( E ) Correlation statistics for localization of endogenous MITOL PMP70 in presence of NMS - 873 . Dots indicate Pearson correlation coefficient data points . In box - plots , center lines show medians , box limits indicate 25th 75th percentiles as determined by R software package , whiskers extend 1 . 5 times interquartile range from 25th 75th percentiles . Means number of samples are shown on box X - axis , respectively . Statistical significance was calculated using a one - Welch ' s t - .

O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Correlation statistics for the localization of endogenous MITOL and PMP70 in the presence of NMS-873. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by the R software package, and whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and the X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( F ) Endogenous MITOL is ubiquitylated following valinomycin treatment . After MITOL - 3Flag KI HCT116 stably expressing HA - Parkin were treated with valinomycin for 3 hours , collected lysates were immunoprecipitated with anti - Flag magnetic beads . immunoprecipitates were blotted using anti - Flag anti - ubiquitin antibodies . Red bars indicate ubiquitylation ; black arrowhead indicates MITOL - 3Flag .

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

(F) Endogenous MITOL is ubiquitylated following valinomycin treatment. After MITOL-3Flag KI HCT116 cells stably expressing HA-Parkin were treated with valinomycin for 3 hours, the collected cell lysates were immunoprecipitated with anti-Flag magnetic beads. The immunoprecipitates were blotted using anti-Flag and anti-ubiquitin antibodies. Red bars indicate ubiquitylation; the black arrowhead indicates MITOL-3Flag.


( A ) Distribution of exogenous 3Flag - MITOL in mitochondria ‐ rich or peroxisome - rich fraction following fractionation . HCT116 stably expressing HA - Parkin 3Flag - MITOL were subjected to fractionation detected using anti ‐ Tom20 , anti ‐ PMP70 , anti ‐ Flag antibodies . Tom20 PMP70 were used as mitochondrial peroxisomal markers , respectively . 3 , 000 g 100 , 000 g pellets represent mitochondria ‐ rich peroxisomes - rich fractions .

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(A) Distribution of exogenous 3Flag-MITOL in the mitochondria‐rich or peroxisome-rich fraction following cellular fractionation. HCT116 cells stably expressing HA-Parkin and 3Flag-MITOL were subjected to fractionation and detected using anti‐Tom20, anti‐PMP70, and anti‐Flag antibodies. Tom20 and PMP70 were used as mitochondrial and peroxisomal markers, respectively. The 3,000 g and 100,000 g pellets represent the mitochondria‐rich and peroxisomes-rich fractions.


( B ) recovery of 3Flag - MITOL between mitochondria - enriched ( 3 , 000 g pellet ) peroxisome - enriched fraction ( 100 , 000 g pellet ) following valinomycin treatment for indicated times . of peroxisome - localized 3Flag - MITOL to mitochondria - localized 3Flag - MITOL increased with valinomycin treatment for 3 hours . Graphic data represent results of two biological replicates . In scatter plot , dots indicate data points . Black dots indicate of 3Flag - MITOL collected in mitochondria - enriched fractions , red dots are of 3Flag - MITOL collected in peroxisome - enriched fractions . Mean values are also shown .

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(B) The recovery ratio of 3Flag-MITOL between the mitochondria-enriched (3,000 g pellet) and peroxisome-enriched fraction (100,000 g pellet) following valinomycin treatment for the indicated times. The ratio of peroxisome-localized 3Flag-MITOL to mitochondria-localized 3Flag-MITOL increased with valinomycin treatment for 3 hours. Graphic data represent results of two biological replicates. In scatter plot, dots indicate individual data points. Black dots indicate the ratio of 3Flag-MITOL collected in the mitochondria-enriched fractions, and red dots are the ratio of 3Flag-MITOL collected in the peroxisome-enriched fractions. Mean values are also shown.


Distribution of endogenous MITOL in mitochondria ‐ rich or peroxisome - rich fraction following fractionation . distribution of endogenous MITOL was examined using MITOL - 3Flag knock - in HCT116 . Treatment of with valinomycin for 3 hours reduced amount of endogenous MITOL in mitochondria - enriched fraction , but concomitantly increased endogenous MITOL in peroxisomes - enriched fraction .

O O O O O O O O O O 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

Distribution of endogenous MITOL in the mitochondria‐rich or peroxisome-rich fraction following cellular fractionation. The distribution of endogenous MITOL was examined using MITOL-3Flag knock-in HCT116 cells. Treatment of cells with valinomycin for 3 hours reduced the amount of endogenous MITOL in the mitochondria-enriched fraction, but concomitantly increased endogenous MITOL in the peroxisomes-enriched fraction.


Distribution of endogenous MITOL in mitochondria ‐ rich or peroxisome - rich fraction following fractionation . distribution of endogenous MITOL was examined using MITOL - 3Flag knock - in HCT116 . Graphic data represent results of two biological replicates . In scatter plot , dots indicate data points . Black dots indicate of MITOL - 3Flag collected in mitochondria - enriched fractions , red dots are of MITOL - 3Flag collected in peroxisome - enriched fractions . Mean values are 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Distribution of endogenous MITOL in the mitochondria‐rich or peroxisome-rich fraction following cellular fractionation. The distribution of endogenous MITOL was examined using MITOL-3Flag knock-in HCT116 cells. Graphic data represent results of two biological replicates. In scatter plot, dots indicate individual data points. Black dots indicate the ratio of MITOL-3Flag collected in mitochondria-enriched fractions, and red dots are the ratio of MITOL-3Flag collected in the peroxisome-enriched fractions. Mean values are also shown.


( A ) HeLa expressing HA - Parkin wild type 3Flag - MITOL or 3Flag - MITOL lacking C - terminal 8 ( ∆ C8 ) were treated with 15 µM CCCP for 3 hours , then subjected to immunocytochemistry with anti - Flag anti - PMP70 antibodies . Expanded peroxisomes were observed in MITOL ∆ C8 - expressing . Higher magnification of boxed regions are shown in small panel . Scale bars , 10 µm .

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(A) HeLa cells expressing HA-Parkin and wild type 3Flag-MITOL or 3Flag-MITOL lacking C-terminal 8 amino acids (∆C8) were treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-Flag and anti-PMP70 antibodies. Expanded peroxisomes were observed in MITOL∆C8-expressing cells. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.


( B ) 3Flag - MITOL ∆ C8 with E3 - inactive Cys65Ser / Cys68Ser ( CS ) H43W mutations were transfected into HeLa stably expressing HA - Parkin , treated with 15 µM CCCP , then subjected to immunocytochemistry with anti - Flag anti - PMP70 antibodies . After 3 hours of CCCP treatment , both CS H43W mutants localized on peroxisomes but expansion was not observed . Scale bars , 10 µm .

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(B) 3Flag-MITOL∆C8 with the E3-inactive Cys65Ser/Cys68Ser (CS) and H43W mutations were transfected into HeLa cells stably expressing HA-Parkin, treated with 15 µM CCCP, and then subjected to immunocytochemistry with anti-Flag and anti-PMP70 antibodies. After 3 hours of CCCP treatment, both the CS and H43W mutants localized on peroxisomes but expansion was not observed. Scale bars, 10 µm.


( C ) HeLa expressing HA - Parkin MITOL wild - type or ∆ C8 were treated with 15 µM CCCP for 3 hours , number of peroxisomes was counted as PMP70 - positive dots per 100 μm2 . In box - plots , dots indicate data points , center lines show medians , box limits indicate 25th 75th percentiles as determined by R software package , whiskers extend 1 . 5 times interquartile range from 25th 75th percentiles . Means number of samples are shown on box X - axis , respectively . Statistical significance was calculated using a one - Welch ' s t - . abundance of peroxisomes in expressing MITOL ∆ C8 was significantly decreased as compared with expressing wild - type MITOL .

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(C) HeLa cells expressing HA-Parkin and MITOL wild-type or ∆C8 were treated with 15 µM CCCP for 3 hours, and the number of peroxisomes was counted as PMP70-positive dots per 100 μm2. In the box-plots, dots indicate individual data points, the center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by the R software package, and whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and the X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test. The abundance of peroxisomes in cells expressing MITOL∆C8 was significantly decreased as compared with cells expressing wild-type MITOL.


( D ) MITOL ∆ C8 causes a drastic expansion of peroxisomes following CCCP treatment . HeLa expressing HA - Parkin wild - type or ∆ C8 MITOL were treated with 15 µM CCCP for 3 hours , approximate of peroxisomes was determined as number of pixels occupied by one peroxisome . PMP70 - positive pixels per 100 µm2 were divided by number of peroxisomes in same . In box - plots , dots indicate data points , center lines show medians , box limits indicate 25th 75th percentiles as determined by R software package , whiskers extend 1 . 5 times interquartile range from 25th 75th percentiles . Means number of samples are shown on box X - axis , respectively . Statistical significance was calculated using a one - Welch ' s t - .

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(D) MITOL∆C8 causes a drastic expansion of peroxisomes following CCCP treatment. HeLa cells expressing HA-Parkin and wild-type or ∆C8 MITOL were treated with 15 µM CCCP for 3 hours, and the approximate size of peroxisomes was determined as the number of pixels occupied by one peroxisome. The PMP70-positive pixels per 100 µm2 were divided by the number of peroxisomes in the same area. In the box-plots, dots indicate individual data points, the center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by the R software package, and whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and the X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( A B ) HeLa expressing HA - Parkin MITOL ∆ C8 ( A ) or MITOL ∆ C8 lacking E3 activity ( B ) were treated with 15 µM CCCP for 3 hours , then subjected to immunocytochemistry with anti - Flag anti - ubiquitin antibodies . Expanded peroxisomes were ubiquitylated upon E3 activity of MITOL . Higher magnification of boxed regions are shown in small panel . Scale bars , 10 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A and B) HeLa cells expressing HA-Parkin and MITOL∆C8 (A) or MITOL∆C8 lacking E3 activity (B) were treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-Flag and anti-ubiquitin antibodies. Expanded peroxisomes were ubiquitylated upon E3 activity of MITOL. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.


