EMBO/SourceData huggingface dataset:
Figure 6A, Graphical representation of FUS synonymous mutations (yellow) found in ALS patients. The canonical FUS mRNA is represented in dark blue (ENST00000254108 or NM_004960). The FUS protein coding sequence is indicated in light blue, and the altFUS protein coding sequence is indicated in green. B, Images by confocal microscopy of TDP-43 (white), FUS (GFP tagged - green) and altFUS (Flag tagged - red) in HeLa cells over-expressing GFP-FUS(Flag), GFP-FUS(P31L-Flag)-S44=, GFP-FUS(A38V-Flag)-G51=, GFP-FUS(A46V-Flag)-G59=, GFP-FUS(R64P-Flag)-S77= (representative images from n=3). White arrows indicate some TDP-43 aggregates. The white scale bar corresponds to 10 μm. C, Quantification of cells with TDP-43 aggregates in HeLa cells (see panel B). The data are represented as the fold-change compared to the GFP-FUS(Flag) expressing cells. Statistical significance is indicated above the bars (n=3 - biological replicates, mean ± SD, **** = p value < 0.0001, two-way ANOVA with Tukey's multiple comparison correction).
Figure 2A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for WT and BsnGT neurons treated with a CDK5 inhibitor roscovitine (rosc, 100µM, gray and pink trace) or vehicle (black and red trace) for 30 minutes before stimulation with 40 and 900 APs in the presence of bafilomycin A. C, D. Plots show mean values of RRP (C) and TRP (D) fraction for both genotypes before and after treatment. E. Roscovitine treatment has a significantly higher effect on RRP and TRP in BsnGT neurons compared to WT. Data information: n corresponding to the number of imaging experiments done on 4 independent cell preparations is given in brackets for each analysis. In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. Significance was assessed by two-way ANOVA with Tukey´s multiple comparison test (C, D) and by Student's t-test (E) *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 2µm.
Figure 3A. Representative immunoblot with antibody against CDK5-dependent pSer551 of Syn1 (pSer551Syn1) and total Syn1 and total protein stain used as loading control for quantification of Syn1 on P2 fractions prepared from hippocampal tissue lysates of WT and BsnGT mice. B, C. Quantification of pSer551Syn1 / Syn1 ratio (in B) and total Syn1 (in C) abundance on blots from A. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independently processed animals in B and C For statistics, student's t-test in B and C were used, *p≤0.05, **p < 0.01, ***p< 0.001.E. Representative images of 19 DIV cultured hippocampal neurons from WT and BsnGT immunostained for pSer551Syn1 and total Syn1. F. Quantification of IF intensity of pSer551Syn1 in synapses from WT, BsnGT, and WT cells treated with roscovitine. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independent images (E, quantified from two (F) independent cultures per condition. For statistics, one-way ANOVA with Sidak´s multiple comparisons test in F were used, *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 5 µm inG. Representative images of 21 DIV hippocampal WT and BsnGT neurons at baseline activity, upon network activity silencing (TTX, 1 µM, 72 h) or in conditions of increased activity (BIC, 30 µM, 48 h) H. Quantification of experiment in G. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independent images , G) quantified from four (H) independent cultures per condition. For statistics two-way ANOVA with Tukey´s multiple comparisons test in H were used, *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 5 µm in G.
Figure 4A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted from WT and BsnGT hippocampal neurons treated with calcineurin inhibitor FK506. Data information: Scale bar is 2 µm.C, D. Quantification of the RRP and TRP fractions in WT and BsnGT neurons from experiment in A, B. Data information: In all plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets for each quantification and reflect the number of analysed imaging experiments done on 3 independently prepared cultures per genotype Statistical significance was assessed using two-way ANOVA with Tukey´s multiple comparison test for C and D significance is depicted as follows: *p≤0.05, **p < 0.01, ***p< 0.001.E. Representative Western blot on P2 fractions prepared from hippocampal tissue of WT and BsnGT mice probed with antibodies against SNAP25 phosphorylated onThr138 and total SNAP25. F. Quantification of blot in E shows lower pThr138SNAP25 to total SNAP25 IF ratio in BsnGT compared to WT. Data information: In all plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets for each quantification and reflect the number of animals per genotype used for WB in F Statistical significance was assessed using Student's t-test in F, significance is depicted as follows: *p≤0.05, **p < 0.01, ***p< 0.001.
