Figure 1 ( 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 .

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Figure 1(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.


Figure 2 ( 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 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 staining .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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 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 staining.


Figure 3 ( 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 the indicated cyclodextrins ( 2 mM ) by HOIL - 1 , visualised by Coomassie staining . Met1 - linked ubiquitin tetramer was also included in these assays .

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Figure 3(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 the indicated cyclodextrins (2 mM) by HOIL-1, visualised by Coomassie staining. Met1-linked ubiquitin tetramer was also included in these assays.


Figure 4 ( 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 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 - 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 - 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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 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-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-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.


Figure 5 ( 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 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 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 ) 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 the LTM , UBL and NZF domains at the N ‑ terminus ) by ubiquitin oligomers .

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Figure 5(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 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 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) 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 the LTM, UBL and NZF domains at the N‑terminus) by ubiquitin oligomers.


Figure 6 ( 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 ) . ( 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 ) .

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Figure 6(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).(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).


Figure 7 ( 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 the reaction components indicated . ( 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 the presence of the indicated ubiquitin tetramers .

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Figure 7(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 the reaction components indicated.(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 the presence of the indicated ubiquitin tetramers.


Figure 1 ( 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 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 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 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 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 .

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Figure 1(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 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 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 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 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.


Figure 2Autaptic 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 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 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 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 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Autaptic 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 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 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 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 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 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).


Figure 3Neurons 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 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 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 ) 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 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 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 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 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 ) 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Neurons 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 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 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) 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 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 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 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 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) 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 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.


Figure 4 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 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 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 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 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 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 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 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 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 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.


Figure 5 ( 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 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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 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 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.


Figure 6 ( 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 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 . 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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 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.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 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.


Figure 7 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 - 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 - 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 - 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 - 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 - 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 - 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7 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-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-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-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-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-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-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).


Figure 8 ( 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 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 ) 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 - 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 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 - 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 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

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(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 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) 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-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 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-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 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


Figure 9 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 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 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 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 - 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 ) .

O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9 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 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 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 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-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).


Figure 1A 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 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 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 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 . E Endogenous LAPTM4B is targeted to both early and late endosomes . MDA ‐ MB ‐ 231 cells were fixed and costained for endogenous LAPTM4B ( red ) and EEA1 or LAMP1 ( green ) . Boxes are selected regions for magnified view . Note : non ‐ specific nuclear staining by the LAPTM4B anti ‐ sera . Scale bar : 10 μm . F Quantification of LAPTM4B colocalization with EEA1 and LAMP1 ( mean + SD ; n ≥ 4 ) . H LAPTM4B partially colocalizes with PIPKIγi5 . MDA ‐ MB ‐ 231 cells expressing Myc ‐ tagged PIPKIγi5 were stained with LAPTM4B anti ‐ sera ( red ) , anti ‐ Myc ( green ) , and DAPI . Box is selected region for magnified view . Scale bar : 10 μm . I MDA ‐ MB ‐ 231 cells stably expressing Flag ‐ LAPTM4B were stained with LAPTM4B anti ‐ sera followed by silver ‐ enhanced immuno ‐ electron microscopy . The early and late MVEs were defined by the number of intraluminal vesicles . N , nucleus ; M , mitochondria ; MVE , multi ‐ vesicular endosome ; and PM , plasma membrane . Scale bars : 2 μm ( left ) ; 200 nm ( middle and right ) .

