( 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 .

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


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

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

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

(E) 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 test , p < 0 . 0004 ) .

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

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

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - cell patch clamp electrophysiology . ( C ) EPSC depression during 10 Hz stimulation is normal .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - cell patch clamp electrophysiology . ( D ) Depression of EPSC charge during 40 Hz stimulation is normal . Inset shows the first 5 pulses .

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - cell patch clamp electrophysiology . ( G ) PDBu was bath applied during 0 . 05 Hz stimulation . Maximum potentiation of EPSC amplitude ( M18WT : 2 . 56 ± 0 . 48 fold , n = 7 ; M18Y473F : 2 . 82 ± 0 . 98 fold , n = 8 , t - test with Welch correction , p = 0 . 8168 ) .

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

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 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 .

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

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 ) .

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

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images from rescued autaptic hippocampal Munc18 - 1 null neurons ( scale bar = 100 nm ) . Neurons expressing M18Y473A have less synaptic vesicles ( SV ) docked at the number of docked SV . Average

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

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


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

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

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


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

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

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


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

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

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


Autaptic excitatory munc18 - 1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies . ( D ) EPSC augmentation is calculated by dividing 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - test , p = 0 . 4049 ) . Average EPSC charge M18Y473A ( naive : 10 . 3 ± 4 . 5 pC , after HFS : 46 . 2 ± 14 . 5 pC , n = 13 , Wilcoxon matched - pairs signed - ranks test , p = 0 . 0005 ) . Average RRP size M18WT ( naive : 1 . 73 ± 0 . 69 nC , after HFS : 1 . 34 ± 0 . 54 nC , n = 12 , paired t - test , p = 0 . 0557 ) Average HFS size M18Y473A ( naive : 0 . 268 ± 0 . 075 nC , after HFS : 0 . 439 ± 0 . 107 nC , n = 13 , paired t - test , p = 0 . 0100 ) . Average Pves M18WT ( EPSC charge / RRP charge * 100 ) ( Naive : 7 . 07 ± 0 . 76 % , after HFS : 10 . 6 ± 1 . 6 % , n = 12 , paired t - test , p = 0 . 0201 ) . Average Pves M18Y473A ( naive : 1 . 98 ± 0 . 72 % , 12 . 2 ± 2 . 8 % , n = 13 , paired t - test , p = 0 . 0021 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) Phase separation over time as monitored by turbidity of 50 μM MBP - N after addition of TEV protease in the presence of salt ( 183 mM NaCl ) and 0 . 3 mg / ml RNA at pH 7 . 4 ( 50 mM Tris ) , pH 6 . 1 , pH 5 . 5 , or pH 4 . 9 ( pH 6 . 1 and below in 20 mM MES ) . Error bars represent standard deviation of three replicates . For samples at B ) pH 7 . 4 or C ) pH 5 . 5 , DIC micrographs of 50 μM MBP - N in the presence of salt ( 183 mM NaCl ) without or with TEV protease ( to cleave MBP from N ) and without or with 0 . 3 mg / mL desalted total torula yeast RNA . Scale bars represent 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Phase separation over time as monitored by turbidity of 50 μM MBP-N after addition of TEV protease in the presence of salt (183 mM NaCl) and 0.3 mg/ml RNA at pH 7.4 (50 mM Tris), pH 6.1, pH 5.5, or pH 4.9 (pH 6.1 and below in 20 mM MES). Error bars represent standard deviation of three replicates. For samples at B) pH 7.4 or C) pH 5.5, DIC micrographs of 50 μM MBP-N in the presence of salt (183 mM NaCl) without or with TEV protease (to cleave MBP from N) and without or with 0.3 mg/mL desalted total torula yeast RNA. Scale bars represent 50 μm.


Phase separation over time as monitored by turbidity of 50 μM MBP - N in 50 mM Tris pH 7 . 4 after addition of TEV protease A ) with varying torula yeast RNA ( at 100 mM NaCl ) or B ) varying NaCl concentrations ( at constant RNA concentration ) . Error bars represent standard deviation of three replicates .

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

Phase separation over time as monitored by turbidity of 50 μM MBP-N in 50 mM Tris pH 7.4 after addition of TEV protease A) with varying torula yeast RNA (at 100 mM NaCl) or B) varying NaCl concentrations (at constant RNA concentration). Error bars represent standard deviation of three replicates.


A ) DIC micrographs of 50 μM N and domain deletion variants in 50 mM Tris 70 mM NaCl pH 7 . 4 without and with TEV protease ( to cleave MBP from N ) and without or with 0 . 5 mg / mL desalted total torula yeast RNA . Scale bars represent 80 μm . Phase separation over time as monitored by turbidity of 50 μM full - length N or deletion variants after addition of TEV protease in the absence ( B ) or presence ( C ) of 0 . 5 mg / ml desalted total torula yeast RNA . Error bars represent standard deviation of three replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) DIC micrographs of 50 μM N and domain deletion variants in 50 mM Tris 70 mM NaCl pH 7.4 without and with TEV protease (to cleave MBP from N) and without or with 0.5 mg/mL desalted total torula yeast RNA. Scale bars represent 80 μm. Phase separation over time as monitored by turbidity of 50 μM full-length N or deletion variants after addition of TEV protease in the absence (B) or presence (C) of 0.5 mg/ml desalted total torula yeast RNA. Error bars represent standard deviation of three replicates.


A ) Turbidity of N is increased in the presence of torula yeast RNA and homopolymeric RNAs . Error bars represent standard deviation of three replicates . B ) DIC micrographs of MBP - N in the absence or presence of TEV ( to cleave MBP from N to initiate with indicated RNA . In the absence of TEV , MBP - N in the presence of torula yeast RNA , polyC , or polyU RNA does not undergo LLPS . However , after cleavage of MBP , N phase separates . polyA RNA increases LLPS of N . Both in the absence and presence of TEV , polyG RNA induces aggregation of MBP - N . Sample conditions : 50 µM MBP - N , 0 . 5 mg / mL RNA / polyX , 70 mM NaCl , 25 ˚ C , 50 mM Tris pH 7 . 4 . Scale bar represents 100 µm .

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

A) Turbidity of N is increased in the presence of torula yeast RNA and homopolymeric RNAs. Error bars represent standard deviation of three replicates. B) DIC micrographs of MBP-N in the absence or presence of TEV (to cleave MBP from N to initiate with indicated RNA. In the absence of TEV, MBP-N in the presence of torula yeast RNA, polyC, or polyU RNA does not undergo LLPS. However, after cleavage of MBP, N phase separates. polyA RNA increases LLPS of N. Both in the absence and presence of TEV, polyG RNA induces aggregation of MBP-N. Sample conditions:50 µM MBP-N, 0.5 mg/mL RNA/polyX, 70 mM NaCl, 25˚C, 50 mM Tris pH 7.4. Scale bar represents 100 µm.


A ) N partitions into hnRNPA2 LC droplets even with the MBP tag attached .

A) N partitions into hnRNPA2 LC droplets even with the MBP tag attached.


B ) N partitions into TDP - 43 CTD droplets even with the MBP tag attached .

B) N partitions into TDP-43 CTD droplets even with the MBP tag attached.


C ) N partitions into FUS LC droplets only after cleavage of the MBP tag .

C) N partitions into FUS LC droplets only after cleavage of the MBP tag.