Figure 5A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for WT and BsnGT neurons without treatment (black and red) and upon treatment with adenylyl cyclase activator forskolin (gray and pink) or PKA inhibitor H89 (light grey and orange). Data information: The scale bar is 2 µm in AC, D. Quantification of the RRP and TRP fraction from experiment in A and B. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size is given in brackets and corresponds to the number of analysed independent imaging experiments performed on three independently prepared culture batches in (C and D), The statistical significance was assessed in C, D using two-way ANOVA with Tukey´s multiple comparison test *p≤0.05, **p < 0.01, ***p< 0.001.E. Representative immunoblot with antibody against pSer9Syn1 and total Syn1 and total protein stain of hippocampal P2 fraction from WT and BsnGT mice prepared in the presence of KN 93 to isolate PKA-dependent regulation of pSer9Syn1. F, G. Quantification of pSer9Syn1/Syn1 ratio (in F) and Syn1 (in G) on blots from E. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size is given in brackets and corresponds to the number of analysed independent imaging experiments samples prepared from individual animals in F and G, The statistical significance was assessed in F and G using Student's t-test as is depicted in graphs as *p≤0.05, **p < 0.01, ***p< 0.001.H. Representative images of hippocampal neurons from WT and BsnGT mice labelled with antibodies against pSer9Syn1 and total Syn1. Neurons were pre-treated (4 µM, 1hr) with KN93 to isolate PKA-dependent phosphorylation and then treated in addition with forskolin or H89 before fixation. I. Quantification of staining in H. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size is given in brackets and corresponds to the independent visual fields obtained from 2 independent culture preparations in I. The statistical significance was assessed in I using two-way ANOVA with Tukey´s multiple comparison test as *p≤0.05, **p < 0.01, ***p< 0.001. The scale bar is 5 µm in H.
Figure 6A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for untreated WT and BsnGT neurons (black and red) and cultures treated with of 6-Bnz-cAMPS (50 µM, 1 hour; gray and pink) and rolipram (1 µM, 30 min, light grey and orange). Data information: Scale bar is 2µm.C, D. Quantifications of RRP and TRP fractions in experiment in A and B. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets and corresponds to the number of imaging experiments performed on three independently prepared cultures Statistical significance was assessed using two-way ANOVA with Tukey´s multiple comparison test in C, D : *p≤0.05, **p < 0.01, ***p< 0.001.H. Quantification of pSer145PDE4B to the total PDE4B ratio from experiment in I. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets and corresponds to the individual animals used for sample preparation Statistical significance was assessed using Student's t-test in H and is given as: *p≤0.05, **p < 0.01, ***p< 0.001.I. Representative immunoblot with antibody against CDK5-dependent pSer145PDE4B and total PDE4B on hippocampal tissue from WT and BsnGT animals.
Figure 1Representative TEM images of normal and injured acinar cells. Scale bar, 2μm. Right (insets)- higher magnification images of boxed regions from left show emergence of double-membraned autophagic vesicles engulfing mature granules, mitochondria, and rough ER 24h after cerulein (CER) injury. Scale bar, 500nm. Arrowheads mark degradation events without double membranes. Graph of the number of acinar cell granules per cell in vehicle vs. 24h CER treatment. Each data point=total granules in a cell in a single cell counted from one mouse. Red line indicates mean. Graph of the number of degradation events scored in vehicle vs. 24h CER treatment. Each data point=total events in a cell in a single cell counted from one mouse. Red line indicates mean.mRNA expression by qRT-PCR of Rab5, Rab7a, Rab7b, and Rab9. Significance determined by one-way ANOVA, Tukey post hoc. Each data point represents the mean of triplicate technical replicates from a single mouse, n=3-4 mice in independent experiments. Error bars denote standard deviation.
