of 2 - - like in cultures treated with increasing concentrations of sodium acetate , sodium L - lactate or D - ribose . Shown are mean ± s . d . of 2 independent cultures , performed across 3 biological replicates . Boxes indicate range between first third quantile , band depicts median whiskers extend no further than 1 . 5 - times interquartile range .

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Percentage of 2-cell-like cells in cultures treated with increasing concentrations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across 3 biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range.


of Zscan4 + in cultures treated with increasing concentrations of sodium acetate , sodium L - lactate or D - ribose . Shown are mean ± s . d . of 2 independent cultures , performed across 3 biological replicates . Boxes indicate range between first third quantile , band depicts median whiskers extend no further than 1 . 5 - times interquartile range .

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Percentage of Zscan4+ cells in cultures treated with increasing concentrations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across 3 biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range.


of Zscan4 + in cultures treated with varying combinations of sodium acetate , sodium L - lactate or D - ribose . Shown are mean ± s . d . of 2 independent cultures , performed across biological replicates . Boxes indicate range between first third quantile , band depicts median whiskers extend no further than 1 . 5 - times interquartile range .

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

Percentage of Zscan4+ cells in cultures treated with varying combinations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range.


of 2 - - like in cultures treated with varying combinations of sodium acetate , sodium L - lactate or D - ribose . Shown are mean ± s . d . of 2 independent cultures , performed across biological replicates . Boxes indicate range between first third quantile , band depicts median whiskers extend no further than 1 . 5 - times interquartile range .

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

Percentage of 2-cell-like cells in cultures treated with varying combinations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range.


A ) Immunofluorescence for OCT4 , ZSCAN4 2C : : tbGFP in control acetate treated ESC cultures . Green arrows indicate 2 - - like , inlets highlight their DAPI structure . Scale bar , 20 μm .

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A) Immunofluorescence staining for OCT4, ZSCAN4 and 2C::tbGFP in control and acetate treated ESC cultures. Green arrows indicate 2-cell-like cells, and inlets highlight their DAPI structure. Scale bar, 20 μm.


B ) RT - qPCR of indicated genes in ESC cultures treated with sodium acetate for 24h . Shown are mean ± s . d . of 3 independent cultures , performed in 2 technical replicates .

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B) RT-qPCR of the indicated genes in ESC cultures treated with sodium acetate for 24h. Shown are the mean ± s.d. of 3 independent cell cultures, performed in 2 technical replicates.


C ) of 2 - - like obtained upon transfection of control or Dux - targeting siRNAs in control conditions or in combination with sodium acetate treatment . Measurements were obtained from 2 independent cultures , performed across 3 independent biological replicates . Boxes indicate range between first third quantile , band depicts median whiskers extend no further than 1 . 5 - times interquartile range . dots indicate measurements obtained in each technical replicate .

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C) Percentage of 2-cell-like cells obtained upon transfection of control or Dux-targeting siRNAs in control conditions or in combination with sodium acetate treatment. Measurements were obtained from 2 independent cell cultures, performed across 3 independent biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range. Individual dots indicate the measurements obtained in each technical replicate.


D ) of 2 - - like obtained upon transfection siRNAs targeting indicated factors in control conditions or in combination with sodium acetate treatment . Shown are mean ± s . d . of 4 independent cultures , performed across 2 independent biological replicates .

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D) Percentage of 2-cell-like cells obtained upon transfection siRNAs targeting the indicated chromatin factors in control conditions or in combination with sodium acetate treatment. Shown are the mean ± s.d. of 4 independent cell cultures, performed across 2 independent biological replicates.


F ) Proportion of Zscan4 + at various timepoints during time - lapse experiment . Shown are mean ± s . d . of 3 independent experiments .

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F) Proportion of Zscan4+ cells at various timepoints during the time-lapse experiment. Shown are the mean ± s.d. of 3 independent experiments.


Glycolytic flux of wildtype ( WT ) TM6 strains strongly correlates with fructose - 1 , 6 - bisphosphate ( FBP ) . glycolytic flux is reported here as flux between metabolites fructose 6 - phosphate ( F6P ) FBP . Glycolytic fluxes were obtained on basis of physiological metabolome data , via a novel method to estimate intracellular fluxes ( Niebel et al , 2019 ) . While on high glucose , WT accomplishes a high glucose uptake rate ( thus glycolytic flux ) , mutant ( TM6 ) only generates a low glucose uptake ( thus glycolytic flux ) . On maltose , also mutant achieves a high glycolytic flux , since maltose is transported by a separate transporter ( Chang et al , 1989 ) . To assess linear correlation between FBP glycolytic flux across studied conditions , we implemented Pearson ' s correlation assisted by bootstrapping . Specifically , we used in total 53 FBP measurements corresponding to six different metabolic conditions ( combinations of strains carbon sources ) , biological technical replicates . We paired each of these FBP measurements with mean standard deviation of model - derived glycolytic flux ( A ) in corresponding metabolic condition . We assumed normal distribution of flux growth rate with given mean standard deviation in every condition , implemented ordinary nonparametric bootstrapping with 100 , 000 iterations by randomly sampling values with replacement from 53 FBP measurements flux or growth - rate distributions to calculate correlation statistics .

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

Glycolytic flux of wildtype (WT) and TM6 strains strongly correlates with fructose-1,6-bisphosphate (FBP) concentration. The glycolytic flux is reported here as the flux between the metabolites fructose 6-phosphate (F6P) and FBP. Glycolytic fluxes were obtained on the basis of physiological and metabolome data, and via a novel method to estimate intracellular fluxes (Niebel et al, 2019). While on high glucose, the WT strain accomplishes a high glucose uptake rate (and thus glycolytic flux), the mutant strain (TM6) only generates a low glucose uptake (and thus glycolytic flux). On maltose, also the mutant strain achieves a high glycolytic flux, since maltose is transported by a separate transporter (Chang et al, 1989). To assess the linear correlation between the FBP concentration and the glycolytic flux across the studied conditions, we implemented Pearson's correlation analysis assisted by bootstrapping. Specifically, we used in total 53 FBP concentration measurements corresponding to six different metabolic conditions (combinations of strains and carbon sources), biological and technical replicates. We paired each of these FBP measurements with the mean and standard deviation of the model-derived glycolytic flux (A) in the corresponding metabolic condition. We assumed the normal distribution of the flux and growth rate with the given mean and standard deviation in every condition, and implemented ordinary nonparametric bootstrapping with 100,000 iterations by randomly sampling values with replacement from the 53 FBP measurements and flux or growth-rate distributions to calculate the correlation statistics.


FBP as a function of growth rate shows weaker correlation . For metabolite growth rates , error bars correspond to standard deviation between three independent experiments ; for glycolytic fluxes to mean standard deviations of sampled flux solution space ( cf . Methods ) . carbon sources were used at a final of 10 gL - 1 are indicated : Glucose ( GLU ) ; Galactose ( GAL ) ; Maltose ( MAL ) Pyruvate ( PYR ) . To assess linear correlation between FBP growth rate ( B ) across studied conditions , we implemented Pearson ' s correlation assisted by bootstrapping . Specifically , we used in total 53 FBP measurements corresponding to six different metabolic conditions ( combinations of strains carbon sources ) , biological technical replicates . We paired each of these FBP measurements with mean standard deviation of growth rate ( B ) in corresponding metabolic condition . We assumed normal distribution of flux growth rate with given mean standard deviation in every condition , implemented ordinary nonparametric bootstrapping with 100 , 000 iterations by randomly sampling values with replacement from 53 FBP measurements flux or growth - rate distributions to calculate correlation statistics .

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

FBP concentration as a function of cellular growth rate shows weaker correlation. For metabolite levels and growth rates, error bars correspond to the standard deviation between three independent experiments; for glycolytic fluxes to the mean and standard deviations of the sampled flux solution space (cf. Methods). The carbon sources were used at a final concentration of 10 gL-1 and are indicated: Glucose (GLU); Galactose (GAL); Maltose (MAL) and Pyruvate (PYR). To assess the linear correlation between the FBP concentration and the growth rate (B) across the studied conditions, we implemented Pearson's correlation analysis assisted by bootstrapping. Specifically, we used in total 53 FBP concentration measurements corresponding to six different metabolic conditions (combinations of strains and carbon sources), biological and technical replicates. We paired each of these FBP measurements with the mean and standard deviation of the growth rate (B) in the corresponding metabolic condition. We assumed the normal distribution of the flux and growth rate with the given mean and standard deviation in every condition, and implemented ordinary nonparametric bootstrapping with 100,000 iterations by randomly sampling values with replacement from the 53 FBP measurements and flux or growth-rate distributions to calculate the correlation statistics.


CggR intracellular abundance in wildtype ( WT ) on glucose strongly varies with promoter used . CggR intracellular were quantified by proteomics in steady - state cultures grown in minimal media with glucose as carbon source at a final of 10 gL - 1 . Error bars represent standard deviation of at least three replicate experiments .

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CggR intracellular abundance in the wildtype (WT) strain on glucose strongly varies with the promoter used. The CggR intracellular levels were quantified by proteomics in steady-state cultures grown in minimal media with glucose as carbon source at a final concentration of 10 gL-1. Error bars represent the standard deviation of at least three replicate experiments.


relative abundance of CggR ( normalized to abundance measured on glucose same promoter ) is almost constant with PTEFmut7 across multiple growth rates in WT TM6 , but not with PTEFmut2 PCMV . CggR intracellular were quantified by proteomics in steady - state cultures grown in minimal media with glucose , galactose , maltose or pyruvate as carbon sources at a final of 10 gL - 1 . WT data includes three promoters , whereas TM6 only includes PTEFmut2 PTEFmut7 data . Error bars represent standard deviation of at least three replicate experiments .

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The relative abundance of CggR (normalized to the abundance measured on glucose and the same promoter) is almost constant with PTEFmut7 across multiple growth rates in WT and TM6 cells, but not with PTEFmut2 and PCMV. The CggR intracellular levels were quantified by proteomics in steady-state cultures grown in minimal media with glucose, galactose, maltose or pyruvate as carbon sources at a final concentration of 10 gL-1. WT data includes all three promoters, whereas TM6 only includes the PTEFmut2 and PTEFmut7 data. Error bars represent the standard deviation of at least three replicate experiments.


