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 .

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 .

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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 .

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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.


A 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

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A 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 experiments


B - 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 .

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B-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 .

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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.


A 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 .

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A 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

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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 shown


E 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 .

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E 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.


B 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 .

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B 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 .

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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 .

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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 .

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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 .

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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.


A 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 .

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A 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 .

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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 .

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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 .

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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.


A ) 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 .

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A) 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 .

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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 .

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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 .

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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.


A ) 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 .

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A) 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 .

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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.


A ) FM4 - 64 and GFP excitation / emission spectra .

A) 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 .

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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 ) .

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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).


C , 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 .

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C,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.


A - 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 .

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A-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.


A - 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-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.


A ) Wildtype ( BY4741 ) cells labeled with FM4 - 64 show extensive contact zones between vacuoles . Scale bar : 2 µm .

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A) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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).


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 )

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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)


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 )

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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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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)


A Representative Hematoxylin & Eosin staining of skeletal muscle in three - month old individuals

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 )

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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

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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

F Western blot analysis of 2D-BNGE of mitochondria from skeletal muscle from one individual of the indicated genotype


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

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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

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

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

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


A APOPT1HA was expressed in S6 and S2 immortalized fibroblasts as shown by the Western blot immunovisualization . The expression levels were tested at different days after transduction . The graph on the right shows the activities of CIV normalized to CS in three biological replicates per cell line . Data are presented as mean ± SEM ( n = 3 ) . The asterisks represent the significance levels calculated by two - way ANOVA with Tukey ' s multiple comparisons test : * * P = 0 . 0030 ( S6 naïve vs S6 APOPT1HA ) , * P = 0 . 0165 ( S6 EV vs S6 APOPT1HA )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A APOPT1HA was expressed in S6 and S2 immortalized fibroblasts as shown by the Western blot immunovisualization. The expression levels were tested at different days after transduction. The graph on the right shows the activities of CIV normalized to CS in three biological replicates per cell line. Data are presented as mean ± SEM (n = 3). The asterisks represent the significance levels calculated by two-way ANOVA with Tukey's multiple comparisons test: **P = 0.0030 (S6 naïve vs S6 APOPT1HA), *P = 0.0165 (S6 EV vs S6 APOPT1HA )


B APOPT1 GFP was expressed in S6 and S2 immortalized fibroblasts as shown by the Western blot immunovisualization . The graph on the right shows the activities of CIV normalized to CS in four biological replicates per cell line . Data are presented as mean ± SEM ( n = 4 ) . The asterisks represent the significance levels calculated by two - way ANOVA with Tukey ' s multiple comparisons test : * * P = 0 . 0066 ( S6 naïve vs S6 APOPT1GFP ) , * * * P = 0 . 0006 ( S6 EV vs S6 APOPT1GFP ) , * P = 0 . 0169 ( S2 naïve vs S2 APOPT1GFP ) , * * * P = 0 . 0008 ( S2 EV vs S2 APOPT1GFP )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B APOPT1 GFP was expressed in S6 and S2 immortalized fibroblasts as shown by the Western blot immunovisualization. The graph on the right shows the activities of CIV normalized to CS in four biological replicates per cell line. Data are presented as mean ± SEM (n = 4). The asterisks represent the significance levels calculated by two-way ANOVA with Tukey's multiple comparisons test: **P = 0.0066 (S6 naïve vs S6 APOPT1GFP), ***P = 0.0006 (S6 EV vs S6 APOPT1GFP), *P = 0.0169 (S2 naïve vs S2 APOPT1GFP), ***P = 0.0008 (S2 EV vs S2 APOPT1GFP)


C Western blot analysis of 1D - BNGE of mitoplasts extracted from the indicated cell lines . Fully assembled COX migrating at 200 kDa was immunovisualized with antibodies recognizing MT - CO1 and MT - CO2 . The presence of the assembly intermediates ' MITRAC ' and ' S3 ' was also detected ( arrows )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Western blot analysis of 1D-BNGE of mitoplasts extracted from the indicated cell lines. Fully assembled COX migrating at 200 kDa was immunovisualized with antibodies recognizing MT-CO1 and MT-CO2. The presence of the assembly intermediates 'MITRAC' and 'S3' was also detected (arrows)


D Western blot analysis of 2D - BNGE of mitoplasts extracted from the indicated cell lines . Fully assembled COX was immunovisualized with antibodies recognizing MT - CO1 and MT - CO2 . The presence of the assembly intermediates ' MITRAC ' and ' S3 ' was also detected ( arrows ) . Anti - GFP immunodetection revealed the presence of the low molecular weight APOPT1GFP complex in the complemented cells expressing the tagged protein

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Western blot analysis of 2D-BNGE of mitoplasts extracted from the indicated cell lines. Fully assembled COX was immunovisualized with antibodies recognizing MT-CO1 and MT-CO2. The presence of the assembly intermediates 'MITRAC' and 'S3' was also detected (arrows). Anti-GFP immunodetection revealed the presence of the low molecular weight APOPT1GFP complex in the complemented cells expressing the tagged protein


