EMBO/SourceData huggingface dataset:
Figure 8Confocal whole-mount immunofluorescence analysis of phosphorylated Akt (pAkt) (A) and phosphorylated p44/42 MAPK (pErk1/2) (B) in Kit+ cells of wild type (32-36sp) and Sl/Sl (31-35sp) E10.5 dorsal aorta, yolk sac and fetal liver. Images are 2.5μm-thick single longitudinal Z slices; dorsal up (DA). Arrowheads point to Kit+ cells with varying levels of pAkt and pErk1/2 expression. A total of 2 wild type and 2 Sl/Sl embryos for each staining were analyzed. Scale bars: 20μm. Quantification of the immunofluorescence in (A, B), showing mean fluorescence intensity (MFI) ratio of pAkt and p44/42 MAPK (pErk1/2) within wild type (+/+) and Sl/Sl CD31+ Kit+ dorsal aorta (DA), yolk sac (YS) and fetal liver (FL) hematopoietic cells. MFI values were normalized to background in the adjacent ventral mesenchyme for DA, to erythrocytes in the YS or stroma in the FL. Additional quantification was performed on sections from 2 wild type and 2 Sl/Sl embryos of similar age range. MFI measurements were taken using ImageJ. Number of cells measured for pAkt: 100 hematopoietic cells and 73 background (+/+); 79 hematopoietic and 52 background (Sl/Sl) in the AGM; 127 hematopoietic cells and 95 background (+/+); 110 hematopoietic and 82 background (Sl/Sl) in the YS; 102 hematopoietic cells and 78 background (+/+); 78 hematopoietic and 83 background (Sl/Sl) in the FL. Number of cells measured for pErk1/2: 94 hematopoietic cells and 82 background (+/+); 114 hematopoietic and 91 background (Sl/Sl) in the AGM; 174 hematopoietic cells and 109 background (+/+); 123 hematopoietic and 82 background (Sl/Sl) in the YS; 100 hematopoietic cells and 70 background (+/+); 63 hematopoietic and 57 background (Sl/Sl) in the FL. N=4 (+/+), 4 (Sl/Sl) embryos analyzed for DA and YS; N=3 (+/+), 2 (Sl/Sl) for FL. Error bars represent SD.Multiplex Fluidigm qRT-PCR analysis of pre-HSC type I (Ter119- VE-Cadherin+ Kit+ CD41+ CD45-), pre-HSC type II/HSC (Ter119- VE-Cadherin+ Kit+ CD41+ CD45+), YS and FL EMP (Ter119- Kit+ CD41+ CD16/32+), FL Early E (Ter119- Kit+ CD41-) and peripheral blood LMPP (Lin- CD19- B220- CD45+ Kit+ Flt3+ IL7Rα+), isolated from E11.5 (11-17 tail somites) wild type and Sl/Sl embryos. 4 biological replicates for wild type (9 total embryos) and 5 biological replicates (9 total embryos) for Sl/Sl were analyzed. Data are mean (±SD).
Figure 1A. Confocal microscopy image of PD myotubes (cl. 3LE8) infected for 72 h with adenovirus containing TFEB (PD + Ad-TFEB 72 h) shows a dramatic reduction in the number of large LAMP1-positive lysosomes (red) compared to that in untreated (PD) or adenovirus (PD + Ad-null)-treated PD myotubes. WT myotubes are shown on the left panel. Nuclei are stained with Hoechst (blue). TFEB was detected with anti-Flag antibody (green).B. Distribution of lysosomal size differs significantly in Ad-null and Ad-TFEB PD myotubes (p = 6.32 × 10−8; Kolmogorov-Smirnov test). Lysosomal size is expressed as number of pixels representing lysosomal area (LAMP1-positive structures). The median lysosomal size of Ad-TFEB infected myotubes (m = 367.13 pixels, n = 703, range 208-2659) was significantly lower than that of Ad-null infected myotubes (m = 491.16 pixels, n = 1395, range 200-2857; p = 6.5 × 10−12; Wilcoxon rank sum test).C. Confocal microscopy image of PD myotubes infected for 24 h with Ad-TFEB shows relocation of lysosomes to the plasma membrane (top). Images showing LAMP1 staining (red) on plasma membrane in a PD myotube infected with Ad-TFEB (bottom; arrows) but not in a non-infected cell (middle). Non-permeabilized cells were incubated with anti-LAMP1 antibody at 4°C for 40 min, followed by fixation and staining with secondary antibody.D. Confocal microscopy images of live non-infected PD myotubes (left) or PD myotubes infected for 72 h with Ad-TFEB (right) show a dramatic reduction in the amount of accumulated glycogen in TFEB-treated cells. The cells were incubated with the fluorescent glucose (2-NBDG; green), extensively washed, and analysed by confocal microscopy. Bar: 10 µm for all panels.
