( 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-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O

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


( A ) representative electron microscopy of ER - mitochondria associations in NSC34 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 B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-CELL_LINE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O

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


( B ) VAPB - PTPIP51 proximity ligation assays of NSC34 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-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR 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 .

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

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


( C ) Exemplary 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 B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

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


( D ) Plots representing 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 B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O 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 B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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


( B ) Plots representing 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 B-SUBCELLULAR O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 B-CELL_TYPE O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O

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


( E ) 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 B-CELL_TYPE O O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O

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


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

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


( D ) Quantitative 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-GENEPROD O O O O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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


( H ) Quantitative representation of 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 B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O

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


( A - G ) Representative frame ( A , D ) 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 B-EXP_ASSAY O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-GENEPROD O O

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


( H , I ) 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 B-SUBCELLULAR O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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


( L ) 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 B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

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


( N ) 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 B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O

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

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


( P ) Sholl 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 B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O

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


( B ) Representative kymographs of dense - 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 B-EXP_ASSAY O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O

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


( C ) 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 B-SUBCELLULAR O O B-SUBCELLULAR O B-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

(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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE 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 B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD 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 B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O

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


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

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

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


( B , E ) Representative 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 B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O

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


( C , D ) 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-GENEPROD O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD 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 B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O

(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 ) .

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(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 ) .

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(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 .

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(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 .

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(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 .

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(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.


( D ) Fluorescence images of cells expressing Chs5 - GFP and TGN marker Sec7 - DsRed in WT , ChAP and strains . Merged images depict the differences in Chs5 - GFP localization to the TGN , cytoplasm and distinct foci ( arrows ) in these strains . The brightness and contrast were adjusted differently in the merged images . Bar , 5 µm . ( E ) Co - localization analysis of Chs5 - GFP and TGN marker Sec7 - DsRed in WT , ChAP and strains . Mander 1 depicts the relative amount of the GFP signal co - localizing with the overall DsRed signal , and Mander 2 vice versa . Manders coefficients were obtained using the JACoP plug - in for ImageJ after background subtraction ( N = 20 ) and are given in Expanded View Table 2 .

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(D) Fluorescence images of cells expressing Chs5-GFP and TGN marker Sec7-DsRed in WT, ChAP and strains. Merged images depict the differences in Chs5-GFP localization to the TGN, cytoplasm and distinct foci (arrows) in these strains. The brightness and contrast were adjusted differently in the merged images. Bar, 5 µm.(E) Co-localization analysis of Chs5-GFP and TGN marker Sec7-DsRed in WT, ChAP and strains. Mander 1 depicts the relative amount of the GFP signal co-localizing with the overall DsRed signal, and Mander 2 vice versa. Manders coefficients were obtained using the JACoP plug-in for ImageJ after background subtraction (N = 20) and are given in Expanded View Table 2.


( F ) Binding kinetics of Chs5 - GFP at the TGN in WT , ChAP and strains . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 . The schematic cartoons depict the predicted majority of the analyzed complexes . The violet ovals represent Chs5 dimer and circles represent ChAPs : green : Bch1 , red : Bud7 , dark blue : Bch2 , light blue : Chs6 .

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(F) Binding kinetics of Chs5-GFP at the TGN in WT, ChAP and strains. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2. The schematic cartoons depict the predicted majority of the analyzed complexes. The violet ovals represent Chs5 dimer and circles represent ChAPs: green:Bch1, red:Bud7, dark blue:Bch2, light blue:Chs6.


( A - B ) Overexpression of Bch2 in absence of other ChAPs results in more efficient Chs5 recruitment and stabilization at the TGN . Fluorescence images of cells expressing Chs5 - GFP ( A ) and binding kinetics of Chs5 - GFP at the TGN ( B ) in WT , BCH2 and GPD - BCH2 strains . Bar , 5 µm . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 . The schematic cartoons depict the predicted majority of the analyzed complexes ; Chs5 dimer : violet ovals , Bch2 : dark blue circles .

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(A-B) Overexpression of Bch2 in absence of other ChAPs results in more efficient Chs5 recruitment and stabilization at the TGN. Fluorescence images of cells expressing Chs5-GFP (A) and binding kinetics of Chs5-GFP at the TGN (B) in WT, BCH2 and GPD-BCH2 strains. Bar, 5 µm. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2. The schematic cartoons depict the predicted majority of the analyzed complexes; Chs5 dimer:violet ovals, Bch2:dark blue circles.