E L - [ 35S ] - Methionine pulse - chase labeling of mtDNA - encoded proteins . After a two - hour exposure with the radioactive label ( pulse ) , cells were cultured in cold medium for the indicated chase times . The graphs show the densitometric quantification of the bands corresponding to MT - CO1 ( left graph ) and MT - CO2 + MT - CO3 ( right graph ) normalized to the ATP6 band over the indicated time points . Graphs represent the values of three biological replicas for each cell line . Data are presented as mean ± SEM ( n = 4 for controls , n = 2 for S2 / S6 / S2 APOPT1GFP / S6 APOPT1GFP ) . The asterisks represent the significance levels calculated by two - way ANOVA with Tukey ' s multiple comparisons test : MT - CO1 - * * P = 0 . 0039 ( 3 . 5 hours , controls vs S6 ) , * P = 0 . 0202 ( 3 . 5 hours , controls vs S2 ) , * P = 0 . 0163 ( 5 hours , S6vs S6 APOPT1GFP ) , * P = 0 . 0118 ( 5 hours , controls vs S2 ) , * * P = 0 . 0011 ( 20 hours , controls vs S6 ) , * P = 0 . 0343 ( 20 hours , S2 vs S2 APOPT1GFP ) , * * * P = 0 . 0007 ( 20 hours , S6 vs S6 APOPT1GFP ) , * * * P = 0 . 0002 ( 20 hours , controls vs S2 ) , * * * * * P < 0 . 0001 ( 20 hours , controls vs S6 ) , MT - CO2 / MT - CO3 - * P = 0 . 0194 ( 3 . 5 hours , S6 vs S6 APOPTGFP ) , * * * P = 0 . 0003 ( 3 . 5 hours , controls vs S6 ) , * P = 0 . 0375 ( 5 hours , S6 vs S6 APOPT1GFP ) , * P = 0 . 0234 ( 20 hours , S2 vs S2 APOPT1GFP ) , * * * * P < 0 . 0001 ( 20 hours , S6 vs S6 APOPT1GFP ) , * * P = 0 . 0013 ( 20 hours , controls vs S2 ) , * * * * P < 0 . 0001 ( 20 hours , controls vs S6 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E L-[35S]-Methionine pulse-chase labeling of mtDNA-encoded proteins. After a two-hour exposure with the radioactive label (pulse), cells were cultured in cold medium for the indicated chase times. The graphs show the densitometric quantification of the bands corresponding to MT-CO1 (left graph) and MT-CO2+MT-CO3 (right graph) normalized to the ATP6 band over the indicated time points. Graphs represent the values of three biological replicas for each cell line. Data are presented as mean ± SEM (n = 4 for controls, n = 2 for S2/S6/S2 APOPT1GFP/S6 APOPT1GFP). The asterisks represent the significance levels calculated by two-way ANOVA with Tukey's multiple comparisons test: MT-CO1 - **P = 0.0039 ( 3.5 hours, controls vs S6), *P = 0.0202 (3.5 hours, controls vs S2), *P = 0.0163 (5 hours, S6vs S6 APOPT1GFP), *P = 0.0118 (5 hours, controls vs S2), **P = 0.0011 (20 hours, controls vs S6), *P = 0.0343 (20 hours, S2 vs S2 APOPT1GFP), ***P = 0.0007 (20 hours, S6 vs S6 APOPT1GFP ), ***P = 0.0002 (20 hours, controls vs S2), *****P < 0.0001 (20 hours, controls vs S6), MT-CO2/MT-CO3 - *P = 0.0194 (3.5 hours, S6 vs S6 APOPTGFP), ***P = 0.0003 (3.5 hours, controls vs S6), *P = 0.0375 (5 hours, S6 vs S6 APOPT1GFP), *P = 0.0234 (20 hours, S2 vs S2 APOPT1GFP), ****P < 0.0001 (20 hours, S6 vs S6 APOPT1GFP), **P = 0.0013 (20 hours, controls vs S2), ****P < 0.0001 (20 hours, controls vs S6)


A Western blot analysis of SDS - PAGE of MGM132 - treated 143B APOPT1HA cells . The graph represents the densitometric quantification of the signals for the precursor and mature protein

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

A Western blot analysis of SDS-PAGE of MGM132-treated 143B APOPT1HA cells. The graph represents the densitometric quantification of the signals for the precursor and mature protein


B The upper part of the panel ( Input ) shows Western blot analyses of APOPT1HA in the MGM132 - treated cells . Note the appearance of higher molecular weight bands upon longer exposure in the samples treated with the proteasome inhibitor . The bottom part of the panel ( Purified fractions ) shows the analysis of fractions from the same cells immunoprecipitated with anti - HA . Note that the higher molecular weight species are cross - reacting with both anti - HA and anti - ubiquitin

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B The upper part of the panel (Input) shows Western blot analyses of APOPT1HA in the MGM132-treated cells. Note the appearance of higher molecular weight bands upon longer exposure in the samples treated with the proteasome inhibitor. The bottom part of the panel (Purified fractions) shows the analysis of fractions from the same cells immunoprecipitated with anti-HA. Note that the higher molecular weight species are cross-reacting with both anti-HA and anti-ubiquitin


C Western blot analysis of SDS - PAGE of total lysates from 143B cells overexpressing tagged APOPT1 ( as indicated ) and exposed to 100 µM H2O2 , as illustrated by the scheme ( H2O2 treatment ) , for the indicated times . The graphs represent the densitometric quantification of the tagged APOPT1 signal at each time point . The graph inset shows that the increase of APOPT1 occurs in the first minutes after the exposure to H2O2