Thermal shift assays were used to determine melting curves of wild - type CggR mutants . Error bars correspond to standard deviation of at least five replicates .

Thermal shift assays were used to determine the melting curves of wild-type CggR and mutants. Error bars correspond to the standard deviation of at least five replicates.


CggR - FBP affinity constants ( KD ' s ) of wildtype mutant variants , determined by fitting a simple cooperative binding model to melting curves data . Error bars indicate 95 % confidence intervals .

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CggR-FBP affinity constants (KD's) of the wildtype and mutant variants, determined by fitting a simple cooperative binding model to the melting curves data. Error bars indicate the 95% confidence intervals.


of CggR binding to . CggR bound to fraction was calculated by dividing of - band by total . background - subtracted total intensities of CggR - free - bands were assessed with ImageJ . Error bars correspond to standard deviation of three replicate experiments for mutants six for wildtype .

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Quantification of CggR binding to DNA. The CggR bound to DNA fraction was calculated by dividing the intensity of the protein-DNA complex band by the total DNA. The background-subtracted total intensities of the CggR-DNA complex and the free-DNA bands were assessed with ImageJ. Error bars correspond to the standard deviation of three replicate experiments for the mutants and six for the wildtype.


four reporter plasmids were transferred to wildtype containing CggR ( R250A ) under control of PTEFmut7 . strength of four promoters was assessed by quantifying YFP ( FL1 ) mCherry ( FL3 ) in exponentially growing wildtype in minimal medium with 10 g l - 1 glucose . FL1 FL3 shown is non - background corrected median of 100 000 events . non - FL control is signal from a wildtype grown under same conditions . background , assessed by wildtype harboring YCplac33 plasmid , was substracted from FL1 FL3 . final reporter activity is of background - corrected YFP mCherry values . Error bars represent standard deviation of three independently determined ratios from three replicate experiments .

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The four reporter plasmids were transferred to the wildtype strain containing the CggR (R250A) under the control of the PTEFmut7. The strength of the four promoters was assessed by quantifying YFP (FL1) and mCherry (FL3) fluorescence in exponentially growing wildtype cells in minimal medium with 10 g l-1 glucose. The FL1 and FL3 fluorescence shown is the non-background corrected median of 100 000 cell events. The non-FL control is the signal from a wildtype strain grown under the same conditions. The background fluorescence, assessed by the wildtype harboring the YCplac33 plasmid, was substracted from FL1 and FL3. The final reporter activity is the ratio of the background-corrected YFP and mCherry values. Error bars represent the standard deviation of three independently determined ratios from three replicate experiments.


Reporter activity of sensor across ( D ) multiple FBP Reporter activity is given by YFP / mCherry , calculated through of YFP mCherry along culture time using flow cytometry . Both YFP mCherry were first corrected for background using same strains harboring YCplac33 plasmid ( Appendix Table S8 ) . control is wildtype TM6 strains expressing only reporter plasmid without CggR . Error bars represent standard deviation of at least three replicate experiments .

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Reporter activity of the sensor across (D) multiple FBP levels Reporter activity is given by the YFP/mCherry ratio, calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry. Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid (Appendix Table S8). The control is the wildtype and TM6 strains expressing only the reporter plasmid without CggR. Error bars represent the standard deviation of at least three replicate experiments.


Reporter activity of sensor across glycolytic fluxes . glycolytic flux is reported as flux between metabolites fructose 6 - phosphate ( F6P ) fructose - 1 , 6 - bisphosphate ( FBP ) . Glycolytic fluxes were here estimated on basis of physiological metabolome data a novel method to estimate intracellular fluxes ( Niebel et al , 2019 ) . Reporter activity is given by YFP / mCherry , calculated through of YFP mCherry along culture time using flow cytometry . Both YFP mCherry were first corrected for background using same strains harboring YCplac33 plasmid ( Appendix Table S8 ) . control is wildtype TM6 strains expressing only reporter plasmid without CggR . Error bars represent standard deviation of at least three replicate experiments .

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Reporter activity of the sensor across glycolytic fluxes. The glycolytic flux is reported as the flux between the metabolites fructose 6-phosphate (F6P) and fructose-1,6-bisphosphate (FBP). Glycolytic fluxes were here estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes (Niebel et al, 2019). Reporter activity is given by the YFP/mCherry ratio, calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry. Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid (Appendix Table S8). The control is the wildtype and TM6 strains expressing only the reporter plasmid without CggR. Error bars represent the standard deviation of at least three replicate experiments.


Fraction of CggR bound to FBP across FBP concentrations . red arrows indicate shift in of CggR bound to FBP achieved in R250A variant . of CggR molecules bound to FBP was calculated after normalizing Tm values for unbound / bound state using Tm at 0 mM FBP as unbound at 36 mM ( corresponding to maximum FBP used ) as total bound states . curve fitting of normalized values of CggR fraction bound to FBP was performed using a one - site specific binding model in GraphPad . solid corresponds to wildtype CggR dashed to R250A variant . Vertical lines delimit physiological FBP range .

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

Fraction of CggR bound to FBP across FBP concentrations. The red arrows indicate the shift in the percentage of CggR bound to FBP achieved in the R250A variant. The percentage of CggR molecules bound to FBP was calculated after normalizing the Tm values for unbound/bound state using the Tm at 0 mM FBP as unbound and at 36 mM (corresponding to maximum FBP concentration used) as total bound states. The curve fitting of the normalized values of CggR fraction bound to FBP was performed using a one-site specific binding model in GraphPad. The solid line corresponds to the wildtype CggR and the dashed line to the R250A variant. Vertical lines delimit the physiological FBP range.


Sub - populations of WT TM6 , grown separately mixed in different fractions as indicated in percentages , can easily be distinguished by flow cytometry , FL1 YFP channel , FL3 mCherry channel . Histogram of single - ratios of FL1 / FL3 intensities of mixed WT TM6 populations analyzed by flow cytometry . Here , a sub - population of minimally 5 % can be distinguished .

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Sub-populations of WT and TM6 cells, grown separately and mixed in different fractions as indicated in percentages, can easily be distinguished by flow cytometry, FL1 YFP channel, FL3 mCherry channel. Histogram of single-cell ratios of FL1/FL3 fluorescence intensities of mixed WT and TM6 populations analyzed by flow cytometry. Here, a sub-population of minimally 5% can be distinguished.


Tukey boxplots showing YFP / mCherry of measured by microscopy as a function of glycolytic flux . At least 35 were analyzed in each condition . glycolytic flux is here reported as flux between metabolites fructose 6 - phosphate ( F6P ) FBP . Glycolytic fluxes were estimated on basis of physiological metabolome data a novel method to estimate intracellular fluxes ( Niebel et al , 2019 ) .

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

Tukey boxplots showing the YFP/mCherry ratio of individual cells measured by microscopy as a function of glycolytic flux. At least 35 cells were analyzed in each condition. The glycolytic flux is here reported as the flux between the metabolites fructose 6-phosphate (F6P) and FBP. Glycolytic fluxes were estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes (Niebel et al, 2019).


YFP / mCherry measured by microscopy in co - existing dividing ( high - flux ) versus non - dividing ( low - flux ) isogenic TM6 on 10 gL - 1 glucose .

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YFP/mCherry ratio measured by microscopy in co-existing dividing (high-flux) versus non-dividing (low-flux) isogenic TM6 cells on 10 gL-1 glucose.


Brightfield ( BF ) , YFP mCherry microscopy for a co - existing dividing ( high - flux ) a non - dividing ( low - flux ) TM6 expressing flux sensor in 10g L - 1 glucose minimal medium .

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Brightfield (BF), YFP and mCherry microscopy images for a co-existing dividing (high-flux) and a non-dividing (low-flux) TM6 cell expressing the flux sensor in 10g L-1 glucose minimal medium.


production rates of YFP mCherry are uncoupled during cycle in biosensor - expressing ( F ) , which reflects - cycle dynamics of intracellular FBP glycolytic flux . In a control , lacking CggR , production rates of YFP mCherry are coupled ( G ) . uncoupling was calculated for - cycle trajectories as difference between YFP mCherry production rates normalised to have same scale ( see more details in Materials methods ; Appendix Figure S10 ) . Each curve represents mean across indicated number of cycles in a replicate experiment . corresponding shaded areas denote 95 % confidence intervals of means ( bootstrapping with 5 000 iterations ) . We smoothed single - - cycle trajectories of YFP mCherry signals as well as volume via Gaussian process regression , used these trajectories to derive YFP mCherry production rates , accounting for - maturation in a first - order kinetics model . To align - cycle trajectories to calculate phase , we used array of three cycle events E { cytokinesis ( cyt ) , budding , next cyt } as reference points . cycles used for had duration in interval between 150 300 minutes , with mean duration presented in parentheses for each replicate experiment . belonged to TM6 were cultivated on 20 gL - 1 glucose in microfluidic device .

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The production rates of YFP and mCherry are uncoupled during the cell cycle in the biosensor-expressing strain (F), which reflects the cell-cycle dynamics of intracellular FBP concentration and glycolytic flux. In a control strain, lacking CggR, the production rates of YFP and mCherry are coupled (G). The uncoupling was calculated for individual cell-cycle trajectories as the difference between the YFP and mCherry production rates normalised to have the same scale (see more details in Materials and methods; Appendix Figure S10). Each curve represents the mean across the indicated number of cell cycles in a replicate experiment. The corresponding shaded areas denote the 95% confidence intervals of the means (bootstrapping with 5 000 iterations). We smoothed the single-cell-cycle trajectories of YFP and mCherry signals as well as cell volume via the Gaussian process regression, and used these trajectories to derive the YFP and mCherry production rates, accounting for fluorescent-protein maturation in a first-order kinetics model. To align the cell-cycle trajectories and to calculate the phase, we used the array of three cell cycle events E {cytokinesis (cyt), budding, next cyt} as reference points. The cell cycles used for the analysis had the duration in the interval between 150 and 300 minutes, with the mean duration presented in parentheses for each replicate experiment. The cells belonged to the TM6 strain and were cultivated on 20 gL-1 glucose in the microfluidic device.


