( A ) Box - - whiskers plot showing CSF p - tau235 concentrations in A - T - participants across preclinical Alzheimer ' s disease ( AD , dichotomised as A + T - A + T + ) . CSF p - tau235 was increased already in A + T - , which represents early cases within preclinical AD . A prominent increase was observed from A + T - to A + T + cases , latter representing late preclinical AD cases . Cut - off value for CSF p - tau235 positivity is displayed with black dashed ( 19 . 92 pg / mL ) .

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(A) Box-and-whiskers plot showing CSF p-tau235 concentrations in A-T- participants and across preclinical Alzheimer's disease (AD, dichotomised as A+T- and A+T+). CSF p-tau235 was increased already in A+T-, which represents the early cases within preclinical AD. A prominent increase was observed from A+T- to A+T+ cases, the latter representing late preclinical AD cases. Cut-off value for CSF p-tau235 positivity is displayed with black dashed line (19.92 pg/mL).


Trajectories of different tau biomarkers in preclinical AD using a local weighted regression method ( Loess curve ) . Changes on biomarkers in CSF are represented as z - scores using Aβ PET in centiloid scale ( CL ) as a proxy of disease progression . Abnormal biomarker were determined as two standard deviations ( SD ) above mean . Incipient Aβ pathology positivity was determined as Aβ PET higher than CL 12 ( Mila - Aloma et al . , 2020 ) . samples were run in singlicates .

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Trajectories of the different tau biomarkers in preclinical AD using a local weighted regression method (Loess curve). Changes on biomarkers levels in CSF are represented as z-scores using Aβ PET in centiloid scale (CL) as a proxy of disease progression. Abnormal biomarker levels were determined as two standard deviations (SD) above the mean. Incipient Aβ pathology positivity was determined as Aβ PET higher than CL 12 (Mila-Aloma et al., 2020). All samples were run in singlicates.


( A ) Stacked bar chart depicting percentages of [ 231 + / 235 + ] , [ 231 + / 235 - ] , [ 231 - / 235 - ] [ 231 - / 235 + ] cases in each preclinical group . Table below indicates exact percentages of participants in each group ( exact number of participants in parenthesis ) . Discordant cases with sequential phosphorylation hypothesis [ 231 - / + 235 ] shown in grey .

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(A) Stacked bar chart depicting the percentages of [231+/235+], [231+/235-], [231-/235-] and [231-/235+] cases in each preclinical group. Table below indicates the exact percentages of participants in each group (exact number of participants in parenthesis). Discordant cases with the sequential phosphorylation hypothesis [231-/+235] shown in grey.


( B ) Correlation between CSF p - tau235 p - tau231 in whole ALFA + cohort ( Spearman ' s rank correlation : rS = 0 . 80 , P ˂ 0 . 0001 ) . cut - offs were determined as mean + 2 SD of A - T - group ( defining p - tau231 p - tau235 positivity or negativity in each participant ) . Cut - off values are indicated in red ( 19 . 92 9 . 59 pg / mL for CSF p - tau235 p - tau231 respectively ) displayed with black dashed lines , resulting in four quadrants , each of them representing four different positive or negatively status for each biomarker . Discordant cases with sequential phosphorylation hypothesis ( [ 231 - / + 235 ] ) showed on lower right quadrant ( 2 . 35 % of total , 9 participants out 383 ) . samples were run in singlicates .

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(B) Correlation between CSF p-tau235 and p-tau231 in the whole ALFA+ cohort (Spearman's rank correlation: rS = 0.80, P˂0.0001). Assay cut-offs were determined as the mean + 2 SD of the A-T- group (defining p-tau231 and p-tau235 positivity or negativity in each participant). Cut-off values are indicated in red (19.92 and 9.59 pg/mL for CSF p-tau235 and p-tau231 respectively) and displayed with black dashed lines, resulting in four quadrants, each of them representing the four different positive or negatively status for each biomarker. Discordant cases with the sequential phosphorylation hypothesis ([231-/+235]) showed on the lower right quadrant (2.35% of the total, 9 participants out 383). All samples were run in singlicates.


A . MDCK were cultured on culture inserts for 4 days . On last day , culture medium was replaced with EV - depleted medium . EVs released from apical basolateral sides of MDCK were purified by PEG precipitation . lysates EV in PEG pellets were analyzed by immunoblotting with antibodies indicated . Note that PEG pellets did not contain mitochondrial TOMM20 , suggesting that PEG pellets were not contaminated by intracellular organelles .

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A. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. EVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation. Cell lysates and EV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated. Note that the PEG pellets did not contain mitochondrial protein TOMM20, suggesting that the PEG pellets were not contaminated by intracellular organelles.


B . PEG pellets were subjected to OptiPrep flotation .

B. PEG pellets were subjected to OptiPrep flotation analysis.


C . MDCK stably expressing human CD63 were cultured sEVs were isolated from pre - cleared medium by direct immunoaffinity capture using anti - CD63 antibody .

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C. MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD63 antibody.


D . sEVs were eluted from beads with a glycine buffer analyzed by nanoparticle tracking ( NTA ) . Representative NTA traces were shown . E . of NTA data obtained in five independent experiments .

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D. sEVs were eluted from the beads with a glycine buffer and analyzed by nanoparticle tracking analysis (NTA). Representative NTA traces were shown. E. Quantification of the NTA data obtained in five independent experiments.


F . MDCK were cultured sEVs were isolated from pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody .

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F. MDCK cells were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody.


G . sEVs were eluted from beads with a glycine buffer analyzed by NTA . Representative NTA traces were shown . H . of NTA data obtained in five independent experiments .

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G. sEVs were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown. H. Quantification of the NTA data obtained in five independent experiments.


A . MDCK were cultured on culture inserts for 4 days . On last day , culture medium was replaced with serum - free medium . sEVs released from apical basolateral sides of MDCK were purified by ultracentrifugation . Apical basolateral sEVs ( P100 ) were analyzed by silver .

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A. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with serum-free medium. sEVs released from the apical and basolateral sides of MDCK cells were purified by ultracentrifugation. Apical and basolateral sEVs (P100) were analyzed by silver staining.


C . MDCK were cultured on culture inserts for 4 days . On last day , culture medium was replaced with EV - depleted medium . sEVs released from apical basolateral sides of MDCK were purified by PEG precipitation . lysates sEV in PEG pellets were analyzed by immunoblotting with antibodies indicated . GPRC5C was detected only in apical sEV

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C. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation. Cell lysates and sEV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated. GPRC5C was detected only in the apical sEV samples


D . PEG pellets were subjected to OptiPrep flotation . GPRC5C was detected only in apical sEV

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D. PEG pellets were subjected to OptiPrep flotation analysis. GPRC5C was detected only in the apical sEV samples


E . MDCK were cultured sEVs were isolated from pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody . GPRC5C was detected only in apical sEV

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E. MDCK cells were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. GPRC5C was detected only in the apical sEV samples


F . MDCK stably expressing human CD63 were cultured sEVs were isolated from pre - cleared medium by direct immunoaffinity capture using anti - CD63 antibody .

F. MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD63 antibody.


A . MDCK were transfected with siControl or siRNAs indicated , were transferred to culture inserts cultured for 4 days . On last day , culture medium was replaced with EV - depleted medium . sEVs were isolated from pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody . lysates sEV samples were analyzed by immunoblotting with antibodies indicated .

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A. MDCK cells were transfected with siControl or the siRNAs indicated, and the cells were transferred to cell culture inserts and cultured for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated.


B . of bands was measured in three independent experiments .

B. The intensity of the bands was measured in three independent experiments.


C . sEVs were eluted from beads with a glycine buffer analyzed by NTA . Representative NTA traces were shown .

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C. sEVs were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown.


D . of NTA data obtained in five independent experiments .

D. Quantification of the NTA data obtained in five independent experiments.


A . MDCK were transfected with siControl or siALIX , were transferred to culture inserts cultured for 4 days . On last day , culture medium was replaced with EV - depleted medium with or without 10 nM GW4869 . sEVs were isolated from pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody . lysates sEV samples were analyzed by immunoblotting with antibodies indicated .

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A. MDCK cells were transfected with siControl or siALIX, and the cells were transferred to cell culture inserts and cultured for 4 days. On the last day, the culture medium was replaced with EV-depleted medium with or without 10 nM GW4869. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated.


Analyses of expression in lysates of various lines by western blotting against indicated antibodies .

Analyses of protein expression in lysates of various cell lines by western blotting against the indicated antibodies.


analyses of GFP - ATG5 or ATG16L1 - GFP stably expressed in ATG5 - / - or ATG16L1 - / - . were acid starved for 2 hr prior to fixation imaging of GFP . Scale bar : 10 μm .

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Fluorescence analyses of GFP-ATG5 or ATG16L1-GFP stably expressed in ATG5-/- or ATG16L1-/-. Cells were amino acid starved for 2 hr prior to fixation and imaging of the GFP fluorescence. Scale bar: 10 μm.


Assessment of ATG16L1 in ( C ) ( M ) fractions in lysates of indicated lines using western blot analyses antibodies against ATG16L1 . ATG16L1 - / - ATG5 - / - stably expressed ATG16L1 - GFP while ATG3 - / - stably expressed Flag - S - ATG16L1 . Antibodies against α - tubulin integrin β1 were used as controls for fractionation . Exogenous ( exo ) ATG16L1 was detected using antibodies against ATG16L1 .