D - F ) N partitions into hnRNPA2 FL ( D ) , TDP - 43 FL ( E ) , FUS FL ( F ) droplets .

O O O O 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-F) N partitions into hnRNPA2 FL (D), TDP-43 FL (E), FUS FL (F) droplets.


A . Scatter plot of the relative timing of cell constriction versus the relative timing of nucleoid separation . The gray scale indicates the density of dots in a given area of the chart . The dotted contours represent the 0 . 5 , 0 . 75 and 0 . 95 probability contours of the 240 WT replicates . The Pearson correlation is ρ = 0 . 65 , with a 95 % CI = [ 0 . 63 , 0 . 66 ] . The black dotted diagonal represents the line where a strain should be if both nucleoid separation and cell constriction happens at the same time . Red dots highlight

A. Scatter plot of the relative timing of cell constriction versus the relative timing of nucleoid separation. The gray scale indicates the density of dots in a given area of the chart. The dotted contours represent the 0.5, 0.75 and 0.95 probability contours of the 240 WT replicates. The Pearson correlation is ρ = 0.65, with a 95% CI = [0.63, 0.66]. The black dotted diagonal represents the line where a strain should be if both nucleoid separation and cell constriction happens at the same time. Red dots highlight strains


C . Average dynamics of nucleoid separation and cell constriction for WT and the ΔmatP mutant strain . The cumulative distributions of the fraction of cells with two nucleoids ( blue ) and of the fraction of cells with a constriction degree above 0 . 15 ( red ) were plotted against cell age . Cell age was calculated according to the rank of each cell based on their cell length with the formula $ \ text { age } _ { i } \ left ( F \ right ) = \ - \ frac { ln ( 1 - \ frac { F } { 2 } ) } { ln ( 2 ) } $ , where F represents the fraction of cells with a cell length equal or below the length of cell i ( Wold et al , 1994 ) . D . Same plots as in C for three strains clustering in island 8 with the ΔmatP strain . The WT curves shown in C were plotted in gray for comparison . E . Same plots as in C for two island - 8 strains , ΔycjV and ΔhslU , and the ΔhslV strain , which does not partition in island 8 .

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

C. Average dynamics of nucleoid separation and cell constriction for WT and the ΔmatP mutant strain. The cumulative distributions of the fraction of cells with two nucleoids (blue) and of the fraction of cells with a constriction degree above 0.15 (red) were plotted against cell age. Cell age was calculated according to the rank of each cell based on their cell length with the formula $\text{age}_{i}\left( F \right) = \ - \frac{ln(1 - \frac{F}{2})}{ln(2)}$, where F represents the fraction of cells with a cell length equal or below the length of cell i (Wold et al, 1994). D. Same plots as in C for three strains clustering in island 8 with the ΔmatP strain. The WT curves shown in C were plotted in gray for comparison. E. Same plots as in C for two island-8 strains, ΔycjV and ΔhslU, and the ΔhslV strain, which does not partition in island 8.


B . The cumulative distribution of the proportion of constricting cells for the ftsA * mutant and its parent were plotted against cell age . We measured the degree of constriction of all cells using Oufti . For each strain , we had two independently acquired image sets ( n = 496 and 1 , 168 cells for the WT replicates and n = 692 and 1 , 506 cells for the ftsA * replicates ) . The distributions of the degree of constriction were not significantly different among the four datasets at a threshold p - value of 0 . 01 using a Kruskal - Wallis multi - comparison test . Moreover , Bonferroni - corrected post - hoc pairwise tests did not allow a distinction between WT and ftsA * samples .

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

B. The cumulative distribution of the proportion of constricting cells for the ftsA* mutant and its parent were plotted against cell age. We measured the degree of constriction of all cells using Oufti. For each strain, we had two independently acquired image sets (n = 496 and 1,168 cells for the WT replicates and n = 692 and 1,506 cells for the ftsA* replicates). The distributions of the degree of constriction were not significantly different among the four datasets at a threshold p-value of 0.01 using a Kruskal-Wallis multi-comparison test. Moreover, Bonferroni-corrected post-hoc pairwise tests did not allow a distinction between WT and ftsA* samples.


C . Scatter plot of the CV of the cell length for the whole population versus the CV of the cell length for constricting cells only . The contour lines represent the 0 . 5 , 075 and 0 . 95 probability envelopes of the 240 independent WT replicates . The gray color levels indicate the density of points in the vicinity of each strain . The ∆ mraZ strain discussed in the text is highlighted in red .

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

C. Scatter plot of the CV of the cell length for the whole population versus the CV of the cell length for constricting cells only. The contour lines represent the 0.5, 075 and 0.95 probability envelopes of the 240 independent WT replicates. The gray color levels indicate the density of points in the vicinity of each strain. The ∆mraZ strain discussed in the text is highlighted in red.


A . Cellular expression and PV incorporation of spike were assessed by Western blotting using an anti - S2 mAb . Lentiviral capsid components were detected using anti - p24 / 55 .

O O O O O O O O O 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. Cellular expression and PV incorporation of spike were assessed by Western blotting using an anti-S2 mAb. Lentiviral capsid components were detected using anti-p24/55.


F . Western blot detection of ACE2 ( rabbit anti - ACE2 ) in cell lysates of the stated cells . ACE2 was detected in HEK 293T samples after loading 3X more lysate and increasing signal exposure time ( labelled in boldface ) .

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

F. Western blot detection of ACE2 (rabbit anti-ACE2) in cell lysates of the stated cells. ACE2 was detected in HEK 293T samples after loading 3X more lysate and increasing signal exposure time (labelled in boldface).


J . Over - expression confirmed by Western blotting , actin and CD81 were used as loading controls for ACE2 and TMPRSS2 respectively .

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

J. Over-expression confirmed by Western blotting, actin and CD81 were used as loading controls for ACE2 and TMPRSS2 respectively.


L . Cellular expression and PV incorporation of the stated spike proteins were assessed by Western blotting . The displayed samples are taken from the same image and have been cropped to remove irrelevant lanes / bands .

L. Cellular expression and PV incorporation of the stated spike proteins were assessed by Western blotting. The displayed samples are taken from the same image and have been cropped to remove irrelevant lanes/bands.


N . Cellular expression and PV incorporation of the stated spike proteins were assessed by Western blotting , note the change in sample order relative to panel M .

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

N. Cellular expression and PV incorporation of the stated spike proteins were assessed by Western blotting, note the change in sample order relative to panel M.


E . Cellular expression and PV incorporation of the stated spike proteins were assessed by Western blotting .

E. Cellular expression and PV incorporation of the stated spike proteins were assessed by Western blotting.


J . Cellular Expression and PV incorporation of the stated spike proteins were assessed by Western blotting .

J. Cellular Expression and PV incorporation of the stated spike proteins were assessed by Western blotting.


D . Cellular Expression and PV incorporation of the stated spike proteins were assessed by Western blotting .