Figure 2Confocal microscopy of live unstained fibres from a GFP-LC3:WT mouse (WT) and from untreated (GAA−/−) or ERT-treated (GAA−/−; +ERT) GFP-LC3:GAA−/− mice. Images show typical centrally located autophagic build-up with multiple clusters of LC3-positive autophagosomes. Structures like these are never seen in WT muscle. Autophagic build-up is present virtually in all fibres derived from EDL or gastrocnemiusmuscles (not shown), but only in 60-70% of FDBfibres. Labelled recombinant human GAA (red) was administered i.v. into 3-4 month-old GFP-LC3:GAA−/− mice (n = 5) at a dose of 100 mg/kg twice with a 24 h interval. Mice were sacrificed the next day, and live fibres (shown for EDLmuscle) were analysed by confocal microscopy. The labelled recombinant GAA was detected almost exclusively in the autophagic vesicles. At least 500 fibres were analysed for each condition. Bar: 10 µm.
Figure 3A. Immunostaining of non-infected cells (day 13 in differentiation medium) and cells infected with a mutant form of TFEB (TFEBmt) with LAMP1 (red) and Flag (green). Ad-TFEBmt was added to the myotubes for 72 h on day 10 in differentiation medium. Ad-TFEBmt-infected cells show massive accumulation of TFEB in the nuclei and significant reduction in lysosomal size, similar to that seen with TFEB.B. Western blot of cell lysates confirms the presence of TFEB in the nuclear fraction; the different intensities of the two bands corresponding to LAMP1 protein in untreated and TFEBmt-treated samples may reflect the differences in the glycosylation pattern.C. Western blot of protein lysates from untreated, Ad-null, TFEB, and TFEBmt-treated myotubes with anti-caspase-3 antibody. No activated (cleaved) products are detected in any condition. α-Tubulin was used as a loading control.D. TUNEL assay shows the presence of some apoptotic cells in Ad-TFEB-infected cultures, but not in control cultures infected with adenovirus alone (n = 3). Bar: 10 µm for all panels.
Figure 4A. Confocal microscopy images of live fibres derived from 3 to 4 month-old GFP-LC3:WT (top left), untreated GFP-LC3:GAA−/− (bottom left) or TFEB-treated GFP-LC3:GAA−/− (right) mice. All fibres were transfected with mCherry-LAMP1 to visualize lysosomes (red). The effects of TFEB are clearly visible - overall reduction in lysosomal size, appearance of normal size lysosomes (similar to those in the WT), and lysosomal docking to the plasma membrane (inset). Bar: 10 µm.B. FDBmuscle of a GAA−/− mouse was transfected with both GFP-TFEB and mCherry-LAMP1 (GAA−/− + GFP-TFEB). Images were taken before (left) and after 4 h (right) of time-lapse microscopy. Lysosomal clearance is visible in the TFEB-transfected fibre at both time points. Lysosomes appear to "exit" the fibre (inset and arrow) at the 4 h time point when TFEB is activated as evidenced by its nuclear translocation (green nuclei). Bar: 10 µm.C. The mean maximum velocity of lysosomes (top) and the number of large (>3.5 µm) lysosomes (bottom) in untreated and TFEB-treated PD fibres (note: all data for Flag- and GFP-TFEB-treated fibres are pooled). Lysosomal velocities were calculated from time-lapse images using ImageJ software with the manual tracking plug-in. For each condition the trajectories of multiple lysosomes were followed (n = 26 untreated; n = 43 TFEB-treated) and the three highest velocity measurements per lysosome were recorded. In TFEB-treated fibres, the maximum velocity of lysosomes was significantly increased (p = 2.07 × 10−17) and the number of large lysosomes was significantly decreased (p = 1.0 × 10−3). Ten untreated and 24 TFEB-treated fibres were analysed for the size calculations. * indicates statistically significant differences (p ≤ 0.001; Student's t-test). Error bars represent 95% confidence intervals.
