EMBO/SourceData huggingface dataset:
Figure 1A Immunoblot of endogenous BiP resolved by native-PAGE from lysates of CHO-K1 S21 wild-type (wt) or FICD-/- cells either transiently overexpressing wild-type FICD (high expression level; Hi) or mutant FICDE234G (E/G) or stably expressing recombinant wild-type FICD (low expression level; Lo). The cells in lanes 1-4 were mock transfected. Where indicated cells were exposed to cycloheximide (CHX; 100 µg/mL) for 3 h before lysis. Unmodified ('A') and AMPylated ('B') monomeric and oligomeric (II and III) forms of BiP are indicated. Immunoblots of the same samples resolved by SDS-PAGE report on FICD, total BiP and eIF2α (loading control). Data representative of four independent experiments are shown.B Wild-type FICD forms homomeric complexes in vivo. Immunoblots of orthogonally-tagged wild-type and Leu258Asp mutant FICD in the input cell lysate and following recovery by pull-down with streptavidin (recognising the AviTag) or anti-FLAG antibody. Proteins were detected with fluorescently-labelled streptavidin (StrepIR800) or FLAG antibody. Data representative of three independent experiments are shown.C Immunoblot of endogenous BiP from transfected CHO-K1 S21 FICD-/- cells (as in A). Note that cells expressing monomeric FICDL258D accumulate AMPylated BiP. Data representative of three independent experiments are shown.D Size-exclusion chromatography (SEC) analysis of wild-type and mutant FICD proteins (each at 20 µM). The elution times of protein standards are indicated as a reference. Note that the Leu258Asp mutation monomerises FICD, while Gly299Ser causes partial monomerisation.E Comparison of the signal-averaged sedimentation coefficients of wild-type (red) and monomeric mutant FICDL258D (blue), as measured by analytical ultracentrifugation. A fit for monomer-dimer association (solid red line), constrained using the average value for the monomeric protein (dashed line, 2.82 S, Sw,20 = 3.02 S), yielded a Kd of 1.2 nM with a 95% confidence interval between 1.1 to 1.4 nM and a value of 4.08 S for the dimer (Sw,20 = 4.36 S). The fitted data points are from three independent experiments.F Autoradiograph of BiP, AMPylated in vitro by the indicated FICD derivatives, with [α-32P]-ATP as a substrate and resolved by SDS-PAGE. Proteins in the gel were visualised by Coomassie staining. A representative result of three independent experiments is shown. The graph shows the quantified relative BiP-AMP signals and SD bars generated by wild-type (wt) FICD and the indicated monomeric mutants.
Figure 2A Monomerising FICD mutations inhibit deAMPylation. Shown is a representative plot of data points and fit curves of the time-dependent deAMPylation of a fluorescent BiPV461F-AMPFAM by the indicated FICD proteins (at 7.5 µM) as detected by a change in fluorescence polarisation (FP). DeAMPylation rates calculated from independent experimentsB-C Dimer interface mutants both AMPylate and deAMPylate BiP. Shown are representative autoradiographs of thin layer chromatography (TLC) plates revealing AMP produced from reactions containing [α-32P]-ATP and the indicated FICD enzymes in the presence or absence of the co-substrate BiP (arrow indicates direction of nucleotide migration). The radioactive signals were quantified and the AMP signals were normalised to the total nucleotide signal in each sample. Plotted below are mean values ± SD from at least three independent experiments. Unpaired t-tests were performed.E Detection of the time-dependent accumulation of AMPylated BiPT229A-V461F in radioactive reactions, containing [α-32P]-ATP and the indicated FICD proteins, in the presence of excess trap. At the specified time-points samples were taken and analysed by SDS-PAGE. The autoradiograph (32P) illustrates the radioactive signals, which represent AMPylated BiP. Proteins were visualised by Coomassie staining. The radioactive signals were quantified and presented in the graph below. Mean values ± SD of three independent experiments are shown.