O B-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A 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 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 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 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.E Endogenous LAPTM4B is targeted to both early and late endosomes. MDA‐MB‐231 cells were fixed and costained for endogenous LAPTM4B (red) and EEA1 or LAMP1 (green). Boxes are selected regions for magnified view. Note: non‐specific nuclear staining by the LAPTM4B anti‐sera. Scale bar: 10 μm.F Quantification of LAPTM4B colocalization with EEA1 and LAMP1 (mean + SD; n ≥ 4).H LAPTM4B partially colocalizes with PIPKIγi5. MDA‐MB‐231 cells expressing Myc‐tagged PIPKIγi5 were stained with LAPTM4B anti‐sera (red), anti‐Myc (green), and DAPI. Box is selected region for magnified view. Scale bar: 10 μm.I MDA‐MB‐231 cells stably expressing Flag‐LAPTM4B were stained with LAPTM4B anti‐sera followed by silver‐enhanced immuno‐electron microscopy. The early and late MVEs were defined by the number of intraluminal vesicles. N, nucleus; M, mitochondria; MVE, multi‐vesicular endosome; and PM, plasma membrane. Scale bars: 2 μm (left); 200 nm (middle and right).


Figure 2A MDA ‐ MB ‐ 231 cells transfected with control or LAPTM4B siRNA were starved and stimulated with 100 ng / ml EGF for indicated time periods , followed by whole ‐ cell lysate harvest for immunoblotting analysis of EGFR levels . B - D Quantification of the levels of EGFR ( B ) , pEGFR ( Y1068 ) ( C ) , and pAKT ( S473 ) ( D ) from the analysis in ( A ) ( mean ± SD ; n = 3 ) . E Control or LAPTM4B siRNA ‐ transfected MDA ‐ MB ‐ 231 cells were starved , pulsed with 25 ng / ml Alexa ‐ 555 ‐ EGF for 3 min , washed , and chased for indicated time periods followed by fixation , DAPI staining , and fluorescence microscopy . Scale bar : 10 μm . F Quantification of the relative amounts of Alexa ‐ 555 ‐ EGF internalized in the indicated conditions ( mean + SD , n = 3 ) . G Quantification of the Alexa ‐ 555 ‐ EGF degradation in control and LAPTM4B knockdown cells in ( E ) ( mean ± SD , n = 3 ) . H Control or LAPTM4B ‐ overexpressing MDA ‐ MB ‐ 231 cells were starved and then stimulated with 100 ng / ml EGF for 1 - 4 h . EGFR degradation and signaling were analyzed by Western blot . Specific antibodies recognizing pEGFR ( Y1068 ) and pAKT ( S473 ) were used . I - K Quantification for the levels of EGFR ( I ) and pEGFR ( J ) normalized to actin and pAKT ( K ) normalized to AKT in control or LAPTM4B ‐ overexpressing cells ( mean ± SD , n = 3 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A MDA‐MB‐231 cells transfected with control or LAPTM4B siRNA were starved and stimulated with 100 ng/ml EGF for indicated time periods, followed by whole‐cell lysate harvest for immunoblotting analysis of EGFR levels.B-D Quantification of the levels of EGFR (B), pEGFR (Y1068)(C), and pAKT (S473)(D) from the analysis in (A) (mean ± SD; n = 3).E Control or LAPTM4B siRNA‐transfected MDA‐MB‐231 cells were starved, pulsed with 25 ng/ml Alexa‐555‐EGF for 3 min, washed, and chased for indicated time periods followed by fixation, DAPI staining, and fluorescence microscopy. Scale bar: 10 μm.F Quantification of the relative amounts of Alexa‐555‐EGF internalized in the indicated conditions (mean + SD, n = 3).G Quantification of the Alexa‐555‐EGF degradation in control and LAPTM4B knockdown cells in (E) (mean ± SD, n = 3).H Control or LAPTM4B‐overexpressing MDA‐MB‐231 cells were starved and then stimulated with 100 ng/ml EGF for 1-4 h. EGFR degradation and signaling were analyzed by Western blot. Specific antibodies recognizing pEGFR (Y1068) and pAKT (S473) were used.I-K Quantification for the levels of EGFR (I) and pEGFR (J) normalized to actin and pAKT (K) normalized to AKT in control or LAPTM4B‐overexpressing cells (mean ± SD, n = 3).


