( G ) A549 , stably expressing lentiviral vectors containing random shRNA or Atg7 shRNA , were infected with WT or P . aeruginosa mutants ( ΔSTY , ΔTY ΔS ) at MOI of 10 . infections were followed by 1 hours of gentamycin treatment . lysates were plated in LB agar for 18 hours CFU were determined . Data information : triangles dots represent ( three technical replicates per ) on control Atg7 knock down from three biological replicates . bars represent means of CFU of each group ( three biological replicates each group ) , error bars represent standard deviation . significance of differences between different was determined using two - student ' s t with Welch ' s correction , NS : not significant ; * * p ≤ 0 . 01 .

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(G) A549 cells, stably expressing lentiviral vectors containing random shRNA or Atg7 shRNA, were infected with WT or P. aeruginosa mutants (ΔSTY, ΔTY and ΔS) at MOI of 10. All infections were followed by 1 hours of gentamycin treatment. Cell lysates were plated in LB agar for 18 hours and CFU were determined. Data information: The triangles and dots represent the individual test (three technical replicates per individual test) on control and Atg7 knock down cells from three biological replicates. The bars represent the means of CFU of each group (three biological replicates each group), error bars represent standard deviation. The significance of differences between different assay was determined using two-tailed student's t test with Welch's correction, NS: not significant; **p ≤ 0.01.


( A ) airway epithelial were isolated from tracheas of Atg7 + / + Atg7 - / - mouse were evaluated by immunoblotting .

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(A) Primary airway epithelial cells were isolated from tracheas of Atg7+/+ and Atg7-/- mouse and were evaluated by immunoblotting.


( B ) Sections of tracheas from Atg7 + / + Atg7 - / - mouse were evaluated by immunofluorescence microscopy following of with DAPI ( Blue ) , ciliated epithelial marker β - tubulin IV ( green ) Atg7 ( red ) . Scale bars : 20 µm .

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(B) Sections of tracheas from Atg7+/+ and Atg7-/- mouse were evaluated by immunofluorescence microscopy following staining of nuclear DNA with DAPI (Blue), ciliated epithelial cell marker β-tubulin IV (green) and Atg7 (red). Scale bars: 20 µm.


( C , D ) Atg7 + / + Atg7 - / - mice were intranasally injected with 3 x107 CFU of WT , ΔS , ΔTY or ΔSTY P . aeruginosa . After 24 hours , lungs ( C ) , spleens livers ( D ) were harvested , homogenized diluted for CFU counting . Each data point represents ( Log10 CFU / g ) from mouse . horizontal lines represent mean value from each group ( 6 mice each group ) , error bars represent standard deviation . significance of differences between different drug treatment was determined using One - way ANOVA Dunn ' s multiple comparisons . , NS : not significant ; * p ≤ 0 .

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(C,D) Atg7+/+ and Atg7-/- mice were intranasally injected with 3 x107 CFU of WT, ΔS, ΔTY or ΔSTY P. aeruginosa. After 24 hours, lungs (C), spleens and livers (D) were harvested, homogenized and diluted for CFU counting. Each data point represents bacterial count (Log10 CFU/g) from individual mouse. The horizontal lines represent the mean value from each group (6 mice each group), error bars represent standard deviation. The significance of differences between different drug treatment was determined using One-way ANOVA and Dunn's multiple comparisons test., NS: not significant; *p ≤ 0.05


( E , F ) Atg7 + / + Atg7 - / - mice were intranasally injected with PBS with or without 1 x109 CFU of ΔTY ( E ) ΔSTY ( F ) . Mice survival was monitored for 10 days post infection . Kaplan - Meyer survival curves were made on observation of 12 - 18 mice / group from two independent experiments . P - values were calculated using log - rank ( ΔTYAtg7 + / + vs ΔTYAtg7 - / - , P = 0 . 2186 ; ΔSTYAtg7 + / + vs ΔSTYAtg7 - / - , P = 0 . 0145 ) .

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(E,F) Atg7+/+ and Atg7-/- mice were intranasally injected with PBS with or without 1 x109 CFU of ΔTY (E) ΔSTY (F). Mice survival was monitored for 10 days post infection. Kaplan-Meyer survival curves were made based on the observation of 12-18 mice/group from two independent experiments. The P-values were calculated using the log-rank test (ΔTYAtg7+/+ vs ΔTYAtg7-/-, P = 0.2186; ΔSTYAtg7+/+ vs ΔSTYAtg7-/-, P=0.0145).


( A A549 were infected for 4 hours with ExoS - deficient P . aeruginosa ( ΔS ) , ExoS containing P . aeruginosa ( SWT ) , P . aeruginosa containing ExoS with loss - of - function mutations in GTPase activating domain ( SG - A + ) , in ADP - ribosylation domain ( SG + A - ) , or in both domains ( SG - A - ) . lysates were evaluated by immunoblotting .

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(A A549 cells were infected for 4 hours with ExoS-deficient P. aeruginosa (ΔS), ExoS containing P. aeruginosa (SWT), P. aeruginosa containing ExoS with loss-of-function mutations in the GTPase activating domain (SG-A+), in the ADP-ribosylation domain (SG+A-), or in both domains (SG-A-). Cell lysates were evaluated by immunoblotting.


( B ) A549 were transfected , for 24hours , with a plasmid expressing empty vector ( EV ) , WT GFP - ExoS , a GFP - ExoS mutant for ADPRT domain ( SG + A - ) or a GFP - ExoS mutant deficient in both GAP ADPRT domains ( SG - A - ) . lysates were evaluated by immunoblotting .

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(B) A549 cells were transfected, for 24hours, with a plasmid expressing empty vector (EV), WT GFP-ExoS, a GFP-ExoS mutant for ADPRT domain (SG+A-) or a GFP-ExoS mutant deficient in both GAP and ADPRT domains (SG-A-). Cell lysates were evaluated by immunoblotting.


( C ) A549 were treated for 24 hours with 100 µM of ADPRT activity inhibitor MIBG then left uninfected ( NI ) or infected with P . aeruginosa containing ExoS with ADPRT activity only ( SG - A + ) . lysates were evaluated by immunoblotting .

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(C) A549 cells were treated for 24 hours with 100 µM of the ADPRT activity inhibitor MIBG and then left uninfected (NI) or infected with P. aeruginosa containing ExoS with ADPRT activity only (SG-A+). Cell lysates were evaluated by immunoblotting.


A549 were infected for 4 hours with WT or P . aeruginosa mutants ( ΔpscD lysates were evaluated by immunoblotting .

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A549 cells were infected for 4 hours with WT or P. aeruginosa mutants (ΔpscD Cell lysates were evaluated by immunoblotting.


A549 were infected for 4 hours with WT or P . aeruginosa mutants STY , ΔS ΔTY ) . lysates were evaluated by immunoblotting .

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A549 cells were infected for 4 hours with WT or P. aeruginosa mutants STY, ΔS and ΔTY). Cell lysates were evaluated by immunoblotting.


F ) A549 were infected for 4 hours with ExoS - deficient P . aeruginosa ( ΔS ) , ExoS containing P . aeruginosa ( SWT ) , P . aeruginosa containing ExoS with loss - of - function mutations in GTPase activating domain ( SG - A + ) , in ADP - ribosylation domain ( SG + A - ) , or in both domains ( SG - A - ) . lysates were evaluated by immunoblotting .

F) A549 cells were infected for 4 hours with ExoS-deficient P. aeruginosa (ΔS), ExoS containing P. aeruginosa (SWT), P. aeruginosa containing ExoS with loss-of-function mutations in the GTPase activating domain (SG-A+), in the ADP-ribosylation domain (SG+A-), or in both domains (SG-A-). Cell lysates were evaluated by immunoblotting.


( A ) A549 were transfected with empty vector ( VE ) vector contains Ras - G12V for 24 hours , followed by a 4 - hours infection with P . aeruginosa ΔSTY or ΔTY . lysates were evaluated by immunoblotting .

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(A) A549 cells were transfected with empty vector (VE) and vector contains Ras-G12V for 24 hours, followed by a 4-hours infection with P. aeruginosa ΔSTY or ΔTY. Cell lysates were evaluated by immunoblotting.


( B ) A549 were transfected , for 24 hours , with empty vector ( VE ) vectors contain Ras - G12V or Ras - G12V & R41K followed by a 4 - hours infection with P . aeruginosa ΔSTY or ΔTY . lysates were evaluated by immunoblotting .

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(B) A549 cells were transfected, for 24 hours, with empty vector (VE) and vectors contain Ras-G12V or Ras-G12V&R41K followed by a 4-hours infection with P. aeruginosa ΔSTY or ΔTY. Cell lysates were evaluated by immunoblotting.


( A ) A549 were transfected , for 48 hours , with a mCherry - DFCP1 plasmid then infected , for 4 hours , with P . aeruginosa ( WT , ΔSTY , ΔS , ΔTY , SG - A - , SG - A + or SG + A - ) mCherry number per was analyzed by microscopy . Representative immunofluorescence were showed , scale bars represent 10 µm . ( B ) Quantitative of mCherry - DFCP1 by visually more than 100 for each sample . Each bar represents mean value of from three independent experiments error bars represent standard deviation . significance of differences between assays was determined using ANOVA with Dunnett multiple - comparison posttest , NS : not significant ; * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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(A) A549 cells were transfected, for 48 hours, with a mCherry-DFCP1 plasmid and then infected, for 4 hours, with P. aeruginosa (WT, ΔSTY, ΔS, ΔTY, SG-A-, SG-A+ or SG+A-) and the mCherry puncta number per cell was analyzed by fluorescence microscopy. Representative immunofluorescence images were showed, and scale bars represent 10 µm. (B) Quantitative analysis of mCherry-DFCP1 puncta by visually count more than 100 cells for each sample. Each bar represents mean value of from three independent experiments and error bars represent standard deviation. The significance of differences between assays was determined using ANOVA with Dunnett multiple-comparison posttest, NS: not significant; *p ≤ 0.05; **p ≤ 0.01.