Figure 3A Autoradiographs of in vitro reactions containing varying concentration of wild-type FICD protein and fixed concentrations of BiPT229A-V461F and [α-32P]-ATP as co-substrates, resolved by SDS-PAGE after the indicated incubation times. The proteins were visualised by Coomassie staining of the gel (bottom). The reactions shown on the right were performed in the presence of an excess of S-SFICDA252C-H363A-C421S (trap) to delay de-modification of BiP. Representative gels are shown, and similar results were observed in three independent experiments. B As in (A) but with 0.2 µM of the indicated FICD variant. The radioactive signals were detected by autoradiography, quantified, and normalised to the signal in lane 6. The mean radioactive signals ± SD from three independent experiments are given. The proteins were visualised by staining with Coomassie. C As in (A) but with dilutions of FICDC421S or covalently linked S-SFICDA252C-C421S. Reactions were preceded by a 16 h incubation of FICD in presence or absence of the reducing agent (DTT). Representative gels are shown of three independent experiments.D Forced dimerisation does not significantly alter deAMPylation rates. Time-dependent deAMPylation of fluorescent BiPV461F-AMPFAM by the indicated FICD proteins (at 7.5 µM) assayed by fluorescence polarisation A representative experiment (data points and fit curves) is shownE Representative autoradiograph of thin layer chromatography (TLC) plates revealing AMP produced from reactions containing [α-32P]-ATP and the indicated FICD enzymes in the presence of the co-substrate BiP. AMP signals were normalised to the total nucleotide signal in each sample and the graph below plots mean values ± SD from at least three independent experiments.
Figure 4B Size-exclusion chromatography elution profile of wild-type and mutant FICD proteins (each at 20 µM). Protein absorbance at 280 nm is plotted against elution time. The elution times of protein standards are indicated as a reference.C Radioactive in vitro AMPylation reactions containing the indicated FICD proteins, [α-32P]-ATP, and BiPT229A-V461F were analysed by SDS-PAGE. The radioactive BiP-AMP signals were detected by autoradiography and proteins were visualised by Coomassie staining of the gel.D Representative autoradiograph of thin layer chromatography (TLC) plates revealing AMP produced from reactions containing [α-32P]-ATP and the indicated FICD enzymes in the presence of the co-substrate BiP. The radioactive signals were quantified and the AMP signals were normalised to the total nucleotide signal in each sample. The graph shows mean AMP values ± SD from three independent experiments.E Melting temperatures (Tm) of the indicated FICD mutants (at 2 µM) were measured by differential scanning fluorimetry (DSF). Shown is the mean Tm ± SD of three independent experiments. The inset shows melt curves with their negative first derivatives from a representative experiment.F A plot of the melting temperature of the indicated FICD proteins in absence (Apo) or presence of nucleotides. Shown are the mean Tm values ± SD of three independent DSF experiments. Monomeric FICDL258D (mFICD) and FICDL258D-E234G (mFICDE/G) as well as dimeric wild-type FICD (dFICD) and FICDE234G (dFICDE/G) were tested. ADP and ATP concentrations in mM are given in parentheses.
Figure 6A BioLayer interferometry (BLI) derived association and dissociation traces of monomeric FICDL258D-H363A (mFICDH363A) or dimeric FICDH363A (dFICDH363A) from immobilised biotinylated BiPT229A-V461F in absence or presence of nucleotides. Unless indicated (*) BiP was saturated with ATP before exposure to FICD variants. A representative experiment of three independent repetitions is shown. B BLI dissociation traces of proteins as in (A). At t = 0 a pre-assembled complex of immobilised, ATP-saturated BiP and the indicated FICD proteins (associated without ATP) were transferred into a solution without or with ATP, as indicated. A representative experiment is shown and the biphasic dissociation kinetics are quantified in (C) and (D). C Graph of the slow dissociation rates (koff,slow) of monomeric FICD from BiP:ATP as shown in (B). Bars represent mean values ± SD of three independent experiments. D The ATP-induced fold change in the percentage of the dissociation phase attributed to a fast dissociation (%Fast), koff,fast, and koff,slow derived from the data represented in (B). Bars represent mean values ± SD of three independent experiments.E (i) BLI assay workflow used to derive FICD dimer off rate data presented in (ii) (ii) Observed dimer off rates under different nucleotide conditions (5 mM, where applicable). ATP, but no ADP, significantly increases the dimer dissociation rate [n.s.: not significant by Tukey test]. Data shown is the mean ± SD of four independent experiments.F Validation of the homo-FRET dimerisation probe. The dimerisation sensitive TMR fluorescence of the labelled FRET dimer (2.5 nM) is de-quenched specifically by equilibration with excess unlabelled dimerisation-competent FICDH363A but not monomeric FICDL258D-H363A. Mean ± SD of three independent experiments.G Homo-FRET measurement of nucleotide dependent modulation of the FICD monomer-dimer equilibrium. ATP increases and ADP decreases the proportion of monomeric FICD. * Plateaus were constrained to a shared best fit value. Data shown is the mean ± SD of four independent experiments.
