nM MiD49 oligomers (red) or monomers (blue). Full curve to 100 µM Mff. Zoom to 20 µM Mff shown in Fig. 2f. d, Blue-native gel electrophoresis of MiD51 cytoplasmic region (50 µM) mixed with 500 µM of the indicated molecules. e, Size-exclusion chromatography (Superose 6) of MiD51 (50 µM) alone (blue) or in the presence of 100 µM palmitoyl-CoA (red). f, Velocity analytical ultracentrifugation of the MiD51 oligomer peak Fig. 3b (left) and the monomer MiD51 peak (right), showing calculated molecular masses. g, Blue-native gel electrophoresis of MiD51 (50 µM) with 500 µM of the indicated molecules, or with 500 µM palmitoyl-CoA + the indicated concentrations of ADP, CoA or acetyl-CoA. h, MANT-ADP binding assay in which 300 nM MANT-ADP is mixed with the indicated concentrations of MiD51 or MiD49 and the fluorescence intensity monitored. i, DRP1 GTPase assays containing DRP1 alone (0.75 µM, black points) or in the presence of 0.5 µM MiD51 oligomers (red) or monomers (blue) added. j, Effect of varying concentrations of Mff on DRP1 GTPase activity (0.75 µM DRP1) in the absence or presence of 250 nM MiD51 oligomers (red) or monomers (blue). Full curve to 100 µM Mff. Zoom to 20 µM Mff shown in Fig. 3j. k, Effect of varying concentrations of actin filaments on DRP1 GTPase activity (0.75 µM DRP1) in the absence (black) or presence of MiD51 oligomers (blue). Source numerical data and unprocessed gels are available in source data.
Extended Data Fig. 6 Expression of MiD49 and MiD51-GFP fusion constructs in HeLa cells.
a, C-terminal GFP fusion constructs of MiD49 or MiD51 were transiently transfected into HeLa cells, then cells were fixed and stained for mitochondria (Tom20). At low expression levels (requiring >500 msec exposure at 100% laser power), the GFP fusions did not alter mitochondrial length, and displayed a punctate appearance on mitochondria. At high expression (<100 msec exposure, 50% laser power), the GFP pattern on mitochondria was uniform and mitochondria were hyperfused. Scale bars: 20 µm (whole cell) and 3 µm (insets). Experiment was repeated independently with similar outcomes 3 times. b, Examples of image processing for quantification of GFP-MiD51 puncta size, and % mitochondrial area covered by GFP-MiD51. Boxed regions are shown in higher magnification below overview images. Binary masks of the respective inset images are shown on the right. These binary masks are representative of the images that were used for analyses in Fig. 5 D and E. Scale bars are 10 µm in full images and 3 µm in insets. c, C-terminal GFP fusion constructs of the indicated MiD51 constructs were transiently transfected into HeLa cells, then cells were fixed and stained for mitochondria (Tom20). Cells were selected for high expression under the criteria described above. Images at right represent indicated boxed regions. Examples for WT and Y185 constructs represent hyperfused phenotype quantified in panel d (blue bar), while example for R342A mutant represents collapsed phenotype (grey bar). Scale bars, 20 µm in full images and 3 µm in insets. d, Graph quantifying % cells displaying hyperfused (blue) or collapsed (grey) mitochondria. Representative examples of each pattern are shown in c. N = 122, 82, and 85 cells were analysed for MiD51-WT, Y185A, R342A, respectively. Error bars in graph represent standard error of means. P values were determined by one-sided ANOVA (Dunnett’s multiple comparisons) test. e, RT-PCR analysis of MiD49/51 knockdown in HeLa cells. Left: agarose gels (SybrSafe-stained) of PCR reactions for GAPDH, MiD49 and MiD51 using cDNAs from three independent control KD and three MiD49/MiD51 KD samples. Lengths of PCR products (in base pairs) indicated at left. Right: Bar graph illustrating relative mRNA levels of MiD49 and MiD51 compared to control siRNA-treated samples. Data were quantified from gels and normalized to the GAPDH signals independently for each sample. Error bars represent standard deviations. P values were determined by unpaired two-sided Student’s t-test. Source numerical data are available in source data.
