( 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 .

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

(B, 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 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 .

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

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 ) 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 .

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

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 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 .

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

(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 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 ) .

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

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

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

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


( 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 ) .

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

(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 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 ) .

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

(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).


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 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 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 O O O O


( 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 ) .

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

(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 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 .

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

(B) 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 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 .

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

(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 ) 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 ) .

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

(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 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 ) .

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

(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 ) 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

(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 , and its interactions with a series of IL - 2Ra proteins were detected .

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

(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 cell lysates were probed with IL - 2Rα ΔSS .

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

(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 ) 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

(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 ( 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 .

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

(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 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 ) .

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

(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 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 .

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

(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 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 .

O 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 B-CONTROLLED_VAR 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

(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 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 .

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

(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 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 .

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

(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 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 .

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

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

(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 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 .

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

(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 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 .

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

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

(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 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 ) .

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

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

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

(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 ) 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 .

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

(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 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 .

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

(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 ) 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 .

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

(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 ) 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 .

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

(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 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 .

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 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

(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 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 .

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

(B) 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 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 .

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

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 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 .

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

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

(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 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 .

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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 SEM .

O O O O O O O O O O O O O O O O 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 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 .

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

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

(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 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

O O O O 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

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

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

(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 ) 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 .

O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O 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 images of a hippocampal neuron ( DIV14 ) immunostained for MAP2 , β - III - tubulin and DCLK1 . Scale bar = 20

O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR 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 ) 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 .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR 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 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 ) .

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

O O O O O 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

(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 ) 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 ) .

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

(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 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

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

(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 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 .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR 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 ) 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 ) .

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

(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 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 .

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

(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 ) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

(N) 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 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 .

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

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

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

(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 ) 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 .

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

O O O O O 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

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

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

(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 and β - III - tubulin . Scale bar = 20 µm .

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

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

(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 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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O 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 ) 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 ) .

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

(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).


( F - I ) Hippocampal neurons were co - transfected with MACF18 - GFP and pSUPER ( control ) or DCLK1 shRNA and the dynamics of microtubules in dendrites was traced using life - cell microscopy . ( F ) Representative kymographs of dynamic microtubules ends growth in dendrites with and without photoablation . Scale bar = 10 µm ( C ) . ( G , I ) Quantification of the fraction of MACF18 events moving retrogradely and anterogradely in a dendrite before ( G ) and after ( I ) photoablation . Eighteen neurons were analyzed per condition ; N = 2 . ( H ) Scheme of live - cell imaging after photoablation .

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

(F-I) Hippocampal neurons were co-transfected with MACF18-GFP and pSUPER (control) or DCLK1 shRNA and the dynamics of microtubules in dendrites was traced using life-cell microscopy. (F) Representative kymographs of dynamic microtubules ends growth in dendrites with and without photoablation. Scale bar = 10 µm (C). (G, I) Quantification of the fraction of MACF18 events moving retrogradely and anterogradely in a dendrite before (G) and after (I) photoablation. Eighteen neurons were analyzed per condition; N=2. (H) Scheme of live-cell imaging after photoablation.


( J , K ) Biotin pull - downs ( PD ) from extracts of HEK293 transfected with biotin - tagged DCLK1 and ( J ) KIF1A - MDC - HA - FRB , KIF1C - MDC - HA - FRB , KIF5B - MDC - HA - FRB constructs and probed for HA / DCLK1 , ( K ) different GFP - tagged KIF1A truncation constructs and probed for GFP / DCLK1 . The ratio input / pellet is 2 % for all pull - down experiments .

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

(J, K) Biotin pull-downs (PD) from extracts of HEK293 transfected with biotin-tagged DCLK1 and (J) KIF1A-MDC-HA-FRB, KIF1C-MDC-HA-FRB, KIF5B-MDC-HA-FRB constructs and probed for HA/DCLK1, (K) different GFP-tagged KIF1A truncation constructs and probed for GFP/DCLK1. The ratio input/pellet is 2% for all pull-down experiments.


( L ) GFP pull - down from extracts of HEK293 transfected with DCLK1 fragments and KIF1A - MDC - HA - FRB , KIF1C - MDC - HA - FRB and KIF1C - MD - HA and probed for HA / GFP . LacZ - HA was used as a negative control .

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

(L) GFP pull-down from extracts of HEK293 transfected with DCLK1 fragments and KIF1A-MDC-HA-FRB, KIF1C-MDC-HA-FRB and KIF1C-MD-HA and probed for HA/GFP. LacZ-HA was used as a negative control.


( A ) Exomer components localize to different extent to the TGN and to the cytoplasm . Fluorescence images of cells expressing GFP - tagged exomer components and TGN marker Sec7 - DsRed . The brightness and contrast were adjusted differently in the GFP and merge images . Bar , 5 µm . ( B ) Quantification of the relative TGN to cytoplasm concentration of the GFP - tagged ChAPs in the cell . The mean fluorescence at the TGN and cytoplasm was determined using the Icy imaging software . 20 - 30 individual cells were analysed . For statistical significance testing the Student ' s T test was used .

O O O O O O 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

(A) Exomer components localize to different extent to the TGN and to the cytoplasm. Fluorescence images of cells expressing GFP-tagged exomer components and TGN marker Sec7-DsRed. The brightness and contrast were adjusted differently in the GFP and merge images. Bar, 5 µm.(B) Quantification of the relative TGN to cytoplasm concentration of the GFP-tagged ChAPs in the cell. The mean fluorescence at the TGN and cytoplasm was determined using the Icy imaging software. 20-30 individual cells were analysed. For statistical significance testing the Student's T test was used.


