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

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(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 ( J ) KIF1A - MDC - HA - FRB , KIF1C - MDC - HA - FRB , KIF5B - MDC - HA - FRB constructs probed for HA / DCLK1 , ( K ) different GFP - tagged KIF1A truncation constructs probed for GFP / DCLK1 . input / pellet is 2 % for pull - down experiments .

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(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 KIF1A - MDC - HA - FRB , KIF1C - MDC - HA - FRB KIF1C - MD - HA probed for HA / GFP . LacZ - HA was used as a negative control .

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(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 TGN to cytoplasm . of expressing GFP - tagged exomer components TGN marker Sec7 - DsRed . brightness contrast were adjusted differently in GFP merge . Bar , 5 µm . ( B ) of relative TGN to cytoplasm of GFP - tagged ChAPs in . mean at TGN cytoplasm was determined using Icy imaging software . 20 - 30 were analysed . For statistical significance testing Student ' s T was used .

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

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

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(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 GFP - tagged Chs5 ChAPs at 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 ) of relative expression of exomer components in GFP - tagged strains . A representative immunoblot of yeast lysates is shown ; Pgk1 serves as loading control . asterisks depict analyzed bands . plot shows average SD of three independent biological experiments . relative expression are normalized to Chs6 ( depicted by red ) .

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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 stable binding of ChAPs at TGN . Differential centrifugation of lysates obtained from Chs6 - GFP WT Δchs5 . TCL , total lysate ; S10 , 10 , 000 g supernatant ; P10 , 10 , 000 g pellet ; S100 , 100 , 000 g supernatant ; P100 , 100 , 000 g pellet . Anp1 serves as Golgi marker Pgk1 as 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 cytoplasm ( open triangles , dashed ) at TGN ( closed circles , full ) . mean of 10 - 20 FRAP measurements from different 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 association in absence of ChAPs . Differential centrifugation of lysates obtained from Chs5 GFP WT ΔΔΔΔ strains . TCL , total lysate ; S10 , 10 , 000 g supernatant ; P10 , 10 , 000 g pellet ; S100 , 100 , 000 g supernatant ; P100 , 100 , 000 g pellet . Anp1 serves as Golgi marker Pgk1 as 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 ) of expressing Chs5 - GFP TGN marker Sec7 - DsRed in WT , ChAP strains . Merged depict differences in Chs5 - GFP localization to TGN , cytoplasm distinct foci ( arrows ) in these strains . brightness contrast were adjusted differently in merged . Bar , 5 µm . ( E ) Co - localization of Chs5 - GFP TGN marker Sec7 - DsRed in WT , ChAP strains . Mander 1 depicts relative amount of GFP signal co - localizing with overall DsRed signal , Mander 2 vice versa . Manders coefficients were obtained using JACoP plug - in for ImageJ after background subtraction ( N = 20 ) 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 TGN in WT , ChAP strains . mean of 20 - 30 FRAP measurements from different is shown . Calculated parameters are shown in Table 2 . schematic cartoons depict predicted majority of analyzed complexes . violet ovals represent Chs5 dimer circles represent ChAPs : green : Bch1 , red : Bud7 , blue : Bch2 , 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 stabilization at TGN . of expressing Chs5 - GFP ( A ) binding kinetics of Chs5 - GFP at TGN ( B ) in WT , BCH2 GPD - BCH2 strains . Bar , 5 µm . mean of 20 - 30 FRAP measurements from different is shown . Calculated parameters are shown in Table 2 . schematic cartoons depict predicted majority of analyzed complexes ; Chs5 dimer : violet ovals , Bch2 : 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 stabilization at TGN . ( C ) of expressing Chs5 - GFP in WT , CHS6 GPD - CHS6 strains . Bar , 5 µm . ( D ) Binding kinetics of Chs5 - GFP at TGN in WT , , CHS6 GPD - CHS6 strains . Data processing was carried out as in ( A - B ) . schematic cartoons depict predicted majority of analyzed complexes ; Chs5 dimer : violet ovals , Chs6 : 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 ) of expressing Chs3 - GFP in WT , CHS6 GPD - CHS6 strains . Arrows point to Chs3 - GFP signals at bud neck . Bar , 5 µm . inset represents a twofold magnification . ( F ) of in ( E ) ; 100 small budded 100 large budded were quantified per experiment . Average 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 was sensitive to calcofluor to a similar extent as WT . Plates were incubated at 30 ° C for 2 - 3 days . A representative drop 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.


of expressing Chs5 - GFP TGN marker Sec7DsRed ( A ) binding kinetics of Chs5 - GFP at TGN ( B ) in WT , CHS6 CHS6BUD7 ( A , B ) brightness contrast were adjusted differently in merged . Bar , 5 µm . mean of 20 - 30 FRAP measurements from different 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.