Figure 2Top - Representative RAB7 expression in base of stomach units (ZCs) in vehicle or 24h HDT by immunohistochemistry. Arrowheads point to RAB7 vesicle aggregates. Scale bar 20µm. Bottom - Representative ATF3 expression in base of stomach units (ZCs) by immunohistochemistry. ATF3 appears in nuclei following 24h high-dose tamoxifen (HDT). Scale bar 20µm. Stomach unit base outlined by dashed black line.Western blot of ATF3 expression over HDT and CER timecourses with β-Actin loading control.Top­ - Representative RAB7 staining in pancreatic acinar cells. Arrowheads indicate RAB7 vesicles. Scale bar 20µm. Bottom - Representative ATF3 expression in pancreatic acinar cells. Nuclei express ATF3 after 24h cerulein (CER) injury. Scale bar 20µm.ChIP-PCR on mouse stomach tissue at vehicle and 6 hour HDT. Chromatin probed with ATF3, Histone H3 antibodies, or a Rabbit IgG control. Also included is the 2% chromatin input and water to control for DNA contamination. Rab7b amplicon is from a conserved, putative ATF3-binding site in the first intron. Amplicon from Atf3 is a positive control from a previously characterized ATF3-binding site; negative control is from a site in Rab9a lacking putative ATF3 binding motifs.
Figure 3mRNA expression of ATF3 by qRT-PCR in control, ATF3 knockdown (KD), and ATF3 overexpression (OE) AGS cell lines generated using a PiggybacTM transposon system. Significance by one-way ANOVA, Dunnett post-hoc. Datapoint = mean of technical triplicates in a single mouse. n=3 mice. Error bars denote standard deviation. mRNA expression of RAB7B by qRT-PCR in AGS cell lines from 3A. Statistics and plotting as for panel A.Top - Immunofluorescence for RAB7 (red) and DAPI (blue, nuclei) in control, ATF3 knockdown (KD), and ATF3 overexpression (OE) AGS cells. Arrowhead indicates aggregated RAB7 vesicular formation similar to those observed in tissue. Bottom - Immunofluorescence for ATF3 (purple) and Cathepsin D (CTSD, green, lysosomes) in AGS cells. Scale bar, 20µm.
Figure 4Representative immunofluorescence images of gastric chief cells after vehicle or 24h HDT treatment stained for Pepsinogen C (PGC, red, granules) and MAP1LC3B (green, autophagy marker). Top row is wild-type, bottom row is Atf3−/−. Scale bar, 50μm. Stomach unit base outlined by dashed white line.Transmission electron micrograph images of zymogenic chief cells (ZCs) after 24h vehicle or HDT treatment in WT (top) and Atf3−/− mice (bottom). Scale bar, 1μm. Arrowheads indicate cell components being degraded. Quantification of degradation events per cell counted from EM images of ZCs after 24h HDT treatment in WT and Atf3−/−mice as shown in panel 3D. Each data point=total granules in a cell in a single TEM section with cells from 2 separate mice plotted together (data point colors indicate cells from a single mouse). Red line indicates mean across all the mice.