( C - D ) Overexpression of Chs6 in absence of other ChAPs results in more efficient Chs5 recruitment and stabilization at the TGN . ( C ) Fluorescence images of cells expressing Chs5 - GFP in WT , CHS6 and GPD - CHS6 strains . Bar , 5 µm . ( D ) Binding kinetics of Chs5 - GFP at the TGN in WT , , CHS6 and GPD - CHS6 strains . Data processing was carried out as in ( A - B ) . The schematic cartoons depict the predicted majority of the analyzed complexes ; Chs5 dimer : violet ovals , Chs6 : light blue circles .

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(C-D) Overexpression of Chs6 in absence of other ChAPs results in more efficient Chs5 recruitment and stabilization at the TGN. (C) Fluorescence images of cells expressing Chs5-GFP in WT, CHS6 and GPD-CHS6 strains. Bar, 5 µm. (D) Binding kinetics of Chs5-GFP at the TGN in WT, , CHS6 and GPD-CHS6 strains. Data processing was carried out as in (A-B). The schematic cartoons depict the predicted majority of the analyzed complexes; Chs5 dimer: violet ovals, Chs6: light blue circles.


( E - G ) Overexpression of Chs6 rescues Chs3 export in absence of other ChAPs . ( E ) Fluorescence images of cells expressing Chs3 - GFP in WT , CHS6 and GPD - CHS6 strains . Arrows point to Chs3 - GFP signals at the bud neck . Bar , 5 µm . The inset represents a twofold magnification . ( F ) Quantification of phenotypes in ( E ) ; 100 small budded and 100 large budded cells were quantified per experiment . Average and SD of three independent biological experiments is shown .

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(E-G) Overexpression of Chs6 rescues Chs3 export in absence of other ChAPs. (E) Fluorescence images of cells expressing Chs3-GFP in WT, CHS6 and GPD-CHS6 strains. Arrows point to Chs3-GFP signals at the bud neck. Bar, 5 µm. The inset represents a twofold magnification.(F) Quantification of phenotypes in (E); 100 small budded and 100 large budded cells were quantified per experiment. Average and SD of three independent biological experiments is shown.


( G ) Chs3 GFP GPD - CHS6 strain was sensitive to calcofluor to a similar extent as the WT strain . Plates were incubated at 30 ° C for 2 - 3 days . A representative drop test of three independent biological experiments is shown .

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(G) Chs3 GFP GPD-CHS6 strain was sensitive to calcofluor to a similar extent as the WT strain. Plates were incubated at 30°C for 2-3 days. A representative drop test of three independent biological experiments is shown.


Fluorescence images of cells expressing Chs5 - GFP and TGN marker Sec7DsRed ( A ) and binding kinetics of Chs5 - GFP at the TGN ( B ) in WT , CHS6 and CHS6BUD7 ( A , B ) The brightness and contrast were adjusted differently in the merged images . Bar , 5 µm . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 .

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Fluorescence images of cells expressing Chs5-GFP and TGN marker Sec7DsRed (A) and binding kinetics of Chs5-GFP at the TGN (B) in WT, CHS6 and CHS6BUD7 (A, B) The brightness and contrast were adjusted differently in the merged images. Bar, 5 µm. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.


Fluorescence images of cells expressing Chs5 - GFP and TGN marker Sec7DsRed ( C ) and binding kinetics of Chs5 - GFP at the TGN ( D ) in WT , or CHS6BCH2 ( C , D ) strains . The brightness and contrast were adjusted differently in the merged images . Bar , 5 µm . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 .

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Fluorescence images of cells expressing Chs5-GFP and TGN marker Sec7DsRed (C) and binding kinetics of Chs5-GFP at the TGN (D) in WT, or CHS6BCH2 (C, D) strains. The brightness and contrast were adjusted differently in the merged images. Bar, 5 µm. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.


( A - D ) ChAPs are differently dependent on each other in terms of binding at the TGN . Binding kinetics of GFP - tagged Chs6 ( A ) , Bud7 ( B ) , Bch2 ( C ) and Bch1 ( D ) at the TGN in the presence ( WT ) or absence of the other ChAPs ( ) . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 .

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(A-D) ChAPs are differently dependent on each other in terms of binding at the TGN. Binding kinetics of GFP-tagged Chs6 (A), Bud7 (B), Bch2 (C) and Bch1 (D) at the TGN in the presence (WT) or absence of the other ChAPs (). The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.