( A ) Representative 5 - day - old seedlings of wild - type Arabidopsis thaliana Col - 0 TF mutants gata12 wri3 grown in . Scale bar , 1 mm .

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(A) Representative 5-day-old seedlings of wild-type Arabidopsis thaliana Col-0 and TF mutants gata12 and wri3 grown in the dark. Scale bar, 1 mm.


( B ) Heat map indicates which TFs significantly affects hypocotyl , root composite trait of root to total in - grown seedlings whether effects of TFs are dependent on condition . TFs are hierarchically clustered using Euclidean .

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(B) Heat map indicates which TFs significantly affects hypocotyl length, root length and the composite trait of root to total length in dark-grown seedlings and whether the effects of TFs are dependent on condition. TFs are hierarchically clustered using Euclidean distance.


( C ) Heat map of average relative effects of TF mutant alleles on hypocotyl , root of root to total reveal that TF lesions significantly perturbed TCA cycle - dependent growth . Mutant alleles are listed in rows traits in columns . of TF mutant alleles in heat map are colored if AGI or AGI : TCA Metabolite linear model terms for each trait are statistically significant ( P < 0 . 05 , two - way ANOVA , 16 - 20 seedlings per genotype per condition across two experiments per genotype ) . Mutant alleles are hierarchically clustered using Euclidean .

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(C) Heat map of the average relative effects of TF mutant alleles on hypocotyl length, root length and the ratio of root to total length reveal that TF lesions significantly perturbed TCA cycle-dependent growth. Mutant alleles are listed in rows and traits in columns. Cells of TF mutant alleles in heat map are colored if the AGI or AGI:TCA Metabolite linear model terms for each trait are statistically significant (P < 0.05, two-way ANOVA, 16-20 seedlings per genotype per condition across two experiments per genotype). Mutant alleles are hierarchically clustered using Euclidean distance.


( A ) Heat map indicates which TFs significantly affects rosette , growth rate , shoot biomass , flowering time seed yield whether effects of TFs are dependent on salt treatment . TFs are hierarchically clustered using Euclidean .

(A) Heat map indicates which TFs significantly affects rosette area, growth rate, shoot biomass, flowering time and seed yield and whether the effects of TFs are dependent on salt treatment. TFs are hierarchically clustered using Euclidean distance.


( C ) Rosette of mutant allele of BPC4 from day 7 to day 21 post - germination . Linear model for two - way ANOVA considers AGI , salt treatment , day post - germination their interactions . AGI AGI : Salt Condition terms are statistically signification ( P < 0 . 001 , P < 0 . 01 , respectively ) . Heat map under plot indicates which term in linear model is statistically significant ( P < 0 . 05 ) using two - way ANOVA for each day from 9 biological replicates per condition . Solid lines , Col - 0 ; dashed , bpc4 ; circle , control condition ; triangle , 50 mM NaCl .

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(C) Rosette area of mutant allele of BPC4 from day 7 to day 21 post-germination. Linear model for two-way ANOVA considers AGI, salt treatment, day post-germination and their interactions. The AGI and AGI:Salt Condition terms are statistically signification (P < 0.001, P < 0.01, respectively). Heat map under line plot indicates which term in linear model is statistically significant (P < 0.05) using two-way ANOVA for each day from 9 biological replicates per condition. Solid lines, Col-0; dashed line, bpc4; circle, control condition; triangle, 50 mM NaCl.


( A ) Hybridization Chain Reaction ( HCR ) detection of CGG repeat along with immunocytochemistry ( ICC ) to detect SRSF1 - FLAG in U2OS . Expanded view of merged channels shows CGG foci ( arrowhead ) an example of SRSF1 - CGG foci co - localization ( arrow ) . DAPI marks nucleus . Scale bars are 10 µm .

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(A) Hybridization Chain Reaction (HCR) detection of CGG repeat RNA along with immunocytochemistry (ICC) to detect SRSF1-FLAG in U2OS cells. Expanded view of merged channels shows the CGG RNA foci (arrowhead) and an example of SRSF1-CGG RNA foci co-localization (arrow). DAPI marks the nucleus. Scale bars are 10 µm.


( B ) Co - localization of CGG ( red traces ) SRSF1 ( green traces ) showing overlaps of CGG SRSF1 . ( a . u . = arbitrary unit )

(B) Co-localization analysis of CGG RNA (red traces) and SRSF1 (green traces) showing overlaps of CGG RNA and SRSF1. (a.u. = arbitrary unit)


( C ) Co - immunoprecipitation of indicated SRSF with PP7 - tagged control ( partial nLUC ) to correct for non - specific binding , AUG CGG reporter RNAs . SRSF1 2 specifically immunoprecipitated with PP7 - tagged CGG reporter .

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(C) Co-immunoprecipitation of indicated SRSF proteins with PP7-tagged control sequence (partial nLUC sequence) to correct for non-specific binding, AUG and CGG reporter RNAs. SRSF1 and 2 specifically immunoprecipitated with PP7-tagged CGG reporter.


( D ) Representative of fly eyes quantitation ( below ) of rough eye scores with fly SRSF1 overexpression ( dSF2 OE ) ; n = 20 - 32 / genotype . Error bars represent mean + / - SD . Data information : For eye scoring , target siRNA lines were compared to non - targeting control siRNA lines using a two - student ' s t with Welch ' s correction for multiple comparisons . ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 . Human orthologs of fly genes are used for labeling .

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(D) Representative photographs of fly eyes and quantitation (below) of rough eye scores with fly SRSF1 overexpression (dSF2 OE); n=20-32/genotype. Error bars represent mean +/- SD. Data information: For eye scoring, target siRNA lines were compared to non-targeting control siRNA lines using a two-tailed student's t test with Welch's correction for multiple comparisons. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Human orthologs of fly genes are used for labeling.


( F ) Quantitation of ( GGGGCC ) 28 - EGFP rough eye phenotype with SRSF modifiers ( n > 30 flies / genotype ) . Error bars represent mean + / - SD . Data information : For eye scoring , target siRNA lines were compared to non - targeting control siRNA lines using a two - student ' s t with Welch ' s correction for multiple comparisons . ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 . Human orthologs of fly genes are used for labeling .

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(F) Quantitation of (GGGGCC)28-EGFP rough eye phenotype with SRSF modifiers (n > 30 flies/ genotype). Error bars represent mean +/- SD. Data information: For eye scoring, target siRNA lines were compared to non-targeting control siRNA lines using a two-tailed student's t test with Welch's correction for multiple comparisons. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Human orthologs of fly genes are used for labeling.


( G ) Representative of fly eyes expressing GMR - GAL4 driven ( GGGGCC ) 28 - EGFP at 29 ˚ C along with quantifications of necrosis eye width . n = 28 - 30 / genotype . Error bars represent mean + / - SD .

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(G) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP at 29˚C along with the quantifications of necrosis and eye width. n=28-30/genotype. Error bars represent mean +/- SD.


( H - I ) Survival assays of flies expressing ( CGG ) 90 - EGFP under Tub5 - GS ( H ) ELAV - GS ( I ) drivers with control or SRSF1 siRNAs . Expression of ( CGG ) 90 - EGFP was initiated with addition of drug starting 1 day post eclosion continued through experiment ( Log - rank Mantel - Cox ; n = 98 - 101 / genotype for Tub - GS n = 120 - 141 / genotype for ELAV - GS flies ) ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 . ( J ) Survival assays of ( GGGGCC ) 28 - EGFP expressing fly under Tub5 - GS driver ( Log - rank Mantel - Cox ; n = 71 - 93 / genotype ) with control or SRSF1 siRNAs . ∗ ∗ p < 0 . 01 .

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(H-I) Survival assays of flies expressing (CGG)90-EGFP under Tub5-GS (H) and ELAV-GS (I) drivers with control or SRSF1 siRNAs. Expression of (CGG)90-EGFP was initiated with addition of drug starting 1 day post eclosion and continued through experiment (Log-rank Mantel-Cox test; n=98-101/genotype for Tub-GS and n=120-141/genotype for ELAV-GS flies) ∗p < 0.05, ∗∗p < 0.01. (J) Survival assays of (GGGGCC)28-EGFP expressing fly under Tub5-GS driver (Log-rank Mantel-Cox test; n=71-93/genotype) with control or SRSF1 siRNAs. ∗∗p < 0.01.


( A , B ) Representative ( A ) of fly eyes ( B ) quantitation with siRNA mediated knockdown of SRPK1 ( dSRPK1 ) ; n = 30 - 34 / genotype . Error bars represent mean + / - SD . Data information : Statistical was performed using two - Student ' s t with Welch ' s correction , ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 .

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(A, B) Representative (A) photographs of fly eyes and (B) quantitation with siRNA mediated knockdown of SRPK1 (dSRPK1); n=30-34/genotype. Error bars represent mean +/- SD. Data information: Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.


( C ) Representative of fly eyes expressing GMR - GAL4 driven ( GGGGCC ) 28 - EGFP with siRNA mediated knockdown of SRPK1 or disruption by insertion . ( D ) Quantitation of rough eye . t - with Welch corrections for comparisons with control ; n = 31 - 34 flies / genotype . Error bars represent mean + / - SD . Data information : Statistical was performed using two - Student ' s t with Welch ' s correction , ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 .

(C) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP with siRNA mediated knockdown of SRPK1 or disruption by insertion. (D) Quantitation of rough eye phenotypes. t-test with Welch corrections for comparisons with the control; n= 31-34 flies/ genotype. Error bars represent mean +/- SD. Data information: Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.


( E ) Representative external eye imaging for detection of GFP caused by ( CGG ) 90 - EGFP transgene expression ( top ) . Converted used to quantify total of GFP ( bottom ) . ( F ) Depletion of SRSF1 or SRPK1 by RNAi results in reduced ( CGG ) 90 - EGFP compared to control siRNA as quantified by total ( a . u . = arbitrary unit ) . n = 13 - 15 flies / genotype . Error bars represent mean + / - SD . Data information : Statistical was performed using two - Student ' s t with Welch ' s correction , ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 .