( C ) FCS measures the in vivo mobility of exomer components in the cytoplasm . FCS analysis was performed on the GFP , Chs5 - GFP and GFP - tagged ChAPs in the wild - type background ( top ) and on GFP - tagged ChAPs in the Δchs5 background ( bottom ) . 10 - 20 FCS measurements were performed for each condition using different cells . The ACF of free GFP is fitted with a one - component anomalous diffusion model . The ACF of Chs5 in WT and ChAPs in WT and Δchs5 background is fitted with a two - component diffusion model . Representative ACF curves ( thin full lines ) and fits ( thick dashed lines ) are shown . Estimated diffusion correlation times , diffusion coefficients and fractions are shown in Table 1 . ( D ) Quantification of the Chs5 independent fraction of ChAPs present in the cytoplasm based on the measurements shown in ( C ) . ( E ) Comparison of the residuals of the one - component and two - component fit ( arrows ) .

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 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 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

(C) FCS measures the in vivo mobility of exomer components in the cytoplasm. FCS analysis was performed on the GFP, Chs5-GFP and GFP-tagged ChAPs in the wild-type background (top) and on GFP-tagged ChAPs in the Δchs5 background (bottom). 10-20 FCS measurements were performed for each condition using different cells. The ACF of free GFP is fitted with a one-component anomalous diffusion model. The ACF of Chs5 in WT and ChAPs in WT and Δchs5 background is fitted with a two-component diffusion model. Representative ACF curves (thin full lines) and fits (thick dashed lines) are shown. Estimated diffusion correlation times, diffusion coefficients and fractions are shown in Table 1.(D) Quantification of the Chs5 independent fraction of ChAPs present in the cytoplasm based on the measurements shown in (C).(E) Comparison of the residuals of the one-component and two-component fit (arrows).


( A ) Comparison of the dynamic behavior of the GFP - tagged Chs5 and Bch1 with the GFP - tagged Sec28 , Cop1 , Clc1 and Apm1 . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 .

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

(A) Comparison of the dynamic behavior of the GFP-tagged Chs5 and Bch1 with the GFP-tagged Sec28, Cop1, Clc1 and Apm1. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.


( B ) Binding kinetics of the GFP - tagged Chs5 and ChAPs at the TGN is different . Data processing was carried out as in ( A ) .

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

(B) Binding kinetics of the GFP-tagged Chs5 and ChAPs at the TGN is different. Data processing was carried out as in (A).


( C ) Quantification of the relative expression levels of the exomer components in GFP - tagged strains . A representative immunoblot of yeast lysates is shown ; Pgk1 serves as loading control . The asterisks depict the analyzed bands . The plot shows average and SD of three independent biological experiments . The relative expression levels are normalized to Chs6 ( depicted by the red 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 O O O O O O O O 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

(C) Quantification of the relative expression levels of the exomer components in GFP-tagged strains. A representative immunoblot of yeast lysates is shown; Pgk1 serves as loading control. The asterisks depict the analyzed bands. The plot shows average and SD of three independent biological experiments. The relative expression levels are normalized to Chs6 (depicted by the red line).


( A ) Chs5 is necessary for recruitment and stable binding of ChAPs at the TGN . Differential centrifugation of cell lysates obtained from Chs6 - GFP WT and Δchs5 strain . TCL , total cell lysate ; S10 , 10 , 000 g supernatant ; P10 , 10 , 000 g pellet ; S100 , 100 , 000 g supernatant ; P100 , 100 , 000 g pellet . Anp1 serves as the Golgi marker and Pgk1 as the cytoplasm marker . A representative immunoblot of three independent biological experiments is shown .

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

(A) Chs5 is necessary for recruitment and stable binding of ChAPs at the TGN. Differential centrifugation of cell lysates obtained from Chs6-GFP WT and Δchs5 strain. TCL, total cell lysate; S10, 10,000 g supernatant; P10, 10,000 g pellet; S100, 100,000 g supernatant; P100, 100,000 g pellet. Anp1 serves as the Golgi marker and Pgk1 as the cytoplasm marker. A representative immunoblot of three independent biological experiments is shown.


( B ) Diffusional behavior of GFP - tagged ChAPs in the cytoplasm ( open triangles , dashed line ) and at the TGN ( closed circles , full line ) . The mean of 10 - 20 FRAP measurements from different cells is shown . Calculated diffusion coefficients are shown in Expanded View Table 1 .

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

(B) Diffusional behavior of GFP-tagged ChAPs in the cytoplasm (open triangles, dashed line) and at the TGN (closed circles, full line). The mean of 10-20 FRAP measurements from different cells is shown. Calculated diffusion coefficients are shown in Expanded View Table 1.


( C ) Chs5 - GFP displays reduced membrane association in the absence of ChAPs . Differential centrifugation of cell lysates obtained from Chs5 GFP WT and ΔΔΔΔ strains . TCL , total cell lysate ; S10 , 10 , 000 g supernatant ; P10 , 10 , 000 g pellet ; S100 , 100 , 000 g supernatant ; P100 , 100 , 000 g pellet . Anp1 serves as the Golgi marker and Pgk1 as the cytoplasm marker . A representative immunoblot of three independent biological experiments is shown .

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

(C) Chs5-GFP displays reduced membrane association in the absence of ChAPs. Differential centrifugation of cell lysates obtained from Chs5 GFP WT and ΔΔΔΔ strains. TCL, total cell lysate; S10, 10,000 g supernatant; P10, 10,000 g pellet; S100, 100,000 g supernatant; P100, 100,000 g pellet. Anp1 serves as the Golgi marker and Pgk1 as the cytoplasm marker. A representative immunoblot of three independent biological experiments is shown.