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR

C Western blot analysis of SDS-PAGE of total lysates from 143B cells overexpressing tagged APOPT1 (as indicated) and exposed to 100 µM H2O2, as illustrated by the scheme (H2O2 treatment), for the indicated times. The graphs represent the densitometric quantification of the tagged APOPT1 signal at each time point. The graph inset shows that the increase of APOPT1 occurs in the first minutes after the exposure to H2O2


D Western blot analysis of SDS - PAGE of total lysates from 143B cells overexpressing tagged APOPT1 ( as indicated ) and exposed to 5 µM MitoParaquat ( MitoPQ ) , as illustrated by the scheme ( MitoPQ treatment ) , for the indicated times . The graphs represent the densitometric quantification of the tagged APOPT1 signal at each time point . The graph inset shows that the increase of APOPT1 occurs in the first minutes after the exposure to MitoPQ

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR

D Western blot analysis of SDS-PAGE of total lysates from 143B cells overexpressing tagged APOPT1 (as indicated) and exposed to 5 µM MitoParaquat (MitoPQ), as illustrated by the scheme (MitoPQ treatment), for the indicated times. The graphs represent the densitometric quantification of the tagged APOPT1 signal at each time point. The graph inset shows that the increase of APOPT1 occurs in the first minutes after the exposure to MitoPQ


A Western blot analysis of SDS - PAGE of different mitochondrial proteins in total lysates from the indicated cell lines treated with 5 µM MitoPQ at the indicated times . UT : untreated cells . B Densitometric quantification of APOPT1GFP signal during the treatment in two biological replicas . C Densitometric quantification of MT - CO1 signal in the non - complemented APOPT1 - less cells ( S6 GFP ) vs . the complemented cells ( S6 APOPT1GFP ) . Three biological replicas ( n = 3 ) were carried out for each cell line . The signals in UT S6 APOPT1 were considered 100 % . The levels of MT - CO1 were significantly lower in the S6 GFP patient cells after 20 hours of MitoPQ treatment compared to the untreated cells ( * P = 0 . 0202 , two - tailed unpaired Student ' s t - test ) . D Densitometric quantification of MT - CO2 signal in the non - complemented APOPT1 - less cells ( S6 GFP ) vs . the complemented cells ( S6 APOPT1GFP ) . Three biological replicas were carried out for each cell line . The signals in UT S6 APOPT1 were considered 100 %

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Western blot analysis of SDS-PAGE of different mitochondrial proteins in total lysates from the indicated cell lines treated with 5 µM MitoPQ at the indicated times. UT: untreated cells. B Densitometric quantification of APOPT1GFP signal during the treatment in two biological replicas. C Densitometric quantification of MT-CO1 signal in the non-complemented APOPT1-less cells (S6 GFP) vs. the complemented cells (S6 APOPT1GFP). Three biological replicas (n=3) were carried out for each cell line. The signals in UT S6 APOPT1 were considered 100%. The levels of MT-CO1 were significantly lower in the S6 GFP patient cells after 20 hours of MitoPQ treatment compared to the untreated cells (*P = 0.0202, two-tailed unpaired Student's t-test). D Densitometric quantification of MT-CO2 signal in the non-complemented APOPT1-less cells (S6 GFP) vs. the complemented cells (S6 APOPT1GFP). Three biological replicas were carried out for each cell line. The signals in UT S6 APOPT1 were considered 100%


A Western blots of eEF2 expression in different brain regions , GAPDH was used as a loading control . B eEF2 expression levels were quantified and represented as fold changes of that in the hippocampus . n = 3 mice for each brain region . Data information : All data are shown as means ± s . e . m . All the t tests are two - tailed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Western blots of eEF2 expression in different brain regions, GAPDH was used as a loading control. B eEF2 expression levels were quantified and represented as fold changes of that in the hippocampus. n=3 mice for each brain region. Data information: All data are shown as means ± s.e.m. All the t tests are two-tailed.


C Immunofluorescent staining of eEF2 ( green ) and different nervous system cells ( red : NeuN / GFAP / IBA1 / OLIG2 / CaMKⅡα / GAD65 / 67 ) in somatosensory cortex of adult mice brains . Arrows indicate highly colocalized cells . Scale bar , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Immunofluorescent staining of eEF2 (green) and different nervous system cells (red: NeuN/GFAP/IBA1/OLIG2/CaMKⅡα/GAD65/67) in somatosensory cortex of adult mice brains. Arrows indicate highly colocalized cells. Scale bar, 10 μm.