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Assessment of ATG16L1 levels in the cytosolic (C) and membrane (M) fractions in lysates of the indicated cell lines using western blot analyses and antibodies against ATG16L1. ATG16L1-/- and ATG5-/- stably expressed ATG16L1-GFP while ATG3-/- stably expressed Flag-S-ATG16L1. Antibodies against α-tubulin and integrin β1 were used as controls for fractionation. Exogenous (exo) ATG16L1 was detected using antibodies against ATG16L1.


Lack of WIPI2 expression is confirmed by western blot analyses in wild type MEFs ( WIPI2 + / + ) WIPI2 - / - .

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Lack of WIPI2 expression is confirmed by western blot analyses in wild type MEFs (WIPI2+/+) and WIPI2-/- cells.


Immunofluorescence analyses of ATG16L1 - / - WIPI2 - / - stably expressing Flag - S - ATG16L1 . were acid starved ( AA starve ) for 2 hr in presence or absence of 3 ' MA ( additional pretreatment for 30 min ) followed by fixation immuno - using antibodies against Flag tag to detect ATG16L1 . Scale bar : 9 μm .

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Immunofluorescence analyses of ATG16L1-/- and WIPI2-/- stably expressing Flag-S-ATG16L1. Cells were amino acid starved (AA starve) for 2 hr in the presence or absence of 3'MA (and additional pretreatment for 30 min) followed by fixation and immuno-staining using antibodies against Flag tag to detect ATG16L1. Scale bar: 9 μm.


Fragments depicted in ( A ) were expressed in U2OS acid starved for 5 hr followed by fixation immunostaining against S tag to detect S - ATG16L1 . Scale bar : 10 μm .

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Fragments depicted in (A) were expressed in U2OS cells and amino acid starved for 5 hr followed by fixation and immunostaining against S tag to detect S-ATG16L1. Scale bar: 10 μm.


U2OS expressing Flag - ATG16L1 ∆ 2 or Flag - ATG16L1 ∆ 2 ∆ 182 - 205 were treated as in ( B ) stained using antibodies against Flag tag ( to detect ATG16L1 , green ) FIP200 ( red ) . Scale bar : 10 μm .

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U2OS cells expressing Flag-ATG16L1∆2 or Flag-ATG16L1∆2∆182-205 were treated as in (B) and stained using antibodies against Flag tag (to detect ATG16L1, green) and FIP200 (red). Scale bar: 10 μm.


- interaction in 293T transiently transfected with indicated Flag - S - tagged ATG16L1 constructs . S tag pull down was performed complexes were analysed by immunoblotting using indicated antibodies .

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Protein-protein interaction assay in 293T cells transiently transfected with the indicated Flag-S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies.


Homodimerisation in 293T transiently transfected with ATG16L1 - GFP indicated Flag - S - tagged ATG16L1 constructs . S tag pull down was performed complexes were analysed by immunoblotting using indicated antibodies .

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Homodimerisation assay in 293T cells transiently transfected with ATG16L1-GFP and the indicated Flag-S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies.


Flag - ATG16L1WT or Flag - ATG16L1 ∆ 182 - 205 were stably expressed in ATG5 - / - analysed by immunofluorescence using antibodies against Flag tag to detect ATG16L1 . Scale bar : 9 μm .

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Flag-ATG16L1WT or Flag-ATG16L1∆182-205 were stably expressed in ATG5-/- cells and analysed by immunofluorescence using antibodies against Flag tag to detect ATG16L1. Scale bar: 9 μm.


Microscopy - - liposome binding . ATG16L1 - GFP immobilised on beads incubated in presence of rhodamine - labelled liposome preparations containing indicated phosphoinositides . Scale bar : 50 μm .

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Microscopy-based protein-liposome binding assay. ATG16L1-GFP immobilised on beads incubated in the presence of rhodamine-labelled liposome preparations containing the indicated phosphoinositides. Scale bar: 50 μm.


Quantification of relative liposome binding


Microscopy - - liposome binding as in ( B ) . ATG16L1WT - ATG16L1LD - GFP immobilised on beads were incubated with rhodamine - labelled , PI3P - positive liposome preparations . Scale bar : 50 μm . Right panel shows of liposome binding relative to ATG16L1WT from 3 independent experiments including values .

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Microscopy-based protein-liposome binding assay as in (B). ATG16L1WT- and ATG16L1LD-GFP immobilised on beads were incubated with rhodamine-labelled, PI3P-positive liposome preparations. Scale bar: 50 μm. Right panel shows quantification of liposome binding relative to ATG16L1WT from 3 independent experiments including SEM values.


- interaction in 293T transiently transfected with indicated S - tagged ATG16L1 constructs . S tag pull down was performed complexes were analysed by immunoblotting using indicated antibodies .

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Protein-protein interaction assay in 293T cells transiently transfected with the indicated S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies.


by immunoblotting using indicated antibodies . Dimerisation in 293T transiently transfected with ATG16L1 - GFP indicated S - tagged ATG16L1 constructs

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by immunoblotting using the indicated antibodies. Dimerisation assay in 293T cells transiently transfected with ATG16L1-GFP and the indicated S-tagged ATG16L1 constructs and analysed


ATG16L1 - / - stably expressing indicated Flag - S - ATG16L1 constructs were acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag ( green ) ATG5 ( red ) . Scale bar : 9 μm . Right panels show quantifications of average number of Flag - ATG5 - positive dots per .

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ATG16L1-/- stably expressing the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag (green) and ATG5 (red). Scale bar: 9 μm. Right panels show quantifications of average number of Flag- and ATG5-positive dots per cell.


ATG16L1 - / - stably expressing indicated Flag - S - ATG16L1 constructs were acid starved for 2 hr prior to immunofluorescence analyses using antibodies against S tag ( to detect ATG16L1 , red ) WIPI2 ( green ) . Scale bar : 9 μm . Lower panel shows of average number of WIPI2 positive dots per .

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ATG16L1-/- stably expressing the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against S tag (to detect ATG16L1, red) and WIPI2 (green). Scale bar: 9 μm. Lower panel shows quantification of average number of WIPI2 positive dots per cell.


Average intensities of Flag - ATG16L1 dots in ( A ) . Underlying grey circles represent data points . Dots were quantified ( n = 1225 for ATG16L1WT ; n = 334 for ATG16L1LD ) .

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Average intensities of individual Flag-ATG16L1 dots in (A). Underlying grey circles represent individual data points. Dots were quantified (n=1225 for ATG16L1WT; n=334 for ATG16L1LD).


WIPI2 - / - reconstituted with indicated Flag - S - ATG16L1 constructs were acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag ( green ) FIP200 ( red ) . Scale bar : 9 μm . Lower panel shows of average number of Flag positive dots per - positive .

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WIPI2-/- cells reconstituted with the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag (green) and FIP200 (red). Scale bar: 9 μm. Lower panel shows quantification of average number of Flag positive dots per puncta-positive cell.


ATG16L1 - / - were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs autophagy assessed during treatment with various stimuli followed by immunoblotting using indicated antibodies . were acid starved ( AA starved ) to induce mTORC1 - dependent autophagy in presence or absence of BafA1 for 2 hr prior to lysis .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. Cells were amino acid starved (AA starved) to induce mTORC1-dependent autophagy in the presence or absence of BafA1 for 2 hr prior to lysis.


ATG16L1 - / - were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs autophagy assessed during treatment with various stimuli followed by immunoblotting using indicated antibodies . Mitophagy was induced by CCCP ( 10 μM ) treatment for 6 hr prior to lysis .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. Mitophagy was induced by CCCP (10 μM) treatment for 6 hr prior to lysis.


ATG16L1 - / - were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs autophagy assessed during treatment with various stimuli followed by immunoblotting using indicated antibodies . Glucose starvation ( Glu starved ) for 20 hr was used to induce mTORC1 - independent autophagy . BafA1 was added to conditions 2 hr prior to lysis .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. Glucose starvation (Glu starved) for 20 hr was used to induce mTORC1-independent autophagy. BafA1 was added to all conditions 2 hr prior to lysis.


ATG16L1 - / - were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs autophagy assessed during treatment with various stimuli followed by immunoblotting using indicated antibodies . LC3 - associated phagocytosis ( LAP ) - like LC3 lipidation was induced by treating with CCCP ( 100 μM ) for 2 hr prior to lysis . Right panel shows of LC3 - II / LC3 - I .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. LC3-associated phagocytosis (LAP)-like LC3 lipidation was induced by treating cells with CCCP (100 μM) for 2 hr prior to lysis. Right panel shows quantification of LC3-II/LC3-I ratio.


ATG16L1 - / - were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs autophagy assessed during treatment with various stimuli followed by immunoblotting using indicated antibodies . LAP - like LC3 lipidation induced by monensin or ammonium chloride ( NH4Cl ) for 2 hr prior to lysis .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. LAP-like LC3 lipidation induced by monensin or ammonium chloride (NH4Cl) for 2 hr prior to lysis.


Coomassie gel of recombinant ATG16L172 - 307 wild type mutants . * indicates

Coomassie gel of recombinant ATG16L172-307 wild type and mutants. * indicates bacterial protein


Liposome co - sedimentation using recombinant ATG16L1 sonicated liposomes . Supernatant ( S ) pellet ( P ) were analysed by immunoblotting against T7 tag to detect ATG16L1 . of in pellet fraction was calculated from 3 independent experiments ( right panel ) .

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Liposome co-sedimentation assay using recombinant ATG16L1 protein and sonicated liposomes. Supernatant (S) and pellet (P) were analysed by immunoblotting against T7 tag to detect ATG16L1. Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments (right panel).


Liposome co - sedimentation , as in ( C ) , using ATG16L172 - 307LE sonicated liposomes that contain or lack PI3P . of in pellet fraction was calculated from 3 independent experiments ( right panel ) with error bars depicting values .