( A A549 , transfected for 48 hours with vector encoding Flag - Atg14L , were infected with P . aeruginosa ( WT , ΔSTY , ΔS , ΔTY Flag - Atg14L - Vps34 was immunoprecipitated ( IP ) by Flag - antibody magnetic beads evaluated by immunoblotting .

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(A A549 cells, transfected for 48 hours with vector encoding Flag-Atg14L, were infected with P. aeruginosa (WT, ΔSTY, ΔS, ΔTY Flag-Atg14L-Vps34 complex was immunoprecipitated (IP) by Flag-antibody magnetic beads and evaluated by immunoblotting.


( B , IP complexes were subjected to in vitro kinase using phosphatidylinositol as substrate . products radiolabeled PI3P were separated by TLC detected by autoradiography . Data are mean ± SD of three independent experiments . significance of differences between different assays was determined using One - way ANOVA followed Dunn ' s multiple - comparison posttest , NS : not significant ; * p ≤ 0 . 05 .

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(B, The IP complexes were subjected to in vitro kinase assay using phosphatidylinositol as substrate. The products radiolabeled PI3P were separated by TLC and detected by autoradiography. Data are mean ± SD of three independent experiments. The significance of differences between different assays was determined using One-way ANOVA analysis followed Dunn's multiple-comparison posttest, NS: not significant; *p ≤ 0.05.


C ) A549 , transfected for 48 hours with vector encoding Flag - Atg14L , were infected with P . aeruginosa , ΔSTY SG - A - , SG - A + or SG + A - ) . Flag - Atg14L - Vps34 was immunoprecipitated ( IP ) by Flag - antibody magnetic beads evaluated by immunoblotting .

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C) A549 cells, transfected for 48 hours with vector encoding Flag-Atg14L, were infected with P. aeruginosa , ΔSTY SG-A-, SG-A+ or SG+A-). Flag-Atg14L-Vps34 complex was immunoprecipitated (IP) by Flag-antibody magnetic beads and evaluated by immunoblotting.


D ) IP complexes were subjected to in vitro kinase using phosphatidylinositol as substrate . products radiolabeled PI3P were separated by TLC detected by autoradiography . Data are mean ± SD of three independent experiments . significance of differences between different assays was determined using One - way ANOVA followed Dunn ' s multiple - comparison posttest , NS : not significant ; * p ≤ 0 . 05 .

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D) The IP complexes were subjected to in vitro kinase assay using phosphatidylinositol as substrate. The products radiolabeled PI3P were separated by TLC and detected by autoradiography. Data are mean ± SD of three independent experiments. The significance of differences between different assays was determined using One-way ANOVA analysis followed Dunn's multiple-comparison posttest, NS: not significant; *p ≤ 0.05.


( A ) UBQLN4 associates with polyubiquitinated proteasomal substrates . Flag - tagged UBQLN4 was affinity - purified from extracts of HeLa that were treated with ( + ) or without ( - ) 20 μM MG - 132 for 4 h before harvesting , Flag - precipitates ( IP : Flag ) were blotted with anti - polyubiquitin ( FK2 ) , anti - BAG6 , anti - Flag antibodies . Mock indicates empty vector transfection . Note that UBQLN4 - BAG6 co - precipitation can be observed exclusively in presence of polyubiquitinated substrates .

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(A) UBQLN4 associates with polyubiquitinated proteasomal substrates. Flag-tagged UBQLN4 protein was affinity-purified from extracts of HeLa cells that were treated with (+) or without (-) 20 μM MG-132 for 4 h before harvesting, and the Flag-precipitates (IP:Flag) were blotted with anti-polyubiquitin (FK2), anti-BAG6, and anti-Flag antibodies. Mock indicates empty vector transfection. Note that UBQLN4-BAG6 co-precipitation can be observed exclusively in the presence of polyubiquitinated substrates.


( B ) Polyubiquitinated associated with UBQLN4 are sensitive to translation inhibitor cycloheximide ( CHX ) . HeLa expressing Flag - tagged UBQLN4 were treated with 5 μM MG - 132 as well as CHX ( either 10 or 25 μg / mL ) for 4 h as indicated , then subjected to immunoprecipitation with an anti - Flag antibody as in ( A ) .

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(B) Polyubiquitinated proteins associated with UBQLN4 are sensitive to the translation inhibitor cycloheximide (CHX). HeLa cells expressing Flag-tagged UBQLN4 were treated with 5 μM MG-132 as well as CHX (either 10 or 25 μg/mL) for 4 h as indicated, and then subjected to immunoprecipitation with an anti-Flag antibody as in (A).


( D ) HeLa were treated as in ( C ) , Flag - precipitates were blotted as in ( B ) .

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(D) HeLa cells were treated as in (C), and Flag-precipitates were blotted as in (B).


( A ) UBQLN4 co - localizes with after puromycin treatment . HeLa expressing Flag - tagged UBQLN4 were treated with 5 µg / mL puromycin for 2 h then followed by 1 % Triton X - 100 extraction 4 % paraformaldehyde fixation . Fixed were stained with anti - polyubiquitin FK2 ( shown in red to detect ubiquitin - positive , ALIS ) anti - Flag antibodies ( shown in green ) .

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(A) UBQLN4 co-localizes with cytoplasmic aggregates after puromycin treatment. HeLa cells expressing Flag-tagged UBQLN4 were treated with 5 µg/mL puromycin for 2 h and then followed by 1% Triton X-100 extraction and 4% paraformaldehyde fixation. Fixed cells were stained with anti-polyubiquitin FK2 (shown in red to detect ubiquitin-positive cytoplasmic aggregates, ALIS) and anti-Flag antibodies (shown in green).


( B , C ) Puromycin - treated with UBQLN4 siRNA contain increased number of ALIS . At 72 h after transfection of two distinct siRNA duplexes for UBQLN4 ( UBQLN4 siRNA # 1 # 2 ) or control siRNA ( 5 nM each ) , were treated with 5 µg / mL puromycin for 2 h , then subjected to immunostaining with an anti - polyubiquitin FK2 antibody ( shown in green , B ) as in ( A ) . number of ALIS was determined . quantified data in ( C ) represent mean ± . n = 76 for control siRNA , n = 89 for UBQLN4 siRNA # 1 , n = 81 for UBQLN4 siRNA # 2 . P ‐ values were calculated by Welch ' s t - between siRNA ‐ transfected respective control condition ( * P < 0 . 01 ) .

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(B, C) Puromycin-treated cells with UBQLN4 siRNA contain increased number of ALIS. At 72 h after transfection of the two distinct siRNA duplexes for UBQLN4 (UBQLN4 siRNA#1 and #2) or control siRNA (5 nM each), the cells were treated with 5 µg/mL puromycin for 2 h, and then subjected to immunostaining with an anti-polyubiquitin FK2 antibody (shown in green, B) as in (A). The number of ALIS was determined. The quantified data in (C) represent mean ± SEM. n = 76 cells for control siRNA, n = 89 cells for UBQLN4 siRNA#1, and n = 81 cells for UBQLN4 siRNA #2. P‐values were calculated by Welch's t-test between the siRNA‐transfected and the respective control condition (*P < 0.01).


( D ) Depletion of UBQLN4 makes HeLa more sensitive to puromycin - induced . After 48 h of UBQLN4 siRNA treatment , were treated with 5 µg / mL puromycin for 15 . 5 h then viability was measured . data represent mean ± SD calculated from 3 independent experiments ( n = 3 ) . P ‐ values were calculated by Student ' s t - compared with puromycin - treated control siRNA ( * P < 0 . 01 ) .

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(D) Depletion of UBQLN4 makes HeLa cells more sensitive to puromycin-induced cell death. After 48 h of UBQLN4 siRNA treatment, the cells were treated with 5 µg/mL puromycin for 15.5 h and then viability was measured. The data represent mean ± SD calculated from 3 independent experiments (n=3). P‐values were calculated by Student's t-test compared with puromycin-treated control siRNA cells (*P < 0.01).


( B ) IL - 2Rα ΔSS mislocalized to fraction . HeLa , expressing Flag - tagged IL - 2Rα WT or ΔSS , were fractionated to ( Cyt . ) ( Mem . ) fractions . Tubulin was used as a marker , while calnexin was used for ER fraction .

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(B) IL-2Rα ΔSS mislocalized to the cytosolic fraction. HeLa cells, expressing Flag-tagged IL-2Rα WT or ΔSS, were fractionated to the cytosolic (Cyt.) and membrane (Mem.) fractions. Tubulin was used as a cytoplasmic marker, while calnexin was used for the ER membrane fraction.


( C ) IL - 2Rα ΔSS was not glycosylated . Flag - tagged IL - 2Rα WT or ΔSS was expressed in HeLa , immunoprecipitated with an anti - Flag antibody . precipitates were incubated with ( + ) or without ( - ) 10 unit of deglycosylation enzyme PNGase F for 2 h , subjected to western blot with an anti - Flag antibody . Glycosylated non - glycosylated signals are indicated .

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(C) IL-2Rα ΔSS was not glycosylated. Flag-tagged IL-2Rα WT or ΔSS proteins was expressed in HeLa cells, and immunoprecipitated with an anti-Flag antibody. The precipitates were incubated with (+) or without (-) 10 unit of the deglycosylation enzyme PNGase F for 2 h, and subjected to western blot analysis with an anti-Flag antibody. Glycosylated and non-glycosylated signals are indicated.


( D ) glycosylated form of IL - 2Rα WT is a stable , while mislocalized IL - 2Rα ΔSS is degraded rapidly via its TMD . A series of IL - 2Ra deletion were expressed in HeLa then chased with 20 μg / mL CHX for indicated periods . Actin was used as a loading control . right panel is a quantified graph of left blot signals represent mean ± SD calculated from 4 independent experiments ( n = 4 ) .