Figure 1A) z-stacks were acquired from FM4-64 labeled vacuoles in wildtype cells (BJ3505), using a spinning disc microscope (step size 200 nm). 3D reconstructions from these stacks reveal a peripherally located fusion pore. Scale bar 2 µm.B) Wildtype (BJ3505) and isogenic pmc1∆/vcx1Δ mutant cells were labeled with FM4-64. The number of vacuoles per cell was determined in 3 independent experiments evaluating 200 cells each. Scale bar: 2 µm. C) Frequency of visible fusion pores. Cells were grown as in (B). The percentage of cells showing visible fusion pore was determined from z-stacks. Means s.d. are shown for 100 stained vacuoles from 3 independent experiments. E) Distribution of Vph1-GFP and FM4-64 in vacuole-vacuole contact sites. Vph1-GFP expressing cells, labeled with FM4-64, were analyzed under a spinning disk confocal microscope equipped with two cameras for the simultaneous acquisition of GFP and FM4-64 fluorescence signals. Separate channels are shown on the right. The entire contact surface, but not the pore, is accessible to both probes. Scale bar: 2 µm.F) Expansion of a vacuolar fusion pore in real time. pmc1∆/vcx1Δ cells were labeled with FM4-64 and immobilized in a 50 µL flow chamber (Ibidi). The osmotic value of the medium was changed by perfusion with water, using a pump with a flow of 30 µL/s. A frame was acquired every 2 s for a total period of the 30 s, using the laser at minimal intensity.
Figure 2A) Vacuolar membranes of wildtype (BY4741), nyv1∆ vam3∆, ypt7∆ or vma16F190Y cells were labeled with the vital dye FM4-64. A vacuole that was in a sufficiently peripheral location to be selectively photobleached was exposed to a laser pulse (white arrows indicate the bleaching area). Recovery of fluorescence in this area was assayed 10 seconds later by spinning disc confocal microscopy. Scale bar: 2 µm. B) Kinetics of FM4-64 recovery in (A). Means and s.d. are shown for 20 vacuole clusters from 3 independent experiments. C) In vivo lipid and content mixing. Vacuoles in living yeast were labeled with the indicated lumenal (CDCFDA) and membrane (FM4-64 or PX-GFP) probes. Note that CRY1 cells lack the ADE2 gene and hence naturally accumulate 5-amino-1-(5-phospho-D-ribosyl)imidazole as fluorescent fluid phase marker in the vacuolar lumen. FRAP-experiments were performed (arrows indicate the bleaching area). 1. Non-bleached lumenal area. 2. Bleached lumenal area. 3. Bleached membrane area. Scale bar: 2 µm. D and E) Kinetics of fluorescence recovery after photobleaching of the probes in (C). Numbers denote the areas shown in (C): 1. Non-bleached lumenal area. 2. Bleached lumenal area. 3. Bleached membrane area. Means and s.d. are shown for 20 vacuole clusters from 3 independent experiments.
Figure 3A) FM4-64 and GFP excitation/emission spectra.C) Assay by microscopy. Half of two cultures of cells expressing Vph1-EGFP or Nyv1-EGFP were labeled with FM4-64, washed and mixed with the other, non-labeled half of the same culture. Cells were analyzed by spinning disc confocal microscopy. The intensities of GFP fluorescence were tracked along the dashed lines and compared between neighboring FM4-64-labeled and unlabeled cells.D) FACS analysis. The strains were labeled washed and analyzes by FACS. Shown are side (SSC-A) and forward scatter (FSC-A), as well as the fluorescence intensity distributions for FM4-64 and for GFP in the absence or presence of FM4-64. E) Comparison of the GFP mean fluorescence from FM4-64-stained cells and unstained cells (20'000 each).