( A - B ) Overexpression of Bch1 displaces Chs6 from the TGN and impairs plasma membrane localization of Chs6 - dependent cargo Chs3 . Fluorescence images of Chs6 - GFP ( A ) and Chs3 - GFP ( B ) in WT and GPD - BCH1 strains . Bar , 5 µm . Arrows point to Chs3 - GFP signals at the bud neck . The inset represents a twofold magnification .

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(A-B) Overexpression of Bch1 displaces Chs6 from the TGN and impairs plasma membrane localization of Chs6-dependent cargo Chs3. Fluorescence images of Chs6-GFP (A) and Chs3-GFP (B) in WT and GPD-BCH1 strains. Bar, 5 µm. Arrows point to Chs3-GFP signals at the bud neck. The inset represents a twofold magnification.


( C ) Quantification of the phenotypes in ( B ) ; 100 small budded and 100 large budded cells were quantified per experiment . Average and SD of three independent biological experiments is shown .

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(C) Quantification of the phenotypes in (B); 100 small budded and 100 large budded cells were quantified per experiment. Average and SD of three independent biological experiments is shown.


( D ) Chitin production is reduced upon Bch1 overexpression . Fluorescence images of chitin stained with calcofluor in Chs3 - GFP WT , chs5 and GPD - BCH1 strains .

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(D) Chitin production is reduced upon Bch1 overexpression. Fluorescence images of chitin stained with calcofluor in Chs3-GFP WT, chs5 and GPD-BCH1 strains.


( E ) Overexpression of Bch1 does not impair plasma membrane localization of the Bch1 - dependent cargo Pin2 . Fluorescence images of Pin2 - GFP in WT and GPD - BCH1 strains .

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(E) Overexpression of Bch1 does not impair plasma membrane localization of the Bch1-dependent cargo Pin2. Fluorescence images of Pin2-GFP in WT and GPD-BCH1 strains.


( F - I ) Overexpression of Chs6 displaces Bch1 and Chs5 from the TGN . Fluorescence images ( F - G ) and binding kinetics at the TGN ( H - I ) of cells expressing Bch1 - GFP ( F , H ) and Chs5 - GFP ( G , I ) upon overexpression of Chs6 under GPD promoter from 2μ plasmid . The insets in ( F , G ) represent two - fold magnifications . The arrows in ( F ) point to residual Bch1 staining at the TGN . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 . Size bars represent 5 µm .

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(F-I) Overexpression of Chs6 displaces Bch1 and Chs5 from the TGN. Fluorescence images (F-G) and binding kinetics at the TGN (H-I) of cells expressing Bch1-GFP (F, H) and Chs5-GFP (G, I) upon overexpression of Chs6 under GPD promoter from 2μ plasmid. The insets in (F, G) represent two-fold magnifications. The arrows in (F) point to residual Bch1 staining at the TGN. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2. Size bars represent 5 µm.


( A ) Overexpression of Pin2 enhances Chs5 stabilization at the TGN . Binding kinetics of Chs5 GFP in WT , bud7bch1 and bud7bch1 strain expressing Bch2 - dependent cargo Pin2 from a centromeric or a 2μ plasmid . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 .

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(A) Overexpression of Pin2 enhances Chs5 stabilization at the TGN. Binding kinetics of Chs5 GFP in WT, bud7bch1 and bud7bch1 strain expressing Bch2-dependent cargo Pin2 from a centromeric or a 2μ plasmid. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.


( B ) Comparison of the expression levels of the endogenous Pin2 with the levels of Pin2 expressed from a centromeric or a 2μ plasmid . Pgk1 serves as loading control . A representative immunoblot of three independent biological experiments is shown .

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(B) Comparison of the expression levels of the endogenous Pin2 with the levels of Pin2 expressed from a centromeric or a 2μ plasmid. Pgk1 serves as loading control. A representative immunoblot of three independent biological experiments is shown.


D , E Images of FRET efficiency in mock - treated ( - ant . A , D ) and Antimycin A - treated ( + ant . A . , E ) ubi - AT1 . 03NL wing discs at time point 2 h . A rainbow colormap is used to indicate the FRET efficiency levels . Scale bars = 50 µm . F Line graphs showing the FRET efficiency in individual mock - treated ( - ant . A , n = 26 ) and ant . A - treated ( + ant . A . , n = 19 ) ubi - AT1 . 03NL wing discs before ( 0 h ) and after 2 h of treatment . Paired t - test , ns = not significant , * * * p ≤ 0 . 001 .