Figure 5Confocal microscopy images of live fibres from GFP-LC3:GAA−/− mice.Muscle was transfected with mCherry-LAMP1 only. The image (a single frame from the time-lapse series presented in Movie 5) shows lysosomes (red), LC3-positive autophagosomes (green) and a number of autolysosomes (yellow).Muscle was transfected with Flag-TFEB and mCherry-LAMP1. Massive formation of autolysosomes is indicated by yellow structures; the three lower panels provide a snapshot of the process of exocytosis. The structures at the plasma membrane are labelled with both LC3 and LAMP1, indicating that they represent amphisomes (a product of fusion between autophagic vesicles and late endosomes) or autolysosomes (a product of fusion between autophagic vesicles and lysosomes). Bar: 10 µm for all panels.
Figure 6Confocal microscopy images of live fibres from muscle-specific autophagy-deficient GAA−/− mice (Atg7:GAA DKO).Muscle was transfected with mCherry-LAMP1 only.Muscle was transfected with GFP-TFEB and mCherry-LAMP1 (Atg7:GAA DKO +GFP-TFEB). The TFEB-transfected fibres show realignment of the lysosomes and membrane detachment (most striking in top and bottom panels) similar to those in TFEB-transfected fibres from PD mice (see Fig 4B inset). Lysosomes can be seen in the space between the fibre and plasma membrane (arrows). Bar: 10 µm for all panels.The mean maximum velocity of lysosomes (top) and the number of large (>3.5 µm) lysosomes (bottom) in untreated and TFEB-treated autophagy-deficient Atg7:GAA DKO fibres. The increase in maximum velocity (41%) is significant (p = 2.729 × 10−18; n = 57 lysosomes for untreated; n = 52 lysosomes for TFEB-treated), but there is only a slight trend toward smaller lysosomal size (p = 7.0 × 10−2; n = 10 fibres for untreated; n = 21 fibres for TFEB-treated). The corresponding values from GAA−/− mice are presented for comparison (for TFEB-treated condition: n = 19 lysosomes for velocity measurements, and n = 10 fibres for the lysosomal size measurements). * indicates statistically significant differences (p ≤ 1.0 × 10−5 for the top panel and p ≤ 0.01 for the lower panel; Student's t-test).
Figure 7A. Glycogen assay in TFEB-injected gastrocnemii and in the contralateral muscles. In TFEB-injected musclesglycogen levels were significantly decreased compared to those in untreated muscles. * indicates statistically significant differences (p = 1.0 × 10−4; n = 6; Student's t-test).B. PAS staining of TFEB-treated muscle shows a reduction of lysosomalglycogen stores (puncta) compared to those in untreated muscle. Original magnification: 20×.C. LAMP1 staining of TFEB-injected gastrocnemii and of the contralateral untreated muscles. In TFEB-treated muscles, the size of LAMP1-positive vesicles was reduced. Bar: 2 µm.
Figure 8A. EM images of muscle injected with either AAV-GFP (untreated) or AAV-TFEB (TFEB-treated). Asterisks indicate glycogen-containing lysosomes. Bar: 1.5 µm (upper panels) and 0.45 µm (lower panels). Higher magnification images (lower panels) show that glycogen particles are less densely packed in TFEB-treated muscle. Black arrows indicate autophagosome profiles; the white empty arrow shows remnants of mitochondria engulfed by the lysosome.B. Graphical presentations of lysosomal length (average ± SE; n = 100 lysosomes; p = 6.31 × 10−5), the number of lysosomes per 5 µm2 area of muscle fibre section (average ± SE; n = 50 fields; p = 4.80 × 10−3), and the number of autophagosomes flanking glycogen-containing lysosomes (average ± SE; n = 100 lysosomes; p = 4.39 × 10−5 < 0.001). Student's t-test was used for each comparison.C. Graphical presentations of mitochondrial size (average ± SE; n = 100) and the number of mitochondria per 5 µm2 area of muscle fibre section (average ± SE; n = 50 fields). The differences were not significant by Student's t-test (p = 3.65 × 10−1 and 4.27 × 10−1, respectively).