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Liposome co-sedimentation assay, as in (C), using ATG16L172-307LE and sonicated liposomes that contain or lack PI3P. Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments (right panel) with error bars depicting SEM values.


ATG16L1 - / - were reconstituted with ATG16L1WT - or ATG16L1LE - expression constructs autophagy assessed during acid starvation ( AA starve ) followed by immunoblotting or immunofluorescence analyses . Immunofluorescence analyses of acid starved ( 2 hr ) using antibodies against WIPI2 ( red ) or ATG16L1 ( green ) . Scale bar : 9 μm . as in ( A ) with addition of 3 ' MA ( pretreatment with drug for 30 min ) . Scale bar : 9 μm .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LE-expression constructs and autophagy assessed during amino acid starvation (AA starve) followed by immunoblotting or immunofluorescence analyses. Immunofluorescence analyses of amino acid starved cells (2 hr) using antibodies against WIPI2 (red) or ATG16L1 (green). Scale bar: 9 μm. Cells as in (A) with the addition of 3'MA (and pretreatment with the drug for 30 min). Scale bar: 9 μm.


Quantification of percentage of cells positive for ATG16L1 puncta


were left untreated , acid starved for 2 hr , or acid starved for 2 hr followed by refeeding with full growth media for 1 hr . formation was assessed by immunofluorescence using antibodies against S tag to detect ATG16L1 . Scale bar : 10 μm .

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Cells were left untreated, amino acid starved for 2 hr, or amino acid starved for 2 hr followed by refeeding with full growth media for 1 hr. Puncta formation was assessed by immunofluorescence staining using antibodies against S tag to detect ATG16L1. Scale bar: 10 μm.


Immunoblot analyses of acid starved in presence of BafA1 for 2 hr . lysates were analysed using indicated antibodies .

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Immunoblot analyses of amino acid starved cells in the presence of BafA1 for 2 hr. Cell lysates were analysed using the indicated antibodies.


WIPI2 - / - reconstituted with indicated ATG16L1 constructs were acid starved for 2 hr . BafA1 treatment was included in conditions for 2 hr prior to lysis . lysates were analysed by immunoblotting using indicated antibodies . of LC3 - II / LC3 - I of three independent experiments is shown on right panel .

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WIPI2-/- cells reconstituted with the indicated ATG16L1 constructs were amino acid starved for 2 hr. BafA1 treatment was included in all conditions for 2 hr prior to lysis. Cell lysates were analysed by immunoblotting using the indicated antibodies. Quantification of LC3-II/LC3-I ratio of three independent experiments is shown on the right panel.


Ferroptosis in ATG16L1 - / - stably expressing ATG16L1WT , ATG16L1LD or ATG16L1LE . were cultured in acid free media ( AA starve ) in presence of 10 % FBS or 10 % dialysed FBS ( diFBS ) for 24 hr . of of PI - positive from at least three independent experiments is shown .

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Ferroptosis assay in ATG16L1-/- cells stably expressing ATG16L1WT, ATG16L1LD or ATG16L1LE. Cells were cultured in amino acid free media (AA starve) in the presence of 10% FBS or 10% dialysed FBS (diFBS) for 24 hr. Quantification of percentage of PI-positive cells from at least three independent experiments is shown.


( a ) Kaplan - Meier survival plot of cohorts of induced Vil Apc , Vil Apc Huwe1het Vil Apc Huwe1hom mice . Deletion of Huwe1 led to a significant reduction in survival of these ( Vil Apc vs Vil Apc Huwe1het / Vil Apc Huwe1hom , Log Rank , p < 0 . 001 , n ≥ 10 ) .

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(a) Kaplan-Meier survival plot of cohorts of induced Vil Apc, Vil Apc Huwe1het and Vil Apc Huwe1hom mice. Deletion of Huwe1 led to a significant reduction in survival of these animals (Vil Apc vs Vil Apc Huwe1het / Vil Apc Huwe1hom, Log Rank, p < 0.001, n ≥ 10).


( b ) Wholemount isolation of small intestines from Vil Apc Vil Apc Huwe1hom mice culled at clinical endpoint . Note huge numbers of tiny macroscopic adenomas visible in Vil Apc Huwe1hom intestine ( red arrows ) .

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(b) Wholemount isolation of small intestines from Vil Apc and Vil Apc Huwe1hom mice culled at clinical endpoint. Note the huge numbers of tiny macroscopic adenomas visible in the Vil Apc Huwe1hom intestine (red arrows).


( c ) H & amp ; amp ; E of intestinal cross sections from Vil Apc Vil Apc Huwe1hom mice demonstrating increased adenoma burden following Huwe1 deletion . Black arrows indicate adenomas . Scale bars = 200 µm .

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(c) H&amp;E staining of intestinal cross sections from Vil Apc and Vil Apc Huwe1hom mice demonstrating the increased adenoma burden following Huwe1 deletion. Black arrows indicate individual adenomas. Scale bars = 200 µm.


( d ) of total tumour numbers per gut in sacrificed Vil Apc , Vil Apc Huwe1het Vil Apc Huwe1hom mice . Deletion of Huwe1 led to a significant increase in number of tumours per gut ( Mann Whitney , n ≥ 5 ) .

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(d) Quantification of total tumour numbers per gut in sacrificed Vil Apc, Vil Apc Huwe1het and Vil Apc Huwe1hom mice. Deletion of Huwe1 led to a significant increase in the number of tumours per gut (Mann Whitney, n ≥ 5).


( a ) H & amp ; amp ; E of control Huwe1 deleted intestinal epithelium . Shortened villi in Huwe1 deficient tissue are indicated . Scale bars = 100 µm . ( b ) BrdU of control Huwe1 deleted intestinal epithelium . Scale bars = 100 µm . ( c ) PAS identifying Goblet . No gross changes were observed . Scale bars = 100 µm . ( d ) Lysozyme ( Paneth marker ) of control Huwe1 deleted small intestine . Note occurrence of lysozyme positive away from crypt base ( black arrows ) . Scale bars = 100 µm .

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(a) H&amp;E staining of control and Huwe1 deleted intestinal epithelium. Shortened villi in Huwe1 deficient tissue are indicated. Scale bars = 100 µm. (b) BrdU IHC of control and Huwe1 deleted intestinal epithelium. Scale bars = 100 µm. (c) PAS staining identifying Goblet cells. No gross changes were observed. Scale bars = 100 µm. (d) Lysozyme staining (Paneth cell marker) of control and Huwe1 deleted small intestine. Note the occurrence of lysozyme positive cells away from the crypt base (black arrows). Scale bars = 100 µm.


( e ) Dual Periodic acid - Schiff / Alcian blue to identify Paneth secretory vesicles ( blue / pink - marked with black arrows ) goblet ( blue / purple - marked with red arrows ) . Note Paneth secretory vesicles are restricted to crypt base in both control Huwe1 deficient small intestines ( inset - black arrows ) . Scale bars = 100µm .

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(e) Dual Periodic acid-Schiff / Alcian blue staining to identify Paneth cell secretory vesicles (light blue / pink - marked with black arrows) and goblet cells (dark blue / purple - marked with red arrows). Note Paneth cell secretory vesicles are restricted to crypt base in both control and Huwe1 deficient small intestines (inset - black arrows). Scale bars = 100µm.


( f ) MMP7 of control Huwe1 deleted small intestine . Note MMP7 is restricted to crypt base in Huwe1 deficient intestines . Scale bars = 100µm .

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(f) MMP7 staining of control and Huwe1 deleted small intestine. Note MMP7 staining is restricted to crypt base in Huwe1 deficient intestines. Scale bars = 100µm.


( a ) HUWE1 MYC Western blot of epithelial extractions from control Huwe1 deficient intestines . of MYC are significantly increased in normal intestines lacking HUWE1 ( Mann Whitney , p = 0 . 04 , n = 3 ) .

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(a) HUWE1 and MYC Western blot analysis of epithelial cell extractions from control and Huwe1 deficient intestines. Levels of MYC protein are significantly increased in normal intestines lacking HUWE1 (Mann Whitney, p = 0.04, n = 3).


( b ) QRT - PCR of Myc expression in control Huwe1 deficient intestines .

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(b) QRT-PCR analysis of Myc expression in control and Huwe1 deficient intestines.


( c ) MYC in tumours from Vil Apc Vil Apc Huwe1hom mice . Scale bars = 50 µm .

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(c) MYC IHC in tumours from Vil Apc and Vil Apc Huwe1hom mice. Scale bars = 50 µm.


( d ) MYC Western blot in extracts from Vil Apc Vil Apc Huwe1hom tumours . of MYC are significantly increased in tumours lacking HUWE1 ( Mann Whitney , p = 0 . 04 , n = 3 ) .

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(d) MYC Western blot in protein extracts from Vil Apc and Vil Apc Huwe1hom tumours. Levels of MYC protein are significantly increased in tumours lacking HUWE1 (Mann Whitney, p = 0.04, n = 3).


( e ) QRT - PCR of Myc expression in tumours from Vil Apc Vil Apc Huwe1hom mice .

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(e) QRT-PCR analysis of Myc expression in tumours from Vil Apc and Vil Apc Huwe1hom mice.


( a ) Kaplan - Meier survival plot of cohorts of induced Vil Apc Pten , Vil Apc Pten Huwe1 , Vil Apc Pten Myc Vil Apc Pten Huwe1 Myc mice . Heterozygous deletion of Myc led to a specific significant increase in survival of Huwe1 deleted ( Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc , Log Rank , p < 0 . 001 , n ≥ 16 ) .