Figure 3A - D Control or LAPTM4B siRNA ‐ transfected MDA ‐ MB ‐ 231 cells were starved , pretreated or not with chloroquine for 2 h , stimulated with 100 ng / ml EGF for 15 min , washed , and chased for indicated time periods before fixation for costaining of EGFR ( red ) with EEA1 ( A , green ) or LAMP1 ( C , green ) . Quantification of the average percentages of EGFR signals colocalized with EEA1 ( B ) and LAMP1 ( D ) at indicated time points ; mean + SD ; n = 3 ; * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 , one ‐ tailed t ‐ test . E MDA ‐ MB ‐ 231 cells were starved , stimulated with 100 ng / ml EGF for 15 or 120 min , fixed , and costained for EGFR and LAPTM4B , followed by fluorescence microscopy . Cells with higher and lower LAPTM4B expression were marked with arrows and arrowheads , respectively . F The amounts of total EGFR staining in individual cells in ( E ) were plotted against LAPTM4B levels at 15 min and 120 min , respectively . Note : for LAPTM4B quantification , the non ‐ specific nuclear staining was not included . Trend lines and Pearson ' s correlation coefficients are shown . AU , arbitrary unit .

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Figure 3A-D Control or LAPTM4B siRNA‐transfected MDA‐MB‐231 cells were starved, pretreated or not with chloroquine for 2 h, stimulated with 100 ng/ml EGF for 15 min, washed, and chased for indicated time periods before fixation for costaining of EGFR (red) with EEA1 (A, green) or LAMP1 (C, green). Quantification of the average percentages of EGFR signals colocalized with EEA1 (B) and LAMP1 (D) at indicated time points; mean + SD; n = 3; *P 0.05, **P 0.01, ***P 0.001, one‐tailed t‐test.E MDA‐MB‐231 cells were starved, stimulated with 100 ng/ml EGF for 15 or 120 min, fixed, and costained for EGFR and LAPTM4B, followed by fluorescence microscopy. Cells with higher and lower LAPTM4B expression were marked with arrows and arrowheads, respectively.F The amounts of total EGFR staining in individual cells in (E) were plotted against LAPTM4B levels at 15 min and 120 min, respectively. Note: for LAPTM4B quantification, the non‐specific nuclear staining was not included. Trend lines and Pearson's correlation coefficients are shown. AU, arbitrary unit.


Figure 4MDA ‐ MB ‐ 231 cells expressing GFP ‐ Rab5Q79L were starved and stimulated with 100 ng / ml Alexa ‐ 555 ‐ EGF for 90 min followed by fixation and immunostaining for LAPTM4B ( blue ) . The percentages of luminal Alexa ‐ 555 ‐ EGF in individual endosomes were plotted against endosomal LAPTM4B levels . Trend lines and Pearson ' s correlation coefficients are shown . Data are representative for three independent experiments . C Control or LAPTM4B knockdown MDA ‐ MB ‐ 231 cells were starved overnight , cell surface EGFR was labeled with immuno ‐ gold on ice . Cells were then stimulated with EGF for 1 h at 37 ° C and fixed for the EM study . Scale bar , 200 nm . See Materials and Methods for details . D Relative amounts of immuno ‐ gold ‐ labeled EGFR in the MVE lumen versus MVE limiting membrane were quantified . Over 80 endosomes for each siRNA treatment from three independent experiments were used for quantification ( mean + SD ; * * * P 0 . 001 , one ‐ tailed t ‐ test ) . MDA ‐ MB ‐ 231 cells were transfected with GFP ‐ Rab5Q79L and Flag ‐ LAPTM4B , starved , and stimulated with Alexa ‐ 555 ‐ EGF for 90 min and followed by intraluminal sorting analysis . See Materials and Methods for details . Quantification of EGF localization in LAPTM4B ‐ positive and LAPTM4B ‐ negative endosomes in ( E ) ( mean + SD ; n = 3 ; * * * P 0 . 0007 , one ‐ tailed t ‐ test ) .

