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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


E Endogenous LAPTM4B is targeted to both early late endosomes . MDA ‐ MB ‐ 231 were fixed costained for endogenous LAPTM4B ( red ) EEA1 or LAMP1 ( green ) . Boxes are selected regions for magnified view . Note : non ‐ specific by LAPTM4B anti ‐ sera . Scale bar : 10 μm . F of LAPTM4B colocalization with EEA1 LAMP1 ( mean + SD ; n ≥ 4 ) .

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

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 expressing Myc ‐ tagged PIPKIγi5 were stained with LAPTM4B anti ‐ sera ( red ) , anti ‐ Myc ( green ) , DAPI . Box is selected region for magnified view . Scale bar : 10 μm .

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

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 stably expressing Flag ‐ LAPTM4B were stained with LAPTM4B anti ‐ sera followed by silver ‐ enhanced immuno ‐ electron microscopy . early late MVEs were defined by number of intraluminal vesicles . N , nucleus ; M , mitochondria ; MVE , multi ‐ vesicular endosome ; PM , plasma . Scale bars : 2 μm ( left ) ; 200 nm ( middle right ) .

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

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).


A MDA ‐ MB ‐ 231 transfected with control or LAPTM4B siRNA were starved stimulated with 100 ng / ml EGF for indicated time periods , followed by whole ‐ lysate harvest for immunoblotting of EGFR . B - D of of EGFR ( B ) , pEGFR ( Y1068 ) ( C ) , pAKT ( S473 ) ( D ) from in ( A ) ( mean ± SD ; n = 3 ) .

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

A 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 were starved , pulsed with 25 ng / ml Alexa ‐ 555 ‐ EGF for 3 min , washed , chased for indicated time periods followed by fixation , DAPI , microscopy . Scale bar : 10 μm . F of relative amounts of Alexa ‐ 555 ‐ EGF internalized in indicated conditions ( mean + SD , n = 3 ) . G of Alexa ‐ 555 ‐ EGF degradation in control LAPTM4B knockdown in ( E ) ( mean ± SD , n = 3 ) .

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

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 were starved then stimulated with 100 ng / ml EGF for 1 - 4 h . EGFR degradation signaling were analyzed by Western blot . Specific antibodies recognizing pEGFR ( Y1068 ) pAKT ( S473 ) were used . I - K for of EGFR ( I ) pEGFR ( J ) normalized to actin pAKT ( K ) normalized to AKT in control or LAPTM4B ‐ overexpressing ( mean ± SD , n = 3 ) .

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

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).


A - D Control or LAPTM4B siRNA ‐ transfected MDA ‐ MB ‐ 231 were starved , pretreated or not with chloroquine for 2 h , stimulated with 100 ng / ml EGF for 15 min , washed , chased for indicated time periods before fixation for costaining of EGFR ( red ) with EEA1 ( A , green ) or LAMP1 ( C , green ) . of average percentages of EGFR signals colocalized with EEA1 ( B ) LAMP1 ( D ) at indicated time points ; mean + SD ; n = 3 ; * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 , one ‐ t ‐ .

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A-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 were starved , stimulated with 100 ng / ml EGF for 15 or 120 min , fixed , costained for EGFR LAPTM4B , followed by microscopy . with higher lower LAPTM4B expression were marked with arrows arrowheads , respectively . F amounts of total EGFR in in ( E ) were plotted against LAPTM4B at 15 min 120 min , respectively . Note : for LAPTM4B , non ‐ specific was not included . Trend lines Pearson ' s correlation coefficients are shown . AU , arbitrary unit .

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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.


MDA ‐ MB ‐ 231 expressing GFP ‐ Rab5Q79L were starved stimulated with 100 ng / ml Alexa ‐ 555 ‐ EGF for 90 min followed by fixation immunostaining for LAPTM4B ( blue ) . percentages of luminal Alexa ‐ 555 ‐ EGF in endosomes were plotted against endosomal LAPTM4B . Trend lines Pearson ' s correlation coefficients are shown . Data are representative for three independent experiments .