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(D) The glycosylated form of IL-2Rα WT is a stable protein, while mislocalized IL-2Rα ΔSS is degraded rapidly via its TMD. A series of IL-2Ra deletion proteins were expressed in HeLa cells and then chased with 20 μg/mL CHX for the indicated periods. Actin was used as a loading control. The right panel is a quantified graph of the left blot signals represent mean ± SD calculated from 4 independent experiments (n=4).


( E ) IL - 2Rα ΔSS is degraded in a proteasome - dependent manner . HeLa expressing Flag - tagged IL - 2Rα ΔSS were treated with 20 μg / mL CHX as well as 0 . 1 % DMSO ( DM . ) , 10 μM MG - 132 ( MG . ) , 2 μM Bortezomib ( Bor . ) , 10 μM Leupeptin ( Leu . ) , 0 . 1 μM Bafilomycin A1 ( Baf . ) , or 10 μg / ml Pepstatin A ( pep . A ) / E64d for indicated periods . right panel indicates that quantified data of left blot signals represent mean ± SD calculated from 3 independent experiments ( n = 3 ) .

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(E) IL-2Rα ΔSS is degraded in a proteasome-dependent manner. HeLa cells expressing Flag-tagged IL-2Rα ΔSS were treated with 20 μg/mL CHX as well as 0.1% DMSO (DM.), 10 μM MG-132 (MG.), 2 μM Bortezomib (Bor.), 10 μM Leupeptin (Leu.), 0.1 μM Bafilomycin A1 (Baf.), or 10 μg/ml Pepstatin A (pep. A)/E64d for the indicated periods. The right panel indicates that the quantified data of the left blot signals represent mean ± SD calculated from 3 independent experiments (n=3).


( F ) Flag - tagged IL - 2Rα ΔSS mutant T7 - tagged ubiquitin were co - expressed in HeLa were treated with ( + ) or without ( - ) 10 μM MG - 132 . After 4 h , Flag - precipitates were blotted with anti - T7 anti - Flag antibodies .

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(F) The Flag-tagged IL-2Rα ΔSS mutant and T7-tagged ubiquitin were co-expressed in HeLa cells and the cells were treated with (+) or without (-) 10 μM MG-132. After 4 h, Flag-precipitates were blotted with anti-T7 and anti-Flag antibodies.


( G ) HeLa expressing Flag - tagged IL - 2RaWT , ΔSS , ΔSSΔTM were treated with ( + ) or without ( - ) 10 μM MG - 132 . At 4 h after MG - 132 treatment , were lysed analyzed by immunoblotting using indicated antibodies . Note that a higher migrating form of non - glycosylated ( thus defective ) IL - 2Ra WT ( indicated by arrowhead ) is detected strongly in presence of MG - 132 .

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(G) HeLa cells expressing Flag-tagged IL-2RaWT, ΔSS, and ΔSSΔTM proteins were treated with (+) or without (-) 10 μM MG-132. At 4 h after MG-132 treatment, the cells were lysed and analyzed by immunoblotting using the indicated antibodies. Note that a higher migrating form of non-glycosylated (and thus defective) IL-2Ra WT (indicated by the arrowhead) is detected strongly in the presence of MG-132.


( A , B ) IL - 2Rα ΔSS is stabilized in UBQLN4 - knockdown . HeLa were transfected with three distinct siRNA duplexes for UBQLN4 ( UBQLN4 siRNA # 1 ~ # 3 ) or control siRNA . At 48 h after siRNA transfection , Flag tagged - IL - 2Rα ΔSS was expressed in . At 24 h after IL - 2RaSS transfection , were chased with 20 μg / mL CHX harvested at indicated time after CHX addition . Anti - Flag signals in control or UBQLN4 siRNA - treated ( siRNA # 1 ~ # 3 ) were quantified at indicated time points . data represent mean ± calculated from 4 independent experiments ( n = 4 ) . UBQLN4 signal is marked by an arrowhead , while band just below it ( indicated by an asterisk ) is a non - specific band , since this signal was never affected by any of UBQLN4 siRNAs . Actin was used as a loading control .

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(A, B) IL-2Rα ΔSS is stabilized in UBQLN4-knockdown cells. HeLa cells were transfected with three distinct siRNA duplexes for UBQLN4 (UBQLN4 siRNA#1~#3) or control siRNA. At 48 h after siRNA transfection, Flag tagged-IL-2Rα ΔSS was expressed in the cells. At 24 h after IL-2RaSS transfection, the cells were chased with 20 μg/mL CHX and harvested at the indicated time after CHX addition. Anti-Flag signals in the control or UBQLN4 siRNA-treated cells (siRNA#1~#3) were quantified at the indicated time points. The data represent mean ± SEM calculated from 4 independent experiments (n=4). The UBQLN4 signal is marked by an arrowhead, while the band just below it (indicated by an asterisk) is a non-specific band, since this signal was never affected by any of the UBQLN4 siRNAs. Actin was used as a loading control.


( C ) Polyubiquitin modification of IL - 2Rα ΔSS was not diminished , but rather strengthened , in UBQLN4 knockdown . At 48 h after transfection of HeLa with siRNA for UBQLN4 or BAG6 , Flag - tagged IL - 2Rα ΔSS T7 - tagged ubiquitin were expressed with ( + ) or without ( - ) 10 mM MG - 132 . From these , Flag - tagged IL - 2Rα ΔSS was affinity - purified using Hot lysis , Flag - precipitates were blotted with an anti - T7 antibody to detect polyubiquitination of IL - 2Rα ΔSS .

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(C) Polyubiquitin modification of IL-2Rα ΔSS was not diminished, but rather strengthened, in UBQLN4 knockdown cells. At 48 h after transfection of HeLa cells with siRNA for UBQLN4 or BAG6, Flag-tagged IL-2Rα ΔSS and T7-tagged ubiquitin were expressed with (+) or without (-) 10 mM MG-132. From these cells, Flag-tagged IL-2Rα ΔSS was affinity-purified using Hot lysis analysis, and Flag-precipitates were blotted with an anti-T7 antibody to detect polyubiquitination of IL-2Rα ΔSS.


( A ) A series of Flag - tagged truncated mutants of IL - 2Ra substrates were expressed in HeLa with T7 - tagged UBQLN4 treated with ( + ) or without ( - ) 10 µM MG - 132 for 4 h . Flag - IL - 2Ra substrates were immunoprecipitated their co - precipitation with UBQLN4 was analyzed .

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(A) A series of Flag-tagged truncated mutants of IL-2Ra substrates were expressed in HeLa cells with T7-tagged UBQLN4 and treated with (+) or without (-) 10 µM MG-132 for 4 h. Flag-IL-2Ra substrates were immunoprecipitated and their co-precipitation with UBQLN4 was analyzed.


( B ) T7 - UBQLN4 was immunoprecipitated , its interactions with a series of IL - 2Ra were detected .

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(B) T7-UBQLN4 was immunoprecipitated, and its interactions with a series of IL-2Ra proteins were detected.


( B ) UBQLN4 dominantly co - precipitates IL - 2Rα ΔSS over UBQLN1 . Precipitates of Flag - tagged UBQLN1 or UBQLN4 from HeLa lysates were probed with IL - 2Rα ΔSS .

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(B) UBQLN4 dominantly co-precipitates IL-2Rα ΔSS over UBQLN1. Precipitates of Flag-tagged UBQLN1 or UBQLN4 from HeLa cell lysates were probed with IL-2Rα ΔSS.


( C ) complete acid sequences of human UBQLN1 UBQLN4 were analyzed using disorder / order structure prediction program PONDR - FIT . STI - II region of UBQLN4 is indicated by red box , that of UBQLN1 is boxed blue . Note that disorder tendency of UBL , STI - I , UBA regions is nearly identical between UBQLN1 UBQLN4 ( indicated by black green boxes ) , while that of STI - II region is quite distinct , STI - II of UBQLN4 shows a high probability of an ordered structure . numbers denote corresponding acid positions in these .

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(C) The complete amino acid sequences of human UBQLN1 and UBQLN4 were analyzed using the disorder/order structure prediction program PONDR-FIT. The STI-II region of UBQLN4 is indicated by the red box, and that of UBQLN1 is boxed blue. Note that the disorder tendency of the UBL, STI-I, and UBA regions is nearly identical between UBQLN1 and UBQLN4 (indicated by black and green boxes), while that of the STI-II region is quite distinct, and the STI-II of UBQLN4 shows a high probability of an ordered structure. The numbers denote the corresponding amino acid positions in these proteins.


( E ) Substitution of STI - II of UBQLN4 with that of UBQLN1 ( designated as 4 - STI - II - 1 mutant ) abolished its high affinity to IL - 2Rα ΔSS client .

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(E) Substitution of the STI-II sequence of UBQLN4 with that of UBQLN1 (designated as the 4-STI-II-1 mutant protein) abolished its high affinity to the IL-2Rα ΔSS client protein.


( F ) Substitution of STI - II of UBQLN1 with that of UBQLN4 ( designated as 1 - STI - II - 4 ) did not enable UBQLN1 to recognize IL - 2Rα ΔSS .

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(F) Substitution of the STI-II sequence of UBQLN1 with that of UBQLN4 (designated as the 1-STI-II-4) did not enable UBQLN1 to recognize IL-2Rα ΔSS.


( B , C D ) wild - type ( WT ) form of Flag - tagged UBQLN4 its truncated derivatives were expressed in HeLa with T7 - tagged IL - 2RαΔSS ( B C ) . At 4 h after addition of 10 μM MG - 132 , UBQLN4 was immunoprecipitated with an anti - Flag antibody precipitates were blotted with an anti - T7 antibody .