of expressing Chs5 - GFP TGN marker Sec7DsRed ( C ) binding kinetics of Chs5 - GFP at TGN ( D ) in WT , or CHS6BCH2 ( C , D ) strains . brightness contrast were adjusted differently in merged . Bar , 5 µm . mean of 20 - 30 FRAP measurements from different 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 TGN . Binding kinetics of GFP - tagged Chs6 ( A ) , Bud7 ( B ) , Bch2 ( C ) Bch1 ( D ) at TGN in presence ( WT ) or absence of other ChAPs ( ) . mean of 20 - 30 FRAP measurements from different 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 TGN impairs plasma localization of Chs6 - dependent cargo Chs3 . of Chs6 - GFP ( A ) Chs3 - GFP ( B ) in WT GPD - BCH1 strains . Bar , 5 µm . Arrows point to Chs3 - GFP signals at bud neck . 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 ) of in ( B ) ; 100 small budded 100 large budded were quantified per experiment . Average 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 . of chitin stained with calcofluor in Chs3 - GFP WT , chs5 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 localization of Bch1 - dependent cargo Pin2 . of Pin2 - GFP in WT 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 Chs5 from TGN . ( F - G ) binding kinetics at TGN ( H - I ) of expressing Bch1 - GFP ( F , H ) Chs5 - GFP ( G , I ) upon overexpression of Chs6 under GPD promoter from 2μ plasmid . insets in ( F , G ) represent two - fold magnifications . arrows in ( F ) point to residual Bch1 at TGN . mean of 20 - 30 FRAP measurements from different is shown . Calculated parameters are shown in Table 2 . 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 TGN . Binding kinetics of Chs5 GFP in WT , bud7bch1 bud7bch1 expressing Bch2 - dependent cargo Pin2 from a centromeric or a 2μ plasmid . mean of 20 - 30 FRAP measurements from different 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 expression of endogenous Pin2 with 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 of FRET efficiency in mock - treated ( - ant . A , D ) Antimycin A - treated ( + ant . A . , E ) ubi - AT1 . 03NL wing discs at time point 2 h . A rainbow colormap is used to indicate FRET efficiency . Scale bars = 50 µm . F graphs showing FRET efficiency in mock - treated ( - ant . A , n = 26 ) ant . A - treated ( + ant . A . , n = 19 ) ubi - AT1 . 03NL wing discs before ( 0 h ) after 2 h of treatment . Paired t - , ns = not significant , * * * p ≤ 0 . 001 .

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

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 dorsal compartment of ubi - AT1 . 03NL wing discs using apGalts driver . of FRET efficiency in control ( G ) , apGalts > PfkRNAi ( H ) , apGalts > GapdhRNAi ( I ) , apGalts > Glo1RNAi ( J ) wing discs after RNAi induction for 48 h ( G ) , 120 h ( H , J ) , 93 hours ( I ) . A rainbow colormap is used to indicate FRET efficiency . Scale bars = 50 µm . K graphs showing FRET efficiency in ventral dorsal compartment of control ( n = 27 ) , apGalts > PfkRNAi ( n = 7 ) , apGalts > GapdhRNAi ( n = 10 ) , apGalts > Glo1RNAi ( n = 6 ) wing discs . Loss of Pfk , Gapdh or Glo1 in dorsal compartment of wing disc reduces of ATP . Paired t - , 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 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 FRET efficiency . Scale bars = 50 µm . M graph showing FRET in dorsal ventral compartment of apGalts > ecdRNAi ( n = 28 ) wing discs . Loss of Ecd in dorsal compartment of wing disc reduces of ATP . Paired t - , * * * p ≤ 0 . 001 .

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

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

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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 dorsal compartment of wing disc . IF of apGalts > PfkRNAi , apGalts > GapdhRNAi , apGalts > Glo1RNAi wing discs , stained for Smo ( C , D , E ) Ci155 ( C ' , D ' , E ' ) . Next to are quantifications of respective in dorsal versus ventral compartment of apGalts > PfkRNAi ( n = 5 ) , apGalts > GapdhRNAi ( n = 5 ) , apGalts > Glo1RNAi ( n = 6 ) wing discs . Graphs show mean ( thick ) ± SD ( thin lines ) . Dashed yellow lines indicate position of A / P boundary . Statistical analyses ( t - ) revealed significant differences in Smo Ci155 expression in dorsal compartment between control 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 on plasma increases range of Ci155 stabilization in 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 B-TISSUE I-TISSUE O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD B-TISSUE I-TISSUE O B-EXP_ASSAY 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 B-GENEPROD O B-GENEPROD 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 B-GENEPROD I-GENEPROD B-EXP_ASSAY 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 B-SUBCELLULAR O O 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