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MDA‐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 were starved overnight , surface EGFR was labeled with immuno ‐ gold on ice . were then stimulated with EGF for 1 h at 37 ° C fixed for EM study . Scale bar , 200 nm . See Materials Methods for details . D Relative amounts of immuno ‐ gold ‐ labeled EGFR in MVE lumen versus MVE limiting were quantified . Over 80 endosomes for each siRNA treatment from three independent experiments were used for ( mean + SD ; * * * P 0 . 001 , one ‐ t ‐ ) .

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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 were transfected with GFP ‐ Rab5Q79L Flag ‐ LAPTM4B , starved , stimulated with Alexa ‐ 555 ‐ EGF for 90 min followed by intraluminal sorting . See Materials Methods for details . of EGF localization in LAPTM4B ‐ positive LAPTM4B ‐ negative endosomes in ( E ) ( mean + SD ; n = 3 ; * * * P 0 . 0007 , one ‐ t ‐ ) .

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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).


A Co ‐ immunoprecipitation ( co ‐ IP ) of full ‐ or N ‐ terminus ‐ deleted LAPTM4B with PIPKIγi5 in HEK293 .

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A Co‐immunoprecipitation (co‐IP) of full‐length or N‐terminus‐deleted LAPTM4B with PIPKIγi5 in HEK293 cells.


B GST ‐ tagged LAPTM4B N ‐ terminus ( 93 ) T7 ‐ tagged PIPKIγi5 C ‐ terminus ( 223 ) were purified from E . coli for in vitro GSTpull ‐ down .

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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 ‐ or N ‐ terminal deletion mutants of LAPTM4B with PIPKIγi5 in HEK293 . ∆ 25 : 1 - 25 deleted .

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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 of purified wild ‐ type mutated LAPTM4B N ‐ termini .

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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 . 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 .

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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 of wild ‐ type or 6RQ mutant of LAPTM4B N ‐ termini with PIPKIγi5 C ‐ terminus .

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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 .

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H Co‐IP of wild‐type or PMB‐mutated LAPTM4B with wild‐type or kinase dead PIPKIγi5 in HEK293 cells.


I Top : GSTpull ‐ down of LAPTM4B N ‐ terminus PIPKIγi5 C ‐ terminus with increased of PtdIns ( 4 , 5 ) P2 addition . Bottom : of relative amounts of PIPKIγi5 C ‐ terminus bound to LAPTM4B N ‐ terminus in top panel ( mean ± SD ; n = 4 ) .

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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 .

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J LAPTM4B N‐terminus binds multiple phosphoinositides including PtdIns(4,5)P2 in liposome‐binding assay.


K Top : GSTpull ‐ down of LAPTM4B N ‐ terminus PIPKIγi5 C ‐ terminus with 0 . 5 μM addition of different phosphoinositides . Bottom : of relative amounts of PIPKIγi5 C ‐ terminus bound to LAPTM4B N ‐ terminus in top panel ( mean + SD ; n = 4 ) .

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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 of wild ‐ type or 6RQ mutant of LAPTM4B N ‐ terminus PIPKIγi5 C ‐ terminus with 0 . 5 μM addition of PtdIns ( 4 , 5 ) P2 . Bottom : of relative amounts of PIPKIγi5 C ‐ terminus bound to LAPTM4B N ‐ terminus in top panel ( mean + SD ; n = 4 ) .

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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).


A Control or LAPTM4B knockdown MDA ‐ MB ‐ 231 were starved stimulated with 100 ng / ml EGF for 30 min , whole ‐ lysates were subject to co ‐ immunoprecipitation ( co ‐ IP ) .

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A 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 were transfected with His ‐ tagged ubiquitin , starved , stimulated with 100 ng / ml EGF for 30 min . Total ubiquitinated were purified from whole ‐ lysates by Ni ‐ NTA agarose analyzed by Western blot .

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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 were transfected with His ‐ tagged ubiquitin myc ‐ tagged Hrs , starved , stimulated with 100 ng / ml EGF for 30 min before whole ‐ lysate harvest . Total ubiquitinated were purified by Ni ‐ NTA agarose analyzed by Western blot . D of Hrs ubiquitination from Western blot in ( C ) ( mean + SD ; n = 5 ) .