D , E Colocalization of eEF2 and each cellular marker was quantified by calculating the Manders ' Colocalization Coefficients . 6 slices from 3 mice in each condition . ( D ) One - way ANOVA with Bonferroni ' s multiple comparisons post hoc , * * * * p < 0 . 0001 ( NeuN + / eEF2 + versus GFAP + / eEF2 + ) , * * * * p < 0 . 0001 ( NeuN + / eEF2 + versus IBA1 + / eEF2 + ) , * * * * p < 0 . 0001 ( NeuN + / eEF2 + versus OLIG2 + / eEF2 + ) . ( E ) Unpaired t test , * * p = 0 . 0065 ( CaMKⅡα + / eEF2 + versus GAD65 / 67 + / eEF2 + ) . Data information : All data are shown as means ± s . e . m . All the t tests are two - tailed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E Colocalization of eEF2 and each cellular marker was quantified by calculating the Manders' Colocalization Coefficients. 6 slices from 3 mice in each condition. (D) One-way ANOVA with Bonferroni's multiple comparisons post hoc, ****p<0.0001 (NeuN+/eEF2+ versus GFAP+/eEF2+), ****p<0.0001 (NeuN+/eEF2+ versus IBA1+/eEF2+), ****p<0.0001 (NeuN+/eEF2+ versus OLIG2+/eEF2+). (E) Unpaired t test, **p=0.0065 (CaMKⅡα+/eEF2+ versus GAD65/67+/eEF2+). Data information: All data are shown as means ± s.e.m. All the t tests are two-tailed.


B Western blots of eEF2 in the PFC , the rest part of cortex , the hippocampus , and the striatum of Eef2 WT and HET mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Western blots of eEF2 in the PFC, the rest part of cortex, the hippocampus, and the striatum of Eef2 WT and HET mice.


C Quantification analysis of ( B ) . n = 5 mice for cortex ( without PFC ) , n = 8 mice for PFC , n = 9 mice for HP , n = 3 mice for striatum . Unpaired t tests . Cortex ( without PFC ) , p = 0 . 9757 ; PFC , * * p = 0 . 0049 ; HP , p = 0 . 1338 ; Striatum , p = 0 . 7301 . Data information : All data are shown as means ± s . e . m . All the tests are two - tailed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Quantification analysis of (B). n=5 mice for cortex (without PFC), n=8 mice for PFC, n=9 mice for HP, n=3 mice for striatum. Unpaired t tests. Cortex (without PFC), p=0.9757; PFC, **p=0.0049; HP, p=0.1338; Striatum, p=0.7301. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.


D Representative merged immunofluorescent staining images of eEF2 ( green ) and CaMKⅡα ( red ) in the mPFC of Eef2 WT and HET mice . Scale bar , 10 μm . E Quantification of mean fluorescent intensity of eEF2 in CaMKⅡα + cells in WT and HET mPFC . n = 918 cells from 3 WT mice , n = 965 cells from 3 HET mice . Unpaired t test . * * * * p < 0 . 0001 . Data information : All data are shown as means ± s . e . m . All the tests are two - tailed .

D Representative merged immunofluorescent staining images of eEF2 (green) and CaMKⅡα (red) in the mPFC of Eef2 WT and HET mice. Scale bar, 10 μm. E Quantification of mean fluorescent intensity of eEF2 in CaMKⅡα+ cells in WT and HET mPFC. n=918 cells from 3 WT mice, n=965 cells from 3 HET mice. Unpaired t test. **** p<0.0001. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.


F Representative merged immunofluorescent staining images of eEF2 ( green ) and GAD65 / 67 ( red ) in the mPFC of Eef2 WT and HET mice . Scale bar , 10 μm . G Quantification of mean fluorescent intensity of eEF2 in GAD65 / 67 + cells in WT and HET mPFC . n = 690 cells from 3 WT mice , n = 686 cells from 3 HET mice . Mann - Whitney test . p = 0 . 6757 . Data information : All data are shown as means ± s . e . m . All the tests are two - tailed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Representative merged immunofluorescent staining images of eEF2 (green) and GAD65/67 (red) in the mPFC of Eef2 WT and HET mice. Scale bar, 10 μm. G Quantification of mean fluorescent intensity of eEF2 in GAD65/67+ cells in WT and HET mPFC. n=690 cells from 3 WT mice, n=686 cells from 3 HET mice. Mann-Whitney test. p=0.6757. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.


H Western blots of de novo global protein synthesis in PFC slices of WT and HET mice , measured by SUnSET . α - tubulin was used as a loading control . I Quantification analysis of puromycin - labeled protein levels . WT , n = 5 mice , HET , n = 4 mice . Unpaired t tests . * * p = 0 . 0011 . Data information : All data are shown as means ± s . e . m . All the tests are two - tailed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H Western blots of de novo global protein synthesis in PFC slices of WT and HET mice, measured by SUnSET. α-tubulin was used as a loading control. I Quantification analysis of puromycin-labeled protein levels. WT, n=5 mice, HET, n=4 mice. Unpaired t tests. **p=0.0011. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.


J Representative images of AHA incorporation ( green ) with CaMKⅡα + staining ( red ) in mPFC of WT and HET mice . Arrows indicate CaMKⅡα + cells . Scale bar , 20 μm . K Quantification of total AHA incorporation . 12 slices from 3 mice for each genotype . Unpaired t test . * p = 0 . 0268 . L Quantification of AHA incorporation in CaMKⅡα + cells . WT , n = 276 cells from 3 WT mice , n = 293 cells from 3 HET mice . Mann - Whitney test . * * * * p < 0 . 0001 . Data information : All data are shown as means ± s . e . m . All the tests are two - tailed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J Representative images of AHA incorporation (green) with CaMKⅡα+ staining (red) in mPFC of WT and HET mice. Arrows indicate CaMKⅡα+ cells. Scale bar, 20 μm. K Quantification of total AHA incorporation. 12 slices from 3 mice for each genotype. Unpaired t test. *p=0.0268. L Quantification of AHA incorporation in CaMKⅡα+ cells. WT, n=276 cells from 3 WT mice, n=293 cells from 3 HET mice. Mann-Whitney test. **** p<0.0001. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.