( A - F ) Representative PAS - stained sections of pons ( A ) hippocampal ( C ) regions of brain heart ( E ) of one year old HOIL - 1 [ C458S ] WT mice are shown . Scale bar = 100 μm . Arrow heads indicate α - amylase - resistant PAS - positive polyglucosan deposits . Graphs showing α - amylase resistant PAS scores of pons ( B ) hippocampal ( D ) regions of brain heart ( F ) of HOIL - 1 [ C458S ] ( red ) WT ( blue ) mice aged 0 . 5 , 1 . 0 1 . 5 years . number of biological replicates analysed at each is indicated . word zero highlighted in blue indicates that no α - amylase resistant , PAS - positive material could be detected in WT mice . error bars show mean + . Statistical significance between genotypes was calculated by using two - way ANOVA Šidák ' s multiple comparison ' s . * * * * denotes p < 0 . 0001 .

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(A-F) Representative PAS-stained sections of the pons (A) and hippocampal (C) regions of the brain and heart (E) of one year old HOIL-1[C458S] and WT mice are shown. Scale bar = 100 μm. Arrow heads indicate α-amylase-resistant PAS-positive polyglucosan deposits. Graphs showing α-amylase resistant PAS scores of the pons (B) and hippocampal (D) regions of the brain and the heart (F) of HOIL-1[C458S] (red) and WT (blue) mice aged 0.5, 1.0 and 1.5 years. The number of biological replicates analysed at each age is indicated. The word zero highlighted in blue indicates that no α-amylase resistant, PAS-positive material could be detected in the WT mice. The error bars show mean + SEM. Statistical significance between the genotypes was calculated by using two-way ANOVA and Šidák's multiple comparison's test. **** denotes p<0.0001.


( A ) HOIL - 1 activity towards purified bovine liver glycogen was measured using Cy5 - labelled ubiquitin . Control assays lacking ATP , UBE2L3 , HOIL - 1 glycogen are indicated , as are subsequent treatments with 50 μg / mL human salivary α ‑ amylase 1 . 5 M hydroxylamine ( NH2OH ) . gel has been stained using periodic acid - Schiff ( PAS ) method to visualise glycogen ( purple - magenta ) Coomassie Blue stain to visualise .

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(A) HOIL-1 activity towards purified bovine liver glycogen was measured using Cy5-labelled ubiquitin. Control assays lacking ATP, UBE2L3, HOIL-1 and glycogen are indicated, as are subsequent treatments with 50 μg/mL human salivary α‑amylase and 1.5 M hydroxylamine (NH2OH). The gel has been stained using the periodic acid-Schiff (PAS) method to visualise glycogen (purple-magenta) and Coomassie Blue stain to visualise protein.


( B ) Prior to PAS Coomassie , gel shown in ( A ) was stained with Flamingo stain ( shown in red ) glycogen ubiquitylation observed by visualising Cy5 - ubiquitin signal ( shown in green ) .

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(B) Prior to PAS and Coomassie staining, the gel shown in (A) was stained with Flamingo fluorescent protein stain (shown in red) and glycogen ubiquitylation observed by visualising the fluorescent Cy5-ubiquitin signal (shown in green).


( D ) In vitro ubiquitylation of maltoheptaose by HOIL - 1 . Assays were treated with α ‑ amylase or hydroxylamine as indicated . Reaction products were detected by Coomassie .

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(D) In vitro ubiquitylation of maltoheptaose by HOIL-1. Assays were treated with α‑amylase or hydroxylamine as indicated. Reaction products were detected by Coomassie staining.


( F ) HOIL - 1 activity against indicated maltosaccharides ( 2 mM ) was assayed visualised by with Coomassie stain . Where indicated , assays were treated with 1 . 5 M hydroxylamine . Met1 - linked ubiquitin tetramer was also included in these assays .

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(F) HOIL-1 activity against the indicated maltosaccharides (2 mM) was assayed and visualised by staining with Coomassie protein stain. Where indicated, assays were treated with 1.5 M hydroxylamine. Met1-linked ubiquitin tetramer was also included in these assays.


( H ) In vitro ubiquitylation of indicated cyclodextrins ( 2 mM ) by HOIL - 1 , visualised by Coomassie . Met1 - linked ubiquitin tetramer was also included in these assays .

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(H) In vitro ubiquitylation of the indicated cyclodextrins (2 mM) by HOIL-1, visualised by Coomassie staining. Met1-linked ubiquitin tetramer was also included in these assays.


( A ) 1 μg of each of indicated recombinant was incubated with amylose resin for 60 min , beads were pelleted by centrifugation , in pellet ( P ) supernatant ( S ) were compared to input ( I ) by means of western blotting with appropriate antibodies .

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(A) 1 μg of each of the indicated recombinant proteins was incubated with amylose resin for 60 min, the beads were pelleted by centrifugation, and proteins in the pellet (P) and supernatant (S) were compared to the protein input (I) by means of western blotting with the appropriate antibodies.


( B ) Bovine liver glycogen potato starch - derived amylopectin were incubated for 60 min with indicated recombinant , centrifuged for 90 min at 100 , 000 × g , in resulting supernatants ( S ) pellets ( P ) analysed by western blotting with appropriate antibodies . without polysaccharide was used as a negative control ( Buffer ) .

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(B) Bovine liver glycogen and potato starch-derived amylopectin were incubated for 60 min with the indicated recombinant proteins, centrifuged for 90 min at 100,000 × g, and protein in the resulting supernatants (S) and pellets (P) analysed by western blotting with the appropriate antibodies. Protein without polysaccharide was used as a negative control (Buffer).


( C ) Amylose binding assays of full - ( 1 - 1072 ) truncated HOIP , visualised by immunoblotting . Shown on right are schematic representations of truncated HOIP constructs used . not drawn to scale . PUB , peptide N - glycanase UBA - UBX - containing domain ; ZF , zinc finger domain ; NZF , Npl4 zinc finger domain ; UBA , ubiquitin - associated domain ; RBR , RING - in - between - RING domain ; LDD , linear ubiquitin chain determining domain .

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(C) Amylose binding assays of full-length (1-1072) and truncated HOIP protein, visualised by immunoblotting. Shown on the right are schematic representations of the truncated HOIP constructs used. Images not drawn to scale. PUB, peptide N-glycanase and UBA- and UBX-containing protein domain; ZF, zinc finger domain; NZF, Npl4 zinc finger domain; UBA, ubiquitin-associated domain; RBR, RING-in-between-RING domain; LDD, linear ubiquitin chain determining domain.


( D ) Amylose binding assays of full - ( 1 - 387 ) truncated Sharpin mutants along with schematic representations of Sharpin constructs used . not shown to scale . PH , pleckstrin homology domain ; LTM , LUBAC - tethering motif ; UBL , ubiquitin - like domain .

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(D) Amylose binding assays of full-length (1-387) and truncated Sharpin mutants along with schematic representations of the Sharpin constructs used. Images not shown to scale. PH, pleckstrin homology domain; LTM, LUBAC-tethering motif; UBL, ubiquitin-like domain.


( A ) Maltoheptaose ubiquitylation by HOIL - 1 was assayed at 30 ° C in presence of Met1 - or Lys63 - linked ubiquitin dimers for indicated times visualised by Coomassie .

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(A) Maltoheptaose ubiquitylation by HOIL-1 was assayed at 30°C in the presence of Met1- or Lys63-linked ubiquitin dimers for the indicated times and visualised by Coomassie staining.


( B ) Maltoheptaose ubiquitylation by HOIL - 1 was assayed at 30 ° C in presence of Met1 - or Lys63 - linked ubiquitin tetramers for indicated times visualised by Coomassie .

(B) Maltoheptaose ubiquitylation by HOIL-1 was assayed at 30°C in the presence of Met1- or Lys63-linked ubiquitin tetramers for the indicated times and visualised by Coomassie staining.


( C ) HOIL - 1 [ T203A / F204A / R210A ] was assayed at 30 ° C in absence or presence of indicated ubiquitin dimers tetramers . These mutations have been reported to impair binding of ubiquitin to NZF domain their approximate location in HOIL - 1 is indicated ( schematic not to scale ) .

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(C) HOIL-1[T203A/F204A/R210A] was assayed at 30°C in the absence or presence of the indicated ubiquitin dimers and tetramers. These mutations have been reported to impair the binding of ubiquitin to the NZF domain and their approximate location in HOIL-1 is indicated (schematic not to scale).


( D ) of HOIL - 1 [ Δ194 - 222 ] lacking NZF domain at 30 ° C in absence or presence of indicated ubiquitin oligomers .

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(D) Assay of HOIL-1[Δ194-222] lacking the NZF core domain at 30°C in the absence or presence of the indicated ubiquitin oligomers.


( E ) Allosteric activation of HOIL - 1 [ 233 - 510 ] ( lacking LTM , UBL NZF domains at N ‑ terminus ) by ubiquitin oligomers .

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(E) Allosteric activation of HOIL-1[233-510] (lacking the LTM, UBL and NZF domains at the N‑terminus) by ubiquitin oligomers.


( B ) Time - course performed at 30 ° C featuring wild - type HOIL - 1 in absence or presence of indicated ubiquitin oligomers , visualised by Coomassie .

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(B) Time-course assay performed at 30°C featuring wild-type HOIL-1 in the absence or presence of the indicated ubiquitin oligomers, visualised by Coomassie staining.


( C ) HOIL - 1 [ I358R ] , assayed as in ( B ) . ( D ) HOIL - 1 [ F381A / E383A ] , assayed as in ( B ) .

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(C) HOIL-1[I358R], assayed as in (B). (D) HOIL-1[F381A/E383A], assayed as in (B).


( E ) truncated HOIL - 1 [ 233 - 510 ; I358R ] mutant was assayed as in ( B ) . ( F ) truncated HOIL - 1 [ 233 - 510 ; F381A / E383A ] mutant was assayed as in ( B ) .

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(E) The truncated HOIL-1[233-510; I358R] mutant was assayed as in (B). (F) The truncated HOIL-1[233-510; F381A/E383A] mutant was assayed as in (B).


( A ) Requirements for en bloc transfer of linear ubiquitin dimer to maltoheptaose were investigated by leaving out reaction components indicated . Reaction products were detected by with Coomassie .

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(A) Requirements for en bloc transfer of linear ubiquitin dimer to maltoheptaose were investigated by leaving out the reaction components indicated. Reaction products were detected by staining with Coomassie.