D. Cellular Expression and PV incorporation of the stated spike proteins were assessed by Western blotting.


A . Expression heat map of netrin - 3 and netrin - 1 . Receptors of netrin - 1 and putative receptors of netrin - 3 : DCC , UNC5 - A , - B , - C , - D , Neogenin , APP , CD146 are presented . The color legend ( right - hand side ) describes all tumor types included in the heat map . Asterisks indicate the two main pathologies in which netrin - 3 expression is detectable . C . Percentage of cell lines positive for netrin - 1 and / or - 3 expression . Co - occurrence of expression was calculated using Fisher ' s exact test . P - value is indicated in the table .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Expression heat map of netrin-3 and netrin-1. Receptors of netrin-1 and putative receptors of netrin-3: DCC, UNC5-A, -B, -C, -D, Neogenin, APP, CD146 are presented. The color legend (right-hand side) describes all tumor types included in the heat map. Asterisks indicate the two main pathologies in which netrin-3 expression is detectable. C. Percentage of cell lines positive for netrin-1 and/or -3 expression. Co-occurrence of expression was calculated using Fisher's exact test. P-value is indicated in the table.


B . Analysis of netrin - 1 and - 3 expression in tumor cell lines and ranked according to netrin - 1 expression

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

B. Analysis of netrin-1 and -3 expression in tumor cell lines and ranked according to netrin-1 expression


B . Analysis of netrin - 1 and - 3 expression in tumor cell lines and ranked according to netrin - 1

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


A . Quantification of netrin - 3 gene expression by q - RT - PCR in a panel of 181 human NB stages 1 , 2 , 3 , 4 and 4S . Number of cases is indicated on the graph . Error bars indicate s . e . m . Statistical treatment of the data was performed using a two - sided Student t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of netrin-3 gene expression by q-RT-PCR in a panel of 181 human NB stages 1, 2, 3, 4 and 4S. Number of cases is indicated on the graph. Error bars indicate s.e.m. Statistical treatment of the data was performed using a two-sided Student t-test.


B . Quantification of netrin - 3 gene expression by q - RT - PCR in a panel of 181 human neuroblastoma patients , defined as low - and high - risk NB . The number of cases is indicated on the graph . Error bars indicate s . e . m . Statistical treatment of the data was performed using a two - sided Student t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of netrin-3 gene expression by q-RT-PCR in a panel of 181 human neuroblastoma patients, defined as low- and high-risk NB. The number of cases is indicated on the graph. Error bars indicate s.e.m. Statistical treatment of the data was performed using a two-sided Student t-test.


C . Netrin - 3 high expression is a marker of poor prognosis in NB . 280 months overall Kaplan - Meier survival curves in a panel of 181 patients of all NB stages . The cohort was dichotomized base on netrin - 3 median expression ( in green and red ) . Statistical treatment of the data : Mantel - Cox ; p value is indicated below the graph .

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

C. Netrin-3 high expression is a marker of poor prognosis in NB. 280 months overall Kaplan-Meier survival curves in a panel of 181 patients of all NB stages. The cohort was dichotomized base on netrin-3 median expression (in green and red). Statistical treatment of the data: Mantel-Cox; p value is indicated below the graph.


A . Gene Set Enrichment Analysis ( GSEA ) in high netrin - 3 NB patients . The cohort was dichotomized with the n = 50 lowest vs 50 highest : amplification the MYCN pathway ( p = 0 . 039 ; fdqr = 0 . 025 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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. Gene Set Enrichment Analysis (GSEA) in high netrin-3 NB patients. The cohort was dichotomized with the n=50 lowest vs 50 highest: amplification the MYCN pathway (p=0.039; fdqr=0.025).


C . Q - RT - PCR analysis of netrin - 3 gene expression in IMR32 ( n = 5 ) and IGRN91 ( n = 5 ) NB cell lines after MYCN silencing by siRNA ( U - test ) . Error bars indicate s . e . m .

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

C. Q-RT-PCR analysis of netrin-3 gene expression in IMR32 (n=5) and IGRN91 (n=5) NB cell lines after MYCN silencing by siRNA (U-test). Error bars indicate s.e.m.


D . ChIP - seq analysis of MYCN binding , active enhancer epigenetic marks H3K27ac , H3K4me1 , and active promoter epigenetic mark H3K4me3 , on netrin - 3 locus ( red line ) . An enrichment of MYCN , associated with active enhancer marks , was detected in three different neuroblastoma cell lines NB - 1643 , COGN415 and LAN5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. ChIP-seq analysis of MYCN binding, active enhancer epigenetic marks H3K27ac, H3K4me1, and active promoter epigenetic mark H3K4me3, on netrin-3 locus (red line). An enrichment of MYCN, associated with active enhancer marks, was detected in three different neuroblastoma cell lines NB-1643, COGN415 and LAN5.


E . Quantitative analysis showing the size of IGRN91 primary tumors implanted on CAM and silenced or not for netrin - 3 ( n = 11 siScr , n = 9 siNetrin - 3 ; U - test ) . Error bars indicate s . e . m . F . Quantitative analysis showing the size of IMR32 primary tumors silenced or not for netrin - 3 ( n = 5 siScr , n = 7 siNetrin - 3 ; U - test ) . Error bars indicate s . e . m .

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E. Quantitative analysis showing the size of IGRN91 primary tumors implanted on CAM and silenced or not for netrin-3 (n=11 siScr, n=9 siNetrin-3; U-test). Error bars indicate s.e.m. F. Quantitative analysis showing the size of IMR32 primary tumors silenced or not for netrin-3 (n=5 siScr, n=7 siNetrin-3; U-test). Error bars indicate s.e.m.


A . Deep analysis of netrin - 3 gene expression in lung cancer cell lines ( n = 76 ; RNA - Seq ) .

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A. Deep analysis of netrin-3 gene expression in lung cancer cell lines (n=76; RNA-Seq).


B . Q - RT - PCR analysis of netrin - 1 and - 3 expression , in lung cancer cell Lines ( Error bars indicate SD , n = 3 ) .

B. Q-RT-PCR analysis of netrin-1 and -3 expression, in lung cancer cell Lines (Error bars indicate SD, n=3).


C . Representative netrin - 3 and - 1 mRNA detection , using RNAscope on SCLC paraffin embedded tumor sections . Negative control DAPB , positive control PPiB . Each brown dot is a unique molecule of mRNA of each targeted gene ( n = 10 , scale bars 20µm ) . D . Quantification of netrin - 3 and - 1 expression in human SCLC paraffin embedded tumor sections ( High ≥ 50 % marked cells ; Low ≤ 5 % ) .

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C. Representative netrin-3 and -1 mRNA detection, using RNAscope on SCLC paraffin embedded tumor sections. Negative control DAPB, positive control PPiB. Each brown dot is a unique molecule of mRNA of each targeted gene (n=10, scale bars 20µm). D. Quantification of netrin-3 and -1 expression in human SCLC paraffin embedded tumor sections (High≥ 50% marked cells; Low≤ 5%).


C . Representative netrin - 3 and - 1 mRNA detection , using RNAscope on SCLC paraffin embedded tumor sections . Negative control DAPB , positive control PPiB . Each brown dot is a unique molecule of mRNA of each targeted gene ( n = 10 , scale bars 20µm ) .

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C. Representative netrin-3 and -1 mRNA detection, using RNAscope on SCLC paraffin embedded tumor sections. Negative control DAPB, positive control PPiB. Each brown dot is a unique molecule of mRNA of each targeted gene (n=10, scale bars 20µm).


A . ChIP - seq analysis of NeuroD1 and ASCL - 1 binding on the netrin - 3 gene promoter . A major binding site was detected ( red and blue peaks for NeuroD1 and ASCL1 respectively ) . Input controls are shown below ( black ) .

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A. ChIP-seq analysis of NeuroD1 and ASCL-1 binding on the netrin-3 gene promoter. A major binding site was detected (red and blue peaks for NeuroD1 and ASCL1 respectively). Input controls are shown below (black).