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(a) Kaplan-Meier survival plot of cohorts of induced Vil Apc Pten, Vil Apc Pten Huwe1, Vil Apc Pten Myc and Vil Apc Pten Huwe1 Myc mice. Heterozygous deletion of Myc led to a specific and significant increase in survival of Huwe1 deleted animals (Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc, Log Rank, p < 0.001, n ≥ 16).


( b ) of total tumour numbers per gut in sacrificed Vil Apc Pten , Vil Apc Pten Huwe1 , Vil Apc Pten Myc Vil Apc Pten Huwe1 Myc mice . Heterozygous deletion of Myc did not reduce number of tumours in Huwe1 deleted mice ( Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc . Mann Whitney , n ≥ 10 ) .

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(b) Quantification of total tumour numbers per gut in sacrificed Vil Apc Pten, Vil Apc Pten Huwe1, Vil Apc Pten Myc and Vil Apc Pten Huwe1 Myc mice. Heterozygous deletion of Myc did not reduce the number of tumours in Huwe1 deleted mice (Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc. Mann Whitney, n ≥ 10).


( c ) Kaplan - Meier survival plot of cohorts of induced AhCre - ERT Apc Pten AhCre - ERT Apc Pten MycT58A mice . Overexpression of proteolytically stabilised MYC led to a significant reduction in survival ( Log Rank , p < 0 . 001 , n = 14 vs 21 ) .

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(c) Kaplan-Meier survival plot of cohorts of induced AhCre-ERT Apc Pten and AhCre-ERT Apc Pten MycT58A mice. Overexpression of proteolytically stabilised MYC led to a significant reduction in survival (Log Rank, p < 0.001, n = 14 vs 21).


( d ) of total tumour numbers per gut in sacrificed Apc Pten Apc Pten MycT58A mice . Overexpression of proteolytically stabilised MYC did not increase number of tumours in Apc Pten mice ( Mann Whitney , n = 9 vs 19 ) .

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(d) Quantification of total tumour numbers per gut in sacrificed Apc Pten and Apc Pten MycT58A mice. Overexpression of proteolytically stabilised MYC did not increase the number of tumours in Apc Pten mice (Mann Whitney, n = 9 vs 19).


( a ) H2AX Western blot in extracts from control Huwe1 deficient intestinal epithelial . Band relative to βactin displayed under each lane ( Mann - Whitney , p = 0 . 04 , n = 3 vs 3 ) .

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(a) H2AX Western blot in protein extracts from control and Huwe1 deficient intestinal epithelial cells. Band intensity relative to βactin displayed under each lane (Mann-Whitney, p = 0.04, n = 3 vs 3).


( b ) γ - H2AX showing increased positivity in Huwe1 deleted intestinal . Scale bars = 50 µm .

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(b) γ-H2AX IHC showing increased positivity in Huwe1 deleted intestinal cells. Scale bars = 50 µm.


( c ) γ - H2AX Western blot in extracts from control Huwe1 deficient intestinal epithelial ( Mann - Whitney , p = 0 . 04 , n = 3 vs 3 ) .

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(c) γ-H2AX Western blot in protein extracts from control and Huwe1 deficient intestinal epithelial cells (Mann-Whitney, p = 0.04, n = 3 vs 3).


( d ) γ - H2AX of Vil Apc Vil Apc Huwe1hom tumours , note increased positivity in Huwe1 deficient tumours . Scale bars = 50 µm .

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(d) γ-H2AX IHC of Vil Apc and Vil Apc Huwe1hom tumours, note increased positivity in Huwe1 deficient tumours. Scale bars = 50 µm.


( a ) Caspase 3 of tumours from Lgr5 Apc Lgr5 Apc Huwe1 mice either untreated or 6h post treatment with 7 . 5mg / kg cisplatin . Arrows identify caspase 3 positive . Scale bars = 50 µm . ( b ) of cisplatin treatment showing a significant increase in apoptosis in Huwe1 deficient tumour ( Mann - Whitney , n = 3 vs 5 ) .

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(a) Caspase 3 IHC of tumours from Lgr5 Apc and Lgr5 Apc Huwe1 mice either untreated or 6h post treatment with 7.5mg/kg cisplatin. Arrows identify caspase 3 positive cells. Scale bars = 50 µm. (b) Quantification of cisplatin treatment showing a significant increase in apoptosis in Huwe1 deficient tumour cells (Mann-Whitney, n = 3 vs 5).


( c ) Comparison of somatic mutation rate ( mutations / Mb ) between human tumours carrying HUWE1 mutations or not . Note significant increase in mutational burden in HUWE1 mutated tumours ( Mann - Whitney , p = 0 . 0002 , n ≥ 15 ) .

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(c) Comparison of somatic mutation rate (mutations / Mb) between human tumours carrying HUWE1 mutations or not. Note the significant increase in mutational burden in HUWE1 mutated tumours (Mann-Whitney, p = 0.0002, n ≥ 15).


( d ) Comparison of somatic mutation rate ( mutations / Mb ) between human tumours carrying HUWE1 mutations or not grouped according to MLH1 status . Note that increased mutation rate is found primarily in tumours where MLH1 is not silenced indicating increased mutation rate ( HUWE1 mutation itself ) is not due to silencing of MLH1 ( Mann - Whitney , p = 0 . 0007 , n ≥ 11 ) .

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(d) Comparison of somatic mutation rate (mutations / Mb) between human tumours carrying HUWE1 mutations or not grouped according to MLH1 status. Note that the increased mutation rate is found primarily in tumours where MLH1 is not silenced indicating increased mutation rate (and HUWE1 mutation itself) is not due to silencing of MLH1 (Mann-Whitney, p = 0.0007, n ≥ 11).


( e ) Survival of colorectal cancer treated with adjuvant chemotherapy divided by HUWE1 expression . Note lowest HUWE1 expressing quartile of respond significantly better to chemotherapy . ( f ) Survival of colorectal cancer not treated with adjuvant chemotherapy divided by HUWE1 expression . Note lowest HUWE1 expressing quartile of do not survive significantly longer than those expressing higher of HUWE1 if not treated with chemotherapy .

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(e) Survival analysis of colorectal cancer patients treated with adjuvant chemotherapy divided by HUWE1 expression levels. Note the lowest HUWE1 expressing quartile of patients respond significantly better to chemotherapy. (f) Survival analysis of colorectal cancer patients not treated with adjuvant chemotherapy divided by HUWE1 expression levels. Note the lowest HUWE1 expressing quartile of patients do not survive significantly longer than those expressing higher levels of HUWE1 if not treated with chemotherapy.


( b ) Kaplan - Meier survival plot of cohorts of induced Lgr5 Apc Lgr5 Apc Huwe1 mice . Deletion of Huwe1 led to a significant reduction in survival of these ( Log Rank , p < 0 . 001 , n ≥ 15 ) .

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(b) Kaplan-Meier survival plot of cohorts of induced Lgr5 Apc and Lgr5 Apc Huwe1 mice. Deletion of Huwe1 led to a significant reduction in survival of these animals (Log Rank, p < 0.001, n ≥ 15).


( c ) Lysozyme demonstrating increased lysozyme positive numbers in tumours from Lgr5 Apc Huwe1 mice . Scale bars = 200 µm ( low magnification ) , 100 µm ( high magnification inset ) . ( d ) OLFM4 demonstrating expanded population in Lgr5 Apc Huwe1 tumours . Scale bars = 200 µm ( low magnification ) , 100 µm ( high magnification inset ) .

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(c) Lysozyme IHC demonstrating increased lysozyme positive cell numbers in tumours from Lgr5 Apc Huwe1 mice. Scale bars = 200 µm (low magnification), 100 µm (high magnification inset). (d) OLFM4 IHC demonstrating expanded stem cell population in Lgr5 Apc Huwe1 tumours. Scale bars = 200 µm (low magnification), 100 µm (high magnification inset).


( a ) MCL1 Western blot in extracts from Vil Apc Vil Apc Huwe1hom tumours . of MCL1 are significantly increased in tumours lacking HUWE1 ( Mann Whitney , p = 0 . 04 , n = 3 ) .

(a) MCL1 Western blot in protein extracts from Vil Apc and Vil Apc Huwe1hom tumours. Levels of MCL1 protein are significantly increased in tumours lacking HUWE1 (Mann Whitney, p = 0.04, n = 3).


( b ) Kaplan - Meier survival plot of cohorts of induced Lgr5 Apc Huwe1 Lgr5 Apc Huwe1 Mcl1 mice . Deletion of one copy of Mcl1 led to a significant increase in survival of these ( Log Rank , p = 0 . 0052 , n = 9 vs 10 ) .

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(b) Kaplan-Meier survival plot of cohorts of induced Lgr5 Apc Huwe1 and Lgr5 Apc Huwe1 Mcl1 mice. Deletion of one copy of Mcl1 led to a significant increase in survival of these animals (Log Rank, p = 0.0052, n = 9 vs 10).


( c ) of adenoma / indolent lesions observed in Lgr5 Apc Huwe1 Lgr5 Apc Huwe1 Mcl1het mice . Note decreased ration of adenoma / indolent lesions observed upon Mcl1 deletion ( Mann - Whitney , p = 0 . 0281 , n = 8 v 8 ) .

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(c) Ratio of adenoma / indolent lesions observed in Lgr5 Apc Huwe1 and Lgr5 Apc Huwe1 Mcl1het mice. Note the decreased ration of adenoma / indolent lesions observed upon Mcl1 deletion (Mann-Whitney, p=0.0281, n = 8 v 8).


A ) GST pulldown used to analyse TBC1D14 interactors . Recombinant GST or GST - TBC1D14 was incubated with or without HEK 293A lysate . Bound were eluted from beads using Laemmli sample buffer subjected to SDS - PAGE , with visible bands being excised from gel analysed by mass spectrometry .