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(B, C and D) The wild-type (WT) form of Flag-tagged UBQLN4 and its truncated derivatives were expressed in HeLa cells with T7-tagged IL-2RαΔSS (B and C). At 4 h after the addition of 10 μM MG-132, UBQLN4 was immunoprecipitated with an anti-Flag antibody and the precipitates were blotted with an anti-T7 antibody.


( B , C D ) wild - type ( WT ) form of Flag - tagged UBQLN4 its truncated derivatives were expressed in HeLa with T7 - tagged IL - 2Rα ΔSS ( B C ) T7 - tagged ubiquitin ( D ) . At 4 h after addition of 10 μM MG - 132 , UBQLN4 was immunoprecipitated with an anti - Flag antibody precipitates were blotted with an anti - T7 antibody .

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(B, C and D) The wild-type (WT) form of Flag-tagged UBQLN4 and its truncated derivatives were expressed in HeLa cells with T7-tagged IL-2Rα ΔSS (B and C) and T7-tagged ubiquitin (D). At 4 h after the addition of 10 μM MG-132, UBQLN4 was immunoprecipitated with an anti-Flag antibody and the precipitates were blotted with an anti-T7 antibody.


( E ) GST - tagged purified UBQLN4 its truncated derivatives ( ΔUBA ΔSTI - II ) were incubated with K48 - linked polyubiquitin chains . After in vitro GST pull - down , precipitates were probed with an anti - polyubiquitin ( FK2 ) antibody .

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(E) GST-tagged and purified UBQLN4 and its truncated derivatives (ΔUBA and ΔSTI-II) were incubated with K48-linked polyubiquitin chains. After in vitro GST pull-down, the precipitates were probed with an anti-polyubiquitin (FK2) antibody.


( A ) UBQLN4 interacts with non - glycosylated ( thus defective ) IL - 2Ra WT in SRP54 - knockdown . After treatment of HeLa with SRP54 siRNA ( 10 nM ) for 48 h , Flag - tagged IL - 2RaWT T7 - tagged UBQLN4 were expressed , were treated with ( + ) or without ( - ) 10 µM MG - 132 at 4 h before harvesting . UBQLN4 was affinity - purified from extracts probed with an anti - Flag antibody . Note that of non - glycosylated IL - 2RaWT ( indicated as Defective ) increased , while glycosylated form of this ( indicated as Assembled ) decreased in SRP54 knockdown .

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(A) UBQLN4 interacts with non-glycosylated (and thus defective) IL-2Ra WT in SRP54-knockdown cells. After treatment of HeLa cells with SRP54 siRNA (10 nM) for 48 h, Flag-tagged IL-2RaWT and T7-tagged UBQLN4 proteins were expressed, and the cells were treated with (+) or without (-) 10 µM MG-132 at 4 h before harvesting. UBQLN4 was affinity-purified from cell extracts and probed with an anti-Flag antibody. Note that the levels of non-glycosylated IL-2RaWT (indicated as Defective) increased, while the glycosylated form of this protein (indicated as Assembled) decreased in SRP54 knockdown cells.


( B , C ) SRP54 knockdown stimulates polyubiquitinated co - precipitation of UBQLN4 . Flag - tagged UBQLN4 T7 - tagged ubiquitin were expressed in SRP54 siRNA - treated , Flag precipitates were probed with an anti - T7 antibody to detect polyubiquitinated clients co - precipitation with UBQLN4 under no addition of MG - 132 ( B ) . Note that interaction of UBQLN4 with polyubiquitinated substrates was further increased in SRP54 knockdown treated with MG - 132 for 4 h before harvesting ( C ) .

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(B, C) SRP54 knockdown stimulates polyubiquitinated proteins co-precipitation of UBQLN4. Flag-tagged UBQLN4 and T7-tagged ubiquitin were expressed in SRP54 siRNA-treated cells, and Flag precipitates were probed with an anti-T7 antibody to detect polyubiquitinated clients co-precipitation with UBQLN4 under no addition of MG-132 (B). Note that the interaction of UBQLN4 with polyubiquitinated substrates was further increased in SRP54 knockdown cells treated with MG-132 for 4 h before harvesting (C).


( A ) expression of FUS does not alter expression of VAPB , PTPIP51 or mitofusin - 2 ( MFN2 ) in transfected NSC34 . Immunoblots of NSC34 transfected with EGFP as a control ( CTRL ) , or wild - type or mutant EGFP - FUS . Transfected were purified via EGFP using a sorter samples probed on immunoblots as indicated . On FUS immunoblot , samples were probed with FUS antibody to show endogenous transfected ; tubulin is shown as a loading control .

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(A) expression of FUS does not alter expression of VAPB, PTPIP51 or mitofusin-2 (MFN2) in transfected NSC34 cells. Immunoblots of NSC34 cells transfected with EGFP as a control (CTRL), or wild-type or mutant EGFP-FUS. Transfected cells were purified via EGFP using a cell sorter and the samples probed on immunoblots as indicated. On the FUS immunoblot, samples were probed with FUS antibody to show endogenous and transfected proteins; tubulin is shown as a loading control.


( B ) representative electron microscopy of ER - mitochondria associations in NSC34 transfected with control EGFP vector ( CTRL ) , EGFP - FUS , EGFP - FUSR521C or EGFP - FUSR518K as indicated ; arrowheads with loops show regions of association . Scale bar = 200 nm . Bar chart shows % of mitochondrial surface closely apposed to ER in different samples . Data were analysed by one - way of variance ( ANOVA ) followed by Tukey ' s multiple comparison . N = 27 - 30 247 - 424 mitochondria , error bars are s . e . m . ; * * * p < 0 . 001 .

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(B) representative electron microscopy of ER-mitochondria associations in NSC34 cells transfected with control EGFP vector (CTRL), EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K as indicated; arrowheads with loops show regions of association. Scale bar=200 nm. Bar chart shows % of the mitochondrial surface closely apposed to ER in the different samples. Data were analysed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. N=27-30 cells and 247-424 mitochondria, error bars are s.e.m.; ***p<0.001.


( C D ) siRNA loss of FUS does not affect ER - mitochondria associations or alter expression of VAPB , PTPIP51 or mitofusin - 2 ( MFN2 ) in NSC34 . ( C ) immunoblots of either mock transfected or treated with control ( CTRL ) or FUS siRNAs ; GAPDH is shown as a loading control .

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(C and D) siRNA loss of FUS does not affect ER-mitochondria associations or alter expression of VAPB, PTPIP51 or mitofusin-2 (MFN2) in NSC34 cells. (C) immunoblots of cells either mock transfected or treated with control (CTRL) or FUS siRNAs; GAPDH is shown as a loading control.


( C D ) siRNA loss of FUS does not affect ER - mitochondria associations or alter expression of VAPB , PTPIP51 or mitofusin - 2 ( MFN2 ) in NSC34 . ( D ) representative electron microscopy of ER - mitochondria associations in control ( CTRL ) FUS siRNA treated . Arrowheads with loops show regions of association . Scale bar = 200 nm . Data analysed by unpaired t - . N = 27 - 28 193 - 202 mitochondria .

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(C and D) siRNA loss of FUS does not affect ER-mitochondria associations or alter expression of VAPB, PTPIP51 or mitofusin-2 (MFN2) in NSC34 cells. (D) representative electron microscopy of ER-mitochondria associations in control (CTRL) and FUS siRNA treated cells. Arrowheads with loops show regions of association. Scale bar=200 nm. Data analysed by unpaired t-test. N=27-28 cells and 193-202 mitochondria.


( A ) FU - induced reductions in ER - mitochondria associations can be detected using SIM . NSC34 were transfected with either EGFP control vector , EGFP - FUS , EGFP - FUSR521C or EGFP - FUSR518K immunostained for TOM20 PDI to label mitochondria ( Mito ) ER respectively ; FUS were detected via their EGFP tags . Merge ( ZOOM ) show zoomed of boxed regions co - localisation shows co - localised pixels . Scale bar = 2 m . Bar chart shows ER - mitochondria co - localisation ( Manders coefficient ) normalized to control in different samples . Data were analysed by one - way ANOVA with Tukey ' s post - hoc . 10 - 14 were analysed per condition from 3 independent experiments ; error bars are s . e . m . , * * p < 0 . 01 * * * p < 0 . 001 .

O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-GENEPROD O O O O B-GENEPROD O O O O O 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-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FU-induced reductions in ER-mitochondria associations can be detected using SIM. NSC34 cells were transfected with either EGFP control vector, EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and immunostained for TOM20 and PDI to label mitochondria (Mito) and ER respectively; FUS were detected via their EGFP tags. Merge (ZOOM) show zoomed images of boxed regions and co-localisation shows co-localised pixels. Scale bar=2 m. Bar chart shows ER-mitochondria co-localisation (Manders coefficient) normalized to control in the different samples. Data were analysed by one-way ANOVA with Tukey's post-hoc test. 10-14 cells were analysed per condition from 3 independent experiments; error bars are s.e.m., **p<0.01 and ***p<0.001.


( B ) ER - mitochondria associations VAPB - PTPIP51 interaction are disrupted by wild - type ALS / FTD mutant FUS . NSC34 were transfected with EGFP control vector ( CTRL ) , EGFP - FUS , EGFP - FUSR521C or EGFP - FUSR518K proximity ligation assays performed using VAPB PTPIP51 antibodies . FUS were detected via their EGFP tags . Scale bar = 10 m . Bar chart shows relative number of proximity ligation signals / . Data were analysed by one - way ANOVA Tukey ' s post - hoc ; n = 47 - 53 from 5 experiments . Error bars are s . e . m . ; * * * p < 0 . 001 .