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(E) Representative external eye imaging for detection of GFP aggregates caused by (CGG)90-EGFP transgene expression (top). Converted images used to quantify total intensity of GFP puncta (bottom). (F) Depletion of SRSF1 or SRPK1 by RNAi results in reduced (CGG)90-EGFP puncta compared to control siRNA as quantified by total intensity (a.u. = arbitrary unit). n=13-15 flies/genotype. Error bars represent mean +/- SD. Data information: Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.


( B ) Anti - FLAG immunoblot of DMSO SRPIN340 pre - treated HEK293T expressing AUG - nLuc - 3xFLAG control or CGG - nLuc - 3xFLAG RAN translation reporters . β - Actin is used as a loading control . To prevent signal saturation , AUG - nLuc lysate was diluted 1 : 3 in sample buffer prior to loading ( n = 3 biological replicates ) . Schematics of AUG - nLUC - 3xFLAG + 1CGG ( 100 ) - nLuc - 3xFLAG reporters presented on top . Data information : Error bars represent mean + / - SD . Statistical was performed using two - Student ' s t with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 ∗ ∗ ∗ ∗ p < 0 . 0001 . To prevent over - exposure , AUG - nLuc lysate was diluted 1 : 3 in sample buffer .

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

(B) Anti-FLAG immunoblot of DMSO and SRPIN340 pre-treated HEK293T cells expressing AUG-nLuc-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. β-Actin is used as a loading control. To prevent signal saturation, AUG-nLuc lysate was diluted 1:3 in sample buffer prior to loading (n=3 biological replicates). Schematics of the AUG-nLUC-3xFLAG and +1CGG(100)-nLuc-3xFLAG reporters presented on top. Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001. To prevent over-exposure, the AUG-nLuc lysate was diluted 1:3 in sample buffer.


( D ) Anti - FLAG immunoblot of DMSO SRPIN340 pre - treated HEK293T expressing GGGGCC - nLuc - 3xFLAG ( GA70 ) + 2CGG - nLuc - 3xFLAG ( FMRpolyA ) RAN translation reporters ( n = 3 biological replicates ) . Schematics of GA70 ( GGGGCCx70 ) + 2CGG reporters presented on top . Data information : Error bars represent mean + / - SD . Statistical was performed using two - Student ' s t with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 ∗ ∗ ∗ ∗ p < 0 . 0001 .

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

(D) Anti-FLAG immunoblot of DMSO and SRPIN340 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3 biological replicates). Schematics of the GA70 (GGGGCCx70) and +2CGG reporters presented on top. Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.


( E ) Immunoblot of DMSO SPHINX31 pre - treated HEK293T expressing AUG - nLuc - 3xFLAG control or CGG - nLuc - 3xFLAG RAN translation reporters . ( n = 3 biological replicates ) . ( F ) Anti - FLAG of DMSO SPHINX31 pre - treated HEK293T expressing GGGGCC - nLuc - 3xFLAG ( GA70 ) + 2CGG - nLuc - 3xFLAG ( FMRpolyA ) RAN translation reporters ( n = 3 biological replicates ) . Data information : Error bars represent mean + / - SD . Statistical was performed using two - Student ' s t with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 ∗ ∗ ∗ ∗ p < 0 . 0001 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot of DMSO and SPHINX31 pre-treated HEK293T cells expressing AUG-nLuc-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. (n=3 biological replicates). (F) Anti-FLAG of DMSO and SPHINX31 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3 biological replicates). Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.


( C , D ) Expression of GGGGCC - nLuc - 3xFLAG ( C ) + 2CGG - nLuc - 3xFLAG ( D ) RAN translation reporters in HEK293T treated with 2 μM TG ( for stress induction ) analyzed by immunoblot ( n = 3 biological replicates ) . To evaluate effects of SRPK1 inhibition were pre - treated with DMSO or SRPIN340 before reporter transfection . Data information : Error bars represent mean + / - SD . Two - Student ' s t with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 ∗ ∗ ∗ ∗ 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 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 O O O O O O O O O O O O O O O O O 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, D) Expression of GGGGCC-nLuc-3xFLAG (C) and +2CGG-nLuc-3xFLAG (D) RAN translation reporters in HEK293T cells treated with 2 μM TG (for stress induction) analyzed by immunoblot (n=3 biological replicates). To evaluate effects of SRPK1 inhibition cells were pre-treated with DMSO or SRPIN340 before reporter transfection. Data information: Error bars represent mean +/- SD. Two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.


( E ) SRPK1 inhibition by SRPIN340 did not alter eIF2α phosphorylation in response to 2 μM TG ( for stress induction ) in HEK293T by immunoblot ( n = 3 biological replicates ) . ( F ) SPRK1 inhibition suppressed eIF2α phosphorylation in HEK293T treated with 2 μM TG ( for stress induction ) in presence of + 1CGG reporter . were pre - treated with DMSO or SRPIN340 before reporter transfection ( n = 3 biological replicates ) . Data information : Error bars represent mean + / - SD . Two - Student ' s t with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 ∗ ∗ ∗ ∗ p < 0 . 0001 .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O 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) SRPK1 inhibition by SRPIN340 did not alter eIF2α phosphorylation in response to 2 μM TG (for stress induction) in HEK293T cells by immunoblot (n=3 biological replicates). (F) SPRK1 inhibition suppressed eIF2α phosphorylation in HEK293T cells treated with 2 μM TG (for stress induction) in the presence of the +1CGG reporter. Cells were pre-treated with DMSO or SRPIN340 before reporter transfection (n=3 biological replicates). Data information: Error bars represent mean +/- SD. Two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.


( B ) ICC of FLAG - tagged SRSF1 after SRPIN340 treatment compared to vehicle ( DMSO ) . shows of of SRSF1 signal Error bars indicate mean + / - 95 % CI ( n = 85 - 126 / condition ) . Statistical was performed using t with Welch ' s correction , ∗ ∗ ∗ ∗ p < 0 . 0001 .

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

(B) ICC images of FLAG-tagged SRSF1 after SRPIN340 treatment compared to vehicle (DMSO). Quantification shows the ratio of nuclear and cytoplasmic intensity of SRSF1 signal Error bars indicate mean +/- 95% CI (n= 85-126 cells/condition). Statistical analysis was performed using t test with Welch's correction, ∗∗∗∗p < 0.0001.


( C ) Nucleocytoplasmic distribution CGG repeat after SRPIN340 treatment compared to vehicle ( DMSO ) as detected by HCR . shows of of CGG signal as parts of whole . Error bars indicate mean + / - 95 % CI ( n = 124 - 151 / condition ) . Statistical was performed using t with Welch ' s correction , ∗ ∗ ∗ ∗ p < 0 . 0001 .

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

(C) Nucleocytoplasmic distribution CGG repeat RNA after SRPIN340 treatment compared to vehicle (DMSO) as detected by HCR. Quantification shows the ratio of nuclear and cytoplasmic intensity of CGG RNA signal as parts of whole. Error bars indicate mean +/- 95% CI (n= 124-151 cells/condition). Statistical analysis was performed using t test with Welch's correction, ∗∗∗∗p < 0.0001.


( D ) Anti - FLAG immunoblot blot of DMSO SRPIN340 pre - treated HEK293T transfected with in vitro transcribed CGG - nLuc - 3xFLAG reporter . β - Actin is used as a loading control . Error bars represent mean + / - SD ( n = 6 biological replicates ) . Statistical was performed using Student ' s t with Welch ' s correction . ∗ p < 0 .

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

(D) Anti-FLAG immunoblot blot of DMSO and SRPIN340 pre-treated HEK293T cells transfected with in vitro transcribed CGG-nLuc-3xFLAG reporter RNA. β-Actin is used as a loading control. Error bars represent mean +/- SD (n=6 biological replicates). Statistical analysis was performed using Student's t test with Welch's correction. ∗p < 0.05


( A , B ) Pharmacological targeting of SPRK1 with ( A ) 40 μM SRPIN340 or ( B ) 8 μM SPHINX31 reduced cumulative risk of in + 1 ( CGG ) 100 - EGFP ( encoding FMRpolyG ) expressing neurons . n = # of neurons quantified for each condition ; Cox proportional hazard , ∗ ∗ ∗ p < 0 . 001 .

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

(A, B) Pharmacological targeting of SPRK1 with (A) 40 μM SRPIN340 or (B) 8 μM SPHINX31 reduced the cumulative risk of death in +1(CGG)100-EGFP (encoding FMRpolyG) expressing neurons. n= # of neurons quantified for each condition; Cox proportional hazard analysis, ∗∗∗p < 0.001.


C Quantitative of lysosomal movement from kymographs . Results were expressed as of total lysosomes ( N = 44 , 49 , 16 neurons for shSc , shLAMTOR1 , shLAMTOR + rLAMTOR1 respectively from 3 - 10 independent experiments ) . Data information : Data with error bars are represented as means ± . Statistical significance was assessed by two - way ANOVA with Tukey ' s * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as compared to shSc ; # P < 0 . 05 , # # # P < 0 . 001 , as compared to shLAMTOR1 n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantitative analysis of lysosomal movement from kymographs. Results were expressed as percent of total lysosomes (N = 44, 49, and 16 neurons for shSc, shLAMTOR1, and shLAMTOR+rLAMTOR1 respectively from 3-10 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's *P < 0.05, **P < 0.01, ***P < 0.001, as compared to shSc; #P < 0.05, ###P < 0.001, as compared to shLAMTOR1 n.s., not significant.