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D, E Images of FRET efficiency in mock-treated (- ant.A, D) and Antimycin A-treated (+ ant. A., E) ubi-AT1.03NL wing discs at time point 2 h. A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars= 50 µm. F Line graphs showing the FRET efficiency in individual mock-treated (- ant. A, n=26) and ant. A-treated (+ ant. A., n=19) ubi-AT1.03NL wing discs before (0 h) and after 2 h of treatment. Paired t-test, ns= not significant, ***p≤0.001.


G - J Time - controlled knock - down of metabolic enzymes in the dorsal compartment of ubi - AT1 . 03NL wing discs using the apGalts driver . Images of FRET efficiency in control ( G ) , apGalts > PfkRNAi ( H ) , apGalts > GapdhRNAi ( I ) , and apGalts > Glo1RNAi ( J ) wing discs after RNAi induction for 48 h ( G ) , 120 h ( H , J ) , and 93 hours ( I ) . A rainbow colormap is used to indicate the FRET efficiency levels . Scale bars = 50 µm . K Line graphs showing the FRET efficiency in the ventral and dorsal compartment of individual control ( n = 27 ) , apGalts > PfkRNAi ( n = 7 ) , apGalts > GapdhRNAi ( n = 10 ) , and apGalts > Glo1RNAi ( n = 6 ) wing discs . Loss of Pfk , Gapdh or Glo1 in the dorsal compartment of the wing disc reduces the levels of ATP . Paired t - test , ns = not significant , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

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G-J Time-controlled knock-down of metabolic enzymes in the dorsal compartment of ubi-AT1.03NL wing discs using the apGalts driver. Images of FRET efficiency in control (G), apGalts>PfkRNAi (H), apGalts>GapdhRNAi (I), and apGalts>Glo1RNAi (J) wing discs after RNAi induction for 48 h (G), 120 h (H, J), and 93 hours (I). A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars = 50 µm. K Line graphs showing the FRET efficiency in the ventral and dorsal compartment of individual control (n=27), apGalts>PfkRNAi (n=7), apGalts>GapdhRNAi (n=10), and apGalts>Glo1RNAi (n=6) wing discs. Loss of Pfk, Gapdh or Glo1 in the dorsal compartment of the wing disc reduces the levels of ATP. Paired t-test, ns= not significant, **p≤0.01, ***p≤0.001.


L Time - controlled knock - down of ecdysoneless ( ecd ) in the dorsal compartment of ubi - AT1 . 03NL wing discs using apGalts > ecdRNAi . Image of FRET efficiency in apGalts > ecdRNAi wing disc 48 h after RNAi induction . A rainbow colormap is used to indicate the FRET efficiency levels . Scale bars = 50 µm . M Line graph showing the FRET in the dorsal and ventral compartment of apGalts > ecdRNAi ( n = 28 ) wing discs . Loss of Ecd in the dorsal compartment of the wing disc reduces the levels of ATP . Paired t - test , * * * p ≤ 0 . 001 .

O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O B-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-TISSUE I-TISSUE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE I-TISSUE O O O B-GENEPROD O O O O O O B-TISSUE I-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

L Time-controlled knock-down of ecdysoneless (ecd) in the dorsal compartment of ubi-AT1.03NL wing discs using apGalts>ecdRNAi. Image of FRET efficiency in apGalts>ecdRNAi wing disc 48 h after RNAi induction. A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars = 50 µm. M Line graph showing the FRET in the dorsal and ventral compartment of apGalts>ecdRNAi (n=28) wing discs. Loss of Ecd in the dorsal compartment of the wing disc reduces the levels of ATP. Paired t-test, ***p≤0.001.