O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O 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) ER-mitochondria associations and the VAPB-PTPIP51 interaction are disrupted by wild-type and ALS/FTD mutant FUS. NSC34 cells were transfected with EGFP control vector (CTRL), EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and proximity ligation assays performed using VAPB and PTPIP51 antibodies. FUS were detected via their EGFP tags. Scale bar=10 m. Bar chart shows relative number of proximity ligation assay signals/cell. Data were analysed by one-way ANOVA and Tukey's post-hoc test; n=47-53 cells from 5 experiments. Error bars are s.e.m.; ***p<0.001.


( C ) overexpression of FUS reduces binding of VAPB to PTPIP51 in transfected . were transfected as indicated with either control empty vector ( CTRL ) , HA - PTPIP51 + CTRL , or HA - PTPIP51 + either EGFP - FUS , EGFP - FUSR521C or EGFP - FUSR518K . PTPIP51 was immunoprecipitated using HA tag amounts of endogenous bound VAPB detected by immunoblotting . Both inputs immunoprecipitations ( IP ) are shown no immunoprecipitating VAPB signals were obtained in absence of HA - PTPIP51 . Bar chart shows relative of VAPB bound to PTPIP51 in immunoprecipitations following of signals from immunoblots . VAPB signals were normalized to immunoprecipitated PTPIP51 - HA signals .

O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O

(C) overexpression of FUS reduces the binding of VAPB to PTPIP51 in transfected cells. Cells were transfected as indicated with either control empty vector (CTRL), HA-PTPIP51+CTRL, or HA-PTPIP51+ either EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K. PTPIP51 was immunoprecipitated using the HA tag and the amounts of endogenous bound VAPB detected by immunoblotting. Both inputs and immunoprecipitations (IP) are shown and no immunoprecipitating VAPB signals were obtained in the absence of HA-PTPIP51. Bar chart shows relative levels of VAPB bound to PTPIP51 in the immunoprecipitations following quantification of signals from immunoblots. VAPB signals were normalized to immunoprecipitated PTPIP51-HA signals.


( A ) overexpression of FUS does not affect expression of VAPB , PTPIP51 or mitofusin - 2 . Immunoblots of spinal cord from three 10 week old FUS transgenic mice three - matched are shown ; FUS was detected via its HA - tag . Tubulin was used as a loading control .

O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O B-GENEPROD O B-ORGANISM O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O

(A) overexpression of FUS does not affect expression of VAPB, PTPIP51 or mitofusin-2. Immunoblots of spinal cord proteins from three 10 week old FUS transgenic mice and three age-matched littermates are shown; FUS was detected via its HA-tag. Tubulin was used as a loading control.


( B ) representative electron microscopy of ER - mitochondria associations in lumbar spinal cord motor neurons of FUS transgenic mice their non - transgenic ; arrowheads with loops show regions of association . Scale bar is 200 nm . Bar chart shows % of mitochondrial surface closely apposed to ER in two samples . Data were analysed by unpaired t - . N = 67 - 88 438 - 749 mitochondria , error bars are s . e . m . ; * * * p < 0 . 001 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-ORGANISM O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

(B) representative electron microscopy of ER-mitochondria associations in lumbar spinal cord motor neurons of FUS transgenic mice and their non-transgenic littermates; arrowheads with loops show regions of association. Scale bar is 200 nm. Bar chart shows % of mitochondrial surface closely apposed to ER in the two samples. Data were analysed by unpaired t-test. N=67-88 cells and 438-749 mitochondria, error bars are s.e.m.; ***p<0.001.


( C ) ER - mitochondria associations VAPB - PTPIP51 interaction are disrupted in lumbar motor neurons in spinal cords of FUS transgenic mice . Representative of proximity ligation signals in 11 week old FUS non - transgenic ( NTg ) littermate mice . Data were analysed by unpaired t - ; N = 40 from 3 FUS 3 non - transgenic ( 10 ) .

O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-TISSUE I-TISSUE O B-GENEPROD O B-ORGANISM O O O O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O

(C) ER-mitochondria associations and the VAPB-PTPIP51 interaction are disrupted in lumbar motor neurons in spinal cords of FUS transgenic mice. Representative images of proximity ligation signals in 11 week old FUS and non-transgenic (NTg) littermate mice. Data were analysed by unpaired t-test; N=40 cells from 3 FUS and 3 non-transgenic littermates (age 10 weeks).


( A B ) FUS disrupts Ca2 + homeostasis . HEK293 were transfected with M3R either control vector ( CTRL ) , FUS , FUSR521C or FUSR518K as indicated . Release of ERCa2 + was induced by treatment of with OxoM . ( A ) shows cytosolicCa2 + with representative Fluo4 traces on left normalized peak values on right . Fluo4 shows a transient increase in cytosolicCa2 + upon OxoM treatment but compared to control , wild - type mutant FUS increase peak cytosolicCa2 + . ( B ) shows mitochondrialCa2 + with representative Rhod2 traces on left normalized peak values on right . Data were analysed by one - way ANOVA Tukey ' s post hoc . ( A ) N = 49 - 52 from 3 experiments ; ( B ) N = 50 - 52 from 5 experiments , error bars are s . e . m . ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O B-GENEPROD O O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FUS disrupts Ca2+ homeostasis. HEK293 cells were transfected with M3R and either control vector (CTRL), FUS, FUSR521C or FUSR518K as indicated. Release of ERCa2+ was induced by treatment of cells with OxoM. (A) shows cytosolicCa2+ levels with representative Fluo4 fluorescence traces on the left and normalized peak values on the right. Fluo4 fluorescence shows a transient increase in cytosolicCa2+ levels upon OxoM treatment but compared to control, wild-type and mutant FUS all increase peak cytosolicCa2+ levels. (B) shows mitochondrialCa2+ levels with representative Rhod2 fluorescence traces on the left and normalized peak values on the right. Data were analysed by one-way ANOVA and Tukey's post hoc test. (A) N=49-52 cells from 3 experiments; (B) N=50-52 cells from 5 experiments, error bars are s.e.m.; *p<0.05, **p<0.01, ***p<0.001.


( C ) FUS reduces mitochondrial ATP production . ATP were measured in NSC34 transfected with ATP indicator AT1 . 03 either control vector ( CTRL ) , HA - FUS , HA - FUSR521C or HA - FUSR518K . were imaged in time - lapse prior to after KCN treatment to inhibit oxidative phosphorylation . Representative traces of YFP / CFP ratios are shown for different samples ; initial YFP / CFP ratios prior to KCN treatment those after KCN treatment are indicated . fall in YFP / CFP ratios correlates with ATP produced by oxidative phosphorylation . Bar chart shows relative ATP produced by oxidative phosphorylation ( OXPHOS ) in different samples .

O O O B-GENEPROD O B-SUBCELLULAR B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-CELL_LINE O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

(C) FUS reduces mitochondrial ATP production. ATP levels were measured in NSC34 cells transfected with the ATP indicator AT1.03 and either control vector (CTRL), HA-FUS, HA-FUSR521C or HA-FUSR518K. Cells were imaged in time-lapse prior to and after KCN treatment to inhibit oxidative phosphorylation. Representative traces of YFP/CFP ratios are shown for the different samples; initial YFP/CFP ratios prior to KCN treatment and those after KCN treatment are indicated. The fall in YFP/CFP ratios correlates with ATP produced by oxidative phosphorylation. Bar chart shows relative ATP levels produced by oxidative phosphorylation (OXPHOS) in the different samples.


( A ) were transfected with either control vector ( CTRL ) , HA - FUS , HA - FUSR521C or HA FUSR518K samples probed on immunoblots for GSK - 3 phosphorylated on serine - 9 ( GSK - 3 - S9 ) , total GSK - 3 , FUS ( using FUS antibody ) tubulin as a loading control . Phosphorylation of GSK - 3 serine - 9 is principal mechanism for regulating its activity ; serine - 9 phosphorylation inhibits GSK - 3 activity . Bar chart shows relative of GSK - 3β serine - 9 phosphorylation following of signals from immunoblots normalization to total GSK - 3β signals . Data were analysed by one - way ANOVA Tukey ' s post hoc . N = 4 , error bars are s . e . m ; * p < 0 . 05 .

O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O 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) cells were transfected with either control vector (CTRL), HA-FUS, HA-FUSR521C or HA FUSR518K and the samples probed on immunoblots for GSK-3 phosphorylated on serine-9 (GSK-3-S9), total GSK-3, FUS (using FUS antibody) and tubulin as a loading control. Phosphorylation of GSK-3 serine-9 is the principal mechanism for regulating its activity; serine-9 phosphorylation inhibits GSK-3 activity. Bar chart shows relative levels of GSK-3β serine-9 phosphorylation following quantification of signals from immunoblots and normalization to total GSK-3β signals. Data were analysed by one-way ANOVA and Tukey's post hoc test. N=4, error bars are s.e.m; *p<0.05.


( B ) immunoblots of total serine - 9 phosphorylated GSK - 3 in spinal cords from three 10 week - old FUS transgenic mice their non - transgenic . Samples were probed for tubulin as a loading control . Bar chart shows relative of GSK - 3β serine - 9 phosphorylation following of signals from immunoblots normalization to total GSK - 3β signals . Data were analysed by unpaired t - , error bars are s . e . m . ; * p < 0 . 05 .

O O O B-EXP_ASSAY O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O 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) immunoblots of total and serine-9 phosphorylated GSK-3 in spinal cords from three 10 week-old FUS transgenic mice and their non-transgenic littermates. Samples were probed for tubulin as a loading control. Bar chart shows relative levels of GSK-3β serine-9 phosphorylation following quantification of signals from immunoblots and normalization to total GSK-3β signals. Data were analysed by unpaired t-test, error bars are s.e.m.; *p<0.05.