G FRAP of dendritic lysosome movement . Quantitative for F ( N = 7 , 7 , 9 neurons for shSc , shLAMTOR1 , shLAMTOR + rLAMTOR1 , respectively , from 3 independent experiments ) . Data information : Data with error bars are represented as means ± . Statistical significance was assessed by Dunnett ' s post - * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as compared to shSc ; # P < 0 . 05 , # # # P < 0 . 001 , as compared to shLAMTOR1 ; n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 FRAP analysis of dendritic lysosome movement. Quantitative analysis for F (N = 7, 7, and 9 neurons for shSc, shLAMTOR1, and shLAMTOR+rLAMTOR1, respectively, from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Dunnett's post-test *P < 0.05, **P < 0.01, ***P < 0.001, as compared to shSc; #P < 0.05, ###P < 0.001, as compared to shLAMTOR1 ; n.s., not significant.


A Representative kymographs of LysoTracker - labeled lysosomes in proximal dendrites of hippocampal neurons infected with scrambled shRNA ( shSc ) , LAMTOR2 shRNA ( shLAMTOR2 ) , or Raptor shRNA ( shRaptor ) . Scale bar , 5 µm . B Quantitative of lysosomal movement from kymographs ( N = 44 , 24 , 19 neurons for shSc , shLAMTOR2 , shRaptor , respectively , from 3 - 10 independent experiments ) . Data information : Data with error bars are represented as means ± . Statistical significance was assessed by two - way ANOVA with Sidak ' s post - * * * P < 0 . 001 as compared to shSc ; n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative kymographs of LysoTracker-labeled lysosomes in proximal dendrites of hippocampal neurons infected with scrambled shRNA (shSc), LAMTOR2 shRNA (shLAMTOR2), or Raptor shRNA (shRaptor). Scale bar, 5 µm. B Quantitative analysis of lysosomal movement from kymographs (N = 44, 24, and 19 neurons for shSc, shLAMTOR2, and shRaptor, respectively, from 3-10 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Sidak's post-test ***P < 0.001 as compared to shSc; n.s., not significant.


C , D FRAP of dendritic lysosomal movement in Raptor KD neurons . C Representative of a neuron at 0 4 min after photobleaching . D of lysosomes in C undergoing retrograde ( red ) or anterograde ( cyan ) transport ( N = 7 8 neurons for shSc shRaptor , respectively , from 3 independent experiments ) . Data information : Data with error bars are represented as means ± . Statistical significance was assessed by Mann - Whitney U * * * P < 0 . 001 as compared to shSc ; n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D FRAP analysis of dendritic lysosomal movement in Raptor KD neurons. C Representative images of a neuron at 0 and 4 min after photobleaching. D Quantification of the lysosomes in C undergoing retrograde (red) or anterograde (cyan) transport (N = 7 and 8 neurons for shSc and shRaptor, respectively, from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Mann-Whitney U test ***P < 0.001 as compared to shSc; n.s., not significant.


E LAMTOR1 KD did not affect trafficking of vesicles labeled with Alexa 594 - conjugated transferrin ( red , Tf 594 ) in dendrites . Shown are dendritic segments ( upper ) kymographs ( lower ) . Scale bar , 5 µm . F Quantitative of vesicular movement from kymographs ( N = 10 neurons from 3 independent experiments ) . Data information : Data with error bars are represented as means ± . Statistical significance was assessed by Mann - Whitney U * * * P < 0 . 001 as compared to shSc ; n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 LAMTOR1 KD did not affect trafficking of vesicles labeled with Alexa 594-conjugated transferrin (red, Tf 594) in dendrites. Shown are dendritic segments (upper) and kymographs (lower). Scale bar, 5 µm. F Quantitative analysis of vesicular movement from kymographs (N = 10 neurons from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Mann-Whitney U test ***P < 0.001 as compared to shSc; n.s., not significant.


A of of lysosomes moving in anterograde or retrograde direction in neurons infected with AAV expressing either LAMTOR1 shRNA ( shLAMTOR1 ) or scrambled shRNA ( shSc ) Accell lyspersin siRNA or control siRNA . Neurons were imaged with LysoTracker to visualize lysosomal trafficking in dendrites . N = 17 - 31 neurons from 3 - 6 independent experiments . Data information : Data with error bars are represented as means ± . * * * P < 0 . 001 compared with shSc or shSc / Accell siControl ; # # P < 0 . 01 , # # # P < 0 . 001 compared with shLAMTOR1 or shLAMTOR1 / Accell siControl ; & & P < 0 . 01 , & & & P < 0 . 001 compared with shSc / Accell siLyspersin ; n . s . , not significant ; two - way ANOVA with Tukey ' s post - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 the percent of lysosomes moving in the anterograde or retrograde direction in neurons infected with AAV expressing either LAMTOR1 shRNA (shLAMTOR1) or scrambled shRNA (shSc) and Accell lyspersin siRNA or control siRNA. Neurons were imaged with LysoTracker to visualize lysosomal trafficking in dendrites. N = 17-31 neurons from 3-6 independent experiments. Data information: Data with error bars are represented as means ± SEM. ***P < 0.001 compared with shSc or shSc/Accell siControl; ##P < 0.01, ###P < 0.001 compared with shLAMTOR1 or shLAMTOR1/Accell siControl; &&P < 0.01, &&&P < 0.001 compared with shSc/Accell siLyspersin; n.s., not significant; two-way ANOVA with Tukey's post-test.


C - F of of lysosomes moving in anterograde or retrograde direction . C , E , F Cultured hippocampal neurons were infected with AAV expressing either shLAMTOR1 or shSc ; they were treated with vehicle control or ML - SI1 ( 20 µM , C , n = 16 - 49 neurons from 3 - 10 independent experiments ) , or ML - SA1 ( 20 µM , E , n = 13 - 49 neurons from 3 - 10 independent experiments ) , or CilioD ( 20 µM , F , n = 12 - 49 neurons from 3 - 10 independent experiments ) , imaged with LysoTracker to visualize lysosomal trafficking in dendrites . D Neurons were infected with shLAMTOR1 or shSc AAV Accell TRPML1 siRNA or control siRNA ; they were imaged as described above . N = 7 - 31 neurons from 3 - 6 independent experiments . Data information : Data with error bars are represented as means ± . * * * P < 0 . 001 compared with shSc or shSc / Accell siControl ; # # P < 0 . 01 , # # # P < 0 . 001 compared with shLAMTOR1 or shLAMTOR1 / Accell siControl ; & & P < 0 . 01 , & & & P < 0 . 001 compared with shSc / Accell siLyspersin ; n . s . , not significant ; two - way ANOVA with Tukey ' s post - .

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

C-F Quantification of the percent of lysosomes moving in the anterograde or retrograde direction. C, E, F Cultured hippocampal neurons were infected with AAV expressing either shLAMTOR1 or shSc; they were treated with vehicle control or ML-SI1 (20 µM, C, n = 16-49 neurons from 3-10 independent experiments), or ML-SA1 (20 µM, E, n = 13-49 neurons from 3-10 independent experiments), or CilioD (20 µM, F, n = 12-49 neurons from 3-10 independent experiments), and imaged with LysoTracker to visualize lysosomal trafficking in dendrites. D Neurons were infected with shLAMTOR1 or shSc AAV and Accell TRPML1 siRNA or control siRNA; they were imaged as described above. N = 7-31 neurons from 3-6 independent experiments. Data information: Data with error bars are represented as means ± SEM. ***P < 0.001 compared with shSc or shSc/Accell siControl; ##P < 0.01, ###P < 0.001 compared with shLAMTOR1 or shLAMTOR1/Accell siControl; &&P < 0.01, &&&P < 0.001 compared with shSc/Accell siLyspersin; n.s., not significant; two-way ANOVA with Tukey's post-test.


F of co - IP results . N = 3 independent experiments . Data information : Data with error bars are represented as means ± . Statistical significance was assessed by one - way ANOVA with Dunnett ' s post - ( F ) . * P < 0 . 05 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification of co-IP results. N = 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test (F). *P < 0.05, ***P < 0.001.


A Interactions between LAMTOR1 TRPML1 in mouse hippocampus . Binding of LAMTOR1 to TRPML1 in vivo was disrupted by systemic administration of TAT - 2031 peptide . Wes with anti - LAMTOR1 - TRPML1 antibodies of immunoprecipitation performed with anti - LAMTOR1 antibodies or negative control anti - HA antibodies using whole hippocampal homogenates from naïve , TAT or TAT - 2031 - treated mice . B of relative abundance of TPRML1 pulled down by LAMTOR1 in naïve , TAT or TAT - 2031 - treated mice . N = 3 mice for each group . Data information : Data with error bars are represented as means ± . Statistical significance was assessed by one - way ANOVA with Dunnett ' s post - * * P < 0 . 01 , * * * P < 0 . 001 .

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

A Interactions between LAMTOR1 and TRPML1 in mouse hippocampus. Binding of LAMTOR1 to TRPML1 in vivo was disrupted by systemic administration of the TAT-2031 peptide. Wes protein analysis with anti-LAMTOR1 and -TRPML1 antibodies of immunoprecipitation performed with anti-LAMTOR1 antibodies or negative control anti-HA antibodies using whole hippocampal homogenates from naïve, TAT or TAT-2031-treated mice. B Quantification of the relative abundance of TPRML1 pulled down by LAMTOR1 in naïve, TAT or TAT-2031-treated mice. N = 3 mice for each group. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test **P < 0.01, ***P < 0.001.


C Representative from proximity ligation ( PLA ) performed on brain slices from naïve , TAT or TAT - 2031 - treated mice . Evidence of proximity between LAMTOR1 TRPML1 is indicated by appearance of red . Nuclei are counterstained with DAPI ( blue ) . Scale bar , 10 μm . D of number of PLA signals in CA1 from naïve , TAT or TAT - 2031 - treated mice . N = 6 mice for each group . Data information : Data with error bars are represented as means ± . Statistical significance was assessed by one - way ANOVA with Dunnett ' s post - * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative images from proximity ligation assay (PLA) performed on brain slices from naïve, TAT or TAT-2031-treated mice. Evidence of proximity between LAMTOR1 and TRPML1 is indicated by the appearance of red puncta. Nuclei are counterstained with DAPI (blue). Scale bar, 10 μm. D Quantification of the number of PLA signals in CA1 from naïve, TAT or TAT-2031-treated mice. N = 6 mice for each group. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test **P < 0.01, ***P < 0.001.