Figure 1B Sequencing chromatograms depicting the identified 22 nucleotide deletion (c.346_*7del) identified in Subject 1 (S1), the heterozygous state of the deletion in the father, and the wild-type NDUFC2 sequence in Control DNA.C Sequencing chromatograms depicting the identified missense variant (c.173A>T; p.(His58Leu)) in Subject 3 (S3) and the wild-type NDUFC2 sequence in Control DNA.
Figure 2A NDUFC2 mRNA transcript levels in fibroblast cell lines of controls (white), Subject 1 (grey) and Subject 3 (black). Quantification of NDUFC2 mRNA transcript levels (normalised to ACTB) showed diminished transcript levels in Subject 1 while transcript levels were unaffected in Subject 3. Bars correspond to standard deviations of two independent experiments, performed in duplicate and analysed by Student's t-test; *p<0.05.B Assessment of respiratory chain enzyme activities, normalised to citrate synthase (CS), in fibroblast cell lines from controls (white), Subject 1 (grey) and Subject 3 (black). Mean residual enzyme activities of control fibroblasts (n = 8) are set at 100% and error bars represent 1 standard deviation of control activities. Asterisks (*) denote enzyme activity below control range.C Assessment of respiratory chain enzyme activities, normalised to citrate synthase (CS), in skeletal muscle from controls (white) and Subject 3 (black). Mean residual enzyme activities of control skeletal muscle (n>100 samples) are set at 100% and bars are 1 standard deviation of control activities. Asterisks (*) denote enzyme activity below control range.D Respiration of Subject 3 and Subject 1 derived fibroblasts (black and grey, respectively) compared to control fibroblasts (white) using high-resolution respirometry. ROX-corrected analysis of basal respiration, Oligomycin inhibited LEAK respiration and maximum uncoupled respiration (ETS). Both subject fibroblasts exhibited a severe mitochondrial respiration defect as measured by oxygen consumption. Data are mean ± SD from at least 3 experiments and analysed by Student's t-test. **p<0.01, ***p<0.001.
Figure 3A Western blot analysis of various structural subunits of complex I in fibroblasts from Subjects 1 and 3 (S1 and S3 respectively) with three age-matched controls (C1, C2, and C3).B BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria from subject and control fibroblasts solubilised using DDM. Immunoblotting was performed using antibodies to structural subunits from each OXPHOS complex (CI [NDUFB8], CII [SDHA], CIII [UQCRC2], CIV [MT-CO1], and CV [ATP5A]).C Western blot analysis of whole cell lysates from controls (C1 and C2) and subject cell lines (S1 and S3) that were either untransduced (-) or transduced with the lentiviral vector (pLVX) containing wild-type NDUFC2 (+). Transduced cell lines were either uninduced or induced with 100 or 200 ng/ml doxycycline (for 48 and 96 hours respectively) where indicated.D BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria isolated from controls (C1 and C2) and subject cell lines that were either untransduced (-) or transduced with the lentiviral vector (pLVX) containing wild-type NDUFC2 (+). Transduced cell lines were either uninduced or induced with 200 ng/ml doxycycline for 72 hours.