( B ) Requirements for en bloc transfer of Met1 - linked ubiquitin tetramer to maltoheptaose were investigated by leaving out reaction components indicated .

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(B) Requirements for en bloc transfer of Met1-linked ubiquitin tetramer to maltoheptaose were investigated by leaving out the reaction components indicated.


( C ) Time - course performed at 30 ° C in presence of Met1 - linked ubiquitin oligomers in which two most C - terminal residues were either glycine ( normal ubiquitin ) or alanine to prevent conjugation to substrates ( GG / AA ) .

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(C) Time-course assay performed at 30°C in the presence of Met1-linked ubiquitin oligomers in which the two most C-terminal residues were either glycine (normal ubiquitin) or alanine to prevent conjugation to substrates (GG/AA).


( D ) Maltoheptaose ubiquitylation was assayed for 60 s at 30 ° C in presence of indicated ubiquitin tetramers .

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(D) Maltoheptaose ubiquitylation was assayed for 60 s at 30°C in the presence of the indicated ubiquitin tetramers.


( A ) Using denaturing immunoprecipitation ( IP ) , Munc18 - 1 was isolated from lysate of HEK293T expressing M18WT , M18Y145F or M18Y473F together with or without n - Src . Munc18 - 1 was immunoblotted for tyrosine phosphorylation using 4G10 antibody ( αPtyr ) , after which blot was stripped re - blotted for Munc18 - 1 . Total lysate ( TL ) is shown as loading control . N - Src was tagged with a flag - tag for visualization .

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(A) Using denaturing immunoprecipitation (IP), Munc18-1 was isolated from cell lysate of HEK293T cells expressing M18WT, M18Y145F or M18Y473F together with or without n-Src. Munc18-1 was immunoblotted for tyrosine phosphorylation using the 4G10 antibody (αPtyr), after which the blot was stripped and re-blotted for Munc18-1. Total lysate (TL) is shown as loading control. N-Src was tagged with a flag-tag for visualization.


( B ) Same as ( A ) but using lysate from HEK293T expressing M18WT or M18Y473F together with or without n - Src , c - Src or Fyn . first two lanes are IPs performed using empty beads to control for non - specific binding of Munc18 or kinases to beads .

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(B) Same as (A) but using lysate from HEK293T cells expressing M18WT or M18Y473F together with or without n-Src, c-Src or Fyn. The first two lanes are IPs performed using empty beads to control for non-specific binding of Munc18 or kinases to the beads.


( F ) Lipid mixing was monitored by increase of Atto 488 for 12 minutes . Data were normalized to maximum after liposome lysis by detergent . Error bars = s . e . m . , N = 3 .

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(F) Lipid mixing was monitored by the increase of Atto 488 fluorescence for 12 minutes. Data were normalized to the maximum fluorescence after liposome lysis by detergent. Error bars = s.e.m., N = 3.


( G ) Same as ( E - F ) but reactions were preincubated on ice for 5 minutes in presence of Complexin II ( CpxII 600 pmol ) . Error bars = s . e . m . , N = 5 .

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(G) Same as (E-F) but the reactions were preincubated on ice for 5 minutes in the presence of Complexin II (CpxII 600 pmol). Error bars = s.e.m., N =5.


( I ) Lipid mixing was monitored by increase of Atto 488 for 60 minutes . Data were normalized to maximum after liposome lysis by detergent . Error bars = s . e . m . , N = 3 .

(I) Lipid mixing was monitored by the increase of Atto 488 fluorescence for 60 minutes. Data were normalized to the maximum fluorescence after liposome lysis by detergent. Error bars = s.e.m., N =3.


Autaptic munc18 - 1 null hippocampal neurons were rescued with a phosphomimetic Munc18 - 1 mutant , Y473D or wild - type Munc18 - 1 as control assessed on synaptic transmission using confocal microscopy ( A ) Typical confocal from neurons stained for MAP2 , Synaptobrevin / VAMP2 Munc18 - 1 .

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Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using confocal microscopy (A) Typical confocal images from neurons stained for MAP2, Synaptobrevin/VAMP2 and Munc18-1.


Autaptic munc18 - 1 null hippocampal neurons were rescued with a phosphomimetic Munc18 - 1 mutant , Y473D or wild - type Munc18 - 1 as control assessed on synaptic transmission using confocal microscopy ( B ) Average synaptic Munc18 - 1 ( M18WT : 389 ± 85 a . u . , n = 21 ; M18Y473D : 261 ± 49 a . u . , n = 20 , t - with Welch correction , p = 0 . 2000 ) , somatic Munc18 - 1 ( M18WT : 328 ± 49 a . u . , n = 21 ; M18Y473D : 188 ± 21 a . u . , n = 20 , t - with Welch correction , p = 0 . 0138 ) , synapse number ( M18WT : 129 ± 14 , n = 21 ; M18Y473D : 159 ± 14 , n = 20 , t - , p = 0 . 1203 ) dendrite ( M18WT : 0 . 433 ± 0 . 028 , n = 21 ; M18Y473D : 0 . 454 ± 0 . 037 , n = 20 , t - , p = 0 . 6613 ) .

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Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using confocal microscopy (B) Average synaptic Munc18-1 intensity (M18WT: 389 ± 85 a.u., n=21; M18Y473D: 261 ± 49 a.u., n=20, t-test with Welch correction, p = 0.2000), somatic Munc18-1 intensity (M18WT: 328 ± 49 a.u., n=21; M18Y473D: 188 ± 21 a.u., n=20, t-test with Welch correction, p = 0.0138), synapse number (M18WT: 129 ± 14, n=21; M18Y473D: 159 ± 14, n=20, t-test, p = 0.1203) and dendrite length (M18WT: 0.433 ± 0.028, n=21; M18Y473D: 0.454 ± 0.037, n=20, t-test, p = 0.6613).


Autaptic munc18 - 1 null hippocampal neurons were rescued with a phosphomimetic Munc18 - 1 mutant , Y473D or wild - type Munc18 - 1 as control assessed on synaptic transmission using whole - patch clamp electrophysiology ( C ) Left : typical examples of evoked release upon action potential stimulation .

Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using whole-cell patch clamp electrophysiology(C) Left: typical examples of evoked release upon action potential stimulation.


Autaptic munc18 - 1 null hippocampal neurons were rescued with a phosphomimetic Munc18 - 1 mutant , Y473D or wild - type Munc18 - 1 as control assessed on synaptic transmission using whole - patch clamp electrophysiology ( D ) Left : typical responses to hyperosmotic sucrose application ( 500 mM , 3 . 5 sec . ) used to assess RRP . Average RRP charge ( M18WT : 1 . 68 ± 0 . 64 nC , n = 13 ; M18Y473D : 0 . 08 ± 0 . 02 nC , n = 10 , t - with Welch correction , p = 0 . 0274 ) .

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Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using whole-cell patch clamp electrophysiology (D) Left: typical responses to hyperosmotic sucrose application (500 mM, 3.5 sec.) used to assess the RRP. Average RRP charge (M18WT: 1.68 ± 0.64 nC, n=13; M18Y473D: 0.08 ± 0.02 nC, n=10, t-test with Welch correction, p = 0.0274).


Autaptic munc18 - 1 null hippocampal neurons were rescued with a phosphomimetic Munc18 - 1 mutant , Y473D or wild - type Munc18 - 1 as control assessed on synaptic transmission using whole - patch clamp electrophysiology ( E ) Average Pves ( EPSC charge / RRP charge ) ( M18WT : 6 . 75 ± 0 . 77 % , n = 13 ; M18Y473D : 0 . 24 ± 0 . 20 % , n = 10 ; Mann - Whitney U , p < 0 . 0001 ) .

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Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using whole-cell patch clamp electrophysiology (E) Average Pves (EPSC charge / RRP charge) (M18WT: 6.75 ± 0.77%, n=13; M18Y473D: 0.24 ± 0.20%, n=10; Mann-Whitney U test, p < 0.0001).


Autaptic munc18 - 1 null hippocampal neurons were rescued with a phosphomimetic Munc18 - 1 mutant , Y473D or wild - type Munc18 - 1 as control assessed on synaptic transmission using whole - patch clamp electrophysiology ( F ) Left : example traces of spontaneous release of single SVs ( mEPSCs ) . Average mEPSC frequency ( M18WT : 20 . 18 ± 5 . 18 Hz , n = 15 ; M18Y473D : 0 . 28 ± 0 . 13 Hz , n = 17 , Mann - Whitney U , p < 0 . 0001 ) , mEPSC amplitude ( M18WT : 26 . 0 ± 1 . 7 pA , n = 15 ; M18Y473D : 27 . 8 ± 2 . 2 pA , n = 17 , t - , p = 0 . 5372 ) mEPSC decay time ( M18WT : 1 . 98 ± 0 . 10 ms , n = 15 ; M18Y473D : 2 . 84 ± 0 . 41 ms , n = 17 , t - with Welch correction , p = 0 . 0549 ) .

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

Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using whole-cell patch clamp electrophysiology (F) Left: example traces of spontaneous release of single SVs (mEPSCs). Average mEPSC frequency (M18WT: 20.18 ± 5.18 Hz, n=15; M18Y473D: 0.28 ± 0.13 Hz, n=17, Mann-Whitney U test, p < 0.0001), mEPSC amplitude (M18WT: 26.0 ± 1.7 pA, n=15; M18Y473D: 27.8 ± 2.2 pA, n=17, t-test, p = 0.5372) and mEPSC decay time (M18WT: 1.98 ± 0.10 ms, n=15; M18Y473D: 2.84 ± 0.41 ms, n=17, t-test with Welch correction, p = 0.0549).


Neurons expressing M18Y473D are not impaired at SV docking . Autaptic munc18 - 1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on chemically fixed samples ( A ) Typical electron microscopy from both groups . Scale bar = 100nm .

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Neurons expressing M18Y473D are not impaired at SV docking. Autaptic munc18-1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on chemically fixed samples (A) Typical electron microscopy images from both groups. Scale bar = 100nm.


