An ER phospholipid hydrolase drives ER-associated mitochondrial constriction for fission and fusion
- Howard Hughes Medical Institute, United States
- Department of Molecular, Cellular and Developmental Biology, University of Colorado, United States
Figures
Figure 1 with 1 supplement
Identification of endoplasmic reticulum (ER)-localized lipid hydrolase, ABHD16A, by proximal proteomics.
(A) Cartoon diagram and domain organization of V5-TurboID-Mfn1 and V5-TurboID-Mfn1E209A (human Mfn1 and indicated amino acid numbers). V5-tagged TurboID was added to the N-terminus of Mfn1. Red E209A indicates the catalytically dead (CD) mutation created in the GTPase domain. TMD: transmembrane domain. (B) Representative images and insets of a HeLa cell expressing GFP-Mfn1 (green), mito-BFP (blue), and immunostained with antibody against the V5 tag (magenta, to detect Mfn1 constructs). Yellow arrows indicate GFP-Mfn1 puncta along mitochondria. (C) Cartoon diagram of the strategy used in HeLa cells to biotinylate ER-mitochondria membrane contact site (MCS) proteins with V5-TurboID-Mfn1 vs. V5-TurboID-Mfn1E209A. OMM: outer mitochondrial membrane (blue); IMM: inner mitochondrial membrane (light blue); Drp1: Dynamin-related protein 1 (fission machinery, pink); Mfn1: Mitofusin 1 (fusion machinery, orange); B: biotin. (D) Immunoblot analysis (anti-V5) shows relative expression of V5-TurboID-Mfn1 and V5-TurboID-Mfn1E209A in HeLa cells. (E) Immunoblot analyses of fractions collected by differential centrifugation including: 7000× g pellet containing mitochondria (TOM20, mitochondria), 20,000× g pellet containing light membrane and ER (Calnexin, ER), and supernatant containing cytosol (GAPDH, cytosol). (F) The 20,000× g pellet was solubilized and probed with streptavidin HRP to reveal biotinylation profiles for each sample prior to mass spectrometry. Asterisk denotes a band size indicative of construct self-biotinylation. (G) Average LFQ intensities of proteins biotinylated and enriched in Mfn1 wild type (WT) vs. Mfn1E209A sample in the 20,000× g pellet. Dashed line indicates equivalent enrichment in the WT vs. E209A mutant sample. Data from LFQ intensity are the average of two technical replicates. CANX: Calnexin; RTN4: Reticulon-4; MFN1: Mitofusin-1; OSBPL8: Oxysterol binding protein like 8; ABHD16A: Alpha/beta hydrolase domain containing phospholipase 16 A. (H) Cartoon diagram of human ABHD16A with motif or domain annotations. (I) Representative inset of a U-2 OS cell expressing low levels of ABHD16A-mNG (green), mCh-Sec61β (ER, blue), and mito-BFP (mitochondria, magenta). Arrows indicate ABHD16A localization to ER (blue) and mitochondria (magenta). Scale bar = 5 µm or 2 µm for insets. See Figure 1—source data 1, Figure 1—source data 2, Figure 1—source data 3, Figure 1—source data 4, Figure 1—source data 5, Figure 1—source data 6, Figure 1—source data 7, Figure 1—source data 8, Figure 1—source data 9, Figure 1—source data 10.
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Figure 1—source data 1
Related To Figure 1D.
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Figure 1—source data 2
Related to Figure 1E.
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Figure 1—source data 3
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Figure 1—source data 4
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Figure 1—source data 5
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Figure 1—source data 6
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Figure 1—source data 7
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Figure 1—source data 8
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Figure 1—source data 9
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Figure 1—source data 10
Related to Figure 1F.
- https://cdn.elifesciences.org/articles/84279/elife-84279-fig1-data10-v2.zip
Figure 1—figure supplement 1
Endogenous ABHD16A localizes to the endoplasmic reticulum (ER) and mitochondria, to a lesser extent (related to Figure 1).