Figure 5Representative immunofluorescence images of gastric units after vehicle or 72h high-dose tamoxifen (HDT) treatment stained for GIF (red, granules), GSII (green, neck cells and SPEM marker), BRDU (white, proliferation) and DAPI (blue, nuclei). Top row is wild-type, bottom row is Atf3−/−. Yellow arrowheads point to areas of base dropout where ZCs have been lost. Scale bar, 50μm. Stomach units outlined by dashed white line.Representative immunohistochemistry of Cleaved-Caspase 3 in pancreatic acinar cells marking apoptosis in vehicle or 24h cerulein (CER) treatment. Top row is WT, bottom row is Atf3−/−. Scale bar, 50μm. Apoptosis quantified following 24h CER injury by the number of Cleaved-Caspase 3+ cells from a 40x field. Each data point is mean of positive cells in 4-5 40x fields in an individual mouse, n=3 mice per treatment group. Significance by unpaired two-tailed t-test. Error bars denote standard deviation.Representative immunohistochemistry of BrdU in pancreatic acinar cells marking proliferation in vehicle or 7 days of repeated cerulein injury. Top row is WT, bottom row is Atf3−/−. Scale bar, 50μm. Quantification of proliferation at peak ADM as counted by the number of BrdU+ cells from a 20x field following 7D cerulein treatment. Plotting and statistical analysis as per panel F.
Figure 1(A) Protein abundance is the normalized signal intensity (LFQ) for a protein divided by its molecular weight. Specificity (enrichment) is the ratio of the protein LFQ intensity in the MIA40FLAG fraction to control samples. The LFQ for proteins that were not detected in the control samples was arbitrarily set to 1 for calculation purposes. LFQ, Label-Free-Quantification; M.W., molecular weight.(B) Flp-In T-REx 293 cells with induced MIA40FLAG expression were solubilized, and the affinity purification of MIA40FLAG was performed. Fractions were analyzed by SDS-PAGE and Western blot. Load: 2.5 %. Eluate: 100 %. Unbound: 2.5 %.(C) Flp-In T-REx 293 cells with induced MIA40FLAG expression were solubilized, and the affinity purification of MIA40FLAG was performed. Eluate fractions were solubilized under reducing (DTT) or non-reducing (IAA) conditions and analyzed by SDS-PAGE and Western blot. DTT, dithiothreitol; IAA, iodoacetamide. Eluate: 100 %.(D) Cellular protein extracts from Flp-In T-REx 293 cells with induced expression of wildtype or mutant MIA40FLAG were subjected to affinity purification. Load and eluate fractions were analyzed by reducing SDS-PAGE and Western blot. Load: 2.5 %. Eluate: 100 %.
Figure 2(A) Schematic representation of the thiol trapping assay. Mitochondria were solubilized in sample buffer with either dithiothreitol (DTT), iodoacetamide (IAA), or 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid (AMS). The samples were analyzed by SDS-PAGE and Western blot.(B) Indirect thiol trapping assay. Mitochondria were pretreated with IAA as indicated to block free cysteine residues, and disulfide bonds were subsequently reduced by DTT. Mitochondria were solubilized in sample buffer with AMS.(C) Localization of mitochondrial proteins analyzed by limited degradation by proteinase K in intact mitochondria (250 mM sucrose), mitoplasts (5 mM sucrose), and mitochondrial lysates (1 % Triton X-100). The samples were analyzed by SDS-PAGE and Western blot. Mitos, mitochondria; Sup, post-mitochondria supernatant; OM, outer membrane; IM, inner membrane; IMS, intermembrane space.(D) N-SIM super-resolution micrographs of one Z-stack (0.15 μm) orthogonal section (XYZ) of HeLa cells or HeLa cells that stably expressed COA7-HA transfected with different markers TOMM20-DsRed (OM), COX8A-DsRed (IM), and matrix targeted photoactivatable GFP (Matrix target) labeled with anti-HA, anti-COA7 and Smac/Diablo (IMS) antibodies. The picture represents the majority population of cells from three independent experiments. Scale bar = 2 μm, scale bar in the magnified insert = 0.5 μm. The panel shows Pearson's coefficient in a co-localized volume of different subcompartment combinations with anti-HA or anti-COA7. The data are expressed as a mean ± SD (n=5). ***p<0.001 [IMSxEnd vs. IMxEnd p=0.0001; IMSxEnd vs. MxEnd p=0.0002], ****p<0.0001 [OMxHA vs. IMSxHA p<0.0001; IMSxHA vs. IMxHA p<0.0001] (one-way ANOVA). M, matrix; End, endogenous COA7; HA, COA7-HA.