D TAT - 2031 treatment ( 10 µM ) increased ML - SA1 - induced TRPML1 Ca2 + release in both control LAMTOR1 KD . Data information : traces represent mean values of each group .

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D TAT-2031 treatment (10 µM) increased ML-SA1-induced TRPML1 Ca2+ release in both control and LAMTOR1 KD cells. Data information: traces represent the mean values of each group.


G Treatment with GPN ( 200 µM ) , BAPTA - AM ( 20 µM ) , or ML - SI1 ( 20 µM ) blocked ML - SA1 - induced TRPML1 - GCaMP6m responses in neurons . Data information : traces represent mean values of each group .

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G Treatment with GPN (200 µM), BAPTA-AM (20 µM), or ML-SI1 (20 µM) blocked ML-SA1-induced TRPML1-GCaMP6m responses in neurons. Data information: traces represent the mean values of each group.


I Both LAMTOR1 KD TAT - 2031 treatment ( 10 µM ) increased ML - SA1 - induced TRPML1 - GCaMP6m responses in neurons . J of peak responses as shown in I . N = 6 - 13 from 3 independent experiments . Data information : Data with error bars are represented as means ± . Statistical significance was assessed by two - way ANOVA with Tukey ' s post hoc * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , # # # P < 0 . 001 , n . s . , not significant . traces represent mean values of each group .

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I Both LAMTOR1 KD and TAT-2031 treatment (10 µM) increased ML-SA1-induced TRPML1-GCaMP6m responses in neurons. J Quantification of peak responses as shown in I. N = 6-13 cells from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's post hoc analysis *P < 0.05, **P < 0.01, ***P < 0.001, ###P < 0.001, n.s., not significant. traces represent the mean values of each group.


K LAMTOR1 KD increased PI ( 3 , 5 ) P2 ( 0 . 5 µM ) - induced TRPML1 - GCaMP6m responses blocking effect of ML - SI1 treatment ( 20 µM ) . L of peak TRPML1 - GCaMP6m responses as shown in K . N = 6 - 16 from 3 independent experiments . Data information : Data with error bars are represented as means ± . Statistical significance was assessed by two - way ANOVA with Tukey ' s post hoc * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , # # # P < 0 . 001 , n . s . , not significant . traces represent mean values of each group .

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

K LAMTOR1 KD increased PI(3,5)P2 (0.5 µM)-induced TRPML1-GCaMP6m responses and the blocking effect of ML-SI1 treatment (20 µM). L Quantification of peak TRPML1-GCaMP6m responses as shown in K. N = 6-16 cells from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's post hoc analysis *P < 0.05, **P < 0.01, ***P < 0.001, ###P < 0.001, n.s., not significant. traces represent the mean values of each group.


A - D Effects of LAMTOR1 KD ML - SI1 treatment on TBS - induced LTP ( A ) or LFS - induced LTD ( C ) in CA1 . B , D Means ± of fEPSPs measured 40 min after TBS ( B , n = 4 - 5 slices from 4 - 5 mice ) or LFS ( D , n = 4 - 8 slices from 4 - 8 mice ) in different groups . Data information : Slopes of fEPSPs were normalized to average values recorded during first 10 min baseline Statistical significance was assessed by two - way ANOVA with Tukey ' s post - * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 compared with shSc P < 0 . 05 , # # P < 0 . 01 , # # # P < 0 . 001 compared with shLAMTOR1 . Insets show representative traces of evoked fEPSPs before 40 min after TBS / LFS . Scale bar 0 . 5 mV / 10 ms .

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A-D Effects of LAMTOR1 KD and ML-SI1 treatment on TBS-induced LTP (A) or LFS-induced LTD (C) in CA1. B, D Means ± SEM of fEPSPs measured 40 min after TBS (B, n = 4-5 slices from 4-5 mice) or LFS (D, n = 4-8 slices from 4-8 mice) in different groups. Data information: Slopes of fEPSPs were normalized to the average values recorded during the first 10 min baseline Statistical significance was assessed by two-way ANOVA with Tukey's post-test *P < 0.05, **P < 0.01, ***P < 0.001 compared with shSc P < 0.05, ##P < 0.01, ###P < 0.001 compared with shLAMTOR1. Insets show representative traces of evoked fEPSPs before and 40 min after TBS/LFS. Scale bar 0.5 mV/10 ms.


G - J Effects of TAT - 2031 treatment on TBS - induced LTP ( G ) or LFS - induced LTD ( I ) in CA1 . H , J Means ± of fEPSPs measured 40 min after TBS ( H , n = 6 slices from 6 mice ) or LFS ( J , n = 3 - 4 slices from 3 - 4 mice ) in different groups . Data information : Slopes of fEPSPs were normalized to average values recorded during first 10 min baseline Statistical significance was assessed by Student ' s t - * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 compared with shSc , Vehicle , or TAT Insets show representative traces of evoked fEPSPs before 40 min after TBS / LFS . Scale bar 0 . 5 mV / 10 ms .

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G-J Effects of TAT-2031 treatment on TBS-induced LTP (G) or LFS-induced LTD (I) in CA1. H, J Means ± SEM of fEPSPs measured 40 min after TBS (H, n = 6 slices from 6 mice) or LFS (J, n = 3-4 slices from 3-4 mice) in different groups. Data information: Slopes of fEPSPs were normalized to the average values recorded during the first 10 min baseline Statistical significance was assessed by Student's t-test *P < 0.05, **P < 0.01, ***P < 0.001 compared with shSc, Vehicle, or TAT Insets show representative traces of evoked fEPSPs before and 40 min after TBS/LFS. Scale bar 0.5 mV/10 ms.


K Effects of LAMTOR1 KD treatment with a calcineurin inhibitor , FK506 , on LFS - induced LTD in CA1 . L Means ± of fEPSPs measured 45 min after LFS in different groups ( n = 3 - 4 slices from 3 - 4 mice ) . Data information : Slopes of fEPSPs were normalized to average values recorded during first 10 min baseline Statistical significance was assessed by two - way ANOVA with Tukey ' s post - * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 compared with shSc , Vehicle , or TAT , # P < 0 . 05 , # # P < 0 . 01 , # # # P < 0 . 001 compared with shLAMTOR1 . Insets show representative traces of evoked fEPSPs before 40 min after LFS . Scale bar 0 . 5 mV / 10 ms .

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

K Effects of LAMTOR1 KD and treatment with a calcineurin inhibitor, FK506, on LFS-induced LTD in CA1. L Means ± SEM of fEPSPs measured 45 min after LFS in different groups (n = 3-4 slices from 3-4 mice). Data information: Slopes of fEPSPs were normalized to the average values recorded during the first 10 min baseline Statistical significance was assessed by two-way ANOVA with Tukey's post-test *P < 0.05, **P < 0.01, ***P < 0.001 compared with shSc, Vehicle, or TAT, #P < 0.05, ##P < 0.01, ###P < 0.001 compared with shLAMTOR1. Insets show representative traces of evoked fEPSPs before and 40 min after LFS. Scale bar 0.5 mV/10 ms.


LAMTOR1 KD significantly reduced of GluA1 phosphorylation total GluA1 as well as of p - GluA1 to GluA1 in hippocampal neurons , this effect was reversed by ML - SI1 treatment . A Representative of CA1 pyramidal neurons stained with anti - p - GluA1 S845 ( magenta ) , anti - GluA1 ( red ) , anti - GFP ( green ) antibodies . Scale bar = 20 µm .

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LAMTOR1 KD significantly reduced levels of GluA1 phosphorylation and total GluA1 as well as the ratio of p-GluA1 to GluA1 in hippocampal neurons, and this effect was reversed by ML-SI1 treatment. A Representative images of CA1 pyramidal neurons stained with anti-p-GluA1 S845 (magenta), anti-GluA1 (red), and anti-GFP (green) antibodies. Scale bar = 20 µm.


E Representative of proximal dendrites of hippocampal neurons stained with anti - GluA1 ( green ) , anti - LAMP2 ( red ) , anti - GFP ( gray ) antibodies . Note that GluA1 labeled with Alexa Fluor 633 secondary antibodies was false - colored green GFP false - colored gray to better show colocalization of GluA1 with LAMP2 . Arrowheads indicate clearly co - localized . Scale bar , 5 µm .

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E Representative images of proximal dendrites of hippocampal neurons stained with anti-GluA1 (green), anti-LAMP2 (red), and anti-GFP (gray) antibodies. Note that GluA1 labeled with Alexa Fluor 633 secondary antibodies was false-colored green and GFP false-colored gray to better show colocalization of GluA1 with LAMP2. Arrowheads indicate clearly co-localized puncta. Scale bar, 5 µm.


A Mice received 10 min of training in an environment with two identical objects received a retention 24 h later in which one object was replaced with a novel one . LAMTOR1 shRNA - injected mice exhibited a significant deficit 24 h after training , which was reversed by ML - SI1 treatment ( N = 7 - 18 mice ) . B % freezing for different experimental groups in context memory ( N = 7 - 19 mice ) . Data information : Data with error bars are represented as means ± . Statistical significance was assessed by two - way ANOVA with Tukey ' s post - . * P < 0 . 05 , * * * P < 0 . 001 , as compared to shSc ; # P < 0 . 05 , # # # P < 0 . 001 , as compared to shLAMTOR1 .

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

A Mice received 10 min of training in an environment with two identical objects and received a retention test 24 h later in which one object was replaced with a novel one. LAMTOR1 shRNA-injected mice exhibited a significant deficit 24 h after training, which was reversed by ML-SI1 treatment (N = 7-18 mice). B % freezing for different experimental groups in context memory (N = 7-19 mice). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's post-test. *P < 0.05, ***P < 0.001, as compared to shSc; #P < 0.05, ###P < 0.001, as compared to shLAMTOR1.