B . ChIP - seq analysis of NeuroD1 and ASCL - 1 chromatin enrichment , which was associated with broad H3K27ac - enriched regions at the netrin - 3 locus ( red line ) , indicating a transcriptionally active region in SCLC cell lines . ( rep = experimental replicates ) .

B. ChIP-seq analysis of NeuroD1 and ASCL-1 chromatin enrichment, which was associated with broad H3K27ac-enriched regions at the netrin-3 locus (red line), indicating a transcriptionally active region in SCLC cell lines. (rep= experimental replicates).


C . Q - RT - PCR analysis of netrin - 3 gene expression in NCI - H82 ( n = 6 ) and NCI - H69 ( n = 4 ) SCLC cell lines after Neurod - 1 and ASCL - 1 silencing by siRNAs ( U - test ) . Error bars indicate s . e . m .

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C. Q-RT-PCR analysis of netrin-3 gene expression in NCI-H82 (n=6) and NCI-H69 (n=4) SCLC cell lines after Neurod-1 and ASCL-1 silencing by siRNAs (U-test). Error bars indicate s.e.m.


A . Bio - layer interferometry analysis of netrin - 2L / receptor interactions . Other members of the netrin family were also tested : netrin - 1 , - 4 and - G2 . ( red : netrin - 2L ; bleu : netrin - 1 ; green netrin - 4 )

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A. Bio-layer interferometry analysis of netrin-2L/receptor interactions. Other members of the netrin family were also tested: netrin-1, -4 and -G2. (red: netrin-2L; bleu: netrin-1; green netrin-4)


C . NCI - H82 CRISPR / Cas9 were subjected to immunoblots . sGx , designates the number of guide RNAs , control cells ( sGc ) .

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C. NCI-H82 CRISPR/Cas9 were subjected to immunoblots. sGx, designates the number of guide RNAs, control cells (sGc).


D . Netrin - 3 silencing delays or inhibits SCLC engraftment in vivo . NMRI nude mice were subcutaneously engrafted with either NCI - H82 - CRIPSR / cas9 - NTN3 with three different guides RNAs ( sG1 p = 0 . 0022 ; sG2 p ≤ 0 . 001 ; and sG3 p ≤ 0 . 001 ) or control cells ( sGc ) n = 6 / group . Positive tumor engraftment was report when tumors reached 50 mm3 ( Mantel Cox ) .

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D. Netrin-3 silencing delays or inhibits SCLC engraftment in vivo. NMRI nude mice were subcutaneously engrafted with either NCI-H82-CRIPSR/cas9-NTN3 with three different guides RNAs (sG1 p=0.0022; sG2 p≤0.001; and sG3 p≤0.001) or control cells (sGc) n=6/group. Positive tumor engraftment was report when tumors reached 50 mm3 (Mantel Cox).


E . NCI - H69 - CRISPR / Cas9 were subjected to immunoblots . sGx , designates the number of guide RNAs , control cells ( sGc )

E. NCI-H69-CRISPR/Cas9 were subjected to immunoblots. sGx, designates the number of guide RNAs, control cells (sGc)


F . NCI - H69 - CRISPR / Cas9 cells presented in E . were subcutaneously engrafted with either NCI - H69 - CRIPSR / cas9 - NTN3 with three different guide RNAs ( sG1 p = 0 . 017 ; sG2 p = 0 . 028 ; and sG3 p = 0 . 001 ) or control cells ( sGc ) n = 6 / group . Positive tumor engraftment was report when tumors reached 20 mm3 ( Mantel Cox ) .

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F. NCI-H69-CRISPR/Cas9 cells presented in E. were subcutaneously engrafted with either NCI-H69- CRIPSR/cas9-NTN3 with three different guide RNAs (sG1 p=0.017; sG2 p=0.028; and sG3 p=0.001) or control cells (sGc) n=6/group. Positive tumor engraftment was report when tumors reached 20 mm3 (Mantel Cox).


B . Analysis of NP137 binding on netrin - 2L , netrin - 1 ; netrin - 4 or netrin - G2 by bio - layer interferometry assays . Kd rhNTN1 : 0 . 96 ± 0 . 30nM ; Kd for rchNTN2 : 1 . 53 ± 0 . 96nM

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B. Analysis of NP137 binding on netrin-2L, netrin-1; netrin-4 or netrin-G2 by bio-layer interferometry assays. Kd rhNTN1: 0.96 ± 0.30nM; Kd for rchNTN2: 1.53 ± 0.96nM


B . Analysis of NP137 binding on netrin - 2L , netrin - 1 ; netrin - 4 or netrin - G2 by bio - layer interferometry assays . Kd rhNTN1 : 0 . 96 ± 0 . 30nM ; Kd for rchNTN2 : 1 . 53 ± 0 .

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C . NP137 inhibits NTN1 / UNC5B or NTN - 2L / UNC5B interaction , detected by Elisa assays . Here the recombinant proteins are the murine forms ( Control IgG = mouse IgG2A , mNP137 = murine Fc , UNCB = rat UN5H2 ) bars indicate SD , n = 3 .

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C. NP137 inhibits NTN1/UNC5B or NTN-2L/UNC5B interaction, detected by Elisa assays. Here the recombinant proteins are the murine forms (Control IgG= mouse IgG2A, mNP137= murine Fc, UNCB=rat UN5H2) bars indicate SD, n=3.


D . NMRI nude mice were engrafted with NCI - H82 cells by subcutaneous injection of 2 million cells . When the mean tumor volume reached approximately 80 mm3 , animals were treated 3 x time / weekly by intra - abdominal injection of PBS ; NP001 ( Human IgG1 , isotype ) ; NP137 ( anti - netrin1 / 3 ) during 20 days . n = 10 animals / group ( * p < 0 . 05 , two - way Anova , Error bars indicate s . e . m . ) . E . NP137 enhances survival of mice engrafted with NCI - H82 cell line ( see D ) . Analysis of Kaplan - Meier survival curves of mice treated or not with NP137 . Mantel Cox test ; n = 10 animals / group .

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D. NMRI nude mice were engrafted with NCI-H82 cells by subcutaneous injection of 2 million cells. When the mean tumor volume reached approximately 80 mm3, animals were treated 3 x time/weekly by intra-abdominal injection of PBS; NP001 (Human IgG1, isotype); NP137 (anti-netrin1/3) during 20 days. n=10 animals/group (*p < 0.05, two-way Anova, Error bars indicate s.e.m.). E. NP137 enhances survival of mice engrafted with NCI-H82 cell line (see D). Analysis of Kaplan-Meier survival curves of mice treated or not with NP137. Mantel Cox test; n=10 animals/group.


D . NMRI nude mice were engrafted with NCI - H82 cells by subcutaneous injection of 2 million cells . When the mean tumor volume reached approximately 80 mm3 , animals were treated 3 x time / weekly by intra - abdominal injection of PBS ; NP001 ( Human IgG1 , isotype ) ; NP137 ( anti - netrin1 / 3 ) during 20 days . n = 10 animals / group ( * p < 0 . 05 , two - way Anova , Error bars indicate s . e . m . ) .