A - D Adult wing phenotype of knock - down of Gapdh ( C765 > GapdhRNAi ) , over - expression of full - length Ecd ( C765 > ecd ) , over - expression of C - terminally truncated form of Ecd ( C765 > ecdDN ) , and knock - down of ecd ( C765 > ecdRNAi ) in the whole wing disc ( see Appendix Fig S3A for the expression pattern of C765 - Gal4 ) . Images of adult wings of control ( A ) , C765 > GapdhRNAi ( A ' ) , C765 > ecd ( B ) , C765 > ecdDN ( C ) , and C765 > ecdRNAi ( D ) male flies . ( A ' ' ) Overlay of the wings shown in A and A ' . ( C ' ) Overlay of control wing and wing shown in C and C ' . Loss of Ecd throughout the wing discs produces adults with vestigal wings ( D ) . In contrast , the effects of ecdDN over - expression or Gapdh knock - down are less severe on tissue survival , giving rise to smaller wings with altered proportions . Images consist of a series of tiles that were stitched together by the microscope software . Scale bar = 500 μm . E , F Tukey box - and - whiskers plot showing quantifications of the area ( E ) and shape ( aspect ratio : major / minor axis , F ) of control , C765 > GapdhRNAi , C765 > ecd , and C765 > ecdDN wings . Lower and upper hinges correspond to the first and third quartiles , vertical lines extend to ± 1 . 5 times the interquartile range . Sample size is indicated for each perturbation . Loss of Gapdh or over - expression of EcdDN leads to smaller wings that are increased along the anterior - posterior ( A / P ) axis compared to the proximal - distal ( P / D ) axis . Statistical analysis was performed using one - way ANOVA , followed by Dunnett ' s multiple comparisons test . * * * p ≤ 0 . 001 .

O O O O B-TISSUE O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O O B-TISSUE O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Adult wing phenotype of knock-down of Gapdh (C765>GapdhRNAi), over-expression of full-length Ecd (C765>ecd), over-expression of C-terminally truncated form of Ecd (C765>ecdDN), and knock-down of ecd (C765>ecdRNAi) in the whole wing disc (see Appendix Fig S3A for the expression pattern of C765-Gal4). Images of adult wings of control (A), C765>GapdhRNAi (A'), C765>ecd (B), C765>ecdDN (C), and C765>ecdRNAi (D) male flies. (A'') Overlay of the wings shown in A and A'. (C') Overlay of control wing and wing shown in C and C'. Loss of Ecd throughout the wing discs produces adults with vestigal wings (D). In contrast, the effects of ecdDN over-expression or Gapdh knock-down are less severe on tissue survival, giving rise to smaller wings with altered proportions. Images consist of a series of tiles that were stitched together by the microscope software. Scale bar= 500 μm. E, F Tukey box-and-whiskers plot showing quantifications of the area (E) and shape (aspect ratio: major/minor axis, F) of control, C765>GapdhRNAi, C765>ecd, and C765>ecdDN wings. Lower and upper hinges correspond to the first and third quartiles, vertical lines extend to ± 1.5 times the interquartile range. Sample size is indicated for each perturbation. Loss of Gapdh or over-expression of EcdDN leads to smaller wings that are increased along the anterior-posterior (A/P) axis compared to the proximal-distal (P/D) axis. Statistical analysis was performed using one-way ANOVA, followed by Dunnett's multiple comparisons test. ***p≤0.001.


A - B ' Time - controlled knock - down of ecd in the dorsal compartment of the wing discs ( see Fig 1C for the expression pattern of apGalts ) . IF of control ( A , A ' ) and apGalts > ecdRNAi ( B , B ' ) wing discs , stained for the Hedgehog ( Hh ) pathway components Smoothened ( Smo , A , B ) , and Ci155 ( A ' , B ' ) . Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control ( n = 5 ) and apGalts > ecdRNAi ( n = 5 ) wing discs . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Statistical analyses ( t - test ) revealed significant differences in Smo and Ci155 expression in the dorsal compartment between control and apGalts > ecdRNAi ( Smo : p ≤ 0 . 001 , Ci155 : p ≤ 0 . 001 ) wing discs . Thus , loss of Ecd elevates Smo levels on the plasma membrane and increases the range of Ci155 stabilization in the anterior compartment . Wing discs were analyzed 48 h after RNAi induction . Scale bars = 50 μm .

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

A-B' Time-controlled knock-down of ecd in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). IF of control (A, A') and apGalts>ecdRNAi (B, B') wing discs, stained for the Hedgehog (Hh) pathway components Smoothened (Smo, A, B), and Ci155 (A', B'). Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control (n=5) and apGalts>ecdRNAi (n=5) wing discs. Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analyses (t-test) revealed significant differences in Smo and Ci155 expression in the dorsal compartment between control and apGalts>ecdRNAi (Smo: p≤0.001, Ci155: p≤0.001) wing discs. Thus, loss of Ecd elevates Smo levels on the plasma membrane and increases the range of Ci155 stabilization in the anterior compartment. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.