Peptide array dot blot to identify LIR motifs in indicated SNARE . for identified LIRs from positive signals are marked on each SNARE . are denoted as acid single letter codes in blot .

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Peptide array dot blot analysis to identify LIR motifs in the indicated SNARE proteins. Amino acids for the identified LIRs from positive signals are marked on each SNARE. Amino acids are denoted as amino acid single letter codes in the blot.


Co - immunoprecipitation ( Co - IP ) of interactions between FLAG - tagged Stx16 / Vti1a / Stx6 EGFP - tagged LC3B or GABARAP overexpressed in HEK293T . * indicates mouse IgG heavy chain of precipitated mouse - anti - FLAG antibody .

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Co-immunoprecipitation (Co-IP) analysis of interactions between FLAG-tagged Stx16/Vti1a/Stx6 and EGFP-tagged LC3B or GABARAP overexpressed in HEK293T cells. * indicates mouse IgG heavy chain of precipitated mouse-anti-FLAG antibody.


GST pull - down of interactions between radiolabeled Myc - Stx16 GST - tagged mAtg8 .

GST pull-down analysis of interactions between radiolabeled Myc-Stx16 and GST-tagged mAtg8 proteins.


GST pull - down of interactions between wild type ( WT ) or LIR - mutant ( L219A / V222A ) Stx16 GST - tagged LC3C or GABARAP . Lower panel shows percentages of WT or LIR - mutant Stx16 bound to GST - LC3C or GST - GABARAP . Data shown as means ± of precipitated Stx16 , n = 3 .

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GST pull-down analysis of interactions between wild type (WT) or LIR-mutant (L219A/V222A) Stx16 and GST-tagged LC3C or GABARAP. Lower panel shows percentages of WT or LIR-mutant Stx16 bound to GST-LC3C or GST-GABARAP. Data shown as means ± SEM of precipitated Stx16, n = 3.


GST pull - down of interactions between WT or different types of LIR - mutant Stx16 GST - tagged LC3C or GABARAP . Lower panel shows percentages of WT or LIR - mutant Stx16 bound to GST - LC3C or GST - GABARAP .

GST pull-down analysis of interactions between WT or different types of LIR-mutant Stx16 and GST-tagged LC3C or GABARAP. Lower panel shows percentages of WT or LIR-mutant Stx16 bound to GST-LC3C or GST-GABARAP.


GST pull - down of interactions between radiolabeled Myc - Stx16 WT or LDS - mutant GABARAP ( GABARAP - Y49A - Y49A / F104A ) . Lower panel shows percentages of Stx16 bound to WT or LDS - mutant GABARAP . Data shown as means ± of precipitated Stx16 , n = 3 .

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GST pull-down analysis of interactions between radiolabeled Myc-Stx16 and WT or LDS-mutant GABARAP (GABARAP-Y49A and -Y49A/F104A). Lower panel shows percentages of Stx16 bound to WT or LDS-mutant GABARAP. Data shown as means ± SEM of precipitated Stx16, n = 3.


WT or STX17 - knockout ( STX17KO ) HeLa were starved with or without presence of bafilomycin A1 ( Baf A1 , 100 nM ) for 2 h , lysates were subjected to Western blot of LC3B p62 . Quantifications of LC3B - II normalized to β - actin from treated as in ( A ) ; Data shown as means ± of LC3B - II β - actin ratios , n = 3 ; * , p < 0 . 05 ( one - way ANOVA ) .

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WT or STX17-knockout (STX17KO) HeLa cells were starved with or without the presence of bafilomycin A1 (Baf A1, 100 nM) for 2 h, and cell lysates were subjected to Western blot analysis of LC3B and p62.   Quantifications of LC3B-II levels normalized to β-actin from cells treated as in (A); Data shown as means ± SEM of LC3B-II and β-actin ratios, n = 3; *, p < 0.05 (one-way ANOVA).


WT , STX16KO , STX17KO or STX16 / STX17 double KO ( STX16 / STX17DKO ) HeLa were starved with or without presence of Baf A1 ( 100 nM ) for 2 h , lysates were subjected to Western blot of LC3B . Quantifications of LC3B - II normalized to β - actin from ( C ) ; Data shown as means ± of LC3B - II β - actin ratios , n = 3 ; † , not significant ; * * , p < 0 . 01 ( one - way ANOVA ) .

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WT, STX16KO, STX17KO or STX16/STX17 double KO (STX16/STX17DKO) HeLa cells were starved with or without the presence of Baf A1 (100 nM) for 2 h, and cell lysates were subjected to Western blot analysis of LC3B.   Quantifications of LC3B-II levels normalized to β-actin from (C); Data shown as means ± SEM of LC3B-II and β-actin ratios, n = 3; †, not significant; **, p < 0.01 (one-way ANOVA).


WT or STX16 / STX17DKO HeLa were starved in EBSS for 2 h , subjected to ultrastructural of autophagic vesicles ( AV ) with electron microscopy . AVi : initial autophagic vacuoles ; AVd : degradative autophagic vacuoles ; G : Golgi apparatus . Image acquisition counting as in Methods . Scale bars , 1 μm 0 . 5 µm ( top sections ) . Quantifications of autophagic vesicles in WT STX16 / STX17DKO HeLa treated as ( E ) ; Data shown as means ± of AV profiles relative to ; † , not significant ; * , p < 0 . 05 ( two - way ANOVA ) . AV profiles from 47 of each samples were counted .

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WT or STX16/STX17DKO HeLa cells were starved in EBSS for 2 h, and subjected to ultrastructural analysis of the autophagic vesicles (AV) with electron microscopy. AVi: initial autophagic vacuoles; AVd: degradative autophagic vacuoles; G: Golgi apparatus. Image acquisition and counting as in Methods. Scale bars, 1 μm and 0.5 µm (top sections).   Quantifications of autophagic vesicles in WT and STX16/STX17DKO HeLa cells treated as (E); Data shown as means ± SEM of AV profiles relative to cytoplasmic area; †, not significant; *, p < 0.05 (two-way ANOVA). AV profiles from 47 images of each samples were counted.


WT or STX16 / STX17DKO HeLa - YFP - Parkin were treated with CCCP ( 10 µM ) or oligomycin A ( 5 µM ) antimycin A ( 10 µM ) ( OA ) for 16 hours , subjected to high - content microscopy ( HCM ) of mitochondria clearance . Masks : blue , nuclei ; red , mitochondria stained with mitochondrial ( mtDNA ) antibody . Scale bar : 20 µm . Quantifications of mtDNA by object or total per in WT or STX16 / STX17DKO HeLa - YFP - Parkin treated as in ( A ) ; Data shown as means ± of mtDNA object ( upper panel ) or object total ( lower panel ) per ; minimum 500 were counted each well from at least 12 wells , 3 independent experiments ; * * , p < 0 . 01 ( two - way ANOVA ) .

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

WT or STX16/STX17DKO HeLa-YFP-Parkin cells were treated with CCCP (10 µM) or oligomycin A (5 µM) and antimycin A (10 µM) (OA) for 16 hours, and subjected to high-content microscopy (HCM) analysis of mitochondria clearance. Masks: blue, nuclei; red, mitochondria stained with mitochondrial DNA (mtDNA) antibody. Scale bar: 20 µm.   Quantifications of mtDNA by object count or total area per cell in WT or STX16/STX17DKO HeLa-YFP-Parkin cells treated as in (A); Data shown as means ± SEM of mtDNA object count (upper panel) or object total area (lower panel) per cell; minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).


WT or STX16 / STX17DKO Huh7 were treated with H2O2 ( 0 . 4 mM ) for indicated time points , autophagic clearance of peroxisomes was measured by of PMP70 , PEX14 p62 . Quantifications of peroxisomal PMP70 PEX14 for WT or STX16 / STX17DKO Huh7 treated as in ( C ) ; Data shown as means ± of PEX14 or PMP70 β - actin ratios , n = 3 ; † , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ( two - way ANOVA ) .

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WT or STX16/STX17DKO Huh7 cells were treated with H2O2 (0.4 mM) for indicated time points, and autophagic clearance of peroxisomes was measured by the protein levels of PMP70, PEX14 and p62.   Quantifications of peroxisomal proteins PMP70 and PEX14 for WT or STX16/STX17DKO Huh7 cells treated as in (C); Data shown as means ± SEM of PEX14 or PMP70 and β-actin ratios, n = 3; †, not significant; *, p < 0.05; **, p < 0.01 (two-way ANOVA).


WT , STX16KO , STX17KO or STX16 / STX17DKO THP - 1 were infected with Mycobacterium tuberculosis ( Mtb ) , followed by starvation to induce autophagy . Autophagic clearance of infected Mtb was measured by Mtb colonies grown on Middlebrook 7H11 agar plates ; Data shown as means ± of Mtb colonies , n = 4 ; * , p < 0 . 05 ; * * , p < 0 . 01 ( two - way ANOVA ) .

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WT, STX16KO, STX17KO or STX16/STX17DKO THP-1 cells were infected with Mycobacterium tuberculosis (Mtb), followed by starvation to induce autophagy. Autophagic clearance of infected Mtb was measured by Mtb colonies grown on Middlebrook 7H11 agar plates; Data shown as means ± SEM of Mtb colonies, n = 4; *, p < 0.05; **, p < 0.01 (two-way ANOVA).


Validation of STX16 / STX17 DKO by Western blot in HEK293 stably expressing RPS3 - Keima ( left panel ) HCT116 stably expressing RPL28 - Keima ( right panel ) .

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Validation of STX16/STX17 DKO by Western blot analysis in HEK293 cells stably expressing RPS3-Keima (left panel) and HCT116 cells stably expressing RPL28-Keima (right panel).