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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 co ‐ IP of Myc ‐ Hrs with Flag ‐ LAPTM4B ‐ WT or 6RQ mutant in HEK293 .

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E The co‐IP of Myc‐Hrs with Flag‐LAPTM4B‐WT or 6RQ mutant in HEK293 cells.


F co ‐ IP between Hrs Nedd4 with Flag ‐ LAPTM4B ‐ WT or 6RQ mutant overexpression in HEK293 .

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F The co‐IP between Hrs and Nedd4 with Flag‐LAPTM4B‐WT or 6RQ mutant overexpression in HEK293 cells.


G effects of LAPTM4B ‐ 6RQ mutant overexpression on EGF ‐ stimulated EGFR degradation signaling . Flag ‐ LAPTM4B ‐ WT or 6RQ mutant overexpression was accomplished by lentivirus ‐ mediated infection approach . with low of expression were selected as polyclonal pools for comparison . Control or overexpressing were starved stimulated with 100 ng / ml EGF for indicated time periods whole ‐ lysates were analyzed by Western blot .

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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 of EGFR pAKT from Western blot in ( G ) ( mean ± SD ; n = 5 ) .

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H Quantification of EGFR and pAKT levels from the Western blot in (G) (mean ± SD; n = 5).


I PY motif mutant LAPTM4B ‐ 2PA loses interaction with Nedd4 but keeps interaction with Hrs in HEK293 cotransfected with indicated .

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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 effects of LAPTM4B ‐ 2PA mutant overexpression on EGF ‐ stimulated EGFR degradation signaling . Overexpression of Flag ‐ LAPTM4B ‐ WT or 2PA mutant overexpression was accomplished by lentivirus ‐ mediated infection approach . with high expression of Flag ‐ LAPTM4B were selected as polyclonal pools for comparison . were starved stimulated with 100 ng / ml EGF for indicated time periods , whole ‐ lysates were analyzed by Western blot . K of EGFR degradation from Western blot in ( J ) ( mean ± SD ; n = 3 ) .

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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).


A - C Co ‐ immunoprecipitation ( co ‐ IP ) of wild ‐ type ( WT ) or C ‐ terminal deletion mutants of HA ‐ PIPKIγi5 with SNX5 ( A ) or LAPTM4B ( B C ) in HEK293 cotransfected with indicated . Δ675 indicates a deletion from acid 675 to C ‐ terminus .

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A-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 cotransfected with indicated were harvested for co ‐ IP to interaction between Myc ‐ SNX5 Flag ‐ LAPTM4B .

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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 cotransfected with indicated were harvested for co ‐ IP to interaction between Myc ‐ Hrs Flag ‐ LAPTM4B .

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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.


( C ) Flag - tagged Raptor was transfected with HA - tagged ArfGAP1 or HA - tagged RFP1 in wildtype ( WT ) or RagA / B knockout ( KO ) HEK293A for 24 hours . 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 , Vinculin loading control .

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(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 were transfected with HA - tagged ArfGAP1 , HA - tagged ArfGAP2 , or HA - tagged RFP1 ( control ) for 72 hours . lysates were immunoprecipitated ( IP ) with anti - HA beads . IP or WCL samples were probed for HA mTORC1 components mTOR , Raptor mLST8 . Vinculin was probed as a control .

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(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.


( D ) ArfGAP1 was stably knocked down in HEK293A using shRNAs against ArfGAP1 ( shArfGAP1 ) or GFP ( shGFP ) control . were cultured under normal condition ( NC ; lanes 1 7 ) , or starved of for 1 hour ( - AA 1h ; lanes 2 8 ) , followed by either maintaining in acid starvation media ( - AA ; lanes 3 9 ) , or addition of 1 mM of Leu ( + Leu ; lanes 4 10 ) , Gln ( + Gln ; lanes 5 11 ) , or Asn ( + Asn ; lanes 6 12 ) for 2 hours . mTORC1 activity was analyzed similar to ( B ) . of ArfGAP1 were confirmed by immunoblotting . S6K1 Actin were probed as controls .