G Representative images of Golgi ‐ stained dendrite spines in the mPFC from WT and HET mice . Scale bar , 5 μm . H Quantification of spine density per 10 μm of dendrites . n = 18 dendrites from 3 WT mice ; n = 23 dendrites from 3 HET mice . Unpaired t test . * * * * p < 0 . 0001 . Data information : All data are shown as means ± s . e . m . All the tests are two - tailed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Representative images of Golgi‐stained dendrite spines in the mPFC from WT and HET mice. Scale bar, 5 μm. H Quantification of spine density per 10 μm of dendrites. n=18 dendrites from 3 WT mice; n=23 dendrites from 3 HET mice. Unpaired t test. ****p<0.0001. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.


I Representative Western blots of expression levels of AMPAR and NMDAR subunits in the synaptosomal fraction of WT and HET PFC . α - tubulin was served as a loading control . J Quantification analysis of synaptosomal expression of different receptor subunits . Unpaired t test . GluA1 , p = 0 . 9346 ; GluA2 , * * p = 0 . 0075 ; GluN1 , p = 0 . 6946 ; GluN2A , p = 0 . 4091 ; GluN2B , p = 0 . 0796 . n . s . , not significant . n = 5 mice for each genotype . Data information : All data are shown as means ± s . e . m . All the tests are two - tailed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Representative Western blots of expression levels of AMPAR and NMDAR subunits in the synaptosomal fraction of WT and HET PFC. α-tubulin was served as a loading control. J Quantification analysis of synaptosomal expression of different receptor subunits. Unpaired t test. GluA1, p=0.9346; GluA2, **p=0.0075; GluN1, p=0.6946; GluN2A, p=0.4091; GluN2B, p=0.0796. n.s., not significant. n=5 mice for each genotype. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.


L Representative immunofluorescent images of eEF2 ( magenta ) and PLA signals ( white ) in lentivirus ( LV ) - scramble - EGFP or LV - Eef2 shRNA - EGFP infected primary cortical neurons ( green , DIV 14 ) . Scale bar , 10 μm . M Quantification of mean fluorescent intensity of eEF2 in EGFP + cells in scramble and Eef2 shRNA groups . 64 cells for scramble group and 51 cells for Eef2 shRNA group from 3 independent experiments . Mann - Whitney test . * * * * p < 0 . 0001 . N Quantification of mean fluorescent intensity of PLA signals in EGFP + cells in the two groups . The PLA signals represent newly synthesized GluA2 . 64 cells for scramble group , 51 cells for Eef2 shRNA group from 3 independent experiments . Mann - Whitney test . * * * * p < 0 . 0001 . Data information : All data are shown as means ± s . e . m . All the tests are two - tailed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

L Representative immunofluorescent images of eEF2 (magenta) and PLA signals (white) in lentivirus (LV)-scramble-EGFP or LV-Eef2 shRNA-EGFP infected primary cortical neurons (green, DIV 14). Scale bar, 10 μm. M Quantification of mean fluorescent intensity of eEF2 in EGFP+ cells in scramble and Eef2 shRNA groups. 64 cells for scramble group and 51 cells for Eef2 shRNA group from 3 independent experiments. Mann-Whitney test. ****p<0.0001. N Quantification of mean fluorescent intensity of PLA signals in EGFP+ cells in the two groups. The PLA signals represent newly synthesized GluA2. 64 cells for scramble group, 51 cells for Eef2 shRNA group from 3 independent experiments. Mann-Whitney test. ****p<0.0001. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.


A PF - 4778574 ( PF ) directly potentiates EPSC amplitude , dashed line represents baseline value of normalized EPSC amplitude . Bottom : representative EPSC traces from baseline ( 1 ) , maximal potentiation ( 2 ) , and recovery ( 3 ) . B , C Quantifications of EPSC peak amplitude ( B ) and decay time ( C ) after PF ( 6 cells from 3 HET mice ) . ( B ) Wilcoxon signed - rank test , * p = 0 . 0313 ( PF v . s . baseline ) and p = 0 . 1600 ( Wash out v . s . baseline ) ; ( C ) Paired t test , * * * * p < 0 . 0001 ( PF v . s . baseline ) and p = 0 . 4423 ( Wash out v . s . baseline ) . Data information : Data are shown as means ± s . e . m . ,

O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A PF-4778574 (PF) directly potentiates EPSC amplitude, dashed line represents baseline value of normalized EPSC amplitude. Bottom: representative EPSC traces from baseline (1), maximal potentiation (2), and recovery (3). B, C Quantifications of EPSC peak amplitude (B) and decay time (C) after PF (6 cells from 3 HET mice). (B) Wilcoxon signed-rank test, *p=0.0313 (PF v.s. baseline) and p=0.1600 (Wash out v.s. baseline); (C) Paired t test, ****p<0.0001 (PF v.s. baseline) and p=0.4423 (Wash out v.s. baseline). Data information: Data are shown as means ± s.e.m., data


D PF rescued AMPAR - EPSC in L5 neurons from HET mice . 25 - 30 % increase at strongest stimulation ( 7 cells from 3 HET mice ) , paired t test , * * p = 0 . 0014 . Bottom : representative traces of the effects of PF on AMPAR - EPSC evoked by different stimulus intensities . Data information : Data are shown as means ± s . e . m .