( C ) FUS activates GSK - 3 more potently than wild type FUS . were transfected with EGFP control , EGFP - FUS or EGFP - FUS lacking its C - terminal localization signal ( FUSC ) . Samples were probed on immunoblots for total GSK - 3 , GSK - 3 phosphorylated on serine - 9 , FUS ( using EGFP antibody ) tubulin as a loading control . Bar chart shows relative of GSK - 3β serine - 9 phosphorylation following of signals from immunoblots normalization to total GSK - 3β signals . Data were analysed by one - way ANOVA Tukey ' s post hoc . N = 3 , error bars are s . e . m . ; * * p < 0 . 01 , * * * p < 0 . 001 .

O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O 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) cytosolic FUS activates GSK-3 more potently than wild type FUS. Cells were transfected with EGFP control, EGFP-FUS or EGFP-FUS lacking its C-terminal nuclear localization signal (FUSC). Samples were probed on immunoblots for total GSK-3, GSK-3 phosphorylated on serine-9, FUS (using EGFP antibody) and tubulin as a loading control. Bar chart shows relative levels of GSK-3β serine-9 phosphorylation following quantification of signals from immunoblots and normalization to total GSK-3β signals. Data were analysed by one-way ANOVA and Tukey's post hoc test. N=3, error bars are s.e.m.; **p<0.01, ***p<0.001.


( D ) FUS reduces VAPB - PTPIP51 interaction more potently than wild type FUS . were transfected with PTPIP51 - HA either EGFP control , EGFP - FUS or EGFP - FUSC . PTPIP51 was immunoprecipitated using HA tag bound endogenous VAPB detected by immunoblotting . Both inputs immunoprecipitations ( IP ) are shown . FUS was detected using EGFP antibody . Bar chart shows relative of VAPB bound to PTPIP51 in immunoprecipitations following of signals from immunoblots . VAPB signals were normalized to immunoprecipitated PTPIP51 - HA signals .

O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O

(D) cytosolic FUS reduces the VAPB-PTPIP51 interaction more potently than wild type FUS. Cells were transfected with PTPIP51-HA and either EGFP control, EGFP-FUS or EGFP-FUSC. PTPIP51 was immunoprecipitated using the HA tag and bound endogenous VAPB detected by immunoblotting. Both inputs and immunoprecipitations (IP) are shown. FUS was detected using EGFP antibody. Bar chart shows relative levels of VAPB bound to PTPIP51 in the immunoprecipitations following quantification of signals from immunoblots. VAPB signals were normalized to immunoprecipitated PTPIP51-HA signals.


( A ) representative electron microscopy of ER - mitochondria associations in NSC34 treated with either vehicle or GSK - 3β inhibitors AR - A014418 ( 1 M ) or CT99021 ( 100 nM ) for 16 h . Arrowheads with loops show regions of association ; scale bar = 200 nm . Bar charts shows % of mitochondrial surface closely apposed to ER in different samples . Data were analysed by one - way ANOVA followed by Tukey ' s multiple comparison . N = 32 181 - 210 mitochondria . Error bars are s . e . m . ; * p < 0 . 05 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O 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-SUBCELLULAR O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O

(A) representative electron microscopy of ER-mitochondria associations in NSC34 cells treated with either vehicle or GSK-3β inhibitors AR-A014418 (1 M) or CT99021 (100 nM) for 16 h. Arrowheads with loops show regions of association; scale bar=200 nm. Bar charts shows % of the mitochondrial surface closely apposed to ER in the different samples. Data were analysed by one-way ANOVA followed by Tukey's multiple comparison test. N=32 cells and 181-210 mitochondria. Error bars are s.e.m.; *p<0.05.


( B ) VAPB - PTPIP51 proximity ligation assays of NSC34 treated with either vehicle 1 M AR - A014418 or 100 nM CT99021 for 16 h . were also stained for nuclei with DAPI . Bar chart shows relative number of proximity ligation signals / .

O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O

(B) VAPB-PTPIP51 proximity ligation assays of NSC34 cells treated with either vehicle 1 M AR-A014418 or 100 nM CT99021 for 16 h. Cells were also stained for nuclei with DAPI. Bar chart shows relative number of proximity ligation assay signals/cell.


Representative electron microscopy of ER - mitochondria associations in NSC34 transfected with EGFP - FUS , EGFP - FUSR521C or EGFP - FUSR518K treated with vehicle or 1 mM AR - A014418 for 16 hours . Arrowheads with loops show regions of association ; scale bar = 200 nm . Bar charts shows % of mitochondrial surface closely apposed to ER in different samples .

O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O

Representative electron microscopy of ER-mitochondria associations in NSC34 cells transfected with EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and treated with vehicle or 1 mM AR-A014418 for 16 hours. Arrowheads with loops show regions of association; scale bar=200 nm. Bar charts shows % of the mitochondrial surface closely apposed to ER in the different samples.


HEK293 were transfected with M3R either control vector ( CTRL ) , FUS , FUSR521C or FUSR518K then treated with vehicle ( DMSO ) or 1 M AR - A014418 for 16 hours as indicated . Release of ER Ca2 + was induced by treatment of with OxoM . Representative Rhod2 traces showing mitochondrial Ca2 + are shown along with bar chart displaying normalized peak values .

B-CELL_LINE O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O

HEK293 cells were transfected with M3R and either control vector (CTRL), FUS, FUSR521C or FUSR518K and then treated with vehicle (DMSO) or 1 M AR-A014418 for 16 hours as indicated. Release of ER Ca2+ was induced by treatment of cells with OxoM. Representative Rhod2 fluorescence traces showing mitochondrial Ca2+ are shown along with bar chart displaying normalized peak values.


( C ) Exemplary of COS - 7 expressing PEX - mRFP - FKBP before ( upper panels ) 30 minutes after ( middle panels ) rapalog addition in presence of a translocating motor - KIF1C - MDC - FRB a non - translocating motor - KIF3B - MDC - FRB . Yellow lines indicate COS7 outline . Lower panel represents overlay of sequential binarized color coded by time ( see also Appendix Figure S1 ) . Scale bar = 20

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

(C) Exemplary images of COS-7 cells expressing PEX-mRFP-FKBP before (upper panels) and 30 minutes after (middle panels) rapalog addition in the presence of a translocating motor - KIF1C-MDC-FRB and a non-translocating motor - KIF3B-MDC-FRB. Yellow lines indicate COS7 cell outline. Lower panel represents the overlay of sequential binarized images color coded by time (see also Appendix Figure S1). Scale bar = 20 µm


( D ) Plots representing time traces of R90 % for COS - 7 transfected with PEX - mRFP - FKBP KIF1C - MDC - FRB ( n = 13 ) or KIF3B - MDC - FRB ( n = 10 ) . R90 % is radius for each time point that includes 90 % of total . Error bars indicate .

O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O 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) Plots representing intensity time traces of R90% for COS-7 cells transfected with PEX-mRFP-FKBP and KIF1C-MDC-FRB (n =13) or KIF3B-MDC-FRB (n =10). R90% is the radius for each time point that includes 90% of the total fluorescent intensity. Error bars indicate SEM.


E ) Quantitative representation of average peroxisome displacement 30 min after addition of rapalog for COS - 7 transfected with kinesin constructs summarized in ( A ) . number of analyzed is summarized in Appendix Table S1 . Kinesins were considered translocators , if they displaced peroxisomes ≥ 5 µm ( marked with a blue dashed ) in 30 minutes after rapalog addition . Error bars indicate .

O O O O O O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O

E) Quantitative representation of average peroxisome displacement 30 min after addition of rapalog for COS-7 cells transfected with kinesin constructs summarized in (A). The number of analyzed cells is summarized in Appendix Table S1. Kinesins were considered translocators, if they displaced peroxisomes ≥ 5 µm (marked with a blue dashed line) in 30 minutes after rapalog addition. Error bars indicate SEM.


( F ) Quantitative representation of average dispersion speed of peroxisomes recruited to translocating kinesins as calculated from time traces of R90 % ( summarized in Appendix figure S2 , Appendix Table S1 ) . Error bars indicate .

O O O O O O O O O O B-SUBCELLULAR O O O O 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) Quantitative representation of average dispersion speed of peroxisomes recruited to translocating kinesins as calculated from time traces of R90% (summarized in Appendix figure S2, Appendix Table S1). Error bars indicate SEM.


( A ) Representative maximum projections of peroxisome distribution before after rapalog addition in DIV14 hippocampal neurons expressing PEX - mRFP - FKBP , KIF1C - MDC - FRB , KIF21A - MDC - FRB or KIF5B - MDC - FRB . of transfected is visualized using a BFP fill . Axons are marked with a blue . Arrows mark peroxisome targeting to dendrites ( gray ) axon ( blue ) 30 min after rapalog addition . Scale bar = 20 µm .

O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O O O O O O

(A) Representative maximum intensity projections of peroxisome distribution before and after rapalog addition in DIV14 hippocampal neurons expressing PEX-mRFP-FKBP, KIF1C-MDC-FRB, KIF21A-MDC-FRB or KIF5B-MDC-FRB. The morphology of transfected cell is visualized using a BFP fill. Axons are marked with a blue line. Arrows mark peroxisome targeting to dendrites (gray) and axon (blue) 30 min after rapalog addition. Scale bar = 20 µm.


( B ) Plots representing time traces of dendrites axons before after rapalog addition for KIF1C - MDC - FRB ( n = 6 neurons ) , KIF21A - MDC - FRB ( n = 4 ) KIF5B - MDC - FRB ( n = 8 ) . Traces were normalized to average before rapalog addition , to background .

O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O

(B) Plots representing intensity time traces of dendrites and axons before and after rapalog addition for KIF1C-MDC-FRB (n = 6 neurons), KIF21A-MDC-FRB (n = 4) and KIF5B-MDC-FRB (n = 8). Traces were normalized to the average intensity before rapalog addition, and to the background.