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(D) ArfGAP1 was stably knocked down in HEK293A cells using shRNAs against ArfGAP1 (shArfGAP1) or GFP (shGFP) control. Cells were cultured under normal condition (NC; lanes 1 and 7), or starved of amino acids for 1 hour (-AA 1h; lanes 2 and 8), followed by either maintaining in amino acid starvation media (-AA; lanes 3 and 9), or addition of 1 mM of Leu (+Leu; lanes 4 and 10), Gln (+Gln; lanes 5 and 11), or Asn (+Asn; lanes 6 and 12) for 2 hours. mTORC1 activity was analyzed similar to (B). Protein levels of ArfGAP1 were confirmed by immunoblotting. S6K1 and Actin were probed as controls.


( E ) RagA / B KO HEK293A were co - transfected with HA - tagged ArfGAP1 , HA - tagged ArfGAP2 , or HA - tagged RFP1 ( control ) with Myc - tagged S6K1 . were starved of ( - AA ) for 2 hours then 4mM of Gln or Asn were added for 2 hours . mTORC1 was analyzed similar to ( B ) . IP or WCL samples were probed for HA , Myc , phosphorylated S6K1 at Thr 389 . Actin serve as a loading control .

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(E) RagA/B KO HEK293A cells were co-transfected with HA-tagged ArfGAP1, HA-tagged ArfGAP2, or HA-tagged RFP1 (control) with Myc-tagged S6K1. Cells were starved of amino acids (-AA) for 2 hours and then 4mM of Gln or Asn were added for 2 hours. mTORC1 was analyzed similar to (B). IP or WCL samples were probed for HA, Myc, and phosphorylated S6K1 at Thr 389. Actin serve as a loading control.


( B ) Lifetime Imaging Microscopy ( FLIM ) Förster resonance energy transfer ( FRET ) were performed in wildtype ( WT , left ) or RagA / B KO ( middle ) HAP1 expressing mCherry - tagged ArfGAP1 endogenously GFP - tagged Raptor . either had ( + AA ) or were starved of ( - AA ) . At least 20 were analyzed in each group . Box plots shows of pixels with ≥ 30 % FRET efficiency of co - transfected . RFP1 was expressed analyzed as a negative control . whiskers of boxplot represent minimum maximum values , while box covers data between 25th 75th percentiles with a in middle plotted at median . Right - Expression level of RagA / B was confirmed Western blot . Actin was used as a control .

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(B) Fluorescence Lifetime Imaging Microscopy (FLIM) and Förster resonance energy transfer (FRET) analysis were performed in wildtype (WT, left) or RagA/B KO (middle) HAP1 cells expressing mCherry-tagged ArfGAP1 and endogenously GFP-tagged Raptor. Cells either had amino acids (+AA) or were starved of amino acids (-AA). At least 20 cells were analyzed in each group. Box plots shows the percentage of pixels with ≥ 30% FRET efficiency of co-transfected cells. RFP1 was expressed and analyzed as a negative control. The whiskers of the boxplot represent the minimum and maximum values, while the box covers the data between 25th and 75th percentiles with a line in the middle plotted at the median. Right- Expression level of RagA/B was confirmed Western blot analysis. Actin was used as a control.


( A ) Mouse embryonic fibroblast ( MEF ) were transfected with HA - tagged ArfGAP1 . Immunofluorescence was assessed for HA - tagged ArfGAP1 ( blue ) , mTOR ( green ) , LAMP2 ( red , lysosomal marker ) . Left - A representative example of immunofluorescence ( scale bar , 10 μm ) . top left image shows of HA - tagged ArfGAP1 , with a outline highlighting region that is positive for HA - tagged ArfGAP1 . This outline was overlaid with mTOR channel ( top right ) LAMP2 channel ( bottom left ) . mTOR LAMP2 channels were then merged processed using Squassh segmentation algorithm ( Rizk et al . , 2014 ; bottom right ) . co - localization of mTOR LAMP2 was analyzed for region within outline ( HA - ArfGAP1 positive ) region outside of outline ( HA - ArfGAP1 - negative ) , respectively . Right - In each microscopy field , image was processed as described above , co - localization between mTOR LAMP2 was quantified normalized by LAMP2 positive using Squassh algorithm ( Rizk et al . , 2014 ) . was performed for subgroups of that are positively or negatively stained for HA - tagged ArfGAP1 , respectively , compared using Student ' s t - . Columns represent Mean ± ( N = 3 ) .