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D PF rescued AMPAR-EPSC in L5 neurons from HET mice. 25-30% increase at strongest stimulation (7 cells from 3 HET mice), paired t test, **p=0.0014. Bottom: representative traces of the effects of PF on AMPAR-EPSC evoked by different stimulus intensities. Data information: Data are shown as means ± s.e.m.


E Representative heatmaps of the two social tasks ( left , social approach ; right , social novelty ) in the Three - chamber test performed by Eef2 HET mice intraperitoneal administered with PF - 4778574 or DMSO ( vehicle control ) . E : empty cup ; S1 : stranger # 1 contained cup ; S2 : stranger # 2 contained cup . F Discrimination score of social approach performed by two groups of mice , calculated by the difference of time spent in sniffing S1 and E . Unpaired t test . p = 0 . 7335 . WT , n = 9 mice ; HET , n = 8 mice . G Discrimination score of social novelty performed by two groups of mice , calculated by the difference of time spent in sniffing S2 and S1 . Unpaired t test . * p = 0 . 0492 . WT , n = 9 mice ; HET , n = 8 mice . Data information : Data F , G , are shown as interquartile range , with line across the box indicating median , whiskers show the highest and lowest values . n . s . , not significant . All the t tests are two - tailed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Representative heatmaps of the two social tasks (left, social approach; right, social novelty) in the Three-chamber test performed by Eef2 HET mice intraperitoneal administered with PF-4778574 or DMSO (vehicle control). E: empty cup; S1: stranger #1 contained cup; S2: stranger #2 contained cup. F Discrimination score of social approach performed by two groups of mice, calculated by the difference of time spent in sniffing S1 and E. Unpaired t test. p=0.7335. WT, n=9 mice; HET, n=8 mice. G Discrimination score of social novelty performed by two groups of mice, calculated by the difference of time spent in sniffing S2 and S1. Unpaired t test. *p=0.0492. WT, n=9 mice; HET, n=8 mice. Data information: Data F, G, are shown as interquartile range, with line across the box indicating median, whiskers show the highest and lowest values. n.s., not significant. All the t tests are two-tailed.


I Representative heatmaps of the two social tasks ( left , social approach ; right , social novelty ) performed by Eef2 HET mice with mPFC infusion of PF - 4778574 or DMSO ( vehicle control ) in the Three - chamber test . E : empty cup ; S1 : stranger # 1 contained cup ; S2 : stranger # 2 contained cup . J Discrimination score of social approach performed by two groups of mice , calculated by the difference of time spent in sniffing S1 and E . Unpaired t test . p = 0 . 3984 . n = 11 mice for each group . K Discrimination score of social novelty performed by two groups of mice , calculated by the difference of time spent in sniffing S2 and S1 . Unpaired t test . * p = 0 . 0489 . n = 11 mice for each group . Data information : Data of J and K are shown as interquartile range , with line across the box indicating median , whiskers show the highest and lowest values . n . s . , not significant . All the t tests are two - tailed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Representative heatmaps of the two social tasks (left, social approach; right, social novelty) performed by Eef2 HET mice with mPFC infusion of PF-4778574 or DMSO (vehicle control) in the Three-chamber test. E: empty cup; S1: stranger #1 contained cup; S2: stranger #2 contained cup. J Discrimination score of social approach performed by two groups of mice, calculated by the difference of time spent in sniffing S1 and E. Unpaired t test. p=0.3984. n=11 mice for each group. K Discrimination score of social novelty performed by two groups of mice, calculated by the difference of time spent in sniffing S2 and S1. Unpaired t test. *p=0.0489. n=11 mice for each group. Data information: Data of J and K are shown as interquartile range, with line across the box indicating median, whiskers show the highest and lowest values. n.s., not significant. All the t tests are two-tailed.


Three independent samples ( Exp 1 to 3 ) of EV secreted by labeled 3T3 - F442A cells were analyzed by mass spectrometry ( in duplicate injections , Inj1 / 2 ) . The percentage of proteins bearing at least one peptide containing a heavy amino acid is indicated .

Three independent samples (Exp 1 to 3) of EV secreted by labeled 3T3-F442A cells were analyzed by mass spectrometry (in duplicate injections, Inj1/2). The percentage of proteins bearing at least one peptide containing a heavy amino acid is indicated.


Two human ( SKMEL28 and 1205Lu ) and a murine ( B16BL6 ) melanoma cell lines were exposed , or not , to the indicated EV from primary murine adipocytes obtained from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) , then FAO was measured ( n = 5 for B16BL6 , SKMEL28 HFD EV and 1205Lu ND EV and n = 6 for other conditions ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Two human (SKMEL28 and 1205Lu) and a murine (B16BL6) melanoma cell lines were exposed, or not, to the indicated EV from primary murine adipocytes obtained from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD), then FAO was measured (n =5 for B16BL6, SKMEL28 HFD EV and 1205Lu ND EV and n=6 for other conditions).