WT or STX16 / STX17DKO HEK293 RPS3 - Keima were cultured in full medium or starved for 8 hours , subjected to HCM of Keima accumulated in autolysosomes with excitation wavelength at 560 nm . Masks : , identified on nuclei ; red , Keima detected with excitation emission wavelengths of 560 nm 620 nm , respectively ; lower panel shows Keima without masks . Scale bar : 20 µm . Quantifications of autolysosomal Keima in WT or STX16 / STX17DKO HEK293 RPS3 - Keima treated as in ( C ) . Data shown as means ± of Keima per , minimum 1000 were counted each well from at least 12 wells , 3 independent experiments ; * * , p < 0 . 01 ( two - way ANOVA ) .

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WT or STX16/STX17DKO HEK293 RPS3-Keima cells were cultured in full medium or starved for 8 hours, and subjected to HCM analysis of Keima puncta accumulated in autolysosomes with the excitation wavelength at 560 nm. Masks: white, cells identified based on nuclei; red, Keima puncta detected with the excitation and emission wavelengths of 560 nm and 620 nm, respectively; lower panel shows Keima puncta without masks. Scale bar: 20 µm.   Quantifications of autolysosomal Keima puncta in WT or STX16 /STX17DKO HEK293 RPS3-Keima cells treated as in (C). Data shown as means ± SEM of Keima puncta per cell, minimum 1000 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).


WT or STX16 / STX17DKO HCT116 RPL28 - Keima were cultured in full medium or starved for 8 hours , subjected to HCM of Keima accumulated in autolysosomes with excitation wavelength at 560 nm . Masks : , identified on nuclei ; red , Keima detected with excitation emission wavelengths of 560 nm 620 nm , respectively ; lower panel shows Keima without masks . Scale bar : 20 µm . Quantifications of autolysosomal Keima in WT or STX16 / STX17DKO HCT116 RPL28 - Keima treated as in ( E ) . Data shown as means ± of Keima per , minimum 1000 were counted each well from at least 12 wells , 3 independent experiments ; † , not significant ; * * , p < 0 . 01 ( two - way ANOVA ) .

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WT or STX16/STX17DKO HCT116 RPL28-Keima cells were cultured in full medium or starved for 8 hours, and subjected to HCM analysis of Keima puncta accumulated in autolysosomes with the excitation wavelength at 560 nm. Masks: white, cells identified based on nuclei; red, Keima puncta detected with the excitation and emission wavelengths of 560 nm and 620 nm, respectively; lower panel shows Keima puncta without masks. Scale bar: 20 µm.   Quantifications of autolysosomal Keima puncta in WT or STX16/STX17DKO HCT116 RPL28-Keima cells treated as in (E). Data shown as means ± SEM of Keima puncta per cell, minimum 1000 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant; **, p < 0.01 (two-way ANOVA).


( A ) WT or STX16KO HeLa were starved in EBSS for 2 h subjected to HCM of LAMP2 LC3 . Masks : , identified on nuclei ; red , LC3 ; green , LAMP2 ; yellow , overlap of LC3 LAMP2 . Scale bar : 20 µm .

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(A) WT or STX16KO HeLa cells were starved in EBSS for 2 h and subjected to HCM analysis of LAMP2 and LC3 puncta. Masks: white, cells identified based on nuclei; red, LC3 puncta; green, LAMP2 puncta; yellow, overlap of LC3 and LAMP2. Scale bar: 20 µm.


( B - D ) Quantifications of LC3 per ( B ) , LC3 LAMP2 overlap per ( C ) , LAMP2 per ( D ) in WT or STX16KO treated Data shown as means ± of or overlap per , minimum 500 were counted each well from at least 12 wells , 3 independent experiments ; * , p < 0 . 05 ; * * , p < 0 . 01 ( two - way ANOVA ) .

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(B-D) Quantifications of LC3 puncta per cell (B), LC3 and LAMP2 overlap area per cell (C), and LAMP2 puncta per cell (D) in WT or STX16KO cells treated Data shown as means ± SEM of puncta or overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (two-way ANOVA).


( E ) Western blot of LAMP1 LAMP2 in WT separate clones of STX16KO HeLa , Huh7 , U2OS .

(E) Western blot analysis of LAMP1 and LAMP2 protein levels in WT and separate clones of STX16KO HeLa, Huh7, and U2OS cells.


( F ) Western blot of LAMP2 in WT separate clones of STX6KO or VTI1AKO HeLa .

(F) Western blot analysis of LAMP2 protein levels in WT and separate clones of STX6KO or VTI1AKO HeLa cells.


( G ) Time course of mTOR inactivation during EBSS starvation in WT vs . STX16KO HeLa analyzed by Western blotting for mTOR substrates ULK1 4E - BP1 .

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(G) Time course of mTOR inactivation during EBSS starvation in WT vs. STX16KO HeLa cells analyzed by Western blotting for mTOR substrates ULK1 and 4E-BP1.


( H ) WT or STX16KO HeLa were starved in EBSS for 1 h subjected to HCM of mTOR . Masks : , identified on nuclei ; red , mTOR . Scale bar : 20 µm . ( I ) Quantifications of mTOR in WT or STX16KO treated as in ( H ) . Data shown as means ± of mTOR per , minimum 500 were counted each well from at least 12 wells , 3 independent experiments ; * * , p < 0 . 01 ( two - way ANOVA ) .

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(H) WT or STX16KO HeLa cells were starved in EBSS for 1 h and subjected to HCM analysis of mTOR puncta. Masks: white, cells identified based on nuclei; red, mTOR puncta. Scale bar: 20 µm. (I) Quantifications of mTOR puncta in WT or STX16KO cells treated as in (H). Data shown as means ± SEM of mTOR puncta per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).


WT or STX16KO HeLa were starved in EBSS for 1 h , followed by starvation with presence of LysoTracker Red DND - 99 ( LTR ) ( 100 nM ) for additional 30 min , subjected to HCM of LTR . Scale bar : 20 µm . Quantifications of LTR per or total per in WT STX16KO HeLa treated as in ( A ) . Data shown as means ± of LTR per or object total per , minimum 500 were counted each well from at least 12 wells , 3 independent experiments ; † , not significant ( two - way ANOVA ) .

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WT or STX16KO HeLa cells were starved in EBSS for 1 h, followed by starvation with the presence of LysoTracker Red DND-99 (LTR) (100 nM) for additional 30 min, and subjected to HCM analysis of LTR. Scale bar: 20 µm.   Quantifications of LTR count per cell or total area per cell in WT and STX16KO HeLa cells treated as in (A). Data shown as means ± SEM of LTR puncta per cell or object total area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant (two-way ANOVA).


WT or STX16KO HeLa were starved in EBSS for 1 h , followed by starvation with presence of LTR ( 100 nM ) for additional 30 min , subjected to HCM of colocalization between LTR TGN46 . Masks : , identified on nuclei ; red , LTR ; green , TGN46 ; yellow , overlap between LTR TGN46 . Scale bar : 20 µm . Quantifications of overlaps between LysoTracker Red TGN46 in WT STX16KO treated as in ( C ) . Data shown as means ± of LTR TGN46 overlap per , minimum 500 were counted each well from at least 12 wells , 3 independent experiments ; * * , p < 0 . 01 ( two - way ANOVA ) .

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WT or STX16KO HeLa cells were starved in EBSS for 1 h, followed by starvation with the presence of LTR (100 nM) for additional 30 min, and subjected to HCM analysis of the colocalization between LTR and TGN46. Masks: white, cells identified based on nuclei; red, LTR puncta; green, TGN46 puncta; yellow, overlap between LTR and TGN46. Scale bar: 20 µm.   Quantifications of overlaps between LysoTracker Red and TGN46 in WT and STX16KO cells treated as in (C). Data shown as means ± SEM of LTR and TGN46 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).


WT or STX16 / STX17DKO HeLa were starved in EBSS for 2 h , subjected to ultrastructural of Golgi apparatus with electron microscopy . VF of Golgi : volume fraction of Golgi relative to total . Scale bar , 2 μm . Red arrows indicate Golgi apparatus .

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WT or STX16/STX17DKO HeLa cells were starved in EBSS for 2 h, and subjected to ultrastructural analysis of the Golgi apparatus with electron microscopy. VF of Golgi: volume fraction of Golgi relative to total cytoplasmic area. Scale bar, 2 μm. Red arrows indicate Golgi apparatus.


WT , LC3TKO , GABARAPTKO or HexaKO HeLa were starved in EBSS for 90 min subjected to HCM of overlaps between LAMP2 Stx16 . Masks : , identified on nuclei ; green , LAMP2 ; red , Stx16 ; yellow , overlap of LAMP2 Stx16 . Scale bar : 20 µm .

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WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells were starved in EBSS for 90 min and subjected to HCM analysis of overlaps between LAMP2 and Stx16. Masks: white, cells identified based on nuclei; green, LAMP2 puncta; red, Stx16 puncta; yellow, overlap of LAMP2 and Stx16. Scale bar: 20 µm.


Quantifications of overlaps between LAMP2 Stx16 in WT , LC3TKO , GABARAPTKO or HexaKO HeLa treated Data shown as means ± of LAMP2 Stx16 overlap per , minimum 500 were counted each well from at least 12 wells , 3 independent experiments ; * , p < 0 . 05 ; * * , p < 0 . 01 ( one - way ANOVA ) .

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Quantifications of overlaps between LAMP2 and Stx16 in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells treated Data shown as means ± SEM of LAMP2 and Stx16 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (one-way ANOVA).


Quantifications of overlaps between LBPA Stx16 in WT , LC3TKO , GABARAPTKO or HexaKO HeLa grown in full medium or treated Data shown as means ± of LBPA Stx16 overlap per , minimum 500 were counted each well from at least 12 wells , 3 independent experiments ; * , p < 0 . 05 ; * * , p < 0 . 01 ( one - way ANOVA ) .

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Quantifications of overlaps between LBPA and Stx16 in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells grown in full medium or treated Data shown as means ± SEM of LBPA and Stx16 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (one-way ANOVA).


WT , LC3TKO , GABARAPTKO or HexaKO HeLa were starved in EBSS for 1 hour , followed by starvation with presence of LTR ( 100 nM ) for additional 30 min , subjected to HCM of LTR . Scale bar : 20 µm .