Neurons expressing M18Y473D are not impaired at SV docking . Autaptic munc18 - 1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on chemically fixed samples ( B ) Average number of docked SV ( M18WT : 7 . 63 ± 0 . 15 SVs ; M18Y473D : 8 . 45 ± 0 . 20 docked SVs , multilevel , p = 0 . 047 ) . ( B - E ) M18WT : N = 6 autaptic neurons , n = 137 synapses ; M18Y473D : N = 8 autaptic neurons , n = 116 synapses .

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

Neurons expressing M18Y473D are not impaired at SV docking. Autaptic munc18-1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on chemically fixed samples (B) Average number of docked SV (M18WT: 7.63 ± 0.15 SVs; M18Y473D: 8.45 ± 0.20 docked SVs, multilevel analysis, p = 0.047).(B-E) M18WT : N=6 autaptic neurons, n=137 synapses; M18Y473D: N=8 autaptic neurons, n=116 synapses.


Neurons expressing M18Y473D are not impaired at SV docking . Autaptic munc18 - 1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on chemically fixed samples ( ( C ) Total number of SVs is not different ( M18WT : 142 ± 11 SVs ; M18Y473D : 148 ± 15 SVs , multilevel , p = 0 . 456 ) . ( B - E ) M18WT : N = 6 autaptic neurons , n = 137 synapses ; M18Y473D : N = 8 autaptic neurons , n = 116 synapses .

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

Neurons expressing M18Y473D are not impaired at SV docking. Autaptic munc18-1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on chemically fixed samples ((C) Total number of SVs is not different (M18WT: 142 ± 11 SVs; M18Y473D: 148 ± 15 SVs, multilevel analysis, p = 0.456).(B-E) M18WT : N=6 autaptic neurons, n=137 synapses; M18Y473D: N=8 autaptic neurons, n=116 synapses.


Neurons expressing M18Y473D are not impaired at SV docking . Autaptic munc18 - 1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on chemically fixed samples ( D ) active zone ( AZ ) is similar ( M18WT : 561 ± 24 nm ; M18Y473D : 646 ± 59 nm , multilevel , p = 0 . 161 ) . ( B - E ) M18WT : N = 6 autaptic neurons , n = 137 synapses ; M18Y473D : N = 8 autaptic neurons , n = 116 synapses .

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

Neurons expressing M18Y473D are not impaired at SV docking. Autaptic munc18-1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on chemically fixed samples (D) The active zone (AZ) length is similar (M18WT: 561 ± 24 nm; M18Y473D: 646 ± 59 nm, multilevel analysis, p = 0.161).(B-E) M18WT : N=6 autaptic neurons, n=137 synapses; M18Y473D: N=8 autaptic neurons, n=116 synapses.


Neurons expressing M18Y473D are not impaired at SV docking . Autaptic munc18 - 1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on chemically fixed samples ( E ) There is no change in number of docked SVs per AZ ( M18WT : 14 . 0 ± 0 . 6 docked SVs / pm AZ ; M18Y473D : 14 . 1 ± 0 . 9 docked SVs / pm AZ , multilevel , p = 0 . 934 ) . ( B - E ) M18WT : N = 6 autaptic neurons , n = 137 synapses ; M18Y473D : N = 8 autaptic neurons , n = 116 synapses .

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

Neurons expressing M18Y473D are not impaired at SV docking. Autaptic munc18-1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on chemically fixed samples (E) There is no change in the number of docked SVs per AZ length (M18WT: 14.0 ± 0.6 docked SVs/pm AZ; M18Y473D: 14.1 ± 0.9 docked SVs/pm AZ, multilevel analysis, p = 0.934). (B-E) M18WT : N=6 autaptic neurons, n=137 synapses; M18Y473D: N=8 autaptic neurons, n=116 synapses.


Neurons expressing M18Y473D are not impaired at SV docking . Autaptic munc18 - 1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on chemically fixed samples ( F ) Typical electron microscopy from both groups . Scale bar = 200nm .

Neurons expressing M18Y473D are not impaired at SV docking. Autaptic munc18-1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on chemically fixed samples (F) Typical electron microscopy images from both groups. Scale bar = 200nm.


Neurons expressing M18Y473D are not impaired at SV docking . Autaptic munc18 - 1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on cryofixed samples ( G ) Average number of docked SV is equal ( M18WT : 10 . 17 ± 0 . 38 SVs ; M18Y473D : 10 . 75 ± 0 . 40 docked SVs , multilevel , p = 0 . 264 ) . ( G - J ) M18WT : N = 3 independent cultures , n = 159 synapses ; M18Y473D : N = 3 independent cultures , n = 156 synapses .

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

Neurons expressing M18Y473D are not impaired at SV docking. Autaptic munc18-1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on cryofixed samples (G) Average number of docked SV is equal (M18WT: 10.17 ± 0.38 SVs; M18Y473D: 10.75 ± 0.40 docked SVs, multilevel analysis, p = 0.264).(G-J) M18WT : N=3 independent cultures, n=159 synapses; M18Y473D: N=3 independent cultures, n=156 synapses.


Neurons expressing M18Y473D are not impaired at SV docking . Autaptic munc18 - 1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on cryofixed samples ( H ) Total number of SVs is similar ( M18WT : 160 . 2 ± 9 . 7 SVs ; M18Y473D : 176 . 7 ± 9 . 2 SVs , multilevel , p = 0 . 212 ) . ( G - J ) M18WT : N = 3 independent cultures , n = 159 synapses ; M18Y473D : N = 3 independent cultures , n = 156 synapses .

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

Neurons expressing M18Y473D are not impaired at SV docking. Autaptic munc18-1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on cryofixed samples (H) Total number of SVs is similar (M18WT: 160.2 ± 9.7 SVs; M18Y473D: 176.7 ± 9.2 SVs, multilevel analysis, p = 0.212).(G-J) M18WT : N=3 independent cultures, n=159 synapses; M18Y473D: N=3 independent cultures, n=156 synapses.


Neurons expressing M18Y473D are not impaired at SV docking . Autaptic munc18 - 1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on cryofixed samples ( I ) active zone ( AZ ) is similar ( M18WT : 740 . 2 ± 29 . 7 nm ; M18Y473D : 771 . 4 ± 28 . 1 nm , multilevel , p = 0 . 871 ) . ( G - J ) M18WT : N = 3 independent cultures , n = 159 synapses ; M18Y473D : N = 3 independent cultures , n = 156 synapses .

Neurons expressing M18Y473D are not impaired at SV docking. Autaptic munc18-1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on cryofixed samples (I) The active zone (AZ) length is similar (M18WT: 740.2 ± 29.7 nm; M18Y473D: 771.4 ± 28.1 nm, multilevel analysis, p = 0.871).(G-J) M18WT : N=3 independent cultures, n=159 synapses; M18Y473D: N=3 independent cultures, n=156 synapses.


Neurons expressing M18Y473D are not impaired at SV docking . Autaptic munc18 - 1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on cryofixed samples ( ( J ) There is no change in number of docked SVs per AZ ( M18WT : 14 . 36 ± 0 . 32 docked SVs / pm AZ ; M18Y473D : 14 . 34 ± 0 . 32 docked SVs / pm AZ , multilevel , p = 0 . 969 ) . ( G - J ) M18WT : N = 3 independent cultures , n = 159 synapses ; M18Y473D : N = 3 independent cultures , n = 156 synapses .

Neurons expressing M18Y473D are not impaired at SV docking. Autaptic munc18-1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on cryofixed samples ( (J) There is no change in the number of docked SVs per AZ length (M18WT: 14.36 ± 0.32 docked SVs/pm AZ; M18Y473D: 14.34 ± 0.32 docked SVs/pm AZ, multilevel analysis, p = 0.969). (G-J) M18WT : N=3 independent cultures, n=159 synapses; M18Y473D: N=3 independent cultures, n=156 synapses.


Synaptic transmission was assessed during immediately after sustained stimulation in autaptic excitatory munc18 - 1 neurons expressing M18WT or M18Y473D . ( A ) Rundown of EPSC amplitude during 100 pulses at 10 Hz . Top : example traces of current evoked by 40Hz stimulation .

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

Synaptic transmission was assessed during and immediately after sustained stimulation in autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473D. (A) Rundown of EPSC amplitude during 100 pulses at 10 Hz. Top: example traces of the current evoked by 40Hz stimulation.


Synaptic transmission was assessed during immediately after sustained stimulation in autaptic excitatory munc18 - 1 neurons expressing M18WT or M18Y473D . ( B ) EPSC charge during 100 pulses at 40 Hz . Top : example traces of current evoked by 40Hz stimulation . Insert shows total charge transferred during entire train ( M18WT : 2 . 63 ± 0 . 38 nC ; M18Y473D : 1 . 48 ± 0 . 24 nC , t with Welch correction , p = 0 . 0177 ) .

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

Synaptic transmission was assessed during and immediately after sustained stimulation in autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473D.(B) EPSC charge during 100 pulses at 40 Hz. Top: example traces of the current evoked by 40Hz stimulation. Insert shows total charge transferred during entire train (M18WT: 2.63 ± 0.38 nC; M18Y473D: 1.48 ± 0.24 nC, t test with Welch correction, p = 0.0177).


Synaptic transmission was assessed during immediately after sustained stimulation in autaptic excitatory munc18 - 1 neurons expressing M18WT or M18Y473D . ( C ) Synaptic transmission was assessed before ( naive ( n ) ) two seconds after ( a ) 40Hz stimulation . Average EPSC charge M18WT ( naive : 70 . 9 ± 19 . 5 pC ; after HFS : 75 . 4 ± 17 . 4 pC , n = 12 , paired t - , p = 0 . 4049 ) . Average EPSC charge M18Y473D ( naive : 0 . 888 ± 0 . 342 pC , after HFS : 25 . 6 ± 9 . 3 pC , n = 9 , paired t - , p = 0 . 0271 ) .

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Synaptic transmission was assessed during and immediately after sustained stimulation in autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473D.(C) Synaptic transmission was assessed before (naive(n)) and two seconds after (a) 40Hz stimulation. Average EPSC charge M18WT (naive: 70.9 ± 19.5 pC; after HFS: 75.4 ± 17.4 pC, n=12, paired t-test, p = 0.4049). Average EPSC charge M18Y473D (naive: 0.888 ± 0.342 pC, after HFS: 25.6 ± 9.3 pC, n=9, paired t-test, p = 0.0271).