(A) Representative image from Figure 1I of a U-2 OS cell expressing low levels of ABHD16A-mNG (green), mCh-Sec61β (ER, blue), and mito-BFP (mitochondria, magenta). White dashed box indicates inset in Figure 1I. (B) Immunoblot of various membrane markers after dounce homogenization, differential centrifugation, and Ficoll mitochondrial purification, yielding a crude mitochondrial fraction (prior to Ficoll spin), pure mitochondrial fraction (after Ficoll spin), mitochondrial associated membrane (MAM) fraction (after Ficoll spin), and cytosol (containing ER and other light membrane fractions). ER = Calnexin, calreticulin. Cytosol = GAPDH. Tom20, COXIV = mitochondria. (C) Top 50 protein candidates sorted by highest LFQ enrichment in Mfn1 wild type (WT) sample and low enrichment (zero) in Mfn1 E209A mutant sample. Scale bar = 5 µm. See Figure 1—figure supplement 1—source data 1, Figure 1—figure supplement 1—source data 2, Figure 1—figure supplement 1—source data 3, Figure 1—figure supplement 1—source data 4, Figure 1—figure supplement 1—source data 5, Figure 1—figure supplement 1—source data 6.
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Figure 1—figure supplement 1—source data 1
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Figure 1—figure supplement 1—source data 2
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Figure 1—figure supplement 1—source data 3
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Figure 1—figure supplement 1—source data 4
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Figure 1—figure supplement 1—source data 5
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Figure 1—figure supplement 1—source data 6
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Figure 2 with 1 supplement
ABHD16A is required for the formation of endoplasmic reticulum (ER)-associated fission and fusion hot spots.
(A) Representative images of U-2 OS cells transfected with GFP-Mfn1 (green), mito-BFP (magenta), mCh-Sec61β (ER, blue), and either control siRNA (n=33 cells, top), ABHD16A siRNA (n=33 cells, middle), or ABHD16A siRNA rescued with ABHD16A-Halo (n=37 cells, bottom). Yellow arrows indicate examples of Mfn1 puncta along mitochondria at ER-mitochondria crossings. (B) Representative images of U-2 OS cells transfected with mCh-Drp1 (green), mito-BFP (magenta), mNG-Sec61β (ER, blue), and either control siRNA (n=32 cells, top), ABHD16A siRNA (n=34 cells, middle), or ABHD16A siRNA rescued with ABHD16A-Halo (n=20 cells, bottom). Yellow arrows indicate examples of Drp1 puncta at ER-mitochondria crossings. (C) Representative images of U-2 OS cells transfected with mCh-Drp1 (magenta), GFP-Mfn1 (green), mito-BFP (not shown), and either control siRNA (n=29 cells, top), ABHD16A siRNA (n=31 cells, middle), or ABHD16A siRNA rescued with ABHD16A-Halo (n=25 cells, bottom). Yellow arrows indicate examples of nodes along mitochondria. (D) Quantification of Mfn1 density along mitochondrial length from experiments shown in (A). (E) Quantification of percent ER crossings containing Drp1 puncta from experiments shown in (B). (F) Quantification of node density along mitochondrial length from experiments shown in (C). All data were taken from three biological replicates; statistical significance was calculated by one-way ANOVA. ***p≤0.001. Scale bar = 2 µm. See Figure 2—source data 1.
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Figure 2—source data 1
Related to Figure 2D–F.
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Figure 2—figure supplement 1
ABHD16A depletion decreases fusion/fission node formation (related to Figure 2).
(A) Representative images of either empty vector (EV) (Halo-N1) or Halo-tagged expression constructs (magenta) were visualized using JF X 646 corresponding to cells from Figure 2A. (B) Representative images of either EV (Halo-N1) or Halo-tagged were expression constructs (magenta) visualized using JF X 646 corresponding to cells from Figure 2B. (C) Representative images of either EV (Halo-N1) or Halo-tagged expression constructs (magenta) were visualized using JF X 646 corresponding to cells from Figure 2C. (D) Representative immunoblot shows efficiency of depletion in U-2 OS cells from Figure 2 treated with control siRNA or ABHD16A siRNA and rescued with siRNA-resistant ABHD16A-Halo. GAPDH serves as a loading control. All data were taken from three biological replicates. Scale bar = 5 µm. See Figure 2—figure supplement 1—source data 1, Figure 2—figure supplement 1—source data 2, Figure 2—figure supplement 1—source data 3.