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Figure 4MDA‐MB‐231 cells expressing GFP‐Rab5Q79L were starved and stimulated with 100 ng/ml Alexa‐555‐EGF for 90 min followed by fixation and immunostaining for LAPTM4B (blue). The percentages of luminal Alexa‐555‐EGF in individual endosomes were plotted against endosomal LAPTM4B levels. Trend lines and Pearson's correlation coefficients are shown. Data are representative for three independent experiments.C Control or LAPTM4B knockdown MDA‐MB‐231 cells were starved overnight, cell surface EGFR was labeled with immuno‐gold on ice. Cells were then stimulated with EGF for 1 h at 37°C and fixed for the EM study. Scale bar, 200 nm. See Materials and Methods for details.D Relative amounts of immuno‐gold‐labeled EGFR in the MVE lumen versus MVE limiting membrane were quantified. Over 80 endosomes for each siRNA treatment from three independent experiments were used for quantification (mean + SD; ***P 0.001, one‐tailed t‐test).MDA‐MB‐231 cells were transfected with GFP‐Rab5Q79L and Flag‐LAPTM4B, starved, and stimulated with Alexa‐555‐EGF for 90 min and followed by intraluminal sorting analysis. See Materials and Methods for details. Quantification of EGF localization in LAPTM4B‐positive and LAPTM4B‐negative endosomes in (E) (mean + SD; n = 3; ***P 0.0007, one‐tailed t‐test).


Figure 5A Co ‐ immunoprecipitation ( co ‐ IP ) of full ‐ length or N ‐ terminus ‐ deleted LAPTM4B with PIPKIγi5 in HEK293 cells . B GST ‐ tagged LAPTM4B N ‐ terminus ( 93 amino acids ) and T7 ‐ tagged PIPKIγi5 C ‐ terminus ( 223 amino acids ) were purified from E . coli for in vitro GSTpull ‐ down assay . C Co ‐ IP of full ‐ length or N ‐ terminal deletion mutants of LAPTM4B with PIPKIγi5 in HEK293 cells . ∆ 25 : amino acids 1 - 25 deleted . E Coomassie Brilliant Blue staining of purified wild ‐ type and mutated LAPTM4B N ‐ termini . F Purified LAPTM4B N ‐ termini from ( E ) were used in PIP strips assay . LPA , lysophosphatidic acid ; LPC , lysophosphocholine ; PE , phosphatidylethanolamine ; PC , phosphatidylcholine ; S1P , sphingosine 1 ‐ phosphate ; PI3 , 4 , 5P3 , PtdIns ( 3 , 4 , 5 ) P3 , phosphatidylinositol ( 3 , 4 , 5 ) ‐ trisphosphate ; PA , phosphatidic acid ; PS , phosphatidylserine . GST pull ‐ down assay of wild ‐ type or 6RQ mutant of LAPTM4B N ‐ termini with PIPKIγi5 C ‐ terminus . H Co ‐ IP of wild ‐ type or PMB ‐ mutated LAPTM4B with wild ‐ type or kinase dead PIPKIγi5 in HEK293 cells . I Top : GSTpull ‐ down assay of LAPTM4B N ‐ terminus and PIPKIγi5 C ‐ terminus with increased concentration of PtdIns ( 4 , 5 ) P2 addition . Bottom : Quantification of the relative amounts of PIPKIγi5 C ‐ terminus bound to LAPTM4B N ‐ terminus in top panel ( mean ± SD ; n = 4 ) . J LAPTM4B N ‐ terminus binds multiple phosphoinositides including PtdIns ( 4 , 5 ) P2 in liposome ‐ binding assay . K Top : GSTpull ‐ down assay of LAPTM4B N ‐ terminus and PIPKIγi5 C ‐ terminus with 0 . 5 μM addition of different phosphoinositides . Bottom : Quantification of the relative amounts of PIPKIγi5 C ‐ terminus bound to LAPTM4B N ‐ terminus in top panel ( mean + SD ; n = 4 ) . L Top : GSTpull ‐ down assay of wild ‐ type or 6RQ mutant of LAPTM4B N ‐ terminus and PIPKIγi5 C ‐ terminus with 0 . 5 μM addition of PtdIns ( 4 , 5 ) P2 . Bottom : Quantification of the relative amounts of PIPKIγi5 C ‐ terminus bound to LAPTM4B N ‐ terminus in top panel ( mean + SD ; n = 4 ) .