Synaptic transmission was assessed during immediately after sustained stimulation in autaptic excitatory munc18 - 1 neurons expressing M18WT or M18Y473D . ( D ) A hypertonic sucrose solution ( 500 mM , 3 . 5 sec . ) was applied to release RRP before ( naive ( n ) ) or two seconds after ( a ) HFS ( 100 pulses at 40 Hz ) .

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

Synaptic transmission was assessed during and immediately after sustained stimulation in autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473D.(D) A hypertonic sucrose solution (500 mM, 3.5 sec.) was applied to release the RRP before (naive (n)) or two seconds after (a) HFS (100 pulses at 40 Hz).


Synaptic transmission was assessed during immediately after sustained stimulation in autaptic excitatory munc18 - 1 neurons expressing M18WT or M18Y473D . ( E ) of RRP Pves ( EPSC charge / RRP charge * 100 ) from C - D . Average RRP M18WT ( naive : 1 . 73 ± 0 . 69 nC , after HFS : 1 . 34 ± 0 . 54 nC , n = 12 , paired t - , p = 0 . 0557 ) . Average RRP M18Y473D ( Naive : 0 . 061 ± 0 . 019 nC , after HFS : 0 . 207 ± 0 . 050 nC , n = 9 , paired t - , p = 0 . 0246 ) . Average Pves M18WT ( naive : 7 . 07 ± 0 . 76 % , after HFS : 10 . 6 ± 1 . 6 % , n = 12 , paired t - , p = 0 . 0201 ) . Average Pves M18Y473D ( naive : 0 . 27 ± 0 . 22 % , after HFS : 10 . 8 ± 2 . 2 % , n = 9 , paired t - , p = 0 . 0012 ) .

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

Synaptic transmission was assessed during and immediately after sustained stimulation in autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473D.(E) Quantification of RRP and Pves (EPSC charge / RRP charge * 100) from C-D. Average RRP size M18WT (naive: 1.73 ± 0.69 nC, after HFS: 1.34 ± 0.54 nC, n=12, paired t-test, p = 0.0557). Average RRP size M18Y473D (Naive: 0.061 ± 0.019 nC, after HFS: 0.207 ± 0.050 nC, n=9, paired t-test, p = 0.0246). Average Pves M18WT (naive: 7.07 ± 0.76 %, after HFS: 10.6 ± 1.6 %, n=12, paired t-test, p = 0.0201). Average Pves M18Y473D (naive: 0.27 ± 0.22 %, after HFS: 10.8 ± 2.2%, n=9, paired t-test, p = 0.0012).


Synaptic transmission was assessed during immediately after sustained stimulation in autaptic excitatory munc18 - 1 neurons expressing M18WT or M18Y473D . ( F ) PDBu application ( 1 µM ) enhanced EPSC in neurons expressing M18Y473D .

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

Synaptic transmission was assessed during and immediately after sustained stimulation in autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473D.(F) PDBu application (1 µM) enhanced EPSC size in neurons expressing M18Y473D.


( B ) GST - Munc18 - 1 constructs were immobilized on glutathione beads incubated with indicated molar excess of Synaptobrevin / VAMP2 ( over Munc18 - 1 ) for 1 . 5 h at 4 ° C . Bound Synaptobrevin / VAMP2 was analyzed by Western blot immune decorating with an anti - Synaptobrevin / VAMP2 antibody quantified by LICORE system ImageJ software . Synaptobrevin / VAMP2 bound to GST - Munc18 - 1 mutants is represented as of Synaptobrevin / VAMP2 bound to GST - Munc18 - 1 WT . Error bars indicate s . e . m . , n = 3 .

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(B) GST-Munc18-1 constructs were immobilized on glutathione beads and incubated with the indicated molar excess of Synaptobrevin/VAMP2 (over Munc18-1) for 1.5 h at 4°C. Bound Synaptobrevin/VAMP2 was analyzed by Western blot and immune decorating with an anti-Synaptobrevin/VAMP2 antibody and quantified by the LICORE system and ImageJ software. Synaptobrevin/VAMP2 bound to the GST-Munc18-1 mutants is represented as percentage of Synaptobrevin/VAMP2 bound to GST-Munc18-1 WT. Error bars indicate s.e.m., n=3.


( D ) Lipid mixing was monitored by increase of Atto 655 for 30 minutes . Data were normalized to maximum after liposome lysis by detergent . minimum was set to 0 % . Error bars = s . e . m . , 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

(D) Lipid mixing was monitored by the increase of Atto 655 fluorescence for 30 minutes. Data were normalized to the maximum fluorescence after liposome lysis by detergent. The minimum fluorescence was set to 0%. Error bars = s.e.m., N = 3.


( E ) Trans - SNARE Formation : t - v - SNARE SUVs were incubated in presence or absence of different Munc18 - 1 constructs for 30min at 4 ° C . After solubilization precipitation of t - SNAREs using nickel beads , presence of full - VAMP2 in precipitates was probed by immunoblotting , which was used as an indicator for trans - SNARE assembly between SUVs .

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(E) Trans-SNARE Formation Assay: t- and v-SNARE SUVs were incubated in the presence or absence of different Munc18-1 constructs for 30min at 4°C. After solubilization and precipitation of the t-SNAREs using nickel beads, presence of full-length VAMP2 in the precipitates was probed by immunoblotting, which was used as an indicator for trans-SNARE assembly between SUVs.


( A ) Typical examples of single EPSCs . EPSC amplitude ( M18WT : 2 . 98 ± 0 . 76 nA , n = 11 ; M18Y473D : 0 . 042 ± 0 . 018 nA , n = 11 , M18Y473D / P335A : 2 . 02 ± 0 . 67 nA , n = 7 , Kruskal - Wallis , p < 0 . 0004 ) .

(A) Typical examples of single EPSCs. EPSC amplitude (M18WT: 2.98 ± 0.76 nA, n=11; M18Y473D: 0.042 ± 0.018 nA, n=11, M18Y473D/P335A: 2.02 ± 0.67 nA, n=7, Kruskal-Wallis test, p < 0.0004).


Evoked spontaneous release in autaptic munc18 - 1 null hippocampal neurons expressing M18Y473D , M18Y473D / P335A or wild - type Munc18 - 1 as control . ( B ) Typical traces of spontaneous release .

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

Evoked and spontaneous release in autaptic munc18-1 null hippocampal neurons expressing M18Y473D, M18Y473D/P335A or wild-type Munc18-1 as control.(B) Typical traces of spontaneous release.


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

Evoked and spontaneous release in autaptic munc18-1 null hippocampal neurons expressing M18Y473D, M18Y473D/P335A or wild-type Munc18-1 as control.(C) mEPSC frequency (M18WT: 8.23 ± 2.29 Hz, n=11; M18Y473D: 0.096 ± 0.056 Hz, n=8, M18Y473D/P335A: 2.18 ± 1.02 Hz, n=11, Kruskal-Wallis test, p = 0.2390), mEPSC amplitude (M18WT: 16.7 ± 1.0 pA, n=11; M18Y473D: 21.6 ± 5.2 pA, n=10, M18Y473D/P335A: 14.8 ± 1.2 pA, n=6, ANOVA, p = 0.1465) and decay time (M18WT: 2.53 ± 0.29 ms, n=11; M18Y473D: 3.79 ± 0.71 ms, n=10, M18Y473D/P335A: 2.85 ± 0.36 pA, n=6, Kruskal-Wallis test, p < 0.0003).


Evoked spontaneous release in autaptic munc18 - 1 null hippocampal neurons expressing M18Y473D , M18Y473D / P335A or wild - type Munc18 - 1 as control . ( D ) Depression of EPSC amplitude during a 10 Hz train . Inset shows first 4 pulses normalized to first EPSC .

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

Evoked and spontaneous release in autaptic munc18-1 null hippocampal neurons expressing M18Y473D, M18Y473D/P335A or wild-type Munc18-1 as control.(D) Depression of EPSC amplitude during a 10 Hz train. Inset shows first 4 pulses normalized to the first EPSC.


Synaptic transmission in autaptic excitatory munc18 - 1 neurons expressing M18WT or a non - phosphorylatable Munc18 - 1 variant , M18Y473F , was tested with whole - patch clamp electrophysiology . ( A ) Evoked release upon action potential stimulation . Average EPSC amplitude ( M18WT : 3 . 78 ± 0 . 58 nA , n = 22 ; M18Y473F : 4 . 10 ± 0 . 61 nA , n = 20 , Mann - Whitney U , p = 0 . 7915 ) . Typical responses are depicted on top .

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

Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology. (A) Evoked release upon action potential stimulation. Average EPSC amplitude (M18WT: 3.78 ± 0.58 nA, n=22; M18Y473F: 4.10 ± 0.61 nA, n=20, Mann-Whitney U test, p = 0.7915). Typical responses are depicted on the top.


Synaptic transmission in autaptic excitatory munc18 - 1 neurons expressing M18WT or a non - phosphorylatable Munc18 - 1 variant , M18Y473F , was tested with whole - patch clamp electrophysiology . ( B ) Average mEPSC frequency ( M18WT : 5 . 42 ± 1 . 86 Hz , n = 17 ; M18Y473F : 4 . 42 ± 1 . 01 Hz , n = 14 , t - with Welch correction , p = 0 . 6424 ) , mEPSC amplitude ( M18WT : 20 . 72 ± 0 . 7 pA , n = 17 ; M18Y473F : 20 , 5 ± 0 . 9 pA , n = 14 , t - , p = 0 . 8373 ) mEPSC decay time ( M18WT : 2 . 54 ± 0 . 20 ms , n = 17 ; M18Y473F : 2 . 46 ± 0 . 18 ms , n = 14 , t - , p = 0 . 7550 ) . Example traces of spontaneous release are depicted on top .

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

Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology.(B) Average mEPSC frequency (M18WT: 5.42 ± 1.86 Hz, n=17; M18Y473F: 4.42 ± 1.01 Hz, n=14, t-test with Welch correction, p = 0.6424), mEPSC amplitude (M18WT: 20.72 ± 0.7 pA, n=17; M18Y473F: 20,5 ± 0.9 pA, n=14, t-test, p = 0.8373) and mEPSC decay time (M18WT: 2.54 ± 0.20 ms, n=17; M18Y473F: 2.46 ± 0.18 ms, n=14, t-test, p = 0.7550). Example traces of spontaneous release are depicted on top.