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

(A) Mouse embryonic fibroblast (MEF) cells were transfected with HA-tagged ArfGAP1. Immunofluorescence staining was assessed for HA-tagged ArfGAP1 (blue), mTOR (green), and LAMP2 (red, lysosomal marker). Left- A representative example of the immunofluorescence staining (scale bar, 10 μm). The top left image shows the staining of HA-tagged ArfGAP1, with a white outline highlighting the region that is positive for HA-tagged ArfGAP1. This outline was overlaid with the mTOR channel (top right) and the LAMP2 channel (bottom left). The mTOR and LAMP2 channels were then merged and processed using the Squassh segmentation algorithm (Rizk et al., 2014; bottom right). The co-localization of mTOR and LAMP2 was analyzed for the region within the outline (HA-ArfGAP1 positive) and the region outside of the outline (HA-ArfGAP1-negative), respectively. Right- In each microscopy field, the image was processed as described above, and the co-localization between mTOR and LAMP2 was quantified and normalized by LAMP2 positive puncta using the Squassh algorithm (Rizk et al., 2014). The quantification was performed for the subgroups of cells that are positively or negatively stained for HA-tagged ArfGAP1, respectively, and compared using Student's t-test. Columns represent Mean ± SEM (N = 3).


( D ) MIA Paca - 2 stably expressing FLAG - tagged or HA - tagged TMEM192 were cultured under normal condition ( NC ) or starved of ( - AA ) , then stimulated with 2 mM glutamine asparagine ( + Gln / Asn ) for 2 hours . HA - tagged TMEM192 was immunoprecipitated ( IP ) with HA beads IP whole lysates ( WCL ) were probed for ArfGAP1 , HA , RagA , or p18 ( LAMTOR1 ) . Flag , pS6K1 ( mTORC1 activity ) , S6K1 , Vinculin were probed for as controls .

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(D) MIA Paca-2 cells stably expressing FLAG-tagged or HA-tagged TMEM192 cells were cultured under normal condition (NC) or starved of amino acids (-AA), and then stimulated with 2 mM glutamine and asparagine (+Gln/Asn) for 2 hours. HA-tagged TMEM192 was immunoprecipitated (IP) with HA beads and IP and whole cell lysates (WCL) were probed for ArfGAP1, HA, RagA, or p18 (LAMTOR1). Flag, pS6K1 (mTORC1 activity), S6K1, and Vinculin were probed for as controls.


( D ) Top - Input of GST , GST - tagged ArfGAP1 , GST - tagged ArfGAP1 L207D / V279D that was us in in vitro binding assays . Bottom - Flag - tagged mTOR , Flag - tagged Raptor , Flag - tagged mLST8 were overexpressed in HEK293A . lysates were immunoprecipitated ( IP ) with anti - Flag beads incubated with either GST ( control ) , GST - tagged ArfGAP1 , or GST - ArfGAP1 L207D / V279D . IP or WCL samples were probed for Flag or GST .

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(D) Top - Input of GST, GST-tagged ArfGAP1, and GST-tagged ArfGAP1 L207D/V279D that was us in the in vitro binding assays. Bottom - Flag-tagged mTOR, Flag-tagged Raptor, and Flag-tagged mLST8 were overexpressed in HEK293A cells. Cell lysates were immunoprecipitated (IP) with anti-Flag beads and incubated with either GST (control), GST-tagged ArfGAP1, or GST-ArfGAP1 L207D/V279D. IP or WCL samples were probed for Flag or GST.


( A ) TCGA cohort of pancreatic cancer were divided into two groups according to median level of ARFGAP1 mRNA expression . Overall survival disease - free survival were compared between these two groups , as shown in Kaplan - Meier curves . Median survivals ( in months ) , log - rank P values , hazard ratios ( HR ) with 95 % confidence intervals are indicated .