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Figure 2—figure supplement 1—source data 1
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Figure 2—figure supplement 1—source data 2
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Figure 2—figure supplement 1—source data 3
Related to Figure 2—figure supplement 1D.
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Figure 3 with 4 supplements
ABHD16A is required for efficient cycles of endoplasmic reticulum (ER)-mediated mitochondrial fission and fusion.
(A) Representative time lapse of a live U-2 OS cell over a 5 min movie expressing mMAPLE-OMP25 and control siRNA (n=30 cells). Top panel: green shows native mMAPLE-OMP25 signal, while magenta shows photoconverted (PC) mMAPLE-OMP25. Bottom panel: fire lookup table (LUT) of PC mMAPLE-OMP25 to show fusion event. Yellow box indicates region of interest (ROI) exposed to 405 nm light. Yellow arrow indicates photoconverted mitochondrion. (B) As in (A) for live U-2 OS cell expressing mMAPLE-OMP25 and ABHD16A siRNA (n=29 cells). Fire LUT and yellow arrow show no fusion occurring over the 5 min movie. (C) As in (A) for live U-2 OS cell expressing mMAPLE-OMP25, ABHD16A siRNA, and rescued with ABHD16A-Halo (n=31 cells). (D) Quantification of fusion rate per mitochondrion per minute from experiments shown in (A–C). (E) Quantification of fission rate per mitochondrion per minute from experiments shown in (A–C). (F) Quantification of the two types of fusion events per mitochondrion per minute from experiments shown in (A–D). Table displays total number of mitochondria, total fusion events, types of fusion events, and rates of fusion events (fusion/mitochondrion/minute). All data were taken from three biological replicates; statistical significance was calculated by one-way ANOVA. n.s., not significant; ***p≤0.001. Scale bar = 2 µm. See Figure 3—source data 1 and Figure 3—video 1, Figure 3—video 2, Figure 3—video 3.
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Figure 3—source data 1
Related to Figure 3D–F.
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Figure 3—figure supplement 1
ABHD16A depletion reduces fusion and fission rate (related to Figure 3).
(A) Cartoon demonstrating mitochondria labeled with mMAPLE-OMP25 (green) undergoing photoconversion upon 405 nm light exposure, converting mMAPLE to red (magenta). Upon fission or fusion, red fluorescence separates or diffuses. (B) Expanded table from Figure 3D for fusion rate per mitochondrion per minute from experiments shown in Figure 3A–C. (C) Expanded table from Figure 3E for fission rate per mitochondrion per minute from experiments shown in Figure 3A–C. All data were taken from three biological replicates; statistical significance was calculated by one-way ANOVA. n.s., not significant; ***p≤0.001.
Figure 3—video 1
siCTRL-treated U-2 OS cell labeled with photoconvertible mMAPLE-OMP25 from Figure 3A.
Representative video of a live U-2 OS cell over a 5 min movie expressing mMAPLE-OMP25 and control siRNA (n=30 cells).
Figure 3—video 2
ABHD16A siRNA treated U-2 OS cell labeled with photoconvertible mMAPLE-OMP25 from Figure 3B.
As in Figure 3—video 1 for live U-2 OS cell expressing mMAPLE-OMP25 and ABHD16A siRNA (n=29 cells).
Figure 3—video 3
ABHD16A siRNA treated U-2 OS cell co-transfected with wild type (WT) ABHD16A-Halo and mMAPLE-OMP25 from Figure 3C.
As in Figure 3—video 1 for live U-2 OS cell expressing mMAPLE-OMP25, ABHD16A siRNA, and rescued with ABHD16A-Halo (n=31 cells).
Figure 4 with 1 supplement
Endoplasmic reticulum (ER)-localized ABHD16A is required to maintain mitochondrial morphology.