Volcano plot of mass spectrometry - based quantitative proteomics results showing relative abundance of proteins in primary murine adipocyte EV from obese mice ( HFD ) , as compared to those from lean mice ( ND ) . The dashed lines indicate cut - off values and points colored in grey indicate proteins that display non - significant fold - change by Welch t - test between both conditions ( n = 3 for ND , 4 for HFD ) . Proteins involved in FAO are indicated by yellow dots .

Volcano plot of mass spectrometry-based quantitative proteomics results showing relative abundance of proteins in primary murine adipocyte EV from obese mice (HFD), as compared to those from lean mice (ND). The dashed lines indicate cut-off values and points colored in grey indicate proteins that display non-significant fold-change by Welch t-test between both conditions (n=3 for ND, 4 for HFD). Proteins involved in FAO are indicated by yellow dots.


Western blot analysis of the indicated FAO enzymes in the EV secreted by primary adipocytes from lean ( ND ) and obese ( HFD ) mice ( top panel ) and from human individuals with varying BMI ( normal weight , NW ; overweight , OW and obese , OB ) ( bottom panel ) . For each blot , extracts from 3 independent batches of murine samples or 3 independent individuals for human samples ( 1 - 3 ) are shown . Flotillin 1 ( FLOT1 ) is used as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis of the indicated FAO enzymes in the EV secreted by primary adipocytes from lean (ND) and obese (HFD) mice (top panel) and from human individuals with varying BMI (normal weight, NW; overweight, OW and obese, OB) (bottom panel). For each blot, extracts from 3 independent batches of murine samples or 3 independent individuals for human samples (1-3) are shown. Flotillin 1 (FLOT1) is used as a loading control.


Western blot analysis of the indicated FAO enzymes in melanoma cells treated , or not , with EV from lean ( ND ) and obese ( HFD ) mice . Tubulin ( TUB ) is used as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis of the indicated FAO enzymes in melanoma cells treated, or not, with EV from lean (ND) and obese (HFD) mice. Tubulin (TUB) is used as a loading control.


RT - qPCR analysis of mRNAs for the indicated genes in 1205Lu cells treated or not with EV secreted by primary adipocytes from lean ( ND ) and obese ( HFD ) mice for 48 h . Results are expressed relative to the corresponding value for control cells ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RT-qPCR analysis of mRNAs for the indicated genes in 1205Lu cells treated or not with EV secreted by primary adipocytes from lean (ND) and obese (HFD) mice for 48 h. Results are expressed relative to the corresponding value for control cells (n=3).


Analysis of FAO levels in 1205Lu cells exposed to 3T3 - F442A EV and treated , or not , with cycloheximide ( CHX ) ( n = 5 ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O

Analysis of FAO levels in 1205Lu cells exposed to 3T3-F442A EV and treated, or not, with cycloheximide (CHX) (n=5).


Lipids were extracted from EV secreted by 3T3 - F442A pre - adipocytes and differentiated 3T3 - F442A adipocytes ( respectively preAd - 3T3 - EV and Ad - 3T3 - EV ) , and FA content was measured ( n = 6 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O

Lipids were extracted from EV secreted by 3T3-F442A pre-adipocytes and differentiated 3T3-F442A adipocytes (respectively preAd-3T3-EV and Ad-3T3-EV), and FA content was measured (n=6).


Indicated melanoma cells were incubated with EV from 3T3 - F442A adipocytes previously loaded with BODIPY FL C16 ( 3T3 - FL C16 - EV ) and , 24h later , cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy .

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Indicated melanoma cells were incubated with EV from 3T3-F442A adipocytes previously loaded with BODIPY FL C16 (3T3-FL C16-EV) and, 24h later, cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy.


Left panel , indicated cells were incubated with EV from 3T3 - F442A adipocytes previously loaded with BODIPY FL C16 and immediately treated , or not , with Etomoxir for 24h . Then , cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy . Right panel , quantification of BODIPY FL C16 staining area per cell ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Left panel, indicated cells were incubated with EV from 3T3-F442A adipocytes previously loaded with BODIPY FL C16 and immediately treated, or not, with Etomoxir for 24h. Then, cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy. Right panel, quantification of BODIPY FL C16 staining area per cell (n=3).


Lipids were extracted from EV secreted by adipocytes from lean ( ND ) and obese ( HFD ) mice ( n = 5 ) ( left panel ) or from human adipose tissue samples from patients with varying BMI ( right panel ) and fatty acid content was measured ( n = 15 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O

Lipids were extracted from EV secreted by adipocytes from lean (ND) and obese (HFD) mice (n=5) (left panel) or from human adipose tissue samples from patients with varying BMI (right panel) and fatty acid content was measured (n=15).


Indicated cells were exposed , or not , to adipocyte EV from primary adipocytes from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) for 24h . Then , cells were fixed , stained with BODIPY and counterstained with DAPI . Left panel , confocal microscopy observation . Right panel , quantification of BODIPY staining area per cell ( n = 5 for SKMEL28 and n = 6 for 1205Lu ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Indicated cells were exposed, or not, to adipocyte EV from primary adipocytes from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) for 24h. Then, cells were fixed, stained with BODIPY and counterstained with DAPI. Left panel, confocal microscopy observation. Right panel, quantification of BODIPY staining area per cell (n=5 for SKMEL28 and n=6 for 1205Lu).