Synaptic transmission in autaptic excitatory munc18 - 1 neurons expressing M18WT or a non - phosphorylatable Munc18 - 1 variant , M18Y473F , was tested with whole - patch clamp electrophysiology . ( C ) EPSC depression during 10 Hz stimulation is normal .

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

Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology.(C) EPSC depression during 10 Hz stimulation is normal.


Synaptic transmission in autaptic excitatory munc18 - 1 neurons expressing M18WT or a non - phosphorylatable Munc18 - 1 variant , M18Y473F , was tested with whole - patch clamp electrophysiology . ( D ) Depression of EPSC charge during 40 Hz stimulation is normal . Inset shows first 5 pulses .

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

Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology.(D) Depression of EPSC charge during 40 Hz stimulation is normal. Inset shows the first 5 pulses.


Synaptic transmission in autaptic excitatory munc18 - 1 neurons expressing M18WT or a non - phosphorylatable Munc18 - 1 variant , M18Y473F , was tested with whole - patch clamp electrophysiology . ( E ) EPSC amplitude recovery two seconds after an RRP depleting 40 Hz train . Average EPSC recovery ( M18WT : 82 . 2 ± 6 . 7 % , n = 17 ; M18Y473F : 65 . 5 ± 11 . 0 % , n = 12 , t - , p = 0 . 1805 ) .

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

Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology.(E) EPSC amplitude recovery two seconds after an RRP depleting 40 Hz train. Average EPSC recovery (M18WT: 82.2 ± 6.7 %, n=17; M18Y473F: 65.5 ± 11.0 %, n=12, t-test, p = 0.1805).


Synaptic transmission in autaptic excitatory munc18 - 1 neurons expressing M18WT or a non - phosphorylatable Munc18 - 1 variant , M18Y473F , was tested with whole - patch clamp electrophysiology . ( F ) amount of DSE - triggered EPSC depression was quantified by of average EPSC amplitude ( taken over 4 pulses given at 0 . 2 Hz ) before or immediately following 10 seconds depolarization to 0mV . Typical EPSCs before following DSE induction are depicted on left . Average DSE ( M18WT : 0 . 57 ± 0 . 04 fold , n = 20 ; M18Y473F : 0 . 53 ± 0 . 04 fold , n = 16 , t - , p = 0 . 5227 ) .

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

Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology.(F) The amount of DSE-triggered EPSC depression was quantified by the ratio of the average EPSC amplitude (taken over 4 pulses given at 0.2 Hz) before or immediately following 10 seconds depolarization to 0mV. Typical EPSCs before and following DSE induction are depicted on the left. Average DSE (M18WT: 0.57 ± 0.04 fold, n=20; M18Y473F: 0.53 ± 0.04 fold, n=16, t-test, p = 0.5227).


Synaptic transmission in autaptic excitatory munc18 - 1 neurons expressing M18WT or a non - phosphorylatable Munc18 - 1 variant , M18Y473F , was tested with whole - patch clamp electrophysiology . ( G ) PDBu was bath applied during 0 . 05 Hz stimulation . Maximum potentiation of EPSC amplitude ( M18WT : 2 . 56 ± 0 . 48 fold , n = 7 ; M18Y473F : 2 . 82 ± 0 . 98 fold , n = 8 , t - with Welch correction , p = 0 . 8168 ) .

Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology.(G) PDBu was bath applied during 0.05 Hz stimulation. Maximum potentiation of EPSC amplitude (M18WT: 2.56 ± 0.48 fold, n=7; M18Y473F: 2.82 ± 0.98 fold, n=8, t-test with Welch correction, p = 0.8168).


( A ) Using denatured IP , Munc18 - 1 was pulled - down from lysate of HEK293T expressing M18WT or M18Y473A together with SFK kinases or an empty vector as control . Munc18 - 1 was then immunoblotted for tyrosine phosphorylation using 4G10 antibody . total amount of Munc18 - 1 was detected after stripping reblotting for Munc18 - 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Using denatured IP, Munc18-1 was pulled-down from cell lysate of HEK293T cells expressing M18WT or M18Y473A together with SFK kinases or an empty vector as control. Munc18-1 was then immunoblotted for tyrosine phosphorylation using the 4G10 antibody. The total amount of Munc18-1 was detected after stripping and reblotting for Munc18-1.


( B - G ) Munc18 - 1 null hippocampal neurons were rescued with wildtype Munc18 - 1a or a Munc18 - 1 mutant , Y473A , in which Y473 was replaced with alanine . ( B ) Typical confocal of neurons stained for MAP2 , Synaptobrevin / VAMP2 Munc18 - 1 . Scale bar = 50πm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Munc18-1 null hippocampal neurons were rescued with wildtype Munc18-1a or a Munc18-1 mutant, Y473A, in which Y473 was replaced with alanine. (B) Typical confocal images of neurons stained for MAP2, Synaptobrevin /VAMP2 and Munc18-1. Scale bar = 50πm.


( B - G ) Munc18 - 1 null hippocampal neurons were rescued with wildtype Munc18 - 1a or a Munc18 - 1 mutant , Y473A , in which Y473 was replaced with alanine . ( C ) of confocal . Average synaptic Munc18 - 1 ( M18WT : 389 ± 85 a . u . , n = 21 ; M18Y473A : 321 ± 48 a . u . , n = 20 , t - with Welch correction , p = 0 . 4868 ) , somatic Munc18 - 1 ( M18WT : 328 ± 49 a . u . , n = 21 ; M18Y473A : 245 ± 42 a . u . , n = 20 , t - , p = 0 . 2810 ) , synapse number ( M18WT : 129 ± 14 , n = 21 ; M18Y473A : 159 ± 16 , n = 20 , t - , p = 0 . 1548 ) dendrite ( M18WT : 0 . 433 ± 0 . 028 , n = 21 ; M18Y473A : 0 . 461 ± 0 . 037 , n = 20 , t - , p = 0 . 5473 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Munc18-1 null hippocampal neurons were rescued with wildtype Munc18-1a or a Munc18-1 mutant, Y473A, in which Y473 was replaced with alanine.(C) Quantification of confocal images. Average synaptic Munc18-1 intensity (M18WT: 389 ± 85 a.u., n=21; M18Y473A: 321 ± 48 a.u., n=20, t-test with Welch correction, p = 0.4868), somatic Munc18-1 intensity (M18WT: 328 ± 49 a.u., n=21; M18Y473A: 245 ± 42 a.u., n=20, t-test, p = 0.2810), synapse number (M18WT: 129 ± 14, n=21; M18Y473A: 159 ± 16, n=20, t-test, p = 0.1548) and dendrite length (M18WT: 0.433 ± 0.028, n=21; M18Y473A: 0.461 ± 0.037, n=20, t-test, p = 0.5473).


( B - G ) Munc18 - 1 null hippocampal neurons were rescued with wildtype Munc18 - 1a or a Munc18 - 1 mutant , Y473A , in which Y473 was replaced with alanine . ( D ) Evoked release upon action potential stimulation . Average EPSC amplitude ( M18WT : 7 . 96 ± 0 . 94 nA , n = 30 ; M18Y473A : 1 . 44 ± 0 . 34 nA , n = 33 , t - with Welch correction , p < 0 . 0001 ) . Typical responses are depicted on right .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Munc18-1 null hippocampal neurons were rescued with wildtype Munc18-1a or a Munc18-1 mutant, Y473A, in which Y473 was replaced with alanine.(D) Evoked release upon action potential stimulation. Average EPSC amplitude (M18WT: 7.96 ± 0.94 nA, n=30; M18Y473A: 1.44 ± 0.34 nA, n=33, t-test with Welch correction, p < 0.0001). Typical responses are depicted on the right.


( B - G ) Munc18 - 1 null hippocampal neurons were rescued with wildtype Munc18 - 1a or a Munc18 - 1 mutant , Y473A , in which Y473 was replaced with alanine . ( E ) Release by hyperosmotic sucrose application ( 500mM , 3 . 5sec . ) was used to assess RRP . Left : RRP charge ( M18WT : 1 . 68 ± 0 . 64 nC , n = 13 ; M18Y473A : 0 . 27 ± 0 . 08 nC , n = 13 , t - with Welch correction , p = 0 . 0487 ) . Typical responses are depicted in middle . Right : vesicular release probability per neuron ( EPSC charge / RRP charge ) . Mean Pves ( M18WT : 6 . 75 ± 0 . 77 % , n = 13 ; M18Y473A : 1 . 98 ± 0 . 72 % , n = 13 , t - , p = 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Munc18-1 null hippocampal neurons were rescued with wildtype Munc18-1a or a Munc18-1 mutant, Y473A, in which Y473 was replaced with alanine.(E) Release by hyperosmotic sucrose application (500mM, 3.5sec.) was used to assess the RRP. Left: RRP charge (M18WT: 1.68 ± 0.64 nC, n=13; M18Y473A: 0.27 ± 0.08 nC, n=13, t-test with Welch correction, p = 0.0487). Typical responses are depicted in the middle. Right: vesicular release probability per neuron (EPSC charge / RRP charge). Mean Pves (M18WT: 6.75 ± 0.77%, n=13; M18Y473A: 1.98 ± 0.72%, n=13, t-test, p = 0.0001).


( B - G ) Munc18 - 1 null hippocampal neurons were rescued with wildtype Munc18 - 1a or a Munc18 - 1 mutant , Y473A , in which Y473 was replaced with alanine . ( F ) Top : example traces of spontaneous release of single vesicles ( mEPSCs ) . Average mEPSC frequency ( M18WT : 20 . 18 ± 5 . 18 Hz , n = 15 ; M18Y473A : 0 . 48 ± 0 . 15 Hz , n = 20 , t - with Welch correction , p = 0 . 0019 ) , amplitude ( M18WT : 26 . 0 ± 1 . 7 pA , n = 15 ; M18Y473A : 24 . 5 ± 1 . 6 pA , n = 20 , t - , p = 0 . 5334 ) decay time ( M18WT : 1 . 98 ± 0 . 10 ms , n = 15 ; M18Y473A : 2 . 13 ± 0 . 17 ms , n = 20 , t - with Welch correction , p = 0 . 4499 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Munc18-1 null hippocampal neurons were rescued with wildtype Munc18-1a or a Munc18-1 mutant, Y473A, in which Y473 was replaced with alanine.(F) Top: example traces of spontaneous release of single vesicles (mEPSCs). Average mEPSC frequency (M18WT: 20.18 ± 5.18 Hz, n=15; M18Y473A: 0.48 ± 0.15 Hz, n=20, t-test with Welch correction, p = 0.0019), amplitude (M18WT: 26.0 ± 1.7 pA, n=15; M18Y473A: 24.5 ± 1.6 pA, n=20, t-test, p = 0.5334) and decay time (M18WT: 1.98 ± 0.10 ms, n=15; M18Y473A: 2.13 ± 0.17 ms, n=20, t-test with Welch correction, p = 0.4499).