(A) Domain organization of chimeric constructs for ER-localized ABHD16A (ABHD16AER) via Stim1’s N-terminal domain (NTD; left) or mitochondrial-localized ABHD16A (ABHD16Amito) via Tom70’s NTD (right). TMD: transmembrane domain. (B) Representative images of mitochondrial morphology (labeled by mito-BFP, gray) of U-2 OS cells transfected with control siRNA (siCTRL, n=41 cells, left) or ABHD16A siRNA (siABHD16A, n=36 cells, right). (C) Representative images of mitochondrial morphology (labeled by mito-BFP, gray) of U-2 OS cells transfected with ABHD16A siRNA and rescued with either siRNA-resistant ABHD16A-mNG (n=28 cells, left), ABHD16AER-mNG (n=36 cells, middle), or ABHD16Amito-mNG (n=34 cells, right). (D) Quantification of mean mitochondrial size (area per mitochondrion in µm2) within a 15×15 µm region of interest (ROI) from (B) and (C). (E) Immunoblot shows efficiency of depletion in cells treated with control siRNA or ABHD16A siRNA and rescued with siRNA-resistant ABHD16A-mNG constructs. GAPDH serves as a loading control. (F) Representative images of mitochondrial morphology of U-2 OS cells (labeled by mito-BFP, gray) and either empty vector (EV, control, n=32 cells), ABHD16A-mNG (n=40 cells), ABHD16AER-mNG (n=38 cells), or ABHD16Amito-mNG (n=34 cells) in green. (G) Quantification of mean mitochondrial size (area per mitochondrion in µm2) within a 15×15 µm ROI from (F). All data were taken from three biological replicates; statistical significance was calculated by one-way ANOVA. n.s., not significant; ***p≤0.001. Scale bar = 5 µm. See Figure 4—source data 1, Figure 4—source data 2, Figure 4—source data 3, Figure 4—source data 4.
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Figure 4—source data 1
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Figure 4—source data 2
Related to Figure 4E.
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Figure 4—source data 3
Related to Figure 4E.
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Figure 4—source data 4
Related to Figure 4E.
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Figure 4—figure supplement 1
ABHD16A is required to maintain mitochondrial morphology in HeLa cells (related to Figure 4).
(A) Representative images of empty vector (EV) constructs (green) corresponding to cells from Figure 4B. (B) Representative images of mNG-tagged expression constructs (green) corresponding to cells from Figure 4C. (C) Representative images of mitochondrial morphology (labeled by mito-BFP, gray) of HeLa cells transfected with control siRNA (n=39 cells, left), ABHD16A siRNA (n=40 cells, middle), or ABHD16A siRNA rescued with siRNA-resistant ABHD16A-mCherry construct (n=30 cells, right). (D) Immunoblot shows efficiency of depletion in HeLa cells from (C) treated with control siRNA or ABHD16A siRNA and rescued with siRNA-resistant ABHD16A-mCherry. GAPDH serves as a loading control. (E) Quantification of mean mitochondrial size (area per mitochondrion in µm2) within a 15×15 µm region of interest (ROI) from (C). (F) Representative mitochondrial morphology (labeled by mito-BFP, gray) of ABHD16A KO U-2 OS cell. Top inset (right) of mitochondria (mito-BFP, magenta) and endoplasmic reticulum (ER; mNG-Sec61β, green) displays the absence of mitochondrial constrictions. Bottom panels display Drp1 absence (mCh-Drp1, gray or green respectively) at ER (mNG-KDEL, blue)-mitochondria (mito-BFP, magenta) crossings. (G) Immunoblot shows deletion of ABHD16A in U-2 OS cells. GAPDH serves as a loading control. All data were taken from three biological replicates; statistical significance was calculated by one-way ANOVA. ***p≤0.001. Scale bar = 2 or 5 µm, respectively. See Figure 4—figure supplement 1—source data 1, Figure 4—figure supplement 1—source data 2, Figure 4—figure supplement 1—source data 3, Figure 4—figure supplement 1—source data 4, Figure 4—figure supplement 1—source data 5, Figure 4—figure supplement 1—source data 6, Figure 4—figure supplement 1—source data 7.
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Figure 4—figure supplement 1—source data 1
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Figure 4—figure supplement 1—source data 2
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Figure 4—figure supplement 1—source data 3
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Figure 4—figure supplement 1—source data 4
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Figure 4—figure supplement 1—source data 5
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Figure 4—figure supplement 1—source data 6
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Figure 4—figure supplement 1—source data 7
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Figure 5 with 1 supplement
ABHD16A is not required for endoplasmic reticulum (ER)-mitochondria membrane contact site (MCS) formation.