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Figure 5A Co‐immunoprecipitation (co‐IP) of full‐length or N‐terminus‐deleted LAPTM4B with PIPKIγi5 in HEK293 cells.B GST‐tagged LAPTM4B N‐terminus (93 amino acids) and T7‐tagged PIPKIγi5 C‐terminus (223 amino acids) were purified from E. coli for in vitro GSTpull‐down assay.C Co‐IP of full‐length or N‐terminal deletion mutants of LAPTM4B with PIPKIγi5 in HEK293 cells. ∆25: amino acids 1-25 deleted.E Coomassie Brilliant Blue staining of purified wild‐type and mutated LAPTM4B N‐termini.F Purified LAPTM4B N‐termini from (E) were used in PIP strips assay. LPA, lysophosphatidic acid; LPC, lysophosphocholine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine 1‐phosphate; PI3,4,5P3, PtdIns(3,4,5)P3, phosphatidylinositol (3,4,5)‐trisphosphate; PA, phosphatidic acid; PS, phosphatidylserine.GST pull‐down assay of wild‐type or 6RQ mutant of LAPTM4B N‐termini with PIPKIγi5 C‐terminus.H Co‐IP of wild‐type or PMB‐mutated LAPTM4B with wild‐type or kinase dead PIPKIγi5 in HEK293 cells.I Top: GSTpull‐down assay of LAPTM4B N‐terminus and PIPKIγi5 C‐terminus with increased concentration of PtdIns(4,5)P2 addition. Bottom: Quantification of the relative amounts of PIPKIγi5 C‐terminus bound to LAPTM4B N‐terminus in top panel (mean ± SD; n = 4).J LAPTM4B N‐terminus binds multiple phosphoinositides including PtdIns(4,5)P2 in liposome‐binding assay.K Top: GSTpull‐down assay of LAPTM4B N‐terminus and PIPKIγi5 C‐terminus with 0.5 μM addition of different phosphoinositides. Bottom: Quantification of the relative amounts of PIPKIγi5 C‐terminus bound to LAPTM4B N‐terminus in top panel (mean + SD; n = 4).L Top: GSTpull‐down assay of wild‐type or 6RQ mutant of LAPTM4B N‐terminus and PIPKIγi5 C‐terminus with 0.5 μM addition of PtdIns(4,5)P2. Bottom: Quantification of the relative amounts of PIPKIγi5 C‐terminus bound to LAPTM4B N‐terminus in top panel (mean + SD; n = 4).