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(A) TCGA cohort of pancreatic cancer patients were divided into two groups according to the median level of ARFGAP1 mRNA expression. Overall survival and disease-free survival were compared between these two groups, as shown in Kaplan-Meier curves. Median survivals (in months), log-rank P values, and hazard ratios (HR) with 95% confidence intervals are indicated.


( D ) MIA Paca - 2 stably expressing short hairpin RNAs ( shRNAs ) against ArfGAP1 or GFP ( control ) were plated treated with mTOR inhibitor Torin 1 ( 250 nM ) or vehicle control for 48 hours prior to counting . Columns represent Mean ± ( N = 6 ) . Student ' s t - shGFP Vehicle vs . shGFP Torin 1 ( P = 2 . 83 E - 07 ) , shGFP Vehicle vs . shArfGAP1 Vehicle ( * P = 0 . 025 ) , shGFP Torin 1 vs , shArfGAP1 Torin 1 ( P = 0 . 139 ) , shArfGAP1 Vehicle vs . shArfGAP1 Torin 1 ( P = 1 . 22 E - 07 ) . " ns " stands for " not significant " .

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(D) MIA Paca-2 cells stably expressing short hairpin RNAs (shRNAs) against ArfGAP1 or GFP (control) were plated and treated with mTOR inhibitor Torin 1 (250 nM) or vehicle control for 48 hours prior to cell counting. Columns represent Mean ± SEM (N = 6). Student's t-test shGFP Vehicle vs. shGFP Torin 1 (P = 2.83 E-07), shGFP Vehicle vs. shArfGAP1 Vehicle (*P = 0.025), shGFP Torin 1 vs, shArfGAP1 Torin 1 (P = 0.139), and shArfGAP1 Vehicle vs. shArfGAP1 Torin 1 (P = 1.22 E-07). "ns" stands for "not significant".


Tau in cortex ( A , C - E ) hippocampus ( B , F - H ) of 4 - 10 - month - old mice were determined by western blot . ( A , B ) Representative western blots of cortical ( A ) hippocampal ( B ) homogenates from mice of indicated genotypes show of hTau ( HT7 ) total tau ( Tau5 ) . Actin served as a loading control . ( C - H ) Quantitation of western blot signals for full - tau detected with mTau / hTau cross - reactive antibodies Tau5 ( C , F ) or EP2456Y ( D , G ) or hTau - specific antibody Tau12 ( E , H ) . n = 3 - 8 mice per group . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by two - unpaired t ( E , H ) or by Welch ' s one - way ANOVA with post - hoc two - Welch ' s t ( C , D , F , G ) . P values were Holm - adjusted for multiple comparisons . Values are means ± .

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Tau levels in the cortex (A, C-E) and hippocampus (B, F-H) of 4-10-month-old mice were determined by western blot analysis. (A, B) Representative western blots of cortical (A) and hippocampal (B) homogenates from mice of the indicated genotypes show levels of hTau (HT7) and total tau (Tau5). Actin served as a loading control. (C-H) Quantitation of western blot signals for full-length tau detected with the mTau/hTau cross-reactive antibodies Tau5 (C, F) or EP2456Y (D, G) or the hTau-specific antibody Tau12 (E, H). n = 3-8 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed unpaired t test (E, H) or by Welch's one-way ANOVA with post-hoc two-tailed Welch's t test (C, D, F, G). P values were Holm-adjusted for multiple comparisons. Values are means ± SEM.


Tau expression in cortex ( A , C , E ) hippocampus ( B , D , F ) of 4 - 10 - month - old mice was determined by quantitative RT - PCR ( A , B ) . ( A , B ) hTau mRNA in tissue homogenates were determined by quantitative RT - PCR .

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Tau expression in the cortex (A, C, E) and hippocampus (B, D, F) of 4-10-month-old mice was determined by quantitative RT-PCR (A, B). (A, B) hTau mRNA levels in tissue homogenates were determined by quantitative RT-PCR.