C - E ' Time - controlled knock - down of metabolic enzymes in the dorsal compartment of the wing disc . IF of apGalts > PfkRNAi , apGalts > GapdhRNAi , and apGalts > Glo1RNAi wing discs , stained for Smo ( C , D , E ) and Ci155 ( C ' , D ' , E ' ) . Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of apGalts > PfkRNAi ( n = 5 ) , apGalts > GapdhRNAi ( n = 5 ) , and apGalts > Glo1RNAi ( n = 6 ) wing discs . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Statistical analyses ( t - test ) revealed significant differences in Smo and Ci155 expression in the dorsal compartment between control and apGalts > PfkRNAi ( Smo : p ≤ 0 . 001 , Ci155 : p ≤ 0 . 01 ) , apGalts > GapdhRNAi ( Smo , Ci155 : p ≤ 0 . 05 ) , or apGalts > Glo1RNAi ( Smo : p ≤ 0 . 001 , Ci155 : p ≤ 0 . 05 ) wing discs . Thus , loss of Pfk , Gapdh or Glo1 elevates Smo levels on the plasma membrane and increases the range of Ci155 stabilization in the anterior compartment . Wing discs were analyzed 120 h after RNAi induction . Scale bars = 50 μm .

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

C-E' Time-controlled knock-down of metabolic enzymes in the dorsal compartment of the wing disc. IF of apGalts>PfkRNAi, apGalts>GapdhRNAi, and apGalts>Glo1RNAi wing discs, stained for Smo (C, D, E) and Ci155 (C', D', E'). Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of apGalts>PfkRNAi (n=5), apGalts>GapdhRNAi (n=5), and apGalts>Glo1RNAi (n=6) wing discs. Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analyses (t-test) revealed significant differences in Smo and Ci155 expression in the dorsal compartment between control and apGalts>PfkRNAi (Smo: p≤0.001, Ci155: p≤0.01), apGalts>GapdhRNAi (Smo, Ci155: p≤0.05), or apGalts>Glo1RNAi (Smo: p≤0.001, Ci155: p≤0.05) wing discs. Thus, loss of Pfk, Gapdh or Glo1 elevates Smo levels on the plasma membrane and increases the range of Ci155 stabilization in the anterior compartment. Wing discs were analyzed 120 h after RNAi induction. Scale bars= 50 μm.


A - D ' ' ' IF of NIH3T3 cells stably expressing Smo - mEos2 treated with mock ( control , A - A ' ' ' ) , the Smo agonist ( + SAG , B - B ' ' ' ) or the glycolytic inhibitors 3 - bromopyruvate ( + 3 - BP , C - C ' ' ' ) and 2 - deoxyglucose ( + 2 - DG , D - D ' ' ' ) . DAPI staining , shown in gray , was used to visualize cell nuclei . Acetylated tubulin ( AcTub ) , used as a ciliary marker , is shown in magenta , Smo - mEos2 is shown in green . Zoomed - in images of cilia marked by white arrows in A ' ' , B ' ' , C ' ' , and D ' ' are shown in A ' ' ' , B ' ' ' , C ' ' ' , and D ' ' ' . Scale bars = 5 μm , but 2 μm in A ' ' ' , B ' ' ' , C ' ' ' , and D ' ' ' . E , F Box plot showing quantification of Smo - mEos2 ciliary localization in NIH3T3 cells upon 3 - BP ( E ) and 2 - DG ( F ) treatment , respectively . Lower and upper hinges correspond to the first and third quartiles , vertical lines extend to the minimum and maximum values . Sample size is indicated for each perturbation . Treatment with glycolytic inhibitors induces Smo - mEos2 localization to the primary cilium . t - test , * * * p ≤ 0 . 001 .