Extended Data Fig. 7 2-bromopalmitate induced mitochondrial fission is MiD-dependent.
a, Fixed-cell fluorescence micrographs of HeLa cells transfected with the indicated siRNAs, treated for 1-hr with either 2-BP or MP and stained for actin (TRITC-phalloidin, red) with Dapi (magenta) and mitochondria (Tom20, green). Experiment was repeated independently with similar outcomes 3 times. b, Quantification of relative mitochondrial lengths between the MP and 2-BP treatments. Data normalized to the MP treatment. Corresponding non-normalized data are in panel c. Error bars in graph represent standard deviations from three independent experiments. P values were determined by unpaired two-sided Student’s t-test. N represents the number of cells analysed per condition (three independent experiments conducted). c, Quantification of mean mitochondrial area between the MP and 2-BP treatments. Corresponding normalized data are in panel b. Box and whisker plots with boxes including 50% (25–75%) and whiskers 80% (10–90%) of all measurements; line within box represents the median and outliers are shown as dots. P values were determined by unpaired two-sided Student’s t-test. N represents the number of cells analysed per condition (three independent experiments conducted). d, Western blot of HeLa cells knocked down for DRP1, confirming the efficiency of the used siRNA. Actin and GAPDH serve as loading controls. Source numerical data and unprocessed blots are available in source data. Experiment was repeated independently with similar outcomes 3 times.
Extended Data Fig. 8 Effects of Triacsin C and Etomoxir on mitochondria morphology.
(a) MEF wild-type cells treated with 5 µM Triacsin C or DMSO for 1 hr were fixed and stained for Tom20 as well as F-actin and Dapi. Boxed regions depict higher magnification images on the right. Scale bars, 20 µm in overview images and 5 µm in zooms. Box and whisker plot on the right showing mean mitochondrial area quantifications for both conditions. Box and whisker plots with boxes including 50% (25–75%) and whiskers 80% (10–90%) of all measurements; line within box represents the median and outliers are shown as dots. P values were determined by unpaired two-sided Student’s t-test. N represents the number of cells analysed per condition (three independent experiments).(b) HeLa cells treated with either 3 or 100 µM Etomoxir (or DMSO as a control) for 1 hr were fixed and stained for Tom20 as well as F-actin and Dapi. Boxed regions depict higher magnification images on the right. Scale bars, 20 µm in overview images and 5 µm in zooms. Box and whisker plot on the right showing mean mitochondrial area quantifications for respective conditions. Box and whisker plots with boxes including 50% (25–75%) and whiskers 80% (10–90%) of all measurements; line within box represents the median and outliers are shown as dots. P values were determined by one-sided ANOVA (Dunnett’s multiple comparisons) test. N represents the number of cells analysed per condition (two independent replicates). Source numerical data are available in source data.
Extended Data Fig. 9 Model for MiD function in activation of fatty acid oxidation.
Step 1. Long-chain fatty acid enters cytoplasm from the extracellular milieu (1 A) or from intracellular lipid droplets (1B). Step 2. Fatty acid is coupled to CoA through fatty acyl-CoA synthetase. Step 3. Fatty acyl-CoA binds to MiD49 and/or MiD51 monomers on the outer mitochondrial membrane (OMM), inducing their oligomerization. Step 4. Oligomerized MiD initiates assembly of active DRP1 oligomers on the OMM. Step 5. DRP1 oligomerization continues on the OMM to create a ring around the mitochondrion. This step might be facilitated (or require) Mff. Step 6. DRP1-mediated constriction of the OMM. Step 7. mitochondrial fission.
Supplementary information
Supplementary Information
Supplementary Video legends 1–4.
Supplementary Video 1
MiD49–GFP dynamics. HeLa cell co-transfected with MiD49–GFP (green) and Mito-Plum (red). Scale bar in whole cell is 10 µm and 3 µm in inset.
Supplementary Video 2
MiD51-WT GFP dynamics. HeLa cell co-transfected with MiD51–GFP (green) and Mito-Plum (red). Scale bar in whole cell is 10 µm and 3 µm in inset.
Supplementary Video 3
MiD51-Y185A GFP dynamics. HeLa cell co-transfected with MiD51-Y185A–GFP (green) and Mito-Plum (red). Scale bar in whole cell is 10 µm and 3 µm in inset.
Supplementary Video 4
MiD51-R342A GFP dynamics. HeLa cell co-transfected with MiD51-R342A–GFP (green) and Mito-Plum (red). Scale bar in whole cell is 10 µm and 3 µm in inset.
Source data
Statistical Source Data
Statistical source data.
Image Source Data
Unprocessed western blots and gels.
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Liu, A., Kage, F., Abdulkareem, A.F. et al. Fatty acyl-coenzyme A activates mitochondrial division through oligomerization of MiD49 and MiD51. Nat Cell Biol 26, 731–744 (2024). https://doi.org/10.1038/s41556-024-01400-3
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DOI: https://doi.org/10.1038/s41556-024-01400-3