Figure 4C,D) FRAP assays for fusion pores. The indicated cells expressing (C) yeGFP-LT or (D) LT-yeGFP were labeled with FM4-64 and subjected to FRAP experiments Cells were imaged before and 0 (bleached) or 30 seconds after photobleaching (recovery). The bleached areas are indicated by an arrow. E,F) FRAP assays for fusion pores were performed as in (C, D), but with nyv1∆ cells. Scale bar: 2 µm. G) The histogram reports the fraction of cells showing FRAP. Means and s.d. are shown from 3 independent experiments, with 100 cells analyzed in each.
Figure 5A-F) The indicated yeast strains were transformed with expression plasmids for FYVE2-GFP and their vacuolar membranes were labeled with FM4-64. FRAP was performed using a spinning disk microscope equipped with an emission beam splitter and two synchronized cameras, allowing to simultaneously analyze FM4-64 and GFP fluorescence. The histograms show the kinetics of signal recovery for (A) wildtype (WT), (B) vam3tsf, (C) ypt7T22N, (D) vma16F190Y, (E) vps33P184L, and (F) nyv1∆ cells. Scale bar: 2 µm. Arrows indicate the bleaching area. Means and s.d. are shown for 20 stained vacuoles from 3 independent experiments.
Figure 6A-D) Wild-type (A, B) or nyv1∆ cells (C,D), which lack the vacuolar R-SNARE Nyv1, were labeled with FM4-64. Cells were immobilized on a chambered slide and subjected to FRAP experiments (C) Vacuole fission induced by hypertonic shift. FRAP experiments were performed before (0') or 15 min after addition of NaCl to 0.5 mol/l. The fluorescence images show the vacuoles inside a single cell. (B, D) Cells in S-phase were selected, which grow a daughter cell and therefore fission their vacuoles into a chain of tubulo-vesicular structures, part of which are transported into the daughter cell. FRAP experiment on vesicles in the inheritance structure were performed. Arrows indicate the bleaching area, dashed orange lines the cell cortex. Scale bar: 2 µm.
Figure 7A) Wildtype (BY4741) cells labeled with FM4-64 show extensive contact zones between vacuoles. Scale bar: 2 µm.C) FRAP analysis of FM4-64-labeled wildtype cells was performed by acquiring pictures in continuous acquisition mode after bleaching, for a total period of 5 s. Three representative frames after photobleaching are shown. The bars in the magnified parts of frames 2 and 3 illustrate the positions at which the recovery of FM4-64 fluorescence was quantified. Scale bar: 2 µm. D) Quantification of recovery experiments in (C). A delay of >50 ms between the appearance of the signal on both sides of the photobleached vacuole was considered as asymmetric recovery. The histogram shows means and s.d. from 3 independent experiments evaluating 50 stained vacuoles each. A Schematic view of the various possible positions of a fusion pore and its consequences for our ability to detect asymmetric FM4-64 recovery. The contact zone between the two vacuoles is tilted by 90° in the middle panel, which gives four examples for the positioning of the pore (shown as a black dot). Asymmetric recovery could only be detected if the pore is sufficiently far from the center of the contact zone and close to the optical plane that is imaged (the cases marked in red).