C ) Quantitative representation of of DIV14 neurons transfected as in ( A ) with constructs showed in Figure 1A , in which peroxisomes after addition of rapalog redistributed to either axons ( " a " , blue bars ) or to axons at least 2 dendrites ( " a + d " , red bars ) . Kinesins were considered " dendrite targeting " if dendrites were targeted in ≥ 50 % of are highlighted in red . Kinesin constructs are transfected as indicated ; number of analyzed is summarized in Appendix Table S1 . Error bars indicate

O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O 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 representation of percentage of DIV14 neurons transfected as in (A) with constructs showed in Figure 1A, in which peroxisomes after addition of rapalog redistributed to either axons ("a", blue bars) or to axons and at least 2 dendrites ("a+d", red bars). Kinesins were considered "dendrite targeting" if dendrites were targeted in ≥ 50% of cells and are highlighted in red. Kinesin constructs are transfected as indicated; the number of analyzed cells is summarized in Appendix Table S1. Error bars indicate SEM


( D ) Representative maximum projections of peroxisome distribution before after rapalog addition in DIV2 hippocampal neurons transfected as in ( A ) . Scale bars : 20

O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O

(D) Representative maximum intensity projections of peroxisome distribution before and after rapalog addition in DIV2 hippocampal neurons transfected as in (A). Scale bars: 20 µm


( E ) of DIV2 neurons transfected with KIF1C - MDC - FRB ( n = 9 neurons ) , KIF21A - MDC - FRB ( n = 14 ) KIF5B - MDC - FRB ( n = 14 ) in which peroxisomes redistributed into at least 2 neurites after addition of rapalog ( F ) of neurites of DIV2 neurons transfected as in ( E ) targeted with peroxisomes after addition of rapalog . Error bars indicate ; Scale bars : 20 µm .

O O O O O B-CELL_TYPE O O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O O

(E) Percentage of DIV2 neurons transfected with KIF1C-MDC-FRB (n=9 neurons), KIF21A-MDC-FRB (n=14) and KIF5B-MDC-FRB (n=14) in which peroxisomes redistributed into at least 2 neurites after addition of rapalog and (F) Percentage of neurites of DIV2 neurons transfected as in (E) targeted with peroxisomes after addition of rapalog. Error bars indicate SEM; Scale bars: 20 µm.


( A ) Representative confocal of a hippocampal neuron ( DIV14 ) immunostained for MAP2 , β - III - tubulin DCLK1 . Scale bar = 20

O O O O B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O

(A) Representative confocal images of a hippocampal neuron (DIV14) immunostained for MAP2, β-III-tubulin and DCLK1. Scale bar = 20 µm


( B ) of average of DCLK1 , MAP1A , MAP1B MAP2 to total tubulin immunofluorescence intensities in dendrites compared to axons . Per condition 6 - 9 were analyzed . Error bars indicate .

O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O

(B) Quantification of the average ratio of DCLK1, MAP1A, MAP1B and MAP2 to total tubulin immunofluorescence intensities in dendrites compared to axons. Per condition 6-9 cells were analyzed. Error bars indicate SEM.


( C ) Representative of DIV10 hippocampal neurons transfected for 4 days with plasmids encoding β - gal for visualizing transfected three different DCLK1 shRNAs or DCLK2 shRNA immunostained for β - gal ( red ) DCLK1 ( green ) .

O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O O

(C) Representative images of DIV10 hippocampal neurons transfected for 4 days with plasmids encoding β-gal for visualizing the transfected cells and three different DCLK1 shRNAs or DCLK2 shRNA and immunostained for β-gal (red) and DCLK1 (green).


( D ) Quantitative of normalized average of DCLK1 in regions of interest of DIV10 neurons transfected with DCLK1 sh # 1 ( n = 16 neurons ) , DCLK1 sh # 2 ( n = 16 ) DCLK1 sh # 3 ( n = 18 ) . Non - transfected were taken as a control ( n = 48 ) . N = 2 . Error bars indicate ; * * * - p < 0 . 001 ( 1 - way ANOVA followed by a Tukey ' s Multiple Comparison ) .

O O O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantitative analysis of normalized average intensity of DCLK1 in regions of interest of DIV10 neurons transfected with DCLK1 sh#1 (n = 16 neurons), DCLK1 sh#2 (n=16) and DCLK1 sh#3 (n=18). Non-transfected cells were taken as a control (n = 48). N=2. Error bars indicate SEM; *** - p < 0.001 (1-way ANOVA followed by a Tukey's Multiple Comparison Test).


( E ) Example of Western blot of extracts of DIV4 cortical neurons electroporated on DIV0 with pSUPER ( control ) or pSUPER encoding one of three DCLK1 shRNAs . of actin were used as an equal loading control .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O

(E) Example of Western blot analysis of protein extracts of DIV4 cortical neurons electroporated on DIV0 with pSUPER (control) or pSUPER encoding one of three DCLK1 shRNAs. Levels of actin were used as an equal loading control.


( F ) of of DCLK1 of samples represented in ( E ) . N = 3 , error bars indicate , * * * - p < 0 . 001 ( 1 - way ANOVA followed by a Tukey ' s Multiple Comparison ) .

O O O O O B-GENEPROD O O O O O O O O O O O O O O O O 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 protein levels of DCLK1 of samples represented in (E). N = 3, error bars indicate SEM, *** - p < 0.001 (1-way ANOVA followed by a Tukey's Multiple Comparison Test).


( G ) Representative maximum projections of peroxisome distribution in DIV14 neurons transfected with PEX - mRFP - FKBP , KIF1C ( MDC ) - FRB indicated shRNA before 30 minutes after rapalog addition . BFP fill was co - transected to visualize of transfected neuron . Axons are marked with a blue . Arrows mark dendrite ( gray ) axon ( blue ) targeting of peroxisomes after rapalog addition . Scale bar = 20

O O O O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O

(G) Representative maximum projections of peroxisome distribution in DIV14 neurons transfected with PEX-mRFP-FKBP, KIF1C (MDC)-FRB and the indicated shRNA before and 30 minutes after rapalog addition. BFP fill was co-transected to visualize the morphology of transfected neuron. Axons are marked with a blue line. Arrows mark dendrite (gray) and axon (blue) targeting of peroxisomes after rapalog addition. Scale bar = 20 µm


( H ) Quantitative representation of of DIV14 neurons transfected with PEX - RFP together with either KIF1C , KIF21A or KIF5B an indicated shRNA . as in ( A ) in which after addition of rapalog peroxisomes redistributed to axons ( " a " , blue bars ) , to both axons dendrites ( " a + d " , red bars ) or did not redistribute into any neuronal compartment ( " nt " - no targeting , gray bars ) . Per condition 19 - 44 neurons were analyzed ; N = 2 .

O O O O O O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O 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-CELL_TYPE O O O O O O O

(H) Quantitative representation of the percentage of DIV14 neurons transfected with PEX-RFP together with either KIF1C, KIF21A or KIF5B and an indicated shRNA. as in (A) in which after addition of rapalog peroxisomes redistributed to axons ("a", blue bars), to both axons and dendrites ("a+d", red bars) or did not redistribute into any neuronal compartment ("nt"-no targeting, gray bars). Per condition 19-44 neurons were analyzed; N = 2.


( A - G ) Representative frame ( A , D ) a kymograph ( B , F ) of a simultaneous two - color movie of a hippocampal neuron expressing NPY - mCherry either KIF1A - GFP ( A , B ) , BDNF - GFP ( D ) or TfR - GFP ( F ) . Scale bar = 20 µm ( A , D ) , 5 µm ( B , F ) . ( C ) of vesicle colocalization in described in ( A , B ) quantifications indicate % of KIF1Avesicles that are NPY positive - left bar quantified from of fixed presented in ( A ) right bar quantified from kymographs as presented in ( B ) ( only moving fraction ) . ( E ) of vesicle colocalization in described in ( D ) - left bar indicates % of NPYvesicles that are BDNF positive , right bar indicates % of BDNFvesicles that are NPY positive . ( G ) of vesicle colocalization in described in ( F ) . left bar indicates % of NPYvesicles that are TfR positive , right bar indicates % of TfR that are NPY positive .

O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O 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-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O

(A-G) Representative frame (A, D) and a kymograph (B, F) of a simultaneous two-color movie of a hippocampal neuron expressing NPY-mCherry and either KIF1A-GFP (A, B), BDNF-GFP (D) or TfR-GFP (F). Scale bar = 20 µm (A, D), 5 µm (B, F). (C) Quantification of vesicle colocalization in cells described in (A, B) The quantifications indicate the % of KIF1Avesicles that are NPY positive - left bar quantified from images of fixed cells presented in (A) and right bar quantified from kymographs as presented in (B) (only moving fraction). (E) Quantification of vesicle colocalization in cells described in (D) - left bar indicates the % of NPYvesicles that are BDNF positive, the right bar indicates the % of BDNFvesicles that are NPY positive. (G) Quantification of vesicle colocalization in cells described in (F). The left bar indicates the % of NPYvesicles that are TfR positive, the right bar indicates the % of TfR that are NPY positive.


( H , I ) of run lengths ( H ) velocities ( I ) of vesicles in dendrites of hippocampal neurons transfected at DIV10 - 14 for 4 days with either NPY - GFP ( n = 924 movements ) or TfR - GFP ( n = 286 ) or KIF1A - GFP ( n = 174 ) together with a marker BFP . N = 2 . Error bars indicate ; * * * p < 0 . 001 ( 1 - way ANOVA followed by a Tukey ' s Multiple Comparison ) .

O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H, I) Quantification of run lengths (H) and velocities (I) of vesicles in dendrites of hippocampal neurons transfected at DIV10-14 for 4 days with either NPY-GFP (n = 924 movements) or TfR-GFP (n = 286) or KIF1A-GFP (n = 174) together with a morphology marker BFP. N = 2. Error bars indicate SEM; *** p < 0.001 (1-way ANOVA followed by a Tukey's Multiple Comparison Test ).