(A) Cartoon depiction of the ER-mitochondria dimerization-dependent fluorescent protein (ddFP) MCS reporter system: one monomer fused to an ER protein (RA-Sec61β) and the other monomer fused to an outer mitochondrial membrane (OMM) protein (B-Mff). When ER and mitochondria come within ~10–30 nm, dimers form and fluoresce red (false colored in green) at ER-mitochondria MCSs. (B) Representative images of ER-mitochondria MCSs in U-2 OS cells transfected with mNG-KDEL (ER, blue), mito-BFP (magenta), RA-Sec61β and B-Mff (ER-mitochondria ddFP MCS pair, green), and either control siRNA (n=24 cells, top), ABHD16A siRNA (n=29 cells, middle), or ABHD16A siRNA rescued with ABHD16A-Halo (n=27 cells, bottom). (C) Quantification of percentage of ER-mitochondria overlap over total mitochondrial area calculated by Mander’s correlation coefficient (MCC) of two binary images taken from 20×20 µm region of interests (ROIs) from images in (B). (D) Quantification of percentage of ddFP-mitochondria overlap over total mitochondrial area calculated by MCC of two binary images taken from 20×20 µm ROIs from images in (B). All data were taken from three biological replicates; statistical significance was calculated by one-way ANOVA. n.s., not significant; ***p≤0.001. Scale bar = 2 µm. See Figure 5—source data 1.
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Figure 5—source data 1
Related to Figure 5C and D.
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Figure 5—figure supplement 1
ABHD16A is not required for endoplasmic reticulum (ER)-mitochondria membrane contact site (MCS) formation (related to Figure 5).
(A) Representative images of either empty vector (EV; SNAP-N1) or SNAP-tagged expression constructs (magenta) were visualized using JF 646, SE corresponding to cells from Figure 5B. (B) Representative immunoblot shows efficiency of depletion in U-2 OS cells from Figure 5B treated with control siRNA or ABHD16A siRNA and rescued with siRNA-resistant ABHD16A-SNAP. GAPDH serves as a loading control. Scale bar = 5 µm. See Figure 5—figure supplement 1—source data 1, Figure 5—figure supplement 1—source data 2, Figure 5—figure supplement 1—source data 3.
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Figure 5—figure supplement 1—source data 1
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Figure 5—figure supplement 1—source data 2
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Figure 5—figure supplement 1—source data 3
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Figure 6 with 1 supplement
Uncoupling ABHD16A’s enzymatic requirements during endoplasmic reticulum (ER)-associated mitochondrial constriction, fission, and fusion.
(A) Representative images and line scans (yellow) of ER-mitochondria crossings in U-2 OS cells transfected with mNG-Sec61β (ER, green), mito-BFP (gray, left; magenta, middle) and either control siRNA (n=30 cells), ABHD16A siRNA (n=29 cells), or ABHD16A siRNA rescued with either wild type (WT) ABHD16A (n=27 cells), the AT mut (acyltransferase motif mutant, n=37 cells), or the α/βHD mut (alpha/beta hydrolase domain mutant, n=26 cells) mNG fusion constructs. A representative line scan (yellow, moved aside for visualization purposes) along a mitochondrion (mito-BFP) shows spatial correlation between constrictions (marked by dips in fluorescence intensity, magenta) and ER crossings (green). Orange and purple arrows mark ER crossings with and without constrictions, respectively. (B) ABHD16A domain organization with indicated amino acids for each motif or domain (top). Mutations are indicated in red for either the AT or α/βHD mutants. (C) Quantification of resolvable ER crossings coincident with a mitochondrial constriction per cell from (A). (D) As in Figure 2B, representative images of U-2 OS cells transfected with mCh-Drp1 (green), mito-BFP (magenta), mNG-Sec61β (ER, blue), and ABHD16A siRNA rescued with either the Halo-tagged ABHD16A AT mut (n=24 cells, top) or α/βHD mut (n=25 cells, bottom). Yellow arrows indicate examples of Drp1 puncta at ER-mitochondria crossings. (E) As in Figure 2A, representative images of U-2 OS cells transfected with GFP-Mfn1 (green), mito-BFP (magenta), mCh-Sec61β (ER, blue), and ABHD16A siRNA rescued with either the Halo-tagged ABHD16A AT mut (n=33 cells, top) or α/βHD mut (n=28 cells, bottom). Yellow arrows indicate examples of Mfn1 puncta along mitochondria at ER-mitochondria crossings. (F) As in Figure 2E, quantification of percent ER crossings containing Drp1 puncta from experiments shown in (D) and Figure 2B. (G) As in Figure 2D, quantification of Mfn1 density along mitochondrial length from experiments shown in (E) and Figure 2A. All data were taken from three biological replicates; statistical significance was calculated by one-way ANOVA. n.s., not significant; ***p≤0.001. Scale bar = 2 µm. See Figure 6—source data 1.
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Figure 6—source data 1
Related to Figure 6C, F and G.
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Figure 6—figure supplement 1
ABHD16A’s acyltransferase motif and alpha/beta hydrolase domain are required for mitochondrial morphology (related to Figure 6).
(A) Representative z-series and line scans (yellow) of endoplasmic reticulum (ER)-mitochondria crossings in U-2 OS cell from Figure 6A transfected with mCh-Sec61β (ER, green), mito-BFP (gray, top; magenta, bottom), and an empty vector (EV) (not shown). Images display examples of mitochondrial focal planes chosen for line scan analysis (z=0 µm). Constrictions at ER crossings are indicated by orange arrows. ER-mitochondria crossings containing no constrictions are indicated by purple arrows. (B) Representative images of either EV (mNG-N1) or mNG-tagged expression constructs (green) corresponding to cells from Figure 6A. (C) Representative immunoblot shows efficiency of depletion in U-2 OS cells from Figure 6A and (G) treated with control siRNA or ABHD16A siRNA and rescued with siRNA-resistant ABHD16A-mNG constructs. GAPDH serves as a loading control. (D) Representative images of Halo-tagged expression constructs (magenta) were visualized using JF X 646 corresponding to cells from Figure 6D. (E) Representative images of Halo-tagged expression constructs (magenta) were visualized using JF X 646 corresponding to cells from Figure 6E. (F) Representative images of mitochondrial morphology (labeled by mito-BFP, gray) of U-2 OS cells transfected with ABHD16A siRNA and either the acyltransferase motif mutant (AT mut, n=29 cells, top) or the alpha/beta hydrolase domain mutant (α/βHD mut, n=28 cells, bottom). (G) Quantification of mean mitochondrial size (area per mitochondrion in µm2) within a 15×15 µm region of interest (ROI) from (F), Figure 4D, and from cells transfected with ABHD16A siRNA and mNG-tagged lipase-like motifs mutant (LLM mut, S176AS306A, n=19 cells). (H) Representative immunoblot shows efficiency of depletion in U-2 OS cells from Figure 6D and E treated with control siRNA or ABHD16A siRNA and rescued with siRNA-resistant ABHD16A-Halo constructs. GAPDH serves as a loading control. All data were taken from three biological replicates; statistical significance was calculated by one-way ANOVA. n.s., not significant; ***p≤0.001. Scale bar = 2 µm and 5 µm, respectively. Figure 6—figure supplement 1—source data 1, Figure 6—figure supplement 1—source data 2, Figure 6—figure supplement 1—source data 3, Figure 6—figure supplement 1—source data 4, Figure 6—figure supplement 1—source data 5, Figure 6—figure supplement 1—source data 6, Figure 6—figure supplement 1—source data 7.
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Figure 6—figure supplement 1—source data 1
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Figure 6—figure supplement 1—source data 2
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Figure 6—figure supplement 1—source data 3
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Figure 6—figure supplement 1—source data 4
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Figure 6—figure supplement 1—source data 5
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Figure 6—figure supplement 1—source data 6
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Figure 6—figure supplement 1—source data 7
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- https://cdn.elifesciences.org/articles/84279/elife-84279-fig6-figsupp1-data7-v2.zip
Figure 7
ORP8 is required to deliver ABHD16A-induced altered phospholipids to mitochondria.
(A) Representative images of mitochondrial morphology (labeled by mito-BFP, gray) of U-2 OS cells transfected with empty vector (EV) (mCherry-N1, magenta) and with either siCTRL and mNG-C1 EV (green, n=28 cells, left); OSBPL8 siRNA and mNG-C1 EV (green, n=21 cells, middle); or OSBPL8 siRNA and siRNA-resistant mNG-ORP8 (green, n=41 cells, right). (B) Representative images of mitochondrial morphology (labeled by mito-BFP, gray) of U-2 OS cells transfected with ABHD16A-mCherry overexpression (OE; magenta) and either siCTRL and mNG-C1 EV (green, n=35 cells, left); OSBPL8 siRNA with mNG-C1 EV (green, n=37 cells, middle); or OSBPL8 siRNA with siRNA-resistant mNG-ORP8 (green, n=37 cells, right). (C) Representative images of mitochondrial morphology (labeled by mito-BFP, gray) of U-2 OS cells transfected with either EV (mCherry-N1, magenta) and siCTRL (n=28 cells); EV and VPS13A/D siRNA (n=30 cells); with ABHD16A-mCherry OE (magenta) and siCTRL (n=36 cells); or with ABHD16A-mCherry OE (magenta) and VPS13A/D siRNA (n=29 cells). (D) Quantification of mean mitochondrial size (area per mitochondrion in µm2) within a 15×15 µm region of interest (ROI) from (A) and (B). (E) Quantification of mean mitochondrial size (area per mitochondrion in µm2) within a 15×15 µm ROI from (C). (F) Representative immunoblot shows efficiency of depletion in U-2 OS cells from (A) and (B) treated with control siRNA or OSBPL8 siRNA and rescued with siRNA-resistant mNG-ORP8. GAPDH serves as a loading control. Asterisk indicates non-specific band. (G) Representative immunoblot shows efficiency of depletion in U-2 OS cells from (C) treated with control siRNA or VPS13A/D siRNA. GAPDH serves as a loading control. All data were taken from three biological replicates; statistical significance was calculated by one-way ANOVA. n.s., not significant; ***p≤0.001. Scale bar = 5 µm. See Figure 7—source data 1, Figure 7—source data 2, Figure 7—source data 3, Figure 7—source data 4, Figure 7—source data 5, Figure 7—source data 6, Figure 7—source data 7, Figure 7—source data 8, Figure 7—source data 9.
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Figure 7—source data 1
Related to Figure 7D and F.
- https://cdn.elifesciences.org/articles/84279/elife-84279-fig7-data1-v2.xlsx
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Figure 7—source data 2
Related to Figure 7F.
- https://cdn.elifesciences.org/articles/84279/elife-84279-fig7-data2-v2.pdf
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Figure 7—source data 3
Related to Figure 7G.
- https://cdn.elifesciences.org/articles/84279/elife-84279-fig7-data3-v2.pdf
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Figure 7—source data 4
Related to Figure 7F.
- https://cdn.elifesciences.org/articles/84279/elife-84279-fig7-data4-v2.zip
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Figure 7—source data 5
Related to Figure 7F.
- https://cdn.elifesciences.org/articles/84279/elife-84279-fig7-data5-v2.zip
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Figure 7—source data 6
Related to Figure 7F.
- https://cdn.elifesciences.org/articles/84279/elife-84279-fig7-data6-v2.zip
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Figure 7—source data 7
Related to Figure 7F.
- https://cdn.elifesciences.org/articles/84279/elife-84279-fig7-data7-v2.zip
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Figure 7—source data 8
Related to Figure 7G.
- https://cdn.elifesciences.org/articles/84279/elife-84279-fig7-data8-v2.zip
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Figure 7—source data 9
Related to Figure 7G.
- https://cdn.elifesciences.org/articles/84279/elife-84279-fig7-data9-v2.zip
Figure 8
Model for how ABHD16A promotes both lysophospholipid and phospholipid formation required for positive and negative membrane curvature to support efficient cycles of fission and fusion.
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An ER phospholipid hydrolase drives ER-associated mitochondrial constriction for fission and fusion
eLife 11:e84279.
https://doi.org/10.7554/eLife.84279