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

A-D''' IF of NIH3T3 cells stably expressing Smo-mEos2 treated with mock (control, A-A'''), the Smo agonist (+ SAG, B-B''') or the glycolytic inhibitors 3-bromopyruvate (+3-BP, C-C''') and 2-deoxyglucose (+ 2-DG, D-D'''). DAPI staining, shown in gray, was used to visualize cell nuclei. Acetylated tubulin (AcTub), used as a ciliary marker, is shown in magenta, Smo-mEos2 is shown in green. Zoomed-in images of cilia marked by white arrows in A'', B'', C'', and D'' are shown in A''', B''', C''', and D'''. Scale bars= 5 μm, but 2 μm in A''', B''', C''', and D'''. E, F Box plot showing quantification of Smo-mEos2 ciliary localization in NIH3T3 cells upon 3-BP (E) and 2-DG (F) treatment, respectively. Lower and upper hinges correspond to the first and third quartiles, vertical lines extend to the minimum and maximum values. Sample size is indicated for each perturbation. Treatment with glycolytic inhibitors induces Smo-mEos2 localization to the primary cilium . t-test, ***p≤0.001.


A - B Time - controlled knock - down of ecd in the dorsal compartment of the wing discs ( see Fig 1C for the expression pattern of apGalts ) . IF of control ( A ) and apGalts > ecdRNAi ( B ) wing discs , stained for Hh . Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control ( n = 4 ) and apGalts > ecdRNAi ( n = 5 ) wing discs . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Statistical analysis ( t - test ) revealed no significant differences in Hh expression in the dorsal compartment between control and apGalts > ecdRNAi wing discs . Thus , loss of Ecd does not affect Hh production and spreading . Wing discs were analyzed 48 h after RNAi induction . Scale bars = 50 μm .

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

A-B Time-controlled knock-down of ecd in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). IF of control (A) and apGalts>ecdRNAi (B) wing discs, stained for Hh. Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control (n=4) and apGalts>ecdRNAi (n=5) wing discs. Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analysis (t-test) revealed no significant differences in Hh expression in the dorsal compartment between control and apGalts>ecdRNAi wing discs. Thus, loss of Ecd does not affect Hh production and spreading. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.


C - F Time - controlled knock - down of ecd in the dorsal compartment of dispatched mutant wing discs ( dispS037707 , apGalts > ecdRNAi , see Fig 1C for the expression pattern of apGalts ) . IF of control ( C ) , apGalts > ecdRNAi ( D ) , dispS037707 ( E ) and dispS037707 , apGalts > ecdRNAi ( F ) wing discs expressing the decapentaplegic ( dpp ) reporter gene dpp - lacZ ( dppZ ) , stained for dppZ . Next to the images are quantifications of the respective stainings ( n = 4 ( C , F ) , n = 5 ( D ) , n = 3 ( E ) ) . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Statistical analyses ( t - test ) revealed a significant difference in dppZ expression in the dorsal compartment between control and apGalts > ecdRNAi ( p ≤ 0 . 01 ) ( C vs D ) , and dispS037707 and dispS037707 , apGalts > ecdRNAi ( p ≤ 0 . 01 ) wing discs ( E vs F ) . Loss of Ecd elevates the expression of dppZ in a broader than normal stripe . However , lack of Ecd function alone is not sufficient to trigger dpp expression , which instead depends on the presence of Hh . Wing discs were analyzed 48 h after RNAi induction . Scale bars = 50 μm .

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

C-F Time-controlled knock-down of ecd in the dorsal compartment of dispatched mutant wing discs (dispS037707, apGalts>ecdRNAi, see Fig 1C for the expression pattern of apGalts). IF of control (C), apGalts>ecdRNAi (D), dispS037707 (E) and dispS037707, apGalts>ecdRNAi (F) wing discs expressing the decapentaplegic (dpp) reporter gene dpp-lacZ (dppZ), stained for dppZ. Next to the images are quantifications of the respective stainings (n=4 (C, F), n=5 (D), n=3 (E)). Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analyses (t-test) revealed a significant difference in dppZ expression in the dorsal compartment between control and apGalts>ecdRNAi (p≤0.01) (C vs D), and dispS037707 and dispS037707, apGalts>ecdRNAi (p≤0.01) wing discs (E vs F). Loss of Ecd elevates the expression of dppZ in a broader than normal stripe. However, lack of Ecd function alone is not sufficient to trigger dpp expression, which instead depends on the presence of Hh. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.


G - J IF of dispS037707 ( G , I ) and dispS037707 , apGalts > ecdRNAi ( H , J ) wing discs , stained for Smo ( G , H ) and Ci155 ( I , J ) . Next to the images are quantifications of the respective stainings ( n = 2 ( G , H ) , n = 3 ( I ) , n = 4 ( J ) ) . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Although statistical analyses ( t - test ) revealed no significant differences in Smo and Ci155 expression in the dorsal compartment between control and dispS037707 , apGalts > ecdRNAi wing discs , loss of Ecd slightly induces an increased Smo accumulation and Ci155 stabilization independently of Hh . Wing discs were analyzed 48 h after RNAi induction . Scale bars = 50 μm .