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A) GST pulldown used to analyse TBC1D14 interactors. Recombinant GST or GST-TBC1D14 was incubated with or without HEK 293A lysate. Bound proteins were eluted from the beads using Laemmli sample buffer and subjected to SDS-PAGE, with visible bands being excised from the gel and analysed by mass spectrometry.


B ) Lysates of HEK293A expressing either GFP or GFP - TBC1D14 were subjected to immunoprecipitation ( IP ) with GFP - Trap , then immunoblotted for GFP , TRAPPC12 TRAPPC4 .

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B) Lysates of HEK293A cells expressing either GFP or GFP-TBC1D14 were subjected to immunoprecipitation (IP) with GFP-Trap, and then immunoblotted for GFP, TRAPPC12 and TRAPPC4.


C ) Lysates of expressing GFP or GFP - TRAPPC3 were subjected to IP with GFP - Trap , then immunoblotted for TRAPPC4 , GFP TBC1D14 .

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C) Lysates of cells expressing GFP or GFP-TRAPPC3 were subjected to IP with GFP-Trap, and then immunoblotted for TRAPPC4, GFP and TBC1D14.


D ) expressing GFP - TBC1D14 , generating a tubulated endosomal compartment , were fed Alexa647 labelled Transferrin ( , blue in merge ) for 15 minutes in full medium , fixed , stained for endogenous TRAPPC4 ( red ) analysed by confocal microscopy . arrowheads indicate transfected , yellow arrowheads depict regions of colocalisation in inset . Scale bars = 10 μm .

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D) Cells expressing GFP-TBC1D14, generating a tubulated endosomal compartment, were fed Alexa647 labelled Transferrin (white, blue in merge) for 15 minutes in full medium, fixed, stained for endogenous TRAPPC4 (red) and analysed by confocal microscopy. White arrowheads indicate transfected cells, yellow arrowheads depict regions of colocalisation in inset. Scale bars = 10 μm.


E ) treated as in D were stained for endogenous TRAPPC12 ( red ) analysed by confocal microscopy . Scale bars = 10

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E) Cells treated as in D were stained for endogenous TRAPPC12 (red) and analysed by confocal microscopy. Scale bars = 10 μm


F ) in complete medium were fixed , stained for endogenous TBC1D14 ( green ) , RAB1B ( red ) TRAPPC4 ( , blue in merge ) analysed by confocal microscopy . Inset shows localisation in juxtanuclear . Yellow arrowheads show regions of colocalisation Scale bars = 10 μm . Data shown are representative from 3 independent experiments .

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F) Cells in complete medium were fixed, stained for endogenous TBC1D14 (green), RAB1B (red) and TRAPPC4 (white, blue in merge) and analysed by confocal microscopy. Inset shows protein localisation in juxtanuclear area. Yellow arrowheads show regions of colocalisation Scale bars = 10 μm. Data shown are representative from 3 independent experiments.


A ) HEK293A transfected with GFP ( upper panels ) or GFP - TBC1D14 ( lower panels ) ( green ) were labelled with anti - RAB11A ( red ) anti - RAB1B ( , blue in merge ) antibodies . Arrows depict transfected . Inset : GFP - TBC1D14 transfected showing triple co - localisation between GFP - TBC1D14 , RAB11A RAB1B ( arrows ) double co - localisation between GFP - TBC1D14 RAB11A ( yellow arrows ) .

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A) HEK293A cells transfected with GFP (upper panels) or GFP-TBC1D14 (lower panels) (green) were labelled with anti-RAB11A (red) and anti-RAB1B (white, blue in merge) antibodies. Arrows depict transfected cells. Inset: GFP-TBC1D14 transfected cell showing triple co-localisation between GFP-TBC1D14, RAB11A and RAB1B (white arrows) and double co-localisation between GFP-TBC1D14 and RAB11A (yellow arrows).


B ) Co - localisation of GFP - TBC1D14 , TRAPPC4 ( red ) RAB11A ( , blue in merge ) . arrowheads indicate regions of triple co - localisation .

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B) Co-localisation of GFP-TBC1D14, TRAPPC4 (red) and RAB11A (white, blue in merge). White arrowheads indicate regions of triple co-localisation.


C ) Co - localisation of GFP - TBC1D14 , TRAPPC4 ( red ) RAB1B ( , blue in merge ) . arrowheads indicate regions of triple co - localisation , yellow arrowheads regions of double co - localisation between GFP - TBC1D14 TRAPPC4 . scale bars = 20 μm .

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C) Co-localisation of GFP-TBC1D14, TRAPPC4 (red) and RAB1B (white, blue in merge). White arrowheads indicate regions of triple co-localisation, yellow arrowheads regions of double co-localisation between GFP-TBC1D14 and TRAPPC4. All scale bars = 20 μm.


B ) HEK 293A lysates ( from approximately 8 × 106 per construct ) expressing indicated GFP fusions were subjected to IP immunoblotting for GFP , TRAPPC12 TRAPPC4 . Data are representative of 3 independent experiments .

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B) HEK 293A cell lysates (from approximately 8 × 106 cells per construct) expressing the indicated GFP fusions were subjected to IP and immunoblotting for GFP, TRAPPC12 and TRAPPC4. Data are representative of 3 independent experiments.


A ) expressing indicated GFP - fusions ( green ) were loaded with Alexa647 - Transferrin ( red ; arrowheads indicate transfected ) for 15 minutes , fixed of transferrin positive recycling endosomes analysed by confocal microscopy . Scale bars = 10 μm . Arrows in inset indicate juxtanuclear

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A) Cells expressing the indicated GFP-fusions (green) were loaded with Alexa647-Transferrin (red; arrowheads indicate transfected cells) for 15 minutes, fixed and the morphology of the transferrin positive recycling endosomes analysed by confocal microscopy. Scale bars = 10 μm. Arrows in inset indicate the juxtanuclear ERC


B ) transfected as in ( A ) were loaded with Alexa - 647 transferrin for 15 minutes transferrin chased out for indicated time periods . At end of timecourse were fixed transferrin content of GFP positive analysed by flow cytometry . Results are expressed as a of Tfn at t = 0 are mean of 3 independent experiments , ± s . e . m .

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B) Cells transfected as in (A) were loaded with Alexa-647 transferrin for 15 minutes and the fluorescent transferrin chased out for the indicated time periods. At the end of the timecourse the cells were fixed and the transferrin content of GFP positive cells analysed by flow cytometry. Results are expressed as a percentage of Tfn fluorescence at t=0 and are the mean of 3 independent experiments, ± s.e.m.


C ) transfected as in ( A ) were stained for cis - Golgi marker GM130 . Arrowheads depict transfected . Scale bars = 10 μm . Bar chart is of fragmented Golgi ( > 100 per condition from three independent experiments , ± s . e . m . * * p < 0 . 01 , one way ANOVA with Sidak ' s post - ) .

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C) Cells transfected as in (A) were stained for the cis-Golgi marker GM130. Arrowheads depict transfected cells. Scale bars = 10 μm. Bar chart is quantification of fragmented Golgi complex (> 100 cells per condition from three independent experiments, ± s.e.m. ** p < 0.01, one way ANOVA with Sidak's post-test).


D ) HeLa C1 transfected with CFP or CFP - TBR were treated with D / D solubiliser ( Clontech ) or D / D solubiliser plus 2 . 5 μg / ml Brefeldin A ( BFA ) for indicated time periods , trypsinised , fixed their GFP content analysed by flow cytometry . Results are expressed as a of GFP at t = 0 , mean of 3 independent experiments ± s . e . m .

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D) HeLa C1 cells transfected with CFP or CFP-TBR were treated with D/D solubiliser (Clontech) or D/D solubiliser plus 2.5 μg/ml Brefeldin A (BFA) for the indicated time periods, trypsinised, fixed and their GFP content analysed by flow cytometry. Results are expressed as a percentage of GFP levels at t=0, and the mean of 3 independent experiments ± s.e.m.


A ) HEK293A expressing GFP , GFP - TBC1D14 224 - 669 or GFP - TBR were treated in duplicate with EBSS , EBSS plus 100 nM BafA1 or not for two hours , lysed subjected to immunoblotting for LC3B , tubulin GFP . amount of LC3B / tubulin for each condition from three independent experiments is shown on bar graph , ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , one way ANOVA with Sidak ' s multiple comparison .

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A) HEK293A cells expressing GFP, GFP-TBC1D14 224-669 or GFP-TBR were treated in duplicate with EBSS, EBSS plus 100 nM BafA1 or not for two hours, lysed and subjected to immunoblotting for LC3B, tubulin and GFP. The amount of LC3B/tubulin for each condition from three independent experiments is shown on the bar graph, ±s.e.m. * p<0.05, ** p<0.01, one way ANOVA with Sidak's multiple comparison test.


B ) HEK293A GFP - LC3B ( 2GL9 ) were transfected with mCherry or mCherry - TBR , treated with EBSS for two hours , fixed analysed by confocal microscopy .

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B) HEK293A GFP-LC3B (2GL9) cells were transfected with mCherry or mCherry-TBR, treated with EBSS for two hours, fixed and analysed by confocal microscopy.


C ) HEK293A were treated as in ( B ) , fixed , stained for WIPI2 analysed by confocal microscopy .

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C) HEK293A cells were treated as in (B), fixed, stained for WIPI2 and analysed by confocal microscopy.


D ) HEK293A stably expressing GFP - DFCP1 were treated as in ( B ) , fixed analysed by confocal microscopy . Scale bars = 20 μm . For of ( B ) , ( C ) ( D ) , 10 fields of view containing transfected were imaged for each of three experiments , number of GFP - LC3B , WIPI2 or GFP - DFCP1 per enumerated using Imaris software ( Bitplane ) .