Transmission electron micrographs of 1205Lu cells exposed , or not , to 3T3 - F442A EV ( 3T3 - EV ) . Scale bar represents 1µm . A zoomed crop of the area with autophagic structures containing lipids ( indicated by an arrow ) is shown ( scale bar : 0 . 5µm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

Transmission electron micrographs of 1205Lu cells exposed, or not, to 3T3-F442A EV (3T3-EV). Scale bar represents 1µm. A zoomed crop of the area with autophagic structures containing lipids (indicated by an arrow) is shown (scale bar: 0.5µm).


1205Lu cells were incubated with EV from 3T3 - F442A adipocytes , previously loaded with BODIPY FL C16 in the presence , or not , of Lalistat 2 . Then , live cells were stained with the LysoTracker probe and observed by confocal microscopy . Arrows indicate colocalization .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

1205Lu cells were incubated with EV from 3T3-F442A adipocytes, previously loaded with BODIPY FL C16 in the presence, or not, of Lalistat 2. Then, live cells were stained with the LysoTracker probe and observed by confocal microscopy. Arrows indicate colocalization.


1205Lu were incubated with EV from 3T3 - F442A adipocytes , previously loaded with BODIPY FL C16 and treated , or not , with Lalistat 2 . Then , cells were fixed and counterstained with DAPI before observation by confocal microscopy . Quantification of BOBIPY FL C16 staining per cell is shown beside ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

1205Lu were incubated with EV from 3T3-F442A adipocytes, previously loaded with BODIPY FL C16 and treated, or not, with Lalistat 2. Then, cells were fixed and counterstained with DAPI before observation by confocal microscopy. Quantification of BOBIPY FL C16 staining per cell is shown beside (n=4).


1205Lu cells were exposed to 3T3 - F442A EV ( 3T3 - EV ) and treated , or not , with Lalistat 2 ( Lal ) . Cell migration was then evaluated in Boyden chamber assays ( left panel ; n = 5 ) or cell motility was tracked by video microscopy ( right panel ; n = 40 cells per group ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

1205Lu cells were exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Lalistat 2 (Lal). Cell migration was then evaluated in Boyden chamber assays (left panel; n=5) or cell motility was tracked by video microscopy (right panel; n=40 cells per group).


1205Lu cells were exposed , or not , to adipocyte EV from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) with , or without , Lalistat 2 ( Lal ) . Cells were then fixed , stained with BODIPY and counterstain with DAPI before observation by confocal microscopy . Quantification of BODIPY staining per cell is shown on the right ( n = 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

1205Lu cells were exposed, or not, to adipocyte EV from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) with, or without, Lalistat 2 (Lal). Cells were then fixed, stained with BODIPY and counterstain with DAPI before observation by confocal microscopy. Quantification of BODIPY staining per cell is shown on the right (n=5).


1205Lu cells were exposed , or not , to adipocyte EV from ND or HFD mice with , or without , Lalistat 2 ( Lal ) . Cell motility was then tracked by video microscopy ( n = 40 cells per group ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

1205Lu cells were exposed, or not, to adipocyte EV from ND or HFD mice with, or without, Lalistat 2 (Lal). Cell motility was then tracked by video microscopy (n=40 cells per group).


1205Lu cells exposed to 3T3 - F442A EV ( 3T3 - EV ) and treated , or not , with Mdivi - 1 , were stained with a MitoTracker probe , fixed and observed by confocal microscopy . Arrows indicate the presence of mitochondria in membrane protrusions . On the images to the right , the outline of cells is shown in dotted lines . Scale bars : 20µm . Right panel , quantification of the distance between mitochondria and nuclei ( n = 15 ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

1205Lu cells exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Mdivi-1, were stained with a MitoTracker probe, fixed and observed by confocal microscopy. Arrows indicate the presence of mitochondria in membrane protrusions. On the images to the right, the outline of cells is shown in dotted lines. Scale bars: 20µm. Right panel, quantification of the distance between mitochondria and nuclei (n=15).


Western blot analysis of the indicated mitochondrial fission proteins in melanoma cells treated , or not , with EV from 3T3 - F442A adipocytes ( 3T3 - EV ) . Tubulin ( TUB ) is used as a loading control .

Western blot analysis of the indicated mitochondrial fission proteins in melanoma cells treated, or not, with EV from 3T3-F442A adipocytes (3T3-EV). Tubulin (TUB) is used as a loading control.


1205Lu cells were exposed to 3T3 - F442A EV ( 3T3 - EV ) and treated , or not , with Mdivi - 1 . Cell migration was then evaluated in Boyden chamber assays ( left panel ; n = 6 ) . Right panel , 3T3 - F442A EV were added to 1205Lu cells , which were immediately treated , or not , with Mdivi - 1 . Cell motility was then tracked by video microscopy ( n = 40 cells per group ) ( right panel ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

1205Lu cells were exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Mdivi-1. Cell migration was then evaluated in Boyden chamber assays (left panel; n=6). Right panel, 3T3-F442A EV were added to 1205Lu cells, which were immediately treated, or not, with Mdivi-1. Cell motility was then tracked by video microscopy (n = 40 cells per group) (right panel).