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

G-J IF of dispS037707 (G, I) and dispS037707, apGalts>ecdRNAi (H, J) wing discs, stained for Smo (G, H) and Ci155 (I, J). Next to the images are quantifications of the respective stainings (n=2 (G, H), n=3 (I), n=4 (J)). Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Although statistical analyses (t-test) revealed no significant differences in Smo and Ci155 expression in the dorsal compartment between control and dispS037707, apGalts>ecdRNAi wing discs, loss of Ecd slightly induces an increased Smo accumulation and Ci155 stabilization independently of Hh. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.


A - A ' ' ' Time - controlled knock - down of ecd or Gapdh in the dorsal compartment of the wing discs ( see Fig 1C for the expression pattern of apGalts ) . The plasma membrane potential was detected using DiBAC4 ( 3 ) . The fluorescence intensity of DiBAC4 ( 3 ) increases upon plasma membrane depolarization . ( A ) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4 ( 3 ) signal intensity in the dorsal to ventral compartment ( control ( n = 19 ) , apGalts > ecdRNAi ( n = 36 ) and apGalts > GapdhRNAi ( n = 18 ) wing discs ) . Error bars indicate ± SD . t - test , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( A ' - A ' ' ' ) Representative images of DiBAC4 ( 3 ) assay in control ( A ' ) , apGalts > ecdRNAi ( A ' ' ) , and apGalts > GapdhRNAi ( A ' ' ' ) wing discs 48 h or 120 h after RNAi induction , respectively . V / D indicates the boundary between the ventral and dorsal compartments . Next to the maximal projections are shown the sum projections of cross - sections along the A / P axis . Loss of Ecd or Gapdh alters the plasma membrane potential . Scale bars = 50 μm .

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

A-A''' Time-controlled knock-down of ecd or Gapdh in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). The plasma membrane potential was detected using DiBAC4(3). The fluorescence intensity of DiBAC4(3) increases upon plasma membrane depolarization. (A) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4(3) signal intensity in the dorsal to ventral compartment (control (n=19), apGalts>ecdRNAi (n=36) and apGalts>GapdhRNAi (n=18) wing discs). Error bars indicate ± SD. t-test, *p≤0.05, ***p≤0.001. (A'-A''') Representative images of DiBAC4(3) assay in control (A'), apGalts>ecdRNAi (A''), and apGalts>GapdhRNAi (A''') wing discs 48 h or 120 h after RNAi induction, respectively. V/D indicates the boundary between the ventral and dorsal compartments. Next to the maximal projections are shown the sum projections of cross-sections along the A/P axis. Loss of Ecd or Gapdh alters the plasma membrane potential. Scale bars= 50 μm.


A - A ' ' ' Time - controlled knock - down of ecd or Gapdh in the dorsal compartment of the wing discs ( see Fig 1C for the expression pattern of apGalts ) . The plasma membrane potential was detected using DiBAC4 ( 3 ) . The fluorescence intensity of DiBAC4 ( 3 ) increases upon plasma membrane depolarization . ( A ) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4 ( 3 ) signal intensity in the dorsal to ventral compartment ( control ( n = 19 ) , apGalts > ecdRNAi ( n = 36 ) and apGalts > GapdhRNAi ( n = 18 ) wing discs ) . Error bars indicate ± SD . t - test , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( A ' - A ' ' ' ) Representative images of DiBAC4 ( 3 ) assay in control ( A ' ) , apGalts > ecdRNAi ( A ' ' ) , and apGalts > GapdhRNAi ( A ' ' ' ) wing

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

A-A''' Time-controlled knock-down of ecd or Gapdh in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). The plasma membrane potential was detected using DiBAC4(3). The fluorescence intensity of DiBAC4(3) increases upon plasma membrane depolarization. (A) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4(3) signal intensity in the dorsal to ventral compartment (control (n=19), apGalts>ecdRNAi (n=36) and apGalts>GapdhRNAi (n=18) wing discs). Error bars indicate ± SD. t-test, *p≤0.05, ***p≤0.001. (A'-A''') Representative images of DiBAC4(3) assay in control (A'), apGalts>ecdRNAi (A''), and apGalts>GapdhRNAi (A''') wing discs