Figure 3(A) Mitochondria were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector. Mitochondria were solubilized and analyzed by reducing SDS-PAGE and Western blot. Mitos, mitochondria.(B) Radiolabeled [35S]TIMM8A and [35S]COX19 precursors were imported into mitochondria that were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 30 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.(C) Mitochondria were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector under reducing (DTT) and non-reducing (IAA) conditions and analyzed for levels of MIA40 by Western blot.(D) Proteins from HEK293 cells transfected with empty plasmid or RESA1HIS were modified with PEG-PCMal. Control cells were pretreated with the thiol oxidizing agent diamide (DAM) or the reductant DTT. The samples were analyzed by SDS-PAGE and Western blot.(E) Flp-In T-REx 293 cells induced to express MIA40FLAG were transfected with a plasmid that encoded COA7HIS or an empty vector. The affinity purification of MIA40FLAG was performed, and eluate fractions were analyzed by SDS-PAGE and Western blot.
Figure 4Radiolabeled [35S]COA7 (A) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.Radiolabeled [ 35S]TIMM8A (B) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.(C) Mitochondria were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG and control cells. The samples were analyzed by SDS-PAGE and Western blot. Mitos, mitochondria.Radiolabeled [35S] COA7 (D) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express ALRFLAG and control cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological experiments were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3).Radiolabeled [35S] TIMM8A (E) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express ALRFLAG and control cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological experiments were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3).(F) Mitochondria were isolated from Flp-In T-REx 293 cells induced to express ALRFLAG and control cells. The samples were analyzed by SDS-PAGE and Western blot.
Figure 5(A) Cellular protein extracts were isolated from HeLa cells transfected with oligonucleotides that targeted different regions of MIA40 mRNA or with control oligonucleotides. The samples were analyzed by reducing SDS-PAGE and Western blot.(B) Cellular protein extracts were isolated from HeLa cells transfected with oligonucleotides that targeted different regions of ALR mRNA or with control oligonucleotides. The samples were analyzed by reducing SDS-PAGE and Western blot.(D) Cellular protein extracts were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells. The samples were analyzed by SDS-PAGE and Western blot.(E) Cellular protein extracts were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells transfected with a plasmid that encoded MIA40 or an empty vector. The samples were subjected to reducing SDS-PAGE and Western blot.Radiolabeled [35S]COA7 (F) precursors were imported into mitochondria that were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.Radiolabeled [ [35S]TIMM8A (G) precursors were imported into mitochondria that were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.
Figure 6(B) Cellular protein extracts were isolated from HEK293 cells that were transfected with a plasmid that encoded wildtype or mutant COA7. The samples were analyzed by reducing SDS-PAGE and Western blot.(C) Cellular fractions were prepared from HEK293 cells that were transfected with a plasmid that encoded wildtype or mutant COA7. The fractions were analyzed by reducing SDS-PAGE and Western blot. T, total; C, cytosol; M, mitochondria.(D) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were solubilized, and the affinity purification of COA7HIS was performed. The samples were analyzed by reducing and non-reducing SDS-PAGE and Western blot. Load: 3 %. Eluate: 100 %.(E) Equal amounts of wildtype and mutant radiolabeled [35S]COA7 precursors were imported into mitochondria isolated from HEK293 cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to wildtype COA7 at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.
Figure 7(B) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were treated with CHX for the indicated times, and protein extracts were isolated. The samples were analyzed by reducing SDS-PAGE and Western blot.(D) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were treated with CHX and/or MG132 for the indicated times, and protein extracts were isolated. The samples were analyzed by reducing SDS-PAGE and Western blot. CHX, cycloheximide.(F) Cellular fractions were prepared from HEK293 cells that transiently expressed mutant COA7HIS and were treated with MG132. The samples were analyzed by reducing SDS-PAGE and Western blot.