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

O O O O O O B-EXP_ASSAY O B-CELL_LINE O 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 B-SMALL_MOLECULE 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 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 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 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 B-GENEPROD O B-GENEPROD B-SUBCELLULAR B-EXP_ASSAY O B-CELL_LINE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR 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 dorsal compartment of wing discs ( see Fig 1C for expression pattern of apGalts ) . IF of control ( A ) apGalts > ecdRNAi ( B ) wing discs , stained for Hh . Next to are quantifications of respective in dorsal versus ventral compartment of control ( n = 4 ) apGalts > ecdRNAi ( n = 5 ) wing discs . Graphs show mean ( thick ) ± SD ( thin lines ) . Dashed yellow lines indicate position of A / P boundary . Statistical ( t - ) revealed no significant differences in Hh expression in dorsal compartment between control apGalts > ecdRNAi wing discs . Thus , loss of Ecd does not affect Hh production 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 B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O O B-EXP_ASSAY 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 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 B-GENEPROD B-EXP_ASSAY 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 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 dorsal compartment of dispatched mutant wing discs ( dispS037707 , apGalts > ecdRNAi , see Fig 1C for expression pattern of apGalts ) . IF of control ( C ) , apGalts > ecdRNAi ( D ) , dispS037707 ( E ) dispS037707 , apGalts > ecdRNAi ( F ) wing discs expressing decapentaplegic ( dpp ) reporter gene dpp - lacZ ( dppZ ) , stained for dppZ . Next to are quantifications of respective stainings ( n = 4 ( C , F ) , n = 5 ( D ) , n = 3 ( E ) ) . Graphs show mean ( thick ) ± SD ( thin lines ) . Dashed yellow lines indicate position of A / P boundary . Statistical analyses ( t - ) revealed a significant difference in dppZ expression in dorsal compartment between control apGalts > ecdRNAi ( p ≤ 0 . 01 ) ( C vs D ) , dispS037707 dispS037707 , apGalts > ecdRNAi ( p ≤ 0 . 01 ) wing discs ( E vs F ) . Loss of Ecd elevates 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 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 B-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O B-GENEPROD 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 B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-TISSUE I-TISSUE B-EXP_ASSAY 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 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 B-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-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 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 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 ) dispS037707 , apGalts > ecdRNAi ( H , J ) wing discs , stained for Smo ( G , H ) Ci155 ( I , J ) . Next to are quantifications of respective stainings ( n = 2 ( G , H ) , n = 3 ( I ) , n = 4 ( J ) ) . Graphs show mean ( thick ) ± SD ( thin lines ) . Dashed yellow lines indicate position of A / P boundary . Although statistical analyses ( t - ) revealed no significant differences in Smo Ci155 expression in dorsal compartment between control dispS037707 , apGalts > ecdRNAi wing discs , loss of Ecd slightly induces an increased Smo accumulation 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 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 B-GENEPROD 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 B-GENEPROD I-GENEPROD B-EXP_ASSAY 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 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 dorsal compartment of wing discs ( see Fig 1C for expression pattern of apGalts ) . plasma potential was detected using DiBAC4 ( 3 ) . of DiBAC4 ( 3 ) increases upon plasma depolarization . ( A ) change in plasma potential upon genetic perturbation was quantified as fold change in of DiBAC4 ( 3 ) signal in dorsal to ventral compartment ( control ( n = 19 ) , apGalts > ecdRNAi ( n = 36 ) apGalts > GapdhRNAi ( n = 18 ) wing discs ) . Error bars indicate ± SD . t - , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( A ' - A ' ' ' ) Representative of DiBAC4 ( 3 ) in control ( A ' ) , apGalts > ecdRNAi ( A ' ' ) , apGalts > GapdhRNAi ( A ' ' ' ) wing discs 48 h or 120 h after RNAi induction , respectively . V / D indicates boundary between ventral dorsal compartments . Next to maximal projections are shown sum projections of cross - sections along A / P axis . Loss of Ecd or Gapdh alters plasma 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 B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O O B-SUBCELLULAR B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR B-EXP_ASSAY 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 B-GENEPROD O B-GENEPROD 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 B-SMALL_MOLECULE 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 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 B-GENEPROD O B-GENEPROD O B-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 dorsal compartment of wing discs ( see Fig 1C for expression pattern of apGalts ) . plasma potential was detected using DiBAC4 ( 3 ) . of DiBAC4 ( 3 ) increases upon plasma depolarization . ( A ) change in plasma potential upon genetic perturbation was quantified as fold change in of DiBAC4 ( 3 ) signal in dorsal to ventral compartment ( control ( n = 19 ) , apGalts > ecdRNAi ( n = 36 ) apGalts > GapdhRNAi ( n = 18 ) wing discs ) . Error bars indicate ± SD . t - , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( A ' - A ' ' ' ) Representative of DiBAC4 ( 3 ) in control ( A ' ) , apGalts > ecdRNAi ( A ' ' ) , 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 B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O O B-SUBCELLULAR B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR B-EXP_ASSAY 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 B-GENEPROD O B-GENEPROD 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 B-SMALL_MOLECULE 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 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


D - E ' ' Dissipation of plasma potential upon treatment of wing discs with gramicidin A ( gA ) . ( D ) of DiBAC4 ( 3 ) signal in mock - treated ( - gA , n = 7 ) gA - treated ( + gA , n = 5 ) wing discs . ( D ' , D ' ' ) Representative of DiