Article Figures & Data
Figures
- Figure 1. LETM1 is required for mitochondrial translation and respiration
A. Immunofluorescence analysis of HeLa cells transfected with either a non‐target dsRNA (NT) or one of the three siRNAs targeting LETM1 (siR1, siR2, or siR3) and labeled with anti‐TOM20 antibody. In siR1‐treated cells, the mitochondria formed a “honeycomb” of swollen distinct organelles; siR3 resulted in giant organelles with a central region distinguished by reduced TOM20; siR2 produced relatively little swelling, and the mitochondrial network was generally well preserved. The pronounced swelling induced by siR1 significantly increased circularity (P = 2.32E‐58; ImageJ analysis), siR1 circularity = 0.683 ± 0.012 compared with 0.414 ± 0.008 for the NT, where 1 = a perfect circle. Over 150 mitochondria were quantified in each cell type; data are mean ± SEM. Scale bar represents 12 μm in the main images and 4 μm in offset magnification.
B. 35S labeling of de novo mitochondrial protein synthesis of HeLa cells transfected as in (A). Polypeptide assignments flank the gel images. Coomassie‐stained gels are used as loading controls, and immunoblots indicate the efficiency of LETM1 knockdown.
C. Quantification of the radiolabeled mitochondrial polypeptides in panel (B) and similar gels, expressed relative to protein synthesis of the NT. Data are expressed as mean ± SEM of n = 6 independent experiments; 1 or 2 rounds of transfection for siR1 and siR3, but exclusively 2 rounds in the case of siR2.
D. Representative immunoblots for the OXPHOS proteins NDUFB8 and COII of HeLa cells transfected with NT or siLETM1 (siR1, siR2, or siR3). Vinculin and GAPDH are shown as loading controls. The mean relative abundances for respiratory subunits COII and NDUFB8 are shown beneath the blots. Data are expressed as mean ± SEM of n = 8 independent experiments.
E. Mitochondrial oxygen consumption rate (OCR) measured using a Seahorse flux analyzer before (basal) and after the addition (maximal) of the uncoupler FCCP, in HeLa cells treated with NT or siLETM1 (siR1 or siR2). Data are expressed as mean ± SEM of n = 5 independent experiments.
Source Data for Figure 1 [emmm201708550-sup-0006-SDataFig1.pdf]
- Figure 2. LETM1 depletion compromises mitochondrial ribosome maintenance and alters the abundance of mitochondrial DNA and RNAs
A. Steady‐state levels of mitochondrial ribosomal structural subunits (MRPL11 and MRPS17) or assembly factor (C7orf30) of HeLa cells transfected with siRNAs for either NT or LETM1 (siR1, siR2, or siR3). Vinculin and GAPDH are shown as loading controls. Data are expressed as mean ± SEM of n = 3 independent experiments for siR1 and siR2, and n = 2 (MRPL11) or 1 (MRPS17 and C7orf30) for siR3.
B. Relative 12S (upper panel) and 16S rRNA (lower panel) levels in HeLa cells after LETM1 siRNA compared to NT, n = 3 independent experiments.
C. Total lysates from HeLa cells treated with NT or a siRNA for LETM1 were separated on 100 mM NaCl, 10–30% isokinetic sucrose gradients, and fractions analyzed by immunoblotting with antibodies to components of the large (39S) and small (28S) subunit of the 55S ribosome. Immunoblots were quantified by ImageJ, and the value for each fraction was expressed as a percentage of the sum of all fractions. Data are expressed as mean ± SEM of n = 3 independent experiments.
D. Relative mtDNA copy number estimated by qPCR. Data are expressed as mean ± SEM of n = 3 independent experiments except for siR3 (n = 2).
E. The combined average mRNA levels of ND1, COII, COIII, Cyt b, and ATP6/8 in HeLa cells treated with either NT or siLETM1 (siR1, siR2, or siR3). Data are mean values ± SEM of n = 3 independent experiments.
Source Data for Figure 2 [emmm201708550-sup-0007-SDataFig2.pdf]
- Figure 3. LETM1 repression perturbs mtDNA and mtRNA organization
A. LETM1 expression was suppressed in HeLa cells by transfection with targeted siRNAs (siR1, siR2, or siR3). A non‐target dsRNA (NT) served as control. Cells were fixed and immunolabeled with anti‐DNA antibody (green). A higher magnification of selected mtDNA foci is shown beside each picture.
B. Quantification of cells in (A) displaying mtDNA abnormalities. At least 50 cells per siRNA were counted from 4 (siR2) and 5 (siR1 or siR3) independent experiments. Data are expressed as mean ± SEM. ***P < 0.001 (one‐way ANOVA).
C. HeLa cells were labeled with bromouridine (BrU) for 60 mins, 144 h after transfection with NT or siR2, and stained with anti‐BrdU (green) and anti‐GRSF1 (red) antibodies. Cell nuclei were stained with DAPI (blue).
D. HeLa cells were labeled with anti‐DNA (green) and anti‐GRSF1 (red) antibodies, 144 h after transfection with NT or siR3. Cell nuclei were stained blue with DAPI.
Source Data for Figure 3 [emmm201708550-sup-0008-SDataFig3.pdf]
- Figure EV1. LETM1 repression perturbs mtDNA organization
A. Further examples of the mtDNA abnormalities are shown in Fig 3A. LETM1 expression was suppressed in HeLa cells by transfection with targeted siRNAs (siR1, siR2, or siR3). A non‐target dsRNA (NT) served as control. Cells were fixed and immunolabeled with anti‐DNA antibody (green). Scale bar: 15 μm.
B. HeLa cells stained with anti‐LETM1 antibody (red) and anti‐BrdU (green) after labeling with 5 mM BrU for 60 min. In other images, LETM1 was stained green and other proteins stained red using antibodies to the RNA granule protein GRSF1, the 55S ribosome component MRPL45, or the outer mitochondrial membrane protein TOM20. Scale bars are 12 μm in the main images and 3 μm in offset magnification.
- Figure 4. LETM1 co‐fractionates with mitochondrial nucleoprotein complexes and is widely distributed in the mitochondrial network, like ribosomes and unlike mtDNA
A, B HeLa cells were immunostained with anti‐DNA (green) and anti‐LETM1 (red) (A), or anti‐LETM1 (green) and anti‐MRPS18 (red) (B).
C. Mitochondrial lysates from HEK293T cells were fractionated on 20–42.5% iodixanol gradients and the migration patterns of LETM1 and selected nucleoid components determined by immunoblotting.
D. HeLa mitochondrial lysates were fractionated on 10–30% sucrose gradients, and the distribution of LETM1 and ribosomal proteins was determined by immunoblotting. 55S corresponds to the assembled mitoribosome and 28S and 39S to its small and large subunits, respectively. Mitochondria were lysed in 100 or 50 mM NaCl (top and bottom panels, respectively).
Source Data for Figure 4 [emmm201708550-sup-0009-SDataFig4.pdf]
- Figure 5. Changes to DRP1 in response to LETM1 depletion§
A. Representative immunoblots of LETM1, DRP1 total protein levels, and activating phosphorylation (DRP1S616) in HeLa cells treated with either NT or LETM1 siR1, siR2, or siR3. GADPH is shown as a loading control. Immunoblot signals were quantified using ImageJ; data are displayed as mean ± SEM of n = 4 independent experiments except for siR3 DRP1S616 (n = 2).
B. One hour 35S pulse‐labeling of mitochondrial translated proteins in HeLa cells silenced for LETM1 (siR1) or DRP1 (siD), or co‐silenced for LETM1 and DRP1 (siR1D) for 144 h. Coomassie‐stained gels are used as loading controls, and polypeptide assignments are indicated to the right.
C. Immunoblotting of cell lysates prepared as in (B) with antibodies to respiratory chain subunits NDUFB8 and COII, and GAPDH as loading control. Data are expressed as mean ± SEM of n = 3 independent experiments.
D. Confocal analysis of HeLa cells silenced for LETM1 (siR1), or for DRP1 (siD), or co‐silenced with both siR1 and siD for 96 h. Mitochondrial network, mtDNA, and cell nuclei were immunostained with anti‐TOM20 (red) anti‐DNA (green), and DAPI (blue), respectively. Scale bars are 6 μm in the main images and 3 μm in offset merged magnifications.
Source Data for Figure 5 [emmm201708550-sup-0010-SDataFig5.pdf]
- Figure 6. LETM1 haploinsufficiency in WHS is associated with abnormal mitochondrial morphology and ultrastructure
A. Steady‐state levels of LETM1 protein in fibroblasts from controls (C1–C3), WHS LETM1+/− (S1, S3, S4), and S5, the sole case of WHS in this study whose deletion does not encompass LETM1 (boxed in blue). GAPDH is shown as a loading control.
B. Immunofluorescence analysis of control (C1), WHS LETM1+/− S1‐S4, and fibroblasts with anti‐TOM20 antibody. Scale bars are 8 μm in the main images and 3 μm in offset merged magnifications.
C. Electron micrographs of controls (C1 and C2) and WHS LETM1+/+ (S5) cells; side panels show zooms. Scale bar: 0.2 μm. Black arrows indicate the areas shown offset at higher magnification.
D. Electron micrographs of WHS LETM1+/− S1, S3, and S4 cells. Black arrows indicate elongated and branched mitochondria; side panels show magnifications. Scale bar: 0.2 μm.
Source data are available online for this figure.
Source Data for Figure 6 [emmm201708550-sup-0011-SDataFig6.pdf]
- Figure EV2. LETM1 haploinsufficiency in WHS is associated with mitochondrial ultrastructure abnormalities
A. Quantification of LETM1 expression in fibroblasts. Data from control cell lines (ctrls) C1–C3 were pooled. All data are expressed as mean ± SEM (n = 4 or 5). **P < 0.01, ***P < 0.001. ns, not statistically significant (one‐way ANOVA).
B. Magnification of WHS LETM1+/− S1 and S4 mitochondria. Black arrows indicate circular cristae and matrix distensions. Scale bar: 0.2 μm.
C. Magnification of control C3 and WHS LETM1+/− S4 mitochondria showing dense matrix and circular cristae exclusively in the latter. Scale bar: 0.2 μm.
D. Electron micrographs of WHS LETM1+/− (S2) fibroblasts. Black arrows indicate circular cristae in the upper 2 images and mitochondria with sparse cristae in the lower images. Scale bar: 0.2 μm.
- Figure 7. WHS LETM1+/− fibroblasts display increased mtDNA copy number and mtDNA clusters
A. Confocal analysis of control C2, WHS LETM1+/− S4, and WHS LETM1+/+ S5 cells, immunolabeled with anti‐TOM20 (red), DNA (green), and DAPI (blue). Scale bar: 8 μm.
B. Confocal images of control C1 and WHS LETM1+/− S3 labeled with TOM20, DNA, and DAPI, shown as merge. Scale bar: 8 μm.
C. Quantification of cells in (A) displaying mtDNA aggregates. At least 50 cells per cell line were counted from three independent experiments. Data are expressed as mean ± SEM.
D. qPCR quantified mtDNA copy number in WHS and control cells. Values are expressed relative to control cells (C1–C3). Data are expressed as mean ± SEM of n = 3 independent experiments.
E. Relative abundance of DRP1 and DRP1S616 in whole‐cell extracts of control and WHS fibroblasts, with GAPDH shown as a loading control. Immunoblot signals were quantified using ImageJ, and data are expressed as mean ± SEM of n = 5 independent experiments.
Source Data for Figure 7 [emmm201708550-sup-0012-SDataFig7.pdf]
- Figure 8. WHS LETM1+/− fibroblasts show reduced PDH phosphorylation and tolerate a strict ketogenic diet
A. Representative immunoblots of mitochondrial lysates of HEK cells separated on a 20–42.5% iodixanol gradients probed for LETM1, TFAM, and PDH.
B. Left panel: immunoblot analysis of total PDH protein levels and the inactive phosphorylated (repressed form) PDHS293, in control (C1, C3), WHS LETM1+/− (S1, S3, S4), and WHS LETM1+/+ (S5) fibroblasts. Center and right panels: Immunoblot analysis of PDK3 in control (C1–C3), WHS LETM1+/− (S1, S3, and S4), and WHS LETM1+/+ (S5) fibroblasts.
C. Left panel: change in cell confluence of control (C1–C3), WHS LETM1+/− (S1‐S4), and WHS LETM1+/+ (S5) fibroblasts grown in BHB over the course of 188 h. Data are expressed as mean ± SEM of n = 3 independent experiments. Right panel: images of control and WHS LETM1+/− S4 cells growing either in the presence of 25 mM glucose (HG) or BHB (KB) for 6 days.
D. Confocal images of control cells (C1) grown for 24 h in the presence of BHB and immunolabeled with anti‐TOM20 (red) and anti‐DNA (green), and DAPI‐stained (blue). Scale bar 6 μm.
E. Proportion of cells that displayed mtDNA aggregation during the KB treatment (panel i—control cells (C1, C2) and WHS LETM1+/+ (S5) fibroblasts; panel ii—LETM1+/− (S1, S2, S4) fibroblasts). At least 50 cells per cell line were counted at the indicated time points. Data are expressed as mean ± SEM of n = 3 independent experiments.
Source data are available online for this figure.
Source Data for Figure 8 [emmm201708550-sup-0013-SDataFig8.pdf]
- Figure EV3. PDH is repressed in WHS LETM1+/− fibroblasts, and unlike controls cells, they are able to grow on medium containing KB in place of glucose and pyruvate
A. Quantification of total PDH, PDHS293, and PDK3 immunoblots. Data are expressed as mean ± SEM of n = 5 independent experiments. *P < 0.05, **P < 0.01. ns, not statistically significant; t‐test with Welch's correction for PDH and PDHS293; one‐way ANOVA for PDK3.
B. Light microscope images of control (C2), WHS LETM1+/+ (S5), and WHS LETM1+/− (S1, S3, and S4) cells growing in 0.3 mM BHB (KB) for 6 days, after a switch from 25 mM glucose and 1 mM pyruvate at 40% confluence. Scale bar: 300 μm.
C. S4 cells grown to confluency in KB medium (as per panel A) were passaged and cultured for a further 6 days.
- Figure 9. Mitochondrial DNA re‐organization in response to nutrient changes
A. Proportion of mtDNA cluster following 48 or 96 h of growth in medium with (+) or without (−) 25 mM glucose (HG), 5 mM glucose (LG), β‐hydroxybutyrate (BHB), or 1 mM pyruvate (pyr). At least 50 cells per cell line were counted per condition from two independent experiments. Data are expressed as mean ± SEM. Representative images appear below the charts. Scale bar: 15 μm.
B. 35S labeling of de novo mitochondrial protein synthesis for 1 h, 72 h after transfection of HeLa cells with either a non‐target dsRNA (NT) or LETM1 siR1; KBs (0.1 mM BHB or acetoacetate (AA)) replaced HG (25 mM glucose, 1 mM pyruvate) 9 h before the labeling reaction. Polypeptide assignments flank the gel image; Coomassie‐stained gels are used as loading controls, and immunoblots indicate the efficiency of LETM1 knockdown.
C. Perceived or actual nutrient availability alters the organization of mitochondrial nucleoprotein complexes. In yeasts, mitochondrial ribosomes are physically linked to mitochondrial nucleoids (Kehrein et al, 2015), and purification studies of a mitochondrial ribosomal protein (Rorbach et al, 2008) and mitochondrial nucleoid proteins (He et al, 2012a) suggest that the same is true of mammalian mitochondria, as illustrated in (a). The current study indicates that changes in nutrient availability (a switch from glucose to ketone body supplemented culture medium) lead to re‐organization of mitochondrial nucleoprotein complexes (MNPCs; panel A and Fig 8D and E, and illustrated in (b)). We infer that MNPC disorganization in LETM1 deficiency is the result of a failure to utilize pyruvate—a defect in nutrient sensing—that can be attributed to repression of pyruvate dehydrogenase (PDH) via PDK3 (Figs 8B and EV3A). Because the bulk of PDH appears to be physically associated with MNPC (Fig 8A), this could be mediated by the LETM1 bound to mitochondrial ribosomes/MNPC (Fig 4C). The direct and marked impact of nutrients on MNPCs is further evidenced by the adaptation of WHS cells (LETM1+/−) to KBs (Figs 8E‐ii and EV3; and Appendix Figs S8 and S9), and the demonstration that KBs mitigate the mitochondrial translation impairment caused by LETM1 depletion (Fig 9B). Hence, WHS cells progressively adapt mtDNA organization to the alternative carbon source (c), whereas control cells are not primed for KB utilization, and in their case, MNPC clustering is followed by cell death (d) (Fig 8C and Movie EV1).
Source data are available online for this figure.
Source Data for Figure 9 [emmm201708550-sup-0014-SDataFig9.pdf]
Supplementary Materials
Expanded View Figures PDF [emmm201708550-sup-0002-EVFigs.pdf]
Movie EV1 [emmm201708550-sup-0003-MovieEV1.mp4]
Movie EV2 [emmm201708550-sup-0004-MovieEV2.mp4]
Movie Legends [emmm201708550-sup-0005-LegendsEV.pdf]