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D) HEK293A cells stably expressing GFP-DFCP1 were treated as in (B), fixed and analysed by confocal microscopy. Scale bars = 20 μm. For quantification of (B), (C) and (D), 10 fields of view containing transfected cells were imaged for each of three experiments, and the number of GFP-LC3B, WIPI2 or GFP-DFCP1 puncta per cell enumerated using Imaris software (Bitplane).


B ) Samples prepared according to ( A ) ( 8 × 107 HEK293A per transfection ) were lysed in TNTE , split equally denatured with 1 % SDS ( D ) or not ( ND ) subjected to 1 hour streptavidin resin pulldown at room . bound were eluted in 2x Laemmli sample buffer plus 3 mM Biotin immunoblotted for indicated .

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B) Samples prepared according to (A) (8 × 107 HEK293A cells per transfection) were lysed in TNTE, split equally and denatured with 1% SDS (D) or not (ND) and subjected to 1 hour streptavidin resin pulldown at room temperature. The bound proteins were eluted in 2x Laemmli sample buffer plus 3 mM Biotin and immunoblotted for the indicated proteins.


C ) siRNA transfected control ( RISC Free ) or TRAPPC8 knockdown ( TRAPPC8 ) ( 1x10cm plate per condition ) were lysed in TNTE used in pulldown assays with immobilised recombinant GST or GST - TBR . Bound were subjected to immunoblot using TRAPPC8 , TRAPPC4 tubulin antibodies . Ponceau was used to verify loading of GST fusion ( GST - TBR indicated with arrowhead ) . bar chart expresses relative amount of TRAPPC4 isolated from siTRAPPC8 compared to siRISC Free control from three independent experiments , error bars are ± s . e . m . * * p < 0 . 01 , unpaired T -

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C) siRNA transfected control cells (RISC Free) or TRAPPC8 knockdown (TRAPPC8) (1x10cm plate per condition) were lysed in TNTE and used in pulldown assays with immobilised recombinant GST or GST-TBR. Bound proteins were subjected to immunoblot analysis using TRAPPC8, TRAPPC4 and tubulin antibodies. Ponceau staining was used to verify loading of the GST fusion proteins (GST-TBR indicated with arrowhead). The bar chart expresses the relative amount of TRAPPC4 isolated from siTRAPPC8 compared to siRISC Free control cells from three independent experiments, error bars are ± s.e.m. ** p<0.01, unpaired T-test


D ) HEK293T stably depleted of TRAPPC8 ( shC8 ) or not ( shCtrl ) were transfected with constructs encoding GFP or GFP - TRAPPC3 subjected to GFP Trap IP . Precipitated were immunoblotted for GFP , TRAPPC4 TRAPPC8 . Bar chart : amount of TRAPPC4 isolated was normalised to amount of GFP - TRAPPC3 precipitated from three independent experiments plotted ± s . e . m . , ns = non - significant , unpaired T - .

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D) HEK293T cells stably depleted of TRAPPC8 (shC8) or not (shCtrl) were transfected with constructs encoding GFP or GFP-TRAPPC3 and subjected to GFP Trap IP. Precipitated proteins were immunoblotted for GFP, TRAPPC4 and TRAPPC8. Bar chart: the amount of TRAPPC4 isolated was normalised to the amount of GFP-TRAPPC3 precipitated from three independent experiments and plotted ± s.e.m., ns = non-significant, unpaired T-test.


A ) HEK293A treated with transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 RAB11A B ( knockdown confirmed by immunoblot ) were fed with Alexa - 647 Transferrin for 15 minutes in full medium , transferrin chased out for indicated time periods . At end of time course , were trypsinised , fixed Alexa - 647 analysed by flow cytometry . Results are plotted as of transferrin at t = 0 for RISC free control , expressed as mean of three independent experiments ± s . e . m .

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A) HEK293A cells treated with transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 and RAB11A and B (knockdown confirmed by immunoblot analysis) were fed with Alexa-647 Transferrin for 15 minutes in full medium, and the fluorescent transferrin chased out for the indicated time periods. At the end of the time course, cells were trypsinised, fixed and the Alexa-647 fluorescence analysed by flow cytometry. Results are plotted as percentage of transferrin fluorescence at t=0 for the RISC free control cells, expressed as the mean of three independent experiments ± s.e.m.


B ) transiently transfected with RISC Free control siRNA , or siRNA duplexes directed against TRAPPC8 or RAB1A B were stained for cis - Golgi markers GM130 or RAB1B analysed by confocal microscopy . Scale bars = 20 μm in main panels , 10 μm in inset panels . Bar graph : > 100 per siRNA were scored for fragmented or normal juxtanuclear Golgi stacks from three independent experiments . Error bars ± s . e . m . , * p < 0 . 05 , * * p < 0 . 01 , one way ANOVA with Sidak ' s multiple comparison .

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B) Cells transiently transfected with RISC Free control siRNA, or siRNA duplexes directed against TRAPPC8 or RAB1A and B were stained for the cis-Golgi markers GM130 or RAB1B and analysed by confocal microscopy. Scale bars = 20 μm in main panels, 10 μm in inset panels. Bar graph: > 100 cells per siRNA were scored for fragmented or normal juxtanuclear Golgi stacks from three independent experiments. Error bars ± s.e.m., * p<0.05, ** p<0.01, one way ANOVA with Sidak's multiple comparison test.


C ) HeLa C1 transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 or RAB1A B ( knockdown confirmed by immunoblot ) , were treated with D / D solubiliser for indicated time periods , trypsinised , fixed their GFP content analysed by flow cytometry . graph shows GFP as a of t = 0 , ± s . e . m . from three independent experiments .

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C) HeLa C1 cells transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 or RAB1A and B (knockdown confirmed by immunoblot analysis), were treated with D/D solubiliser for the indicated time periods, trypsinised, fixed and their GFP content analysed by flow cytometry. The line graph shows the GFP fluorescence as a percentage of t=0, ± s.e.m. from three independent experiments.


A ) ATG9 positive membranes were immunoisolated from approximately 2 × 107 fed ( F ) or starved ( S ) HEK293A using a monoclonal hamster anti - ATG9 ( ATG9 ) or control hamster IgM immunoblotted for ATG9 using a rabbit polyclonal antibody , TRAPPC8 , TRAPPC4 actin .

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A) ATG9 positive membranes were immunoisolated from approximately 2 × 107 fed (F) or starved (S) HEK293A cells using a monoclonal hamster anti-ATG9 (ATG9) or control hamster IgM and immunoblotted for ATG9 using a rabbit polyclonal antibody, TRAPPC8, TRAPPC4 and actin.


B ) HEK293A transfected with GFP or GFP - TBR ( green ) were stained for ATG9 using hamster anti - ATG9 ( red ) analysed by confocal microscopy . Scale bar = 20

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B) HEK293A cells transfected with GFP or GFP-TBR (green) were stained for ATG9 using hamster anti-ATG9 (red) and analysed by confocal microscopy. Scale bar = 20 μm


C ) HEK293A transiently transfected with RISC Free siRNA or siRNA against TRAPPC8 were stained for RAB1B ( green ) ATG9 using hamster anti - ATG9 ( red ) imaged using confocal microscopy .

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C) HEK293A cells transiently transfected with RISC Free siRNA or siRNA against TRAPPC8 were stained for RAB1B (green) and ATG9 using hamster anti-ATG9 (red) and imaged using confocal microscopy.


D ) Stills at t = 30 minutes from time lapse imaging of 9B9 ( stably expressing GFP - LC3 mRFP - ATG9 ) expressing either CFP or CFP - TBR ( blue ) . Scale bars = 20 μm . Inset panels show mRFP - ATG9 GFP - LC3 contact events at indicated time points . Bar chart shows number of times a GFP - LC3 spot was within 1 μm of an mRFP - ATG9 spot ( a contact event ) , expressed as total mRFP - ATG9 contacts per GFP - LC3 spot , n = 18 per condition pooled from 4 independent experiments .

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D) Stills at t = 30 minutes from time lapse imaging of 9B9 cells (stably expressing GFP-LC3 and mRFP-ATG9) expressing either CFP or CFP-TBR (blue). Scale bars = 20 μm. Inset panels show mRFP-ATG9 and GFP-LC3 contact events at the indicated time points. Bar chart shows number of times a GFP-LC3 spot was within 1 μm of an mRFP-ATG9 spot (a contact event), expressed as total mRFP-ATG9 contacts per GFP-LC3 spot, n = 18 cells per condition pooled from 4 independent experiments.


A ) depleted of ULK1 ( siULK1 ) or not ( RISC Free ) were transfected with GFP or GFP - TBR ( green ) , starved ( EBSS ) or not ( - EBSS ) stained for ATG9 using hamster anti - ATG9 ( red ) . Arrows indicate transfected . Western blot indicates ULK1 knockdown . Scale bars = 20 μm .

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A) Cells depleted of ULK1 (siULK1) or not (RISC Free) were transfected with GFP or GFP-TBR (green), starved (EBSS) or not (-EBSS) and stained for ATG9 using hamster anti-ATG9 (red). Arrows indicate transfected cells. Western blot indicates ULK1 knockdown. Scale bars = 20 μm.


B ) depleted of ULK1 ( siULK1 ) or not ( RISC Free ) were transfected with myc - RAB1B or myc - RAB1B ( S22N ) ( green ) , starved ( EBSS ) or not ( - EBSS ) stained for ATG9 using hamster anti - ATG9 ( red ) Arrows indicate transfected . Western blot indicates ULK1 knockdown . Scale bars = 20 μm .