Figure 4A Complexome profiling identified assembly intermediates in subject cells with NDUFC2 variants. Complexes from mitochondrial membranes of Control, Subject 1 and Subject 3 fibroblasts were separated by BN-PAGE (Fig EV2) and analysed by complexome profiling. Sample preparation, mass spectrometry, data processing and raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Vizcaíno et al., 2013) with the dataset identifier <PXD014936>. Assignment of complexes: III, complex III; IV, complex IV; III/IV, supercomplex containing dimer of complex III and 1-2 copies of complex IV; S, supercomplex containing complex I, dimer of complex III and 1-4 copies of complex IV. Assembly intermediates are highlighted in dashed boxes indicating stalled complex I assembly. In subject complexomes, various stalled complex I intermediates are assembled: ND4 module (blue dashed box); Q-TIMMDC1-NDUFA13 complex I intermediate (orange dashed box); and Q-TIMMDC1-NDUFA13-MCIA (green dashed box). In control complexome, the Q module (orange dashed box) is assembled without TIMMDC1 or NDUFA13. Complex I modules are denoted according to Stroud and colleagues (Stroud et al., 2016).B Abundance data for all the subunits of complex I were averaged to plot an abundance profile of complex I. Heatmap (upper panel) and profile plot (lower panel) compare complex I between control (black), Subject 1 (red) and Subject 3 (blue). C Profile plot of complex III (average of all subunits). Black arrows indicate shifted complex III to the position of the individual complex and small supercomplex (III/IV) in Subjects 1 and 3. D Profile plot of complex IV (average of all subunits) indicate less complex IV at the position of the supercomplexes for Subject 1 and Subject 3, but higher amounts of individual complex IV and small supercomplex (III/IV, black arrow). E Details of Q module-containing assembly intermediates of Subject 3. Intensity based absolute quantification (iBAQ) values of assembly factors, single subunit and averaged Q-module subunits were plotted to show abundance profiles. The cartoon (lower panel) indicate assembled intermediate complexes relative to the plot.F Details about ND4 module assembly intermediate of Subject 3. Abundance profile of averaged iBAQ values of ND4 subunits and assembly factors show accumulation of a complex containing the ND4 module, FOXRED1 and TMEM70.
Figure 1(D) Top: Schematic of GFP expression reporter system, with the CBASS promoter, capH, and capP genes from E. coli MS115-1 and the CBASS core genes replaced with GFP. Bottom: Western blot showing GFP expression in cells with the wild-type GFP reporter or constructs lacking either capP or capH genes. RNAP: RNA polymerase loading control.(E) Western blots of the CBASS expression reporter system with FLAG-NucC showing FLAG-NucC expression after infection with phage λ cI- (multiplicity of infection: 10). MPI: minutes post infection. α-RNAP: anti-RNA polymerase loading control. Low RNAP expression at later time points is due to cell death.
Figure 2(B) Fluorescence polarization assay showing binding of E. coli MS115-1 CapH (His6-MBP tagged) to three 40-bp DNAs: 1-40 (black circles), 31-70 (Site 1; green squares), and 81-120 (Site 2; orange triangles). Fit Kd and Hill coefficient for each DNA is shown. Error bars indicate standard deviation from three technical replicates.(D) Fluorescence polarization assay showing binding of E. coli MS115-1 CapH (His6-MBP tagged; wild-type or indicated point mutants) to the Site 2 DNA (bases 81-120 in panel A). Fit Kd and Hill coefficient for each DNA is shown.(E) GFP expression reporter assay showing loss of suppression upon mutation of CapH. RNAP: RNA polymerase loading control.
Figure 3(D) Fluorescence polarization assay showing binding of E. coli MS115-1 CapH (His6-MBP tagged; full-length (black circles) or NTD(green squares)) to the Site 2 DNA. WT Kd = 0.30 +/- 0.08 μM, Hill coefficient = 2.0 +/- 0.7; no binding detected for CapHNTD). Error bars indicate standard deviation from three technical replicates. (E) Fluorescence polarization assay showing binding of E. coli MS115-1 CapH (His6-MBP tagged) to the Site 1 (bases 31-70) and Site 2 (bases 81-120) DNAs. Wild-type CapH binding Site 1 is shown in orange triangles, and binding Site 2 is shown in blue diamonds. CapH(I99M) binding Site 1 is shown in black circles, and binding Site 2 is shown in green squares. Fit Kd and Hill coefficient for each combination is shown.