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WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells were starved in EBSS for 1 hour, followed by starvation with the presence of LTR (100 nM) for additional 30 min, and subjected to HCM analysis of LTR. Scale bar: 20 µm.


Quantifications of LTR in WT , LC3TKO , GABARAPTKO or HexaKO HeLa treated Data shown as means ± of LTR per , minimum 500 were counted each well from at least 12 wells , 3 independent experiments ; † , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ( one - way ANOVA ) .

Quantifications of LTR puncta in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells treated Data shown as means ± SEM of LTR puncta per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant; *, p < 0.05; **, p < 0.01 (one-way ANOVA).


a , ( wt , ∆ atg18 or ∆ atg18 / ∆ atg21 / ∆ hsv2 ( ) ) , with without a plasmid expressing wt or a mutant form of Atg18 ) were grown in YPD , vacuoles were stained with FM4 - 64 imaged by confocal microscopy . At least 10 confocal sections spaced at 300 nm were assembled into maximum projections . show before ( 0 min ) 15 , 30 60 min after induction of vacuolar fragmentation by addition of 0 . 4 M NaCl . Red : FM4 - 64 . Gray : DIC . Scale bar 5 µm .

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a, Cells (wt, ∆atg18 or ∆atg18/∆atg21/∆hsv2 ()), with and without a plasmid expressing wt or a mutant form of Atg18) were grown in YPD, vacuoles were stained with FM4-64 and imaged by confocal microscopy. At least 10 confocal sections spaced at 300 nm were assembled into maximum projections. Images show cells before (0 min) and 15, 30 and 60 min after induction of vacuolar fragmentation by addition of 0.4 M NaCl. Red: FM4-64. Gray: DIC. Scale bar 5 µm.


b , number of vacuoles per was quantified for experiment in a . 150 from three independent experiments were counted for each condition . Values represent mean , error bars .

b, The number of vacuoles per cell was quantified for the experiment in a. 150 cells from three independent experiments were counted for each condition. Values represent the mean, error bars the SEM.


atg18 expressing plasmids carrying Atg18 - GFP or Atg18FGGG - GFP , a mutant in lipid binding sites , were stained with FM4 - 64 . a , Confocal z - stacks were taken before 2 , 6 10 min after addition of 0 . 4 M NaCl . Stacks were processed into maximum projections using ImageJ . Scale bar : 5 µm .

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atg18 cells expressing plasmids carrying Atg18-GFP or Atg18FGGG-GFP, a mutant in the lipid binding sites, were stained with FM4-64. a, Confocal z-stacks were taken before and 2, 6 and 10 min after addition of 0.4 M NaCl. Stacks were processed into maximum projections using ImageJ. Scale bar: 5 µm.


b , Same experiment as in a , but in a fab1 or vps34 background .

b, Same experiment as in a, but in a fab1 or vps34 background.


d , Localisation of Atg18 - GFP 5 min after salt shock . Experiment as in a . Scale bar : 1 µm .

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d, Localisation of Atg18-GFP 5 min after salt shock. Experiment as in a. Scale bar: 1 µm.


b , c Their secondary structure was analyzed by circular dichroism spectroscopy in presence of increasing concentrations of hexafluoro - 2 - propanol ( HFIP ) , in presence of 6 mM small unilamellar vesicles ( SUVs , 70 % egg PC 30 % PS , total phospholipid approx . 6 mM ) , or of or control buffer only ( blue ) . Spectra are shown for b , wildtype

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

b,c Their secondary structure was analyzed by circular dichroism spectroscopy in the presence of increasing concentrations of hexafluoro-2-propanol (HFIP), in the presence of 6 mM small unilamellar vesicles (SUVs, 70% egg PC and 30 % PS, total phospholipid concentration approx. 6 mM), or of or control buffer only (blue). Spectra are shown for b, the wildtype peptide


b , c Their secondary structure was analyzed by circular dichroism spectroscopy in presence of increasing concentrations of hexafluoro - 2 - propanol ( HFIP ) , in presence of 6 mM small unilamellar vesicles ( SUVs , 70 % egg PC 30 % PS , total phospholipid approx . 6 mM ) , or of or control buffer only ( blue ) . Spectra are shown for c , SLoop peptide .

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b,c Their secondary structure was analyzed by circular dichroism spectroscopy in the presence of increasing concentrations of hexafluoro-2-propanol (HFIP), in the presence of 6 mM small unilamellar vesicles (SUVs, 70% egg PC and 30 % PS, total phospholipid concentration approx. 6 mM), or of or control buffer only (blue). Spectra are shown for c, the SLoop peptide.


d , Vacuole of expressing Atg18Sloop - GFP , imaged before after osmotic shock as in Fig 1a . Scale bar : 5 µm .

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d, Vacuole morphology of cells expressing Atg18Sloop-GFP, imaged before and after osmotic shock as in Fig 1a. Scale bar: 5 µm.


e , Number of vacuoles per in d , assayed as in Figure 1b . n = 3 .

e, Number of vacuoles per cell in d, assayed as in Figure 1b. n=3.


f , Vacuole of expressing Atg18DLoop - GFP , imaged as in d .

f, Vacuole morphology of cells expressing Atg18DLoop-GFP, imaged as in d.


g , Number of vacuoles per in e , assayed as in Fig . 1b . n = 3 .

g, Number of vacuoles per cell in e, assayed as in Fig. 1b. n=3.


Binding of recombinant Atg18 variants to liposomes . SUVs were made of EPC with 5 % of indicated phosphoinositides , or of EPC indicated fraction of PS . Cholesterol had been added to 20 mol % of total phospholipid amount . Liposomes were incubated with purified for 30 min at room ( 25 ° C ) , centrifuged supernatants ( S ) pellets ( P ) analyzed by SDS - PAGE Coomassie . Binding is shown for a ,

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Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for a, Atg18wt


Binding of recombinant Atg18 variants to liposomes . SUVs were made of EPC with 5 % of indicated phosphoinositides , or of EPC indicated fraction of PS . Cholesterol had been added to 20 mol % of total phospholipid amount . Liposomes were incubated with purified for 30 min at room ( 25 ° C ) , centrifuged supernatants ( S ) pellets ( P ) analyzed by SDS - PAGE Coomassie . Binding is shown for b , Atg18FGGG ;

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Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for b, Atg18FGGG;


Binding of recombinant Atg18 variants to liposomes . SUVs were made of EPC with 5 % of indicated phosphoinositides , or of EPC indicated fraction of PS . Cholesterol had been added to 20 mol % of total phospholipid amount . Liposomes were incubated with purified for 30 min at room ( 25 ° C ) , centrifuged supernatants ( S ) pellets ( P ) analyzed by SDS - PAGE Coomassie . Binding is shown for c , Atg18DLoop ;

Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for c, Atg18DLoop;


Binding of recombinant Atg18 variants to liposomes . SUVs were made of EPC with 5 % of indicated phosphoinositides , or of EPC indicated fraction of PS . Cholesterol had been added to 20 mol % of total phospholipid amount . Liposomes were incubated with purified for 30 min at room ( 25 ° C ) , centrifuged supernatants ( S ) pellets ( P ) analyzed by SDS - PAGE Coomassie . Binding is shown for d , Atg18SLoop .

Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for d, Atg18SLoop.


e , bands from a - d were quantified from 3 independent experiments . mean with is shown .

e, The bands from a-d were quantified from 3 independent experiments. The mean with SEM is shown.


a . Autophagy measured with pho8 ∆ 60 in atg18 complemented with wt , FGGG , SLoop or DLoop alleles of ATG18 .

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a. Autophagy measured with the pho8∆60 assay in atg18 cells complemented with wt, FGGG, SLoop or DLoop alleles of ATG18.


b . Same as in a , but in atg18 atg21 complemented with wt or SLoop versions of ATG18 ATG21 . Atg21SLoop contains K426F F429K substitutions . Helical wheel projections for both Atg21 helices are given . arrows indicate magnitude of hydrophobic moment .

b. Same assay as in a, but in atg18 atg21 cells complemented with wt or SLoop versions of ATG18 and ATG21. Atg21SLoop contains K426F and F429K substitutions. Helical wheel projections for both Atg21 helices are given. The arrows indicate the magnitude of the hydrophobic moment.


c . Vacuole fragmentation in atg2 , assayed 15 min after a moderate hypertonic shock as in Fig . 1 .

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c. Vacuole fragmentation in atg2 cells, assayed 15 min after a moderate hypertonic shock as in Fig. 1.


d . Number of vacuoles per in c , assayed as in Figure 1b . n = 3 .

d. Number of vacuoles per cell in c, assayed as in Figure 1b. n=3.


a , Time lapse of Atg18 added to liposomes . 10 µL of 10 µM purified recombinant Atg18wt was added to 100 µL buffer containing GUVs with 5 % PI3P 1 % PI ( 3 , 5 ) P2 . sample was imaged on a spinning disc confocal microscope . Most of tubules underwent scission into small vesicles ( See movie EV6 ) . Scale bar 5 µm . Areas in boxes 1 2 of c are shown at higher magnification in b c . Scale bars : 2 . 5 µm .

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a, Time lapse analysis of Atg18 added to liposomes. 10 µL of 10 µM purified recombinant Atg18wt was added to 100 µL buffer containing GUVs with 5% PI3P and 1% PI(3,5)P2. The sample was imaged on a spinning disc confocal microscope. Most of the tubules underwent scission into small vesicles (See movie EV6). Scale bar 5 µm. Areas in boxes 1 and 2 of c are shown at higher magnification in b and c. Scale bars: 2.5 µm.


d , of fission activity . For each movie , whole frame was divided into squares of 10 per 10 µm number of small vesicles per 100 µm2 counted . mean of three independent experiments is shown with .

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d, Quantification of fission activity. For each movie, the whole frame was divided into squares of 10 per 10 µm and the number of small vesicles per 100 µm2 counted. The mean of three independent experiments is shown with SEM.