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B) Cells depleted of ULK1 (siULK1) or not (RISC Free) were transfected with myc-RAB1B or myc-RAB1B (S22N) (green), starved (EBSS) or not (-EBSS) and stained for ATG9 using hamster anti-ATG9 (red) Arrows indicate transfected cells. Western blot indicates ULK1 knockdown. Scale bars = 20 μm.


( B ) ParB clusters on F plasmid in vivo . Typical E . coli ( DLT3594 ) displays foci of ParBF - mVenus ( top ) expressed from endogenous genetic locus of F plasmid ( F1 - 10B - mVenus ) . nucleoid is labelled with Hu - mCherry ( central ) . overlay ( bottom ) combines two channels . Over 99 % of harbor ParBF foci . bars : 1

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(B) ParB clusters on F plasmid in vivo. Typical E. coli cells (DLT3594) displays foci of ParBF-mVenus protein (top) expressed from the endogenous genetic locus of the F plasmid (F1-10B-mVenus). The nucleoid is labelled with Hu-mCherry (central). The overlay (bottom) combines the two fluorescent channels. Over 99 % of cells harbor ParBF foci. White bars: 1 µm


( C ) ParBF binding outside parSF on F plasmid is compatible with a power - law decay . High resolution ChIP - seq performed on DLT3586 carrying F plasmid ( F1 - 10B ) . ParB density , normalized to 1 at first bp downstream last parSF binding repeat after background subtraction , is displayed over 14 - Kbp on right side of parSF . Monte Carlo simulations analytic formula are represented in red dotted black lines , respectively . MC simulations were performed with a Freely - Jointed Chain of linear L = 15 - Kbp a cluster radius σ = 75nm . two other parameters , Kuhn a = 10 - bp total number of on F plasmid Nt = 360 ( related to normalization constant of κ = 0 . 41 ) were fitted from ChIP - seq data ( see text Fig . EV2 ) . As a benchmark for simulations , analytics are obtained from Eq . ( 1 ) with same parameters . Inset ; ParBF binding profile ( black ) is represented as number of nucleotide reads over 80 - Kbp centered at parS . number of reads in Input sample ( grey ) is normalized to total number of reads in IP

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(C) ParBF binding outside parSF on the F plasmid is compatible with a power-law decay. High resolution ChIP-seq performed on DLT3586 carrying the F plasmid (F1-10B). The ParB density, normalized to 1 at the first bp downstream the last parSF binding repeat after background subtraction, is displayed over 14-Kbp on the right side of parSF. Monte Carlo simulations and analytic formula are represented in red and dotted black lines, respectively. MC simulations were performed with a Freely-Jointed Chain of linear length L=15-Kbp and a cluster radius σ=75nm. The two other parameters, the Kuhn length a=10-bp and the total number of proteins on the F plasmid Nt=360 (related to the normalization constant of the protein concentration κ=0.41) were fitted from the ChIP-seq data (see text and Fig. EV2). As a benchmark for simulations, the analytics are obtained from Eq.(1) with the same parameters. Inset; The ParBF binding profile (black line) is represented as the number of nucleotide reads over 80-Kbp centered at parS. The number of reads in the Input sample (grey line) is normalized to the total number of reads in the IP sample


Typical E . coli displays foc expressed from endogenous genetic locus of F plasmid ( F1 - 10B - mVenus ) . nucleoid is labelled with Hu - mCherry ( central ) . overlay ( bottom ) combines two channels parSF inserted at xylE locus on E . coli chromosome from

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

Typical E. coli cell displays foc expressed from the endogenous genetic locus of the F plasmid (F1-10B-mVenus). The nucleoid is labelled with Hu-mCherry (central). The overlay (bottom) combines the two fluorescent channels parSF inserted at the xylE locus on E. coli chromosome from DLT358


ParBF binding outside parSF on F plasmid is compatible with a power - law decay . High resolution ChIP - seq performed on DLT3586 carrying F plasmid ( F1 - 10B ) . ParB density , normalized to 1 at first bp downstream last parSF binding repeat after background subtraction , is displayed over 14 - Kbp on right side of parSF . Monte Carlo simulations analytic formula are represented in red dotted black lines , respectively . MC simulations were performed with a Freely - Jointed Chain of linear L = 15 - Kbp a cluster radius σ = 75nm . two other parameters , Kuhn a = 10 - bp total number of on F plasmid Nt = 360 ( related to normalization constant of κ = 0 . 41 ) were fitted from ChIP - seq dat parSF inserted at xylE locus on E . coli chromosome fro

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

ParBF binding outside parSF on the F plasmid is compatible with a power-law decay. High resolution ChIP-seq performed on DLT3586 carrying the F plasmid (F1-10B). The ParB density, normalized to 1 at the first bp downstream the last parSF binding repeat after background subtraction, is displayed over 14-Kbp on the right side of parSF. Monte Carlo simulations and analytic formula are represented in red and dotted black lines, respectively. MC simulations were performed with a Freely-Jointed Chain of linear length L=15-Kbp and a cluster radius σ=75nm. The two other parameters, the Kuhn length a=10-bp and the total number of proteins on the F plasmid Nt=360 (related to the normalization constant of the protein concentration κ=0.41) were fitted from the ChIP-seq dat parSF inserted at the xylE locus on E. coli chromosome fro DLT207


( A ) Normalized rescaled ParBF binding profiles at different ParBF / parSF . ChIP - seq density on right side of parSF inserted at xylE were measured in DLT2075 induced ( 16 , 28 ) or not ( 0 . 4 ) with IPTG ( 100 500 µM ) , or carrying HCN plasmids pZC302 ( 0 . 04 ) or pJYB57 ( 0 . 016 ) , normalized as in Fig . 1C , E with amplitudes of curves rescaled by indicated factors ( 1 . 2 , 10 or 50 ) to overlap with curves of highest amplitude . ParB / parS is calculated relative to one of F plasmid as determined from Western blot analyses ( Appendix Fig . S2B ) . Monte Carlo simulations analytical formula are plotted with same parameters as in Fig . 1E . Note ( i ) that dips at ~ 9 - kbp are not visible for low of available ParB since signal is close to basal level ( ii ) that ChIP - seq data at 100 µM IPTG induction ( 16 ) are same as in Fig . 1E . Inset ; Same as in main to display density without rescaling . ( B ) ParBF are dispersed in upon titration by HCN plasmids . ParBF - mVenus expressed from pJYB294 were imaged as in Fig . 1D in DLT3577 ( left ) DLT3576 ( right ) carrying pZC302 pJYB57 , respectively . number of extra parSF per , indicated on top of each raw , are estimated from copy number per of HCN plasmids carrying 10 specific binding sites . bars : 1 µm . ( C ) of ParBF clusters is independent of intracellular ParBF . We considered two possible evolutions of cluster upon variations of ParB amount in framework of " Nucleation & caging " with corresponding schematics drawn on right . For direct comparison with ( A ) , curves are displayed with a rescaling of amplitude corresponding to WT expression level . Top : constant ParB ; supposing that clusters are compact , cluster radius σ would depend on number m of ParB like $ \ \ sigma = m ^ { \ frac { 1 } { 3 } } $ . Predictions profiles , plotted at different of ParB / parS , vary within range of experimental tested . Bottom : constant cluster ; ParB concentrations vary but range of exploration remains same resulting in

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

(A) Normalized and rescaled ParBF binding profiles at different ParBF/parSF ratio. ChIP-seq density on the right side of parSF inserted at xylE were measured in DLT2075 induced (16, 28) or not (0.4) with IPTG (100 and 500 µM), or carrying HCN plasmids pZC302 (0.04) or pJYB57 (0.016), normalized as in Fig. 1C, E with the amplitudes of the curves rescaled by the indicated factors (1.2, 10 or 50) to overlap with the curves of highest amplitude. The ParB/parS ratio is calculated relative to the one of F plasmid as determined from Western blot analyses (Appendix Fig. S2B). Monte Carlo simulations and analytical formula are plotted with the same parameters as in Fig. 1E. Note (i) that the dips at ~9-kbp are not visible for the low levels of available ParB since the signal is close to the basal level and (ii) that the ChIP-seq data at 100 µM IPTG induction (16) are the same as in Fig. 1E. Inset; Same as in the main to display the density without rescaling. (B) ParBF are dispersed in the cell upon titration by HCN plasmids. ParBF-mVenus expressed from pJYB294 were imaged as in Fig. 1D in DLT3577 (left) and DLT3576 (right) carrying pZC302 and pJYB57, respectively. The number of extra parSF per cell, indicated on top of each raw, are estimated from the copy number per cell of HCN plasmids carrying 10 specific binding sites. White bars: 1 µm. (C) The size of ParBF clusters is independent of the intracellular ParBF concentration. We considered two possible evolutions of the cluster size upon variations of ParB amount in the framework of "Nucleation & caging" with corresponding schematics drawn on the right. For direct comparison with (A), all curves are displayed with a rescaling of the amplitude corresponding to the WT expression level. Top: constant ParB concentration; supposing that clusters are compact, the cluster radius σ would depend on the number m of ParB like$\ \sigma = m^{\frac{1}{3}}$. Predictions profiles, plotted at different ratio of ParB/parS, vary within the range of the experimental levels tested. Bottom: constant cluster size; ParB concentrations vary but the range of exploration remains the same resulting in overlappin