Figure 6A Control or LAPTM4B knockdown MDA ‐ MB ‐ 231 cells were starved and stimulated with 100 ng / ml EGF for 30 min , and whole ‐ cell lysates were subject to co ‐ immunoprecipitation ( co ‐ IP ) assay . B Control or LAPTM4B knockdown MDA ‐ MB ‐ 231 cells were transfected with His ‐ tagged ubiquitin , starved , and stimulated with 100 ng / ml EGF for 30 min . Total ubiquitinated proteins were purified from whole ‐ cell lysates by Ni ‐ NTA agarose and analyzed by Western blot . C Control or LAPTM4B ‐ WT / 6RQ ‐ overexpressing MDA ‐ MB ‐ 231 cells were transfected with His ‐ tagged ubiquitin and myc ‐ tagged Hrs , starved , and stimulated with 100 ng / ml EGF for 30 min before whole ‐ cell lysate harvest . Total ubiquitinated proteins were purified by Ni ‐ NTA agarose and analyzed by Western blot . D Quantification of Hrs ubiquitination levels from the Western blot in ( C ) ( mean + SD ; n = 5 ) . E The co ‐ IP of Myc ‐ Hrs with Flag ‐ LAPTM4B ‐ WT or 6RQ mutant in HEK293 cells . F The co ‐ IP between Hrs and Nedd4 with Flag ‐ LAPTM4B ‐ WT or 6RQ mutant overexpression in HEK293 cells . G The effects of LAPTM4B ‐ 6RQ mutant overexpression on EGF ‐ stimulated EGFR degradation and signaling . Flag ‐ LAPTM4B ‐ WT or 6RQ mutant overexpression was accomplished by lentivirus ‐ mediated infection approach . Cells with low levels of expression were selected as polyclonal pools for comparison . Control or overexpressing cells were starved and stimulated with 100 ng / ml EGF for indicated time periods and whole ‐ cell lysates were analyzed by Western blot . H Quantification of EGFR and pAKT levels from the Western blot in ( G ) ( mean ± SD ; n = 5 ) . I The PY motif mutant LAPTM4B ‐ 2PA loses interaction with Nedd4 but keeps interaction with Hrs in HEK293 cells cotransfected with the indicated proteins . J The effects of LAPTM4B ‐ 2PA mutant overexpression on EGF ‐ stimulated EGFR degradation and signaling . Overexpression of Flag ‐ LAPTM4B ‐ WT or 2PA mutant overexpression was accomplished by lentivirus ‐ mediated infection approach . Cells with high expression of Flag ‐ LAPTM4B were selected as polyclonal pools for comparison . Cells were starved and stimulated with 100 ng / ml EGF for the indicated time periods , and whole ‐ cell lysates were analyzed by Western blot . K Quantification of EGFR degradation from Western blot in ( J ) ( mean ± SD ; n = 3 ) .

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Figure 6A Control or LAPTM4B knockdown MDA‐MB‐231 cells were starved and stimulated with 100 ng/ml EGF for 30 min, and whole‐cell lysates were subject to co‐immunoprecipitation (co‐IP) assay.B Control or LAPTM4B knockdown MDA‐MB‐231 cells were transfected with His‐tagged ubiquitin, starved, and stimulated with 100 ng/ml EGF for 30 min. Total ubiquitinated proteins were purified from whole‐cell lysates by Ni‐NTA agarose and analyzed by Western blot.C Control or LAPTM4B‐WT/6RQ‐overexpressing MDA‐MB‐231 cells were transfected with His‐tagged ubiquitin and myc‐tagged Hrs, starved, and stimulated with 100 ng/ml EGF for 30 min before whole‐cell lysate harvest. Total ubiquitinated proteins were purified by Ni‐NTA agarose and analyzed by Western blot.D Quantification of Hrs ubiquitination levels from the Western blot in (C) (mean + SD; n = 5).E The co‐IP of Myc‐Hrs with Flag‐LAPTM4B‐WT or 6RQ mutant in HEK293 cells.F The co‐IP between Hrs and Nedd4 with Flag‐LAPTM4B‐WT or 6RQ mutant overexpression in HEK293 cells.G The effects of LAPTM4B‐6RQ mutant overexpression on EGF‐stimulated EGFR degradation and signaling. Flag‐LAPTM4B‐WT or 6RQ mutant overexpression was accomplished by lentivirus‐mediated infection approach. Cells with low levels of expression were selected as polyclonal pools for comparison. Control or overexpressing cells were starved and stimulated with 100 ng/ml EGF for indicated time periods and whole‐cell lysates were analyzed by Western blot.H Quantification of EGFR and pAKT levels from the Western blot in (G) (mean ± SD; n = 5).I The PY motif mutant LAPTM4B‐2PA loses interaction with Nedd4 but keeps interaction with Hrs in HEK293 cells cotransfected with the indicated proteins.J The effects of LAPTM4B‐2PA mutant overexpression on EGF‐stimulated EGFR degradation and signaling. Overexpression of Flag‐LAPTM4B‐WT or 2PA mutant overexpression was accomplished by lentivirus‐mediated infection approach. Cells with high expression of Flag‐LAPTM4B were selected as polyclonal pools for comparison. Cells were starved and stimulated with 100 ng/ml EGF for the indicated time periods, and whole‐cell lysates were analyzed by Western blot.K Quantification of EGFR degradation from Western blot in (J) (mean ± SD; n = 3).