Figure 1 C Relative mRNA expression of Apopt1 normalized to the expression of GAPDH in skeletal muscle and liver of wild-type, heterozygous and knock-out mice of three months of age. Data are presented as mean ± SEM (n = 6 mice per genotype). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: Muscle - ***P = 0.0001 (WT vs KO), *P = 0.0310 (WT vs het), *P = 0.0395 (het vs KO), Liver - **P = 0.0022 (WT vs KO), *P = 0.0101 (WT vs het)
Figure 2 A Body weight measured in female and male animals at 3, 6 and 12 months of age. Data are presented as mean ± SEM (n = 5 per group) Distance run by the tested female and male mice on the treadmill at 3 and 12 months of age. Data are presented as mean ± SEM (n = 6 per group). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: Females - ****P < 0.0001 (3 months), ***P = 0.0004 (12 months, Males - ****P < 0.0001 (3 months), **P = 0.0019 (12 months)Time in seconds spent by the female and male mice on the Rotarod cylinders before falling at different ages. Data are presented as mean ± SEM (n = 5 per group). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: Females - **P = 0.0059 (3 months), **P = 0.0083 (6 months), *P = 0.0391 (12 months), Males - **P = 0.0069 (12 months)Number of entries in each arm of the Y maze performed at different ages. Data are presented as mean ± SEM (n = 10 per group). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: ****P < 0.0001 E Total spontaneous horizontal and vertical movements of 12-month old mice measured in an activity cage. Data are presented as mean ± SEM (n = 6 per group). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: ****P < 0.0001 (horizontal)
Figure 3 A Representative Hematoxylin & Eosin staining of skeletal muscle in three-month old individuals  B Histochemical reaction specific to COX and SDH in skeletal muscle, cerebellar cortex and kidney of the same individuals  C COX (CIV) enzymatic activity normalized to the activity of citrate synthase (CS) in five animals per genotype as indicated at three and twelve months of age. Data are presented as mean ± SEM (n = 6 per group). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: Muscle - ****P < 0.0001 (het vs KO), ***P = 0.0001 (WT vs KO), *P = 0.0185 (WT vs het), Kidney - ****P < 0.0001 (het vs KO), ***P = 0.0002 (WT vs KO), Heart - ***P = 0.0003 (het vs KO), ***P = 0.0001 (WT vs KO), Brain and Liver - ****P < 0.0001 D Western blot analysis of SDS-PAGE of total lysates from liver, skeletal muscle and brain from the indicated genotypes, each lane showing the results for one animal. The graph shows the densitometric quantification of the signals obtained in the liver. Data are presented as mean ± SEM (n=2 WT; n=2 het; n=3 KO). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons: ***P = 0.0002 and ****P < 0.0001 (WT vs KO)  E Western blot analysis of 1D-BNGE of mitochondria from skeletal muscle from the indicated genotypes, each lane showing the results from one animal  F Western blot analysis of 2D-BNGE of mitochondria from skeletal muscle from one individual of the indicated genotype
Figure 4 A Western blot of SDS-PAGE of different fractions from 143B cells expressing APOPT1HA. Tot: total lysate. Cyto: post-mitochondrial fraction (cytoplasm). Mt: isolated mitochondria. Mt sol: Soluble mitochondrial fraction. Mt memb: mitochondrial membranes. CO32- pellet: Pellet after carbonate extraction with 0.1 M Na2CO3, pH 10.5 for 30 minutes. CO32- sol: soluble fraction after the carbonate extraction  B Western blot of SDS-PAGE of mitochondria used for protease protection assay after digitonin treatment. The experiment was carried out in isolated mitochondria from 143B cells expressing APOPT1HA exposed to increasing amounts of digitonin (expressed in µg) and 50 µg/ml trypsin. Complete solubilization with 1% Triton X-100 was used as a control of protease sensitivity. TOM20: Translocase of the outer membrane (OM) 20 kDa. ACO2: Aconitase 2 (mitochondrial isoform). AIF: Apoptosis inducing factor. AK2: Adenylate kinase 2. OM: outer mitochondrial membrane. IM: inner mitochondrial membrane. IMS: intermembrane space  C Western blot of SDS-PAGE of mitochondria used for protease protection assay after hypotonic shock. The experiment was carried out in isolated mitochondria from 143B cells expressing APOPT1HA incubated either in isotonic (Iso) or hypotonic buffers for 5 minutes (Hypo 5') or 15 minutes (Hypo 15') and 50 µg/ml trypsin. Complete solubilization with 1% Triton X-100 was used as a control of protease sensitivity  D N-SIM super-resolution micrographs showing 0.8 μm Maximum Intensity Projection (0.15 μm for each Z-stack) of 143B cells expressing APOPT1GFP shown in green, specific markers for each mitochondrial compartment: TOMM20 (OM), COX8A (IM), MitoTracker (IM+M) and matrix-target (mScarlet) shown in red. Scale bar: 5 μm. The graph shows Pearson's Coefficient in colocalized volume of different sub-compartment combinations with APOPT1GFP. Data show mean ± SEM (n = 5). One-way ANOVA (*P = 0.0298 and **P = 0.0076) was employed for the statistical analysis