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D. NMRI nude mice were engrafted with NCI-H82 cells by subcutaneous injection of 2 million cells. When the mean tumor volume reached approximately 80 mm3, animals were treated 3 x time/weekly by intra-abdominal injection of PBS; NP001 (Human IgG1, isotype); NP137 (anti-netrin1/3) during 20 days. n=10 animals/group (*p < 0.05, two-way Anova, Error bars indicate s.e.m.).


F . Quantification of cell death ( cleaved PARP ) in H82 treated tumors in D . Tumors were analyzed 7 days after the first treatment ( ie : 400mm3 ; n = 3 / group . U - test , bars indicate SD ) .

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F. Quantification of cell death (cleaved PARP) in H82 treated tumors in D. Tumors were analyzed 7 days after the first treatment (ie: 400mm3; n=3/group. U-test, bars indicate SD).


Isosurface of the subtomogram average ( in pink ) with an applied C5 symmetry and 0 . 69 nm tomographic slices ( A - E ) at the indicated heights . Scale bars 10 nm . The average is shown in side view , whereas the slices represent perpendicular slices . The 5 pairs of pillars formed a narrow central channel and separated into 10 arches towards the base ( base not visible at the used threshold ) . The division of the structure into subparts was adapted from ( Durand et al . , 2015 ) . Subvolumes were extracted from cryotomograms of ghost cells and FIB - milled intact E . coli BL21 cells heterologously expressing TssJLM Slice ( 0 . 69 nm ) through the non - symmetrized average . Note that the C5 symmetry was

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Isosurface of the subtomogram average (in pink) with an applied C5 symmetry and 0.69 nm tomographic slices (A-E) at the indicated heights. Scale bars 10 nm. The average is shown in side view, whereas the slices represent perpendicular slices. The 5 pairs of pillars formed a narrow central channel and separated into 10 arches towards the base (base not visible at the used threshold). The division of the structure into subparts was adapted from (Durand et al., 2015). Subvolumes were extracted from cryotomograms of ghost cells and FIB-milled intact E. coli BL21 cells heterologously expressing TssJLM Slice (0.69 nm) through the non-symmetrized average. Note that the C5 symmetry was visible


Slice ( 9 . 7 nm ) through a cryotomogram of a FIB - milled E . coli BL21 cell expressing TssJLM . The average shown in Figure 1 was placed back at the positions and orientations of the individual subvolumes that were used to generate the final average . The zoomed - in area highlights the position of the TssJLM particles within the inner and the outer membrane . Some particles were found in cytoplasmic membrane invaginations , as indicated by white arrows . Scale bar 100

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Slice (9.7 nm) through a cryotomogram of a FIB-milled E. coli BL21 cell expressing TssJLM. The average shown in Figure 1 was placed back at the positions and orientations of the individual subvolumes that were used to generate the final average. The zoomed-in area highlights the position of the TssJLM particles within the inner and the outer membrane. Some particles were found in cytoplasmic membrane invaginations, as indicated by white arrows. Scale bar 100 nm


Cryotomographic slices ( 9 . 7 nm ) showing side views of TssJLM . In these examples , the basal parts of TssJLM ( red arrows ) could be seen extending into the cytoplasm . Scale bar 10

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Cryotomographic slices (9.7 nm) showing side views of TssJLM. In these examples, the basal parts of TssJLM (red arrows) could be seen extending into the cytoplasm. Scale bar 10 nm


Fluorescence of prey cells ( green ) indicating their survival against attacker cells after 4 h . The image of a representative bacterial competition spot is shown on the upper part . The relative fluorescent level ( in AU ) and the number of recovered E . coli recipient cells ( in log10 of colony forming units ( cfu ) ) are indicated in the lower graphs . The assays were performed from at least three independent cultures , with technical triplicates and a representative technical triplicate is shown . Error bars in the left graph represent ± standard deviation ( SD ) from technical triplicate . The circles in the right graph indicate values from technical triplicate , and the average is indicated by the bar .

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Fluorescence of prey cells (green) indicating their survival against attacker cells after 4 h. The image of a representative bacterial competition spot is shown on the upper part. The relative fluorescent level (in AU) and the number of recovered E. coli recipient cells (in log10 of colony forming units (cfu)) are indicated in the lower graphs. The assays were performed from at least three independent cultures, with technical triplicates and a representative technical triplicate is shown. Error bars in the left graph represent ± standard deviation (SD) from technical triplicate. The circles in the right graph indicate values from technical triplicate, and the average is indicated by the bar.


Time - lapse fluorescence microscopy recordings showing localization and dynamics of the sfGFPTssM fusion protein in the parental ( WT ) and TssJ mutated strains ( TssJ D97K ) . Individual images were taken every 60 seconds . The corresponding time of each micrograph is indicated in the lower right part of the image . Stable foci in the WT strain and less stable foci in the TssJ D97K strain are respectively indicated by white and blue arrowheads . Scale bars , 1 µm .

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Time-lapse fluorescence microscopy recordings showing localization and dynamics of the sfGFPTssM fusion protein in the parental (WT) and TssJ mutated strains (TssJ D97K). Individual images were taken every 60 seconds. The corresponding time of each micrograph is indicated in the lower right part of the image. Stable foci in the WT strain and less stable foci in the TssJ D97K strain are respectively indicated by white and blue arrowheads. Scale bars, 1 µm.


Fluorescence of prey cells ( green ) indicating their survival against attacker cells after 4h . The image of a representative bacterial competition spot is shown on the upper part . The relative fluorescent level ( in AU ) and the number of recovered E . coli recipient cells ( in log10 of colony forming units ( cfu ) ) are indicated in the lower graphs . The assays were performed from at least three independent cultures , with technical triplicates and a representative technical triplicate is shown . Error bars in the left graph represent ± standard deviation ( SD ) from technical triplicate . The circles in the right graph indicate values from technical triplicate , and the average is indicated by the bar .

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Fluorescence of prey cells (green) indicating their survival against attacker cells after 4h. The image of a representative bacterial competition spot is shown on the upper part. The relative fluorescent level (in AU) and the number of recovered E. coli recipient cells (in log10 of colony forming units (cfu)) are indicated in the lower graphs. The assays were performed from at least three independent cultures, with technical triplicates and a representative technical triplicate is shown. Error bars in the left graph represent ± standard deviation (SD) from technical triplicate. The circles in the right graph indicate values from technical triplicate, and the average is indicated by the bar.


Fluorescence microscopy recordings showing sfGFPTssM foci in the parental ( WT ) and TssM mutated strains ( TssM Q779C / N780C , TssM ∆ 777 - 783 ) . TssM foci containing cells are indicated by arrowheads . Microscopy analyses were performed independently three times , each in technical triplicate , and a representative experiment is shown . Scale bars , 1 µm .

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Fluorescence microscopy recordings showing sfGFPTssM foci in the parental (WT) and TssM mutated strains (TssM Q779C/N780C, TssM ∆777-783). TssM foci containing cells are indicated by arrowheads. Microscopy analyses were performed independently three times, each in technical triplicate, and a representative experiment is shown. Scale bars, 1 µm.


Fluorescence microscopy recordings showing TssBsfGFP sheath in the parental ( WT ) and TssM mutated strains ( TssM Q779C / N780C , TssM ∆ 777 - 783 ) . Fluorescent sheath containing cells are indicated by arrowheads . Microscopy analyses were performed independently three times , each in technical triplicate , and a representative experiment is shown . Scale bars , 1 µm .