( K ) Representative kymographs of NPY - GFP - labeled dense vesicle motility in dendrites of transfected neurons . DIV10 - 14 hippocampal neurons were cotransfected , for 4 days , with plasmids encoding NPY - GFP , BFP fill a KIF21 shRNA mix ( n = 30 ) , KIF1shRNA mix ( n = 31 ) , KIF1A shRNA ( n = 19 ) , KIF1B shRNA ( n = 19 ) or KIF1C shRNA ( n = 11 ) . pSUPER was used as a control ( n = 15 ) . Scale bar = 5 µm .

O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O B-SUBCELLULAR O O B-CELL_TYPE O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O 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) Representative kymographs of NPY-GFP-labeled dense core vesicle motility in primary dendrites of transfected neurons. DIV10-14 hippocampal neurons were cotransfected, for 4 days, with plasmids encoding NPY-GFP, BFP fill and a KIF21 shRNA mix (n = 30), KIF1shRNA mix (n = 31), KIF1A shRNA (n = 19), KIF1B shRNA (n = 19) or KIF1C shRNA (n = 11). pSUPER was used as a control (n = 15). Scale bar = 5 µm.


( L ) of average number of dense vesicle entries into dendrite in 100 s after photobleaching . N = 2 - 3 . Error bars indicate ; * * * - p < 0 . 001 ( 1 - way ANOVA followed by a Tukey ' s Multiple Comparison ) .

O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(L) Quantification of the average number of dense core vesicle entries into the primary dendrite in 100 s after photobleaching. N = 2-3. Error bars indicate SEM; *** - p < 0.001 (1-way ANOVA followed by a Tukey's Multiple Comparison Test).


( M ) Representative kymographs of TfR - GFP - labeled recycling endosome motility in dendrites . DIV10 - 14 Hippocampal neurons were cotransfected with TfR - GFP together with a BFP fill a KIF1shRNA mix ( n = 11 ) . pSUPER was used as a control ( n = 20 ) . Scale bar = 5 µm .

O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

(M) Representative kymographs of TfR-GFP-labeled recycling endosome motility in primary dendrites. DIV10-14 Hippocampal neurons were cotransfected with TfR-GFP together with a BFP fill and a KIF1shRNA mix (n = 11). pSUPER was used as a control (n = 20). Scale bar = 5 µm.


( N ) of average number of recycling endosome entries into dendrite in 50 s . N = 2 . Error bars indicate . ns - not significant ( Mann - Whitney ) .

O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O

(N) Quantification of the average number of recycling endosome entries into the primary dendrite in 50 s. N = 2. Error bars indicate SEM. ns - not significant (Mann-Whitney test).


( O ) Representative of hippocampal neurons ( DIV4 ) cotransfected with an empty pSUPER ( control ) or pSUPER encoding an indicated shRNA together with a GFP fill fixed at DIV8 immunostained for MAP2 sodium channels ( AIS ) . Scale bar = 50 µm .

O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O

(O) Representative images of hippocampal neurons (DIV4) cotransfected with an empty pSUPER (control) or pSUPER encoding an indicated shRNA together with a GFP fill fixed at DIV8 and immunostained for MAP2 and sodium channels (AIS). Scale bar = 50 µm.


( P ) Sholl of described in ( O ) Error bars indicate ; * - p < 0 . 05 , * * - p < 0 . 01 , * * * - p < 0 . 001 ( two - way ANOVA followed by Bonferroni ' s post hoc ) . ( R ) of total dendritic of described in ( O ) Error bars indicate ; * - p < 0 . 05 , * * - p < 0 . 01 * * * - p < 0 . 001 ( 1 - way ANOVA followed by a Tukey ' s Multiple Comparison ) . Per condition 21 - 24 neurons ( n ) were analyzed ; N = 2 .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O O O O O O O

(P) Sholl analysis of cells described in (O) Error bars indicate SEM; * - p < 0.05, ** - p < 0.01, and *** - p < 0.001 (two-way ANOVA test followed by Bonferroni's post hoc test). (R) Analysis of the total dendritic length of cells described in (O) Error bars indicate SEM; * - p < 0.05, ** - p < 0.01 and *** - p < 0.001 (1-way ANOVA followed by a Tukey's Multiple Comparison Test). Per condition 21-24 neurons (n) were analyzed; N = 2.


( B ) Representative kymographs of dense - vesicle motility in proximal dendrites of transfected neurons . DIV 10 - 14 hippocampal neurons were co - transfected for 4 days with NPY - GFP pSUPER ( control ) or pSUPER encoding DCLK1 shRNA in absence or presence of DCLK1 truncation constructs dense vesicle motility in dendrites was recorded during live - imaging . Scale bar = 5 µm .

O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(B) Representative kymographs of dense-core vesicle motility in proximal dendrites of transfected neurons. DIV 10-14 hippocampal neurons were co-transfected for 4 days with NPY-GFP and pSUPER (control) or pSUPER encoding DCLK1 shRNA in the absence or presence of DCLK1 truncation constructs and dense core vesicle motility in dendrites was recorded during live-cell imaging. Scale bar = 5 µm.


( C ) of average number of dense - vesicle entries into dendrites of neurons transfected as in ( B ) during 100 s of live recording after photobleaching . Per condition 10 - 32 were analyzed . Error bars indicate ; * * * - p < 0 . 001 ( Kruskal - Wallis followed by post hoc Dunn ' s ) . N = 2 .

O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-CELL_TYPE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O 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) Quantification of the average number of dense-core vesicle entries into dendrites of neurons transfected as in (B) during 100 s of the live recording after photobleaching. Per condition 10-32 cells were analyzed. Error bars indicate SEM; *** - p < 0.001 (Kruskal-Wallis test followed by post hoc Dunn's test). N = 2.


( D , E ) DIV10 - 14 hippocampal neurons were co - transfected for 4 days with either pSUPER ( control , n = 20 neurons ) or DCLK1 shRNA together with TfR - GFP ( n = 16 ) . ( D ) Representative kymographs of recycling endosome motility in dendrites . Scale bar = 5 µm . ( E ) of average number of recycling endosome entries into dendrites during 50 s of live recording after photobleaching . Error bars indicate ; ns - not significant ( Mann - Whitney ) . N = 2 .

O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

(D, E) DIV10-14 hippocampal neurons were co-transfected for 4 days with either pSUPER (control, n = 20 neurons) or DCLK1 shRNA together with TfR-GFP (n = 16).(D) Representative kymographs of recycling endosome motility in dendrites. Scale bar = 5 µm. (E) Quantification of the average number of recycling endosome entries into dendrites during 50 s of the live recording after photobleaching. Error bars indicate SEM; ns - not significant (Mann-Whitney test). N = 2.


( A ) Example of Western blot of DCLK1 DCX in extracts obtained from DIV1 , DIV7 , DIV14 DIV21 hippocampal neurons .

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(A) Example of Western blot analysis of DCLK1 and DCX levels in protein extracts obtained from DIV1, DIV7, DIV14 and DIV21 hippocampal neurons.


( B ) Representative image of a hippocampal neuron ( DIV14 ) immunostained for DCLK1 β - III - tubulin . Scale bar = 20 µm .

O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

(B) Representative image of a hippocampal neuron (DIV14) immunostained for DCLK1 and β-III-tubulin. Scale bar = 20 µm.


( C , D ) Representative image of a COS - 7 transfected with DCLK1 - GFP ( C ) or DCLK1 ( ΔKD ) - GFP ( D ) stained for detyrosinated , tyrosinated or α - tubulin . ( E ) Representative image of a COS - 7 transfected with DCLK1 - GFP TagRFP - EB3 recorded during live - imaging . Representative GFP was drawn ( over a region marked with boxes ) showing that DCLK1 - GFP colocalizes with EB3 - TagRFP - T . Scale bars = 20 um .

O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O

(C, D) Representative image of a COS-7 cell transfected with DCLK1-GFP (C) or DCLK1(ΔKD)-GFP (D) and stained for detyrosinated, tyrosinated or α-tubulin.(E) Representative image of a COS-7 cell transfected with DCLK1-GFP and TagRFP-EB3 and recorded during live-cell imaging. Representative GFP was drawn (over a region marked with white boxes) showing that DCLK1-GFP colocalizes with EB3-TagRFP-T. Scale bars = 20 um.


( A ) Extracts of DIV 7 hippocampal neurons electroporated with pSuper control or with three different DCLK1 shRNAs at DIV0 . Samples were analyzed by western blot with indicated antibodies .

O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O

(A) Extracts of DIV 7 hippocampal neurons electroporated with pSuper control or with three different DCLK1 shRNAs at DIV0. Samples were analyzed by western blot with indicated antibodies.


( B , E ) Representative of a hippocampal transfected at DIV10 with RFP ( to visualize transfected ) , pSUPER ( control ) or DCLK1 shRNA , fixed at DIV14 stained for EB3 . Black lines indicate neuronal soma outline . Scale bars = 10 µm .

O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O

(B, E) Representative images of a hippocampal cell transfected at DIV10 with RFP (to visualize transfected cells), pSUPER (control) or DCLK1 shRNA, fixed at DIV14 and stained for EB3. Black lines indicate the neuronal soma outline. Scale bars = 10 µm.


( C , D ) of number of EB3 comets in dendrite ( C ) in soma ( D ) . 25 - 26 neurons were analyzed per condition , N = 2 . Error bars indicate , * * * - p < 0 . 001 , * * - p < 0 , 01 ( unpaired t - ) .

O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O 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) Quantification of the number of EB3 comets in the dendrite (C) and in the soma (D). 25-26 neurons were analyzed per condition, N=2. Error bars indicate SEM, *** - p < 0.001, ** - p<0,01 (unpaired t-test).