Figure 7A - C Co ‐ immunoprecipitation ( co ‐ IP ) of wild ‐ type ( WT ) or C ‐ terminal deletion mutants of HA ‐ PIPKIγi5 with SNX5 ( A ) or LAPTM4B ( B and C ) in HEK293 cells cotransfected with indicated proteins . Δ675 indicates a deletion from amino acid 675 to the C ‐ terminus . D PIPKIγi5 promotes SNX5 association with LAPTM4B . HEK293 cells cotransfected with indicated proteins were harvested for co ‐ IP to assay the interaction between Myc ‐ SNX5 and Flag ‐ LAPTM4B . E SNX5 inhibits Hrs association with LAPTM4B . HEK293 cells cotransfected with indicated proteins were harvested for co ‐ IP to assay the interaction between Myc ‐ Hrs and Flag ‐ LAPTM4B .

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Figure 7A-C Co‐immunoprecipitation (co‐IP) of wild‐type (WT) or C‐terminal deletion mutants of HA‐PIPKIγi5 with SNX5 (A) or LAPTM4B (B and C) in HEK293 cells cotransfected with indicated proteins. Δ675 indicates a deletion from amino acid 675 to the C‐terminus.D PIPKIγi5 promotes SNX5 association with LAPTM4B. HEK293 cells cotransfected with indicated proteins were harvested for co‐IP to assay the interaction between Myc‐SNX5 and Flag‐LAPTM4B.E SNX5 inhibits Hrs association with LAPTM4B. HEK293 cells cotransfected with indicated proteins were harvested for co‐IP to assay the interaction between Myc‐Hrs and Flag‐LAPTM4B.


Figure 1 ( C ) Flag - tagged Raptor was transfected with HA - tagged ArfGAP1 or HA - tagged RFP1 in wildtype ( WT ) or RagA / B knockout ( KO ) HEK293A cells for 24 hours . Cell lysates were immunoprecipitated ( IP ) with anti - Flag beads . IP or WCL samples were probed for HA - tagged ArfGAP1 or HA - tagged RFP1 , Flag - tagged Raptor , RagA , RagB , and Vinculin the loading control . ( G ) RagA / B KO HEK293A cells were transfected with HA - tagged ArfGAP1 , HA - tagged ArfGAP2 , or HA - tagged RFP1 ( control ) for 72 hours . Cell lysates were immunoprecipitated ( IP ) with anti - HA beads . IP or WCL samples were probed for HA and mTORC1 components mTOR , Raptor and mLST8 . Vinculin was probed as a control .

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Figure 1(C) Flag-tagged Raptor was transfected with HA-tagged ArfGAP1 or HA-tagged RFP1 in wildtype (WT) or RagA/B knockout (KO) HEK293A cells for 24 hours. Cell lysates were immunoprecipitated (IP) with anti-Flag beads. IP or WCL samples were probed for HA-tagged ArfGAP1 or HA-tagged RFP1, Flag-tagged Raptor, RagA, RagB, and Vinculin the loading control.(G) RagA/B KO HEK293A cells were transfected with HA-tagged ArfGAP1, HA-tagged ArfGAP2, or HA-tagged RFP1 (control) for 72 hours. Cell lysates were immunoprecipitated (IP) with anti-HA beads. IP or WCL samples were probed for HA and mTORC1 components mTOR, Raptor and mLST8. Vinculin was probed as a control.