Figure 5(A) Anti-GFP western blot showing coexpression in E. coli of an MBP-CapH-GFP fusion construct with wild-type or catalytic-dead (E98Q) CapP. Full-length MBP-CapH-GFP is indicated with a yellow arrowhead, and the C-terminal product of CapP cleavage is indicated with a white arrowhead. RNAP: RNA polymerase loading control.(B) In vitro cleavage of purified MBP-CapH-GFP (yellow arrowhead) into N-terminal and C-terminal products (white arrowheads) by CapP is stimulated by DNA. For both ssDNA and dsDNA, the highest concentration is 350 ng/μL, followed by three 5-fold dilutions. Red asterisk indicates the band that was analyzed by Edman degradation.(E) In vitro cleavage of MBP-CapH-GFP (wild-type or R83A mutant) by CapP, in the presence of 10 μM ssDNA.(F) GFP reporter assay showing effect of a CapP E98Q catalytic-dead mutant, CapH R83A mutant, or removal of CapH residues 83-107 (capH 1-82) on GFP expression. RNAP: RNA polymerase loading control.(G) CBASS expression reporter system with FLAG-NucC, showing effect of a CapH R83A mutant, or removal of CapH residues 83-107 (capH 1-82) on FLAG-NucC expression. α-RNAP: anti-RNA polymerase loading control.
Figure 6(A) Anti-GFP western blot showing coexpression in E. coli of an MBP-CapH-GFP fusion construct with wild-type CapP after exposure to zeocin (100 μg/mL). Full-length MBP-CapH-GFP is indicated with a yellow arrowhead, and the C-terminal product of CapP cleavage is indicated with a white arrowhead. α-RNAP: anti-RNA polymerase loading control.(B) Western blot of the CBASS expression reporter system with FLAG-NucC, showing FLAG-NucC expression after exposure to zeocin (100 μg/mL). α-RNAP: anti-RNA polymerase loading control. (C) Western blot of the CBASS expression reporter system with FLAG-NucC, showing FLAG-NucC expression after exposure to mitomycin C (1 μg/mL). α-RNAP: anti-RNA polymerase loading control.
figf1The percentage of dead cells was determined at the indicated timepoints by trypan blue exclusion assay (mean ± s.d.; n = 3). (a) Bif-1+/+ (wild type; WT) and Bif-1−/− (knockout; KO) MEFs were cultured in EBSS for 0, 3, 6 or 12 h. (b) Bif-1−/− MEFs stably transfected with a Bif-1 expression plasmid or control empty vector were incubated in EBSS for 0 or 12 h. Recovery of Bif-1 expression in Bif-1−/− cells was determined by immunoblot analysis (inset).(c) Bif-1+/+ and Bif-1−/− MEFs were cultured in EBSS in the presence of 50 μM z-VAD-fmk or control DMSO for 0, 3, 6 or 12 h.(d) Bax+/+Bak+/+ (WT) and Bax−/−Bak−/− (DKO) MEFs were cultured in EBSS in the presence of 50 μM z-VAD-fmk or control DMSO for 0, 6 or 12 h.
figf2(a, b) Bif-1+/+ and Bif-1−/− MEFs or Bax+/+Bak+/+ and Bax−/−Bak−/− MEFs were infected with Beclin 1 shRNA or control shRNA (SCR shRNA) lentiviruses. Twenty-four hours after infection, the medium was replaced with fresh culture medium and the cells were allowed to recover for an additional 48 h. The cells were then cultured in EBSS for 0, 6 or 12 h, and the percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3). Knockdown of Beclin 1 expression was confirmed by immunoblot analysis (insets).(c, d) Bif-1+/+ and Bif-1−/− MEFs were pre-treated with 10 mM 3-methyladenine (3-MA), 0.2 μM wortmannin (WM) or control DMSO for 30 min followed by culture in EBSS for the indicated times. The percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3).(c, d) Bif-1+/+ and Bif-1−/− MEFs were pre-treated with 10 mM 3-methyladenine (3-MA), 0.2 μM wortmannin (WM) or control DMSO for 30 min followed by culture in EBSS for the indicated times. The percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3). Immunoblot analyses were performed to detect LC3 modification and caspase-3 activation.(e) Atg5+/+ and Atg5−/− MEFs were cultured in EBSS in the presence of 50 μM of z-VAD-fmk or control DMSO for 0, 6 or 12 h. The percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3).(f) Total cell lysates (TCL) were prepared from Atg5+/+ and Atg5−/− MEFs cultured in EBSS for the indicated times and subjected to immunoblot analyses. Uncropped images of the immunoblots in a, b, d and f are shown in the Supplementary Information, Fig. S5.