Fluorescence microscopy recordings showing TssBsfGFP sheath in the parental (WT) and TssM mutated strains (TssM Q779C/N780C, TssM ∆777-783). Fluorescent sheath containing cells are indicated by arrowheads. Microscopy analyses were performed independently three times, each in technical triplicate, and a representative experiment is shown. Scale bars, 1 µm.


( A ) EGFR degradation was followed in MSD , MPS ‐ IIIA and WT MEFs by treating the cells with EGF for the indicated time to stimulate EGFR internalization . The cells were then immediately lysed and subjected to anti ‐ EGFR blotting . The amount of remaining EGFR was quantified by densitometry analysis ( Image J ) of the blot and expressed in the chart as % of the EGFR amount present at time T0 ( 100 % ) . The values in the chart represent the mean ± s . e . m . values of three independent experiments .

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(A) EGFR degradation was followed in MSD, MPS‐IIIA and WT MEFs by treating the cells with EGF for the indicated time to stimulate EGFR internalization. The cells were then immediately lysed and subjected to anti‐EGFR blotting. The amount of remaining EGFR was quantified by densitometry analysis (Image J) of the blot and expressed in the chart as % of the EGFR amount present at time T0 (100%). The values in the chart represent the mean±s.e.m. values of three independent experiments.


( B ) MSD , MPS ‐ IIIA and WT MEFs cells loaded with dextran ( alexafluor ‐ 594 conjugated ) were labelled with anti ‐ LAMP1 antibody and the percentage of dextran co ‐ localizing with LAMP1 was evaluated . The chart displays merge values ( means ± s . e . m . ) that represent the percentage of dextran co ‐ localizing with LAMP1 measured in 15 different cells of triplicate experiments .

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(B) MSD, MPS‐IIIA and WT MEFs cells loaded with dextran (alexafluor‐594 conjugated) were labelled with anti‐LAMP1 antibody and the percentage of dextran co‐localizing with LAMP1 was evaluated. The chart displays merge values (means±s.e.m.) that represent the percentage of dextran co‐localizing with LAMP1 measured in 15 different cells of triplicate experiments.


( C ) The rate of lysosome fusion with autophagosomes was monitored in MSD , MPS ‐ IIIA and WT MEFs transfected with a tandem fluorescently tagged LC3 ( Kimura et al , 2007 ) . The rate of autophagosome maturation reflected the percentage of the LC3 ' unfused ' ( green / red fluorescence ratio ) at each time ( 1 and 3 h ) after bafilomycin removal ( T0 ) . The percentage of LC3 ' unfused ' was displayed versus the value at T0 ( assumed to be 100 % ) . Values are represented as means ± s . e . m . of triplicate experiments . * P0 . 05 , Student ' s t ‐ test : ( A ) : WT versus MSD and WT versus MPS ‐ IIIA ; ( B , C ) : WT versus MSD and WT versus MPS ‐ IIIA at each time point . Scale bar : 10 μm ( B , C ) .

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(C) The rate of lysosome fusion with autophagosomes was monitored in MSD, MPS‐IIIA and WT MEFs transfected with a tandem fluorescently tagged LC3 ( Kimura et al, 2007). The rate of autophagosome maturation reflected the percentage of the LC3 'unfused' (green/red fluorescence ratio) at each time (1 and 3 h) after bafilomycin removal (T0). The percentage of LC3 'unfused' was displayed versus the value at T0 (assumed to be 100%). Values are represented as means±s.e.m. of triplicate experiments. *P0.05, Student's t‐test: (A): WT versus MSD and WT versus MPS‐IIIA; (B, C): WT versus MSD and WT versus MPS‐IIIA at each time point. Scale bar: 10 μm (B, C).


( A ) Cholesterol levels were measured in the indicated endolysosomal membrane samples containing equal amount of proteins and expressed as ng of cholesterol per μg of protein ( see Materials and methods section for details ) .

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(A) Cholesterol levels were measured in the indicated endolysosomal membrane samples containing equal amount of proteins and expressed as ng of cholesterol per μg of protein (see Materials and methods section for details).


( B ) Filipin staining showing cholesterol accumulation in the endolysosomal compartment of MSD and MPS ‐ IIIA MEFs ( arrowheads and enlarged images ) .

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(B) Filipin staining showing cholesterol accumulation in the endolysosomal compartment of MSD and MPS‐IIIA MEFs (arrowheads and enlarged images).


( C , D ) Total lipids extracted from the indicated endolysosomal membrane samples ( 30 μg of proteins ) were either ( C ) subjected to a phosphate assay to quantify the bulk of phospholipids or ( D ) separated by TLC . Phospholipids and cholesterol on TLC plates were revealed by molybdenum blue staining . CHOL , cholesterol ; LBPA lysobisphosphatidic acid ; PC , phosphatidylcoline ; PE phosphatidylethanolamine ; PI phosphatidyinositol ; SM sphingomyelin . ( A , C ) Values represent the mean ± s . e . m . values of three independent experiments . * P0 . 05 , Student ' s t ‐ test : ( A , C ) : WT versus MSD and WT versus MPS ‐ IIIA . Scale bar , 10 μm ( B ) .

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(C, D) Total lipids extracted from the indicated endolysosomal membrane samples (30 μg of proteins) were either (C) subjected to a phosphate assay to quantify the bulk of phospholipids or (D) separated by TLC. Phospholipids and cholesterol on TLC plates were revealed by molybdenum blue staining. CHOL, cholesterol; LBPA lysobisphosphatidic acid; PC, phosphatidylcoline; PE phosphatidylethanolamine; PI phosphatidyinositol; SM sphingomyelin. (A, C) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus MSD and WT versus MPS‐IIIA. Scale bar, 10 μm (B).


Cholesterol accumulation inhibits lysosomal fusion . Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either ( A ) WT MEFs loaded with cholesterol or in

B-CONTROLLED_VAR 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

Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD


Cholesterol accumulation inhibits lysosomal fusion . Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either ( A ) WT MEFs loaded with cholesterol or in MSD and ( D ) MPS ‐ IIIA MEFs treated with MβCD . Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol ‐ loaded WT MEFs . After treatments the rate of autophagosome maturation ( B , E ) and the transport of fluorescent dextran to lysosomes ( C , F ) were also analysed as in Figure 1 . WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1 . ( A - F ) Values represent the mean ± s . e . m . values of three independent experiments . * P0 . 05 , Student ' s t ‐ test : ( A , C ) : WT versus WT + cholesterol ; ( B ) : WT versus WT + cholesterol for each time point ; ( D , F ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD ; ( E ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD for each time point . Scale

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

Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale bar


Cholesterol accumulation inhibits lysosomal fusion . Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either ( A ) WT MEFs loaded with cholesterol or in MSD and ( D ) MPS ‐ IIIA MEFs treated with MβCD . Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol ‐ loaded WT MEFs . After treatments the rate of autophagosome maturation ( B , E ) and the transport of fluorescent dextran to lysosomes ( C , F ) were also analysed as in Figure 1 . WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1 . ( A - F ) Values represent the mean ± s . e . m . values of three independent experiments . * P0 . 05 , Student ' s t ‐ test : ( A , C ) : WT versus WT + cholesterol ; ( B ) : WT versus WT + cholesterol for each time point ; ( D , F ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD ; ( E ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD for each time point . Scale bar : 10 μm ( A , C , D , F ) .

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

Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale bar: 10 μm (A, C, D, F).


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


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


( A ) Endolysosomal membranes from MSD , MPS ‐ IIIA and WT MEFs were treated with 1 % Triton X ‐ 114 and loaded on a sucrose gradient . Immunoblots with Flotillin ‐ 1 identified DRMs in fractions 2 , 3 and 4 ( arrows ) . The fractions at the bottom of the gradient ( 12 and 13 ) correspond to high ‐ density detergent soluble fractions , whereas the remaining ones were defined as intermediate fractions ( intermediate ‐ I : 5 , 6 , 7 8 ; intermediate ‐ II : 9 , 10 and 11 ) . The percentage of Flotillin ‐ 1 in DRMs was calculated from the densitometric quantification of immunoblots .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Endolysosomal membranes from MSD, MPS‐IIIA and WT MEFs were treated with 1% Triton X‐114 and loaded on a sucrose gradient. Immunoblots with Flotillin‐1 identified DRMs in fractions 2, 3 and 4 (arrows). The fractions at the bottom of the gradient (12 and 13) correspond to high‐density detergent soluble fractions, whereas the remaining ones were defined as intermediate fractions (intermediate‐I: 5, 6, 7 8; intermediate‐II: 9, 10 and 11). The percentage of Flotillin‐1 in DRMs was calculated from the densitometric quantification of immunoblots.


( B ) Immuno ‐ EM of GM1 lipid was carried out in WT , MSD and MPS ‐ IIIA MEFs by staining cells with anti ‐ cholera toxin B antibodies ( see Materials and methods section ) . The number of GM1 ‐ positive dots was measured in 25 cells from three independent experiments and displayed as fold to WT .

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

(B) Immuno‐EM of GM1 lipid was carried out in WT, MSD and MPS‐IIIA MEFs by staining cells with anti‐cholera toxin B antibodies (see Materials and methods section). The number of GM1‐positive dots was measured in 25 cells from three independent experiments and displayed as fold to WT.


( C ) Endolysosomal membranes from MSD , MPS ‐ IIIA and WT MEFs were stained with C ‐ laurdan and subsequently analysed by fluorescence spectrophotometry to calculate the GP value ( see Materials and methods section for details ) . Distribution of cholesterol was also measured throughout the gradient ad expressed as percentage of total cholesterol in raft ( DRMs ) and soluble fractions .

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

(C) Endolysosomal membranes from MSD, MPS‐IIIA and WT MEFs were stained with C‐laurdan and subsequently analysed by fluorescence spectrophotometry to calculate the GP value (see Materials and methods section for details). Distribution of cholesterol was also measured throughout the gradient ad expressed as percentage of total cholesterol in raft (DRMs) and soluble fractions.


( D ) Equal aliquots from either DRMs or soluble fractions were pooled , the protein content determined and displayed as percentage of total protein in DRM and soluble gradient regions .

O O O O O O O O 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) Equal aliquots from either DRMs or soluble fractions were pooled, the protein content determined and displayed as percentage of total protein in DRM and soluble gradient regions.


( E ) Immunoblotting profiles of the transferrin receptor and LAMP1 in the sucrose gradient . Values represent the mean ± s . e . m . values of three experiments ( A - D ) . * P0 . 05 , Student ' s t ‐ test : ( A - C ) : WT versus MSD and WT versus MPS ‐ IIIA ; ( D ) : WT versus MSD and WT versus MPS ‐ IIIA for each fraction . Scale bar : 0 . 3 μm ( B ) .

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

(E) Immunoblotting profiles of the transferrin receptor and LAMP1 in the sucrose gradient. Values represent the mean±s.e.m. values of three experiments (A-D). *P0.05, Student's t‐test: (A-C): WT versus MSD and WT versus MPS‐IIIA; (D): WT versus MSD and WT versus MPS‐IIIA for each fraction. Scale bar: 0.3 μm (B).


( A ) VAMP7 , Vti1b and syntaxin 7 distributions in endo ‐ lysosomal membranes from MSD , MPSIIIA and WT MEFs was evaluated by immonoblotting analysis of gradient fractions . To simplify the analysis the DRM , the intermediate ‐ I , the intermediate ‐ II and soluble fractions of the gradient were pooled separately and then subjected to immunoblotting . SNARE distribution was also analysed after loading WT cells with cholesterol and after MβCD treatment . In MSD and MPS ‐ IIIA MEFs , all analysed SNAREs abnormally accumulate in DRMs of lysosomal membranes . Cholesterol modulation results in a change of SNARE distribution .

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

(A) VAMP7, Vti1b and syntaxin 7 distributions in endo‐lysosomal membranes from MSD, MPSIIIA and WT MEFs was evaluated by immonoblotting analysis of gradient fractions. To simplify the analysis the DRM, the intermediate‐I, the intermediate‐II and soluble fractions of the gradient were pooled separately and then subjected to immunoblotting. SNARE distribution was also analysed after loading WT cells with cholesterol and after MβCD treatment. In MSD and MPS‐IIIA MEFs, all analysed SNAREs abnormally accumulate in DRMs of lysosomal membranes. Cholesterol modulation results in a change of SNARE distribution.


( B ) The percentage of each analysed SNARE observed in DRM fractions was quantified from blots ( ImageJ densitometry analysis ) and displayed as relative amount versus WT .

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

(B) The percentage of each analysed SNARE observed in DRM fractions was quantified from blots (ImageJ densitometry analysis) and displayed as relative amount versus WT.


( C , E ) Immunoblots and relative quantification showing SNARE protein levels along with LAMP1 protein levels in ( C ) endolysosomal membranes and ( E ) total cell lysates from WT ( untreated and cholesterol treated ) and MSD MEFs ( untreated and MβCD treated ) . In the graphs , the protein levels were displayed as relative amount versus WT .

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

(C, E) Immunoblots and relative quantification showing SNARE protein levels along with LAMP1 protein levels in (C) endolysosomal membranes and (E) total cell lysates from WT (untreated and cholesterol treated) and MSD MEFs (untreated and MβCD treated). In the graphs, the protein levels were displayed as relative amount versus WT.


( D ) WT and MSD MEFs transfected with GFP - VAMP7 were stained with anti ‐ GFP for immuno ‐ EM . The enlarged image shows internalization of GFP - VAMP7 particles ( arrow ) .

O O O O O O O O O O O O O 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) WT and MSD MEFs transfected with GFP-VAMP7 were stained with anti‐GFP for immuno‐EM. The enlarged image shows internalization of GFP-VAMP7 particles (arrow).


( F ) Syntaxin 5 , Sec22 and SNAP23 distribution in total membrane derived from control WT and MSD MEFs . Syntaxin 5 immunoblot shows two bands ( * , 35 kDa and * * , 42 kDa ) corresponding to the two isoforms of the protein .

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

(F) Syntaxin 5, Sec22 and SNAP23 distribution in total membrane derived from control WT and MSD MEFs. Syntaxin 5 immunoblot shows two bands (*, 35 kDa and **, 42 kDa) corresponding to the two isoforms of the protein.


( G ) Distribution profile of Rab7 in WT and MSD lysosomal membranes . Values represent the mean ± s . e . m . values of three independent experiments ( B , C , E ) . * P0 . 05 , Student ' s t ‐ test : ( B ) : WT versus MSD , WT versus MPS ‐ IIIA , WT versus WT + cholesterol , MSD versus MSD + MβCD , and MPS ‐ IIIA versus MβCD for each analysed SNARE ; ( C , E ) : WT versus MSD , WT versus WT + cholesterol and MSD versus MSD + MβCD for each analysed protein . Scale bar : 0 . 3 μm ( D ) .

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

(G) Distribution profile of Rab7 in WT and MSD lysosomal membranes. Values represent the mean±s.e.m. values of three independent experiments (B, C, E). *P0.05, Student's t‐test: (B): WT versus MSD, WT versus MPS‐IIIA, WT versus WT+cholesterol, MSD versus MSD+MβCD, and MPS‐IIIA versus MβCD for each analysed SNARE; (C, E): WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD for each analysed protein. Scale bar: 0.3 μm (D).


( A ) SDS ‐ resistant complexes containing Vti1b were detected by immunoblotting analysis of nonboiled samples corresponding to total , detergent insoluble ( DRM ) and detergent soluble ( Sol . ) endo ‐ lysosomal membrane fractions derived from MSD and WT MEFs . The SDS ‐ resistant complexes were also visualized after loading WT MEFs with cholesterol and after treating MSD MEFs with MβCD . Immunoblots revealed the presence of low molecular weight complexes ( * , 50 - 60 kDa ) and high molecular weight complexes ( * * , > 80 kDa ) . The percentage of Vti1b in SDS ‐ resistant complexes in total endolysosomal membranes ( bottom ‐ left chart ) and the amount of Vti1b ‐ containing SDS ‐ resistant complexes in DRM and soluble fractions ( bottom ‐ right chart ) were calculated by the densitometric quantification of the correspondent immunoblots ( ImageJ ) . Values represent the mean ± s . e . m . values of three independent measurements . * P0 . 05 , Student ' s t ‐ test : WT versus MSD , WT versus WT + cholesterol and MSD versus MSD + MβCD ( bottom ‐ left chart ) ; WT versus MSD , WT versus WT + cholesterol and MSD versus MSD + MβCD for each fraction ( bottom ‐ right chart ) .

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

(A) SDS‐resistant complexes containing Vti1b were detected by immunoblotting analysis of nonboiled samples corresponding to total, detergent insoluble (DRM) and detergent soluble (Sol.) endo‐lysosomal membrane fractions derived from MSD and WT MEFs. The SDS‐resistant complexes were also visualized after loading WT MEFs with cholesterol and after treating MSD MEFs with MβCD. Immunoblots revealed the presence of low molecular weight complexes (*, 50-60 kDa) and high molecular weight complexes (**, >80 kDa). The percentage of Vti1b in SDS‐resistant complexes in total endolysosomal membranes (bottom‐left chart) and the amount of Vti1b‐containing SDS‐resistant complexes in DRM and soluble fractions (bottom‐right chart) were calculated by the densitometric quantification of the correspondent immunoblots (ImageJ). Values represent the mean±s.e.m. values of three independent measurements. *P0.05, Student's t‐test: WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD (bottom‐left chart); WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD for each fraction (bottom‐right chart).


( B ) Syntaxin 7 and VAMP7 were co ‐ immunoprecipitated with Vti1b using anti ‐ Vti1b antibodies in WT ( untreated or cholesterol treated ) and in MSD ( not treated or MβCD treated ) MEFs . The amount of Vti1b precipitated in each cell line is also shown .

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

(B) Syntaxin 7 and VAMP7 were co‐immunoprecipitated with Vti1b using anti‐Vti1b antibodies in WT (untreated or cholesterol treated) and in MSD (not treated or MβCD treated) MEFs. The amount of Vti1b precipitated in each cell line is also shown.


( C ) SDS ‐ resistant complexes are decorated by anti ‐ syntaxin 7 antibodies in total endolysosomal membrane fraction from WT and MSD MEFs .

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

(C) SDS‐resistant complexes are decorated by anti‐syntaxin 7 antibodies in total endolysosomal membrane fraction from WT and MSD MEFs.


( D ) Membrane ‐ associated α ‐ SNAP and its release in the cytosol were evaluated by western blot analysis on total cell lysates ( total ) , intracellular membranes recovered after centrifugation from a post ‐ nuclear supernatant fraction ( membrane associated ) and cell lysates devoid of membranes ( cytosolic released ) derived from MSD ( untreated or MβCD treated ) and WT ( untreated or cholesterol treated ) MEFs .

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

(D) Membrane‐associated α‐SNAP and its release in the cytosol were evaluated by western blot analysis on total cell lysates (total), intracellular membranes recovered after centrifugation from a post‐nuclear supernatant fraction (membrane associated) and cell lysates devoid of membranes (cytosolic released) derived from MSD (untreated or MβCD treated) and WT (untreated or cholesterol treated) MEFs.


( A ) MSD , MPS ‐ IIIA and WT MEFs were subjected to a triple labelling with anti ‐ VAMP7 , anti ‐ Vti1b and anti ‐ LAMP1 antibodies . The merges between VAMP7 and Vti1b ( double merges in yellow ) and between VAMP7 , Vti1b and LAMP1 ( triple merges in white ) are shown ( see also enlarged images showing the extent of co ‐ localization in different regions of the cells ) . The VAMP7 - Vti1b co ‐ localization was quantified in 15 different cells and displayed as % of Vti1b co ‐ localizing with VAMP7 ( means ± s . e . m . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) MSD, MPS‐IIIA and WT MEFs were subjected to a triple labelling with anti‐VAMP7, anti‐Vti1b and anti‐LAMP1 antibodies. The merges between VAMP7 and Vti1b (double merges in yellow) and between VAMP7, Vti1b and LAMP1 (triple merges in white) are shown (see also enlarged images showing the extent of co‐localization in different regions of the cells). The VAMP7-Vti1b co‐localization was quantified in 15 different cells and displayed as % of Vti1b co‐localizing with VAMP7 (means±s.e.m.).


( B ) Co ‐ localization of Vti1b with epsinR was quantified by double ‐ labelling experiments in MSD and MPS ‐ IIIA ( untreated and MβCD treated ) and in control WT ( untreated and cholesterol treated ) MEFs . The chart displays merge values ( means ± s . e . m . ) that represent the percentage of Vti1b co ‐ localizing with epsinR measured in 15 different cells .

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

(B) Co‐localization of Vti1b with epsinR was quantified by double‐labelling experiments in MSD and MPS‐IIIA (untreated and MβCD treated) and in control WT (untreated and cholesterol treated) MEFs. The chart displays merge values (means±s.e.m.) that represent the percentage of Vti1b co‐localizing with epsinR measured in 15 different cells.


( C ) Vti1b trafficking was monitored by FRAP analysis in WT ( untreated and cholesterol treated ) and MSD ( untreated and MβCD treated ) MEFs transfected with GFP - Vti1b ( see Materials and methods section for details ) . FRAP data are displayed as percentage of recovery with respect to the fluorescence before bleach ( 100 % ) and are representative of 10 recordings from different cells .

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

(C) Vti1b trafficking was monitored by FRAP analysis in WT (untreated and cholesterol treated) and MSD (untreated and MβCD treated) MEFs transfected with GFP-Vti1b (see Materials and methods section for details). FRAP data are displayed as percentage of recovery with respect to the fluorescence before bleach (100%) and are representative of 10 recordings from different cells.


b , Representative phase images of the morphology of WT and Apc - / - organoids before mRNA isolation .

b, Representative phase images of the morphology of WT and Apc-/- organoids before mRNA isolation.