( B - G ) Munc18 - 1 null hippocampal neurons were rescued with wildtype Munc18 - 1a or a Munc18 - 1 mutant , Y473A , in which Y473 was replaced with alanine . ( G ) Typical electron microscopy from rescued autaptic hippocampal Munc18 - 1 null neurons ( scale bar = 100 nm ) . Neurons expressing M18Y473A have less synaptic vesicles ( SV ) docked at number of docked SV . Average

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Munc18-1 null hippocampal neurons were rescued with wildtype Munc18-1a or a Munc18-1 mutant, Y473A, in which Y473 was replaced with alanine.(G) Typical electron microscopy images from rescued autaptic hippocampal Munc18-1 null neurons (scale bar = 100 nm). Neurons expressing M18Y473A have less synaptic vesicles (SV) docked at the number of docked SV. Average


Autaptic excitatory munc18 - 1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies . ( A ) Absolute EPSC amplitudes during a 10 Hz train .

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

Autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies. (A) Absolute EPSC amplitudes during a 10 Hz train.


Autaptic excitatory munc18 - 1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies . ( B ) EPSC amplitudes during a 10 Hz train normalized to first EPSC .

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

Autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies.(B) EPSC amplitudes during a 10 Hz train normalized to the first EPSC.


Autaptic excitatory munc18 - 1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies . ( C ) Top : example traces of currents evoked by 40 Hz stimulation . Bottom right : first 5 pulses of 40 Hz train . Far right : transferred charge during 40 Hz train . Insert shows total charge transferred during entire 40 Hz train ( M18WT : 2 . 63 ± 0 . 38 nC ; M18Y473A : 2 . 93 ± 0 . 54 nC , t , p = 0 . 6630 ) .

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

Autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies.(C) Top: example traces of the currents evoked by 40 Hz stimulation. Bottom right: first 5 pulses of the 40 Hz train. Far right: transferred charge during 40 Hz train. Insert shows total charge transferred during entire 40 Hz train (M18WT: 2.63 ± 0.38 nC; M18Y473A: 2.93 ± 0.54 nC, t test, p = 0.6630).


Autaptic excitatory munc18 - 1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies . ( D ) EPSC augmentation is calculated by dividing EPSC amplitude of a single after a stimulation train by amplitude of first EPSC within this train . Mean augmentation after 5 Hz train ( M18WT : 0 . 54 ± 0 . 05 , n = 8 ; M18Y473A : 2 . 88 ± 0 . 47 , n = 10 ; Unpaired t - with Welch correction , p = 0 . 0008 ) , 10 Hz train ( M18WT : 0 . 70 ± 0 . 10 , n = 8 ; M18Y473A : 5 . 39 ± 0 . 77 , n = 10 ; Unpaired t - with Welch correction , p = 0 . 0002 ) 40 Hz train ( M18WT : 0 . 93 ± 0 . 11 , n = 8 ; M18Y473A : 8 . 77 ± 1 . 52 , n = 10 ; Unpaired t - with Welch correction , p = 0 . 0006 ) .

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

Autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies.(D) EPSC augmentation is calculated by dividing the EPSC amplitude of a single pulse after a stimulation train by the amplitude of the first EPSC within this train. Mean augmentation after 5 Hz train (M18WT: 0.54 ± 0.05, n=8; M18Y473A: 2.88 ± 0.47, n=10; Unpaired t-test with Welch correction, p = 0.0008), 10 Hz train (M18WT: 0.70 ± 0.10, n=8; M18Y473A: 5.39 ± 0.77, n=10; Unpaired t-test with Welch correction, p = 0.0002) and 40 Hz train (M18WT: 0.93 ± 0.11, n=8; M18Y473A: 8.77 ± 1.52, n=10; Unpaired t-test with Welch correction, p = 0.0006).


Autaptic excitatory munc18 - 1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies . ( E ) A single action potential or sucrose application ( 500 mM , 3 . 5 sec . ) was given before ( naive ) or two seconds after HFS ( 100 pulses at 40 Hz ) . Average EPSC charge M18WT ( Naive : 70 . 9 ± 19 . 5 pC , after HFS : 75 . 4 ± 17 . 4 pC , n = 12 , Paired t - , p = 0 . 4049 ) . Average EPSC charge M18Y473A ( naive : 10 . 3 ± 4 . 5 pC , after HFS : 46 . 2 ± 14 . 5 pC , n = 13 , Wilcoxon matched - pairs signed - ranks , p = 0 . 0005 ) . Average RRP M18WT ( naive : 1 . 73 ± 0 . 69 nC , after HFS : 1 . 34 ± 0 . 54 nC , n = 12 , paired t - , p = 0 . 0557 ) Average HFS M18Y473A ( naive : 0 . 268 ± 0 . 075 nC , after HFS : 0 . 439 ± 0 . 107 nC , n = 13 , paired t - , p = 0 . 0100 ) . Average Pves M18WT ( EPSC charge / RRP charge * 100 ) ( Naive : 7 . 07 ± 0 . 76 % , after HFS : 10 . 6 ± 1 . 6 % , n = 12 , paired t - , p = 0 . 0201 ) . Average Pves M18Y473A ( naive : 1 . 98 ± 0 . 72 % , 12 . 2 ± 2 . 8 % , n = 13 , paired t - , p = 0 . 0021 ) .

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

Autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies.(E) A single action potential or sucrose application (500 mM, 3.5 sec.) was given before (naive) or two seconds after HFS (100 pulses at 40 Hz). Average EPSC charge M18WT (Naive: 70.9 ± 19.5 pC, after HFS: 75.4 ± 17.4 pC, n=12, Paired t-test, p = 0.4049). Average EPSC charge M18Y473A (naive: 10.3 ± 4.5 pC, after HFS: 46.2 ± 14.5 pC, n=13, Wilcoxon matched-pairs signed-ranks test, p = 0.0005). Average RRP size M18WT (naive: 1.73 ± 0.69 nC, after HFS: 1.34 ± 0.54 nC, n=12, paired t-test, p = 0.0557) Average HFS size M18Y473A (naive: 0.268 ± 0.075 nC, after HFS: 0.439 ± 0.107 nC, n=13, paired t-test, p = 0.0100). Average Pves M18WT (EPSC charge / RRP charge * 100) (Naive: 7.07 ± 0.76 %, after HFS: 10.6 ± 1.6 %, n=12, paired t-test, p = 0.0201). Average Pves M18Y473A (naive: 1.98 ± 0.72 %, 12.2 ± 2.8 %, n=13, paired t-test, p = 0.0021).


A Endogenous PIPKIγi5 LAPTM4B were immunoprecipitated from whole ‐ lysates of MDA ‐ MB ‐ 231 followed by immunoblotting to examine co ‐ immunoprecipitatedPIPKIγi5 LAPTM4B .

O O 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 Endogenous PIPKIγi5 and LAPTM4B were immunoprecipitated from the whole‐cell lysates of MDA‐MB‐231 cells followed by immunoblotting to examine the co‐immunoprecipitatedPIPKIγi5 and LAPTM4B.


B PIPKIγi5 specifically interacts with LAPTM4B , but not LAPTM4A or LAPTM5 . Top : schematic diagram of three LAPTM members . Bottom : each Flag ‐ tagged LAPTM was immunoprecipitated from HEK293 cotransfected with Myc ‐ tagged PIPKIγi5 empty vector or Flag ‐ tagged LAPTM , co ‐ immunoprecipitatedPIPKIγi5 was examined by immunoblotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 PIPKIγi5 specifically interacts with LAPTM4B, but not LAPTM4A or LAPTM5. Top: schematic diagram of all three LAPTM members. Bottom: each Flag‐tagged LAPTM protein was immunoprecipitated from HEK293 cells cotransfected with Myc‐tagged PIPKIγi5 and empty vector or Flag‐tagged LAPTM, and the co‐immunoprecipitatedPIPKIγi5 was examined by immunoblotting.


C LAPTM4B selectively associates with PIPKIγi5 . Top : schematic diagram of four PIPKI isoforms . Bottom : Myc ‐ tagged PIPKIγi5 was immunoprecipitated from HEK293 expressing Flag ‐ tagged LAPTM4B each isoform of PIPKI , followed by immunoblotting to examine co ‐ immunoprecipitatedLAPTM4B .

O O O O O O O O O O O O O O O O O O O O O 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 LAPTM4B selectively associates with PIPKIγi5. Top: schematic diagram of four PIPKI isoforms. Bottom: Myc‐tagged PIPKIγi5 was immunoprecipitated from HEK293 cells expressing Flag‐tagged LAPTM4B and each isoform of PIPKI, followed by immunoblotting to examine the co‐immunoprecipitatedLAPTM4B.


D Kinase activity of PIPKIγi5 is required for LAPTM4B association . HEK293 expressing LAPTM4B wild ‐ type ( WT ) or kinase ‐ dead ( KD ) PIPKIγi5 were starved overnight , stimulated or not with 100 ng / ml EGF for 15 min , harvested for immunoprecipitation with anti ‐ myc . co ‐ immunoprecipitatedLAPTM4B was detected by immunoblotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Kinase activity of PIPKIγi5 is required for LAPTM4B association. HEK293 cells expressing LAPTM4B and wild‐type (WT) or kinase‐dead (KD) PIPKIγi5 were starved overnight, stimulated or not with 100 ng/ml EGF for 15 min, and harvested for immunoprecipitation with anti‐myc. The co‐immunoprecipitatedLAPTM4B was detected by immunoblotting.