( A ) formation of secondary ParBF - complexes requires box II motif . EMSA were performed with a 144 - bp 32P - labelled fragments ( C144 ) carrying a single 16 - bp parS binding motif . Reaction mixtures containing 100 µg . ml - 1 sonicated salmon sperm were incubated in absence ( - ) or presence of increasing concentrations ( grey triangle ; 10 , 30 , 100 , 300 , 1000 nM ) of ParBF or ParBF - 3R * . Positions of free bound probes are indicated on left . B1 represents complexes involving specific interaction on 16 - bp binding site , while B " 2 B " 3 complexes represent secondary complexes involving parSF site with one or two additional nsDNA - binding interactions , respectively ( Sanchez et al ,

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

(A) The formation of secondary ParBF-DNA complexes requires the box II motif. EMSA were performed with a 144-bp 32P-labelled DNA fragments (C144) carrying a single 16-bp parS binding motif. Reaction mixtures containing 100 µg.ml-1 sonicated salmon sperm DNA were incubated in the absence (-) or the presence of increasing concentrations (grey triangle; 10, 30, 100, 300, and 1000 nM) of ParBF or ParBF-3R*. Positions of free and bound probes are indicated on the left. B1 represents complexes involving the specific interaction on the 16-bp binding site, while B"2 and B"3 complexes represent secondary complexes involving the parSF site with one or two additional nsDNA-binding interactions, respectively (Sanchez et al,


( B ) ParBF cluster formation requires box II motif . Epifluorescence microscopy of ParBF - 3R * - mVenus from DLT3566 is displayed as in Fig . 1D . bars : 1

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(B) ParBF cluster formation requires the box II motif. Epifluorescence microscopy of ParBF-3R*-mVenus from DLT3566 is displayed as in Fig. 1D. White bars: 1 µm


( C ) ParBF in vivo binding in vicinity of parSF sites requires box II motif . ChIP - seq was performed on DLT3726 carrying parSF in xylE chromosomal locus expressing ParBF - 3R * variant . ParBF - 3R * binding profile displayed number of nucleotide reads as a function of E . coli genomic coordinates . peak at parSF covered approximately 950 - bp , which corresponds to 402 - bp between 1st 10th specific binding sites ~ 280 - bp on each sides ( representing average of library ; see Appendix Fig . S3E ) . No ParBF - 3R * enrichment was found on parSF - flanking elsewhere on chromosome . Inset , zoom in on right side of parSF over 5 - Kbp with ParB density , normalized to 1 at first bp after last parS binding repeat , plotted as a function of from parSF . Note that a highly similar binding pattern is obtained with ParBF - 3R * - mVenus ( DLT3566 ; Appendix Fig . S3C )

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

(C) ParBF in vivo DNA binding in the vicinity of parSF sites requires the box II motif. ChIP-seq was performed on DLT3726 carrying parSF in the xylE chromosomal locus and expressing ParBF-3R* variant. ParBF-3R* DNA binding profile displayed the number of nucleotide reads as a function of the E. coli genomic coordinates. The peak at parSF covered approximately 950-bp, which corresponds to the 402-bp between the 1st and 10th specific binding sites and ~280-bp on each sides (representing the average size of the DNA library; see Appendix Fig. S3E). No ParBF-3R* enrichment was found on parSF-flanking DNA and elsewhere on the chromosome. Inset, zoom in on the right side of parSF over 5-Kbp with the ParB density, normalized to 1 at the first bp after the last parS binding repeat, plotted as a function of the distance from parSF. Note that a highly similar DNA binding pattern is obtained with ParBF -3R*-mVenus (strain DLT3566; Appendix Fig. S3C)


( A ) Schematic representation of genomic locus of chromosome 1 of V . cholerae with three parS sites , named parS1 - 3 . rRNA operon ( blue rectangle ) spans genomic coordinates 53823 to 59123 ( B ) ChIP - seq performed on N16961 is displayed as number of nucleotide reads in function of genomic coordinates . Correspondence to parS1 - 3 location represented in ( A ) is indicated by grey dotted lines . number of reads in Input sample ( grey ) is normalized to total number of reads in IP sample . ( C ) We modeled ChIP - Seq data as in Fig . 1C - E by means of MC simulations with a Freely jointed chain of N = 2000 monomers of a = 16 - bp . Data are normalized after background subtraction to read value at parS1 ( genomic coordinate 62438 ) . best fit was achieved with σ = 25nm an amplitude κ = 0 . 15 leading to Nt ~ 50 ParB on chromosome . In MC simulation , we accounted for finite width of distribution around parS sites by including average fragment of

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

(A) Schematic representation of the genomic locus of the chromosome 1 of V. cholerae with the three parS sites, named parS1-3. The rRNA operon (blue rectangle) spans the genomic coordinates 53823 to 59123 (B) ChIP-seq performed on strain N16961 is displayed as the number of nucleotide reads in function of the genomic coordinates. Correspondence to the parS1-3 location represented in (A) is indicated by grey dotted lines. The number of reads in the Input sample (grey line) is normalized to the total number of reads in the IP sample. (C) We modeled the ChIP-Seq data as in Fig.1C-E by means of MC simulations with a Freely jointed chain of size N=2000 monomers of size a=16-bp. Data are normalized after background subtraction to the read value at parS1 (genomic coordinate 62438). The best fit was achieved with σ=25nm and an amplitude κ=0.15 leading to Nt~50 ParB on the chromosome. In the MC simulation, we accounted for the finite width of the distribution around parS sites by including the average fragment size of the DNA librar


ChIP - sequencing assays were performed on DLT2075 ( xylE : : parSF ) expressing ParBF grown in exponential ( expo ) or stationary ( stat ) phases with addition of rifampicin when indicated ( + Rif ) . assays have been performed in duplicate for + Rif once for stationary phase experiments . ( A ) ParBF binding around parSF is independent of active transcription . color - coded ParBF profiles are represented over 50 - Kbp as relative ParB density normalized to 1 at first bp after last parSF binding site . Loci A , C , E F are defined in Appendix Fig . S1A . ( B ) dips peaks are highly similar in three indicated conditions . Same as in ( A ) with zoom in on right side of parSF up to 9 - Kpb normalization to 1 at genomic coordinate 230 . dotted corresponds to analytics description of " Nucleation caging " ( see details in Fig . 1C - E ) . ( C ) ParBF binding profile upstream of locus A . Same as in ( A ) with zoom in from - 6 . 5 to - 16 . 5 - Kbp by normalization to 1 at genomic coordinate - 6 . 5 - Kbp ( upstream of dip at locus A ) . ParBF binding profile remains compatible to a power - law , represented by analytics description ( dotted ) , upstream of locus A in stationary phase ( black ) in exponential phase ( blue ) . Also , dips peaks are highly similar in both conditions . These data are not in favor of " 1D - spreading " or " Spreading bridging " models that predicts a basal uniform distribution or a linear decrease after a barrier , respectively ( Broedersz et al , 2014 ) . ( D ) promoter region at locus A prevents ParBF binding . Chip - seq assays were performed in isogenic xylE : : parSF strains ( DLT2075 ; black curve ) in which locus A is replaced by a kanamycin gene ( DLT3651 ; red curve ) . in ∆ ( locus A ) genomic context has been performed once . relative ParB density as a function of from parSF

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

ChIP-sequencing assays were performed on DLT2075 (xylE::parSF) expressing ParBF grown in exponential (expo) or stationary (stat) phases with addition of rifampicin when indicated (+Rif). The assays have been performed in duplicate for the +Rif and once for the stationary phase experiments. (A) ParBF DNA binding around parSF is independent of active transcription. The color-coded ParBF profiles are represented over 50-Kbp as the relative ParB density normalized to 1 at the first bp after the last parSF binding site. Loci A, C, E and F are defined in Appendix Fig. S1A. (B) The dips and peaks are highly similar in the three indicated conditions. Same as in (A) with zoom in on the right side of parSF up to 9-Kpb and normalization to 1 at genomic coordinate 230. The dotted line corresponds to the analytics description of "Nucleation and caging" (see details in Fig. 1C-E). (C) ParBF binding profile upstream of the locus A. Same as in (A) with zoom in from -6.5 to -16.5-Kbp by normalization to 1 at genomic coordinate -6.5-Kbp (upstream of the dip at the locus A). The ParBF DNA binding profile remains compatible to a power-law, represented by the analytics description (dotted line), upstream of the locus A in stationary phase (black) and in exponential phase (blue). Also, the dips and peaks are highly similar in both conditions. These data are not in favor of the "1D-spreading" or the "Spreading and bridging" models that predicts a basal uniform distribution or a linear decrease after a barrier, respectively (Broedersz et al, 2014). (D) The promoter region at locus A prevents ParBF DNA binding. Chip-seq assays were performed in isogenic xylE::parSF strains (DLT2075; black curve) in which the locus A is replaced by a kanamycin gene (DLT3651; red curve). The assay in the ∆(locus A) genomic context has been performed once. The relative ParB density as a function of the distance from parSF is


ParBF exchange between foci was measured by FRAP an from two - foci of DLT1215 carrying pJYB234 A - Representative of a photobleached during a FRAP experiment . 488 nm was pulsed ( Bleach ) on one of two foci at ~ 2 . 4 sec ( black arrow ) . Red blue arrows correspond to bleached unbleached focus , respectively . Time is indicated in second ( upper right ) . outline is drawn in red . Scale bar : 1

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

ParBF exchange between foci was measured by FRAP an from two-foci cell of DLT1215 carrying pJYB234 A- Representative images of a photobleached cell during a FRAP experiment. The 488 nm laser was pulsed (Bleach) on one of the two foci at ~2.4 sec (black arrow). Red and blue arrows correspond to the bleached and unbleached focus, respectively. Time is indicated in second (upper right). The cell outline is drawn in red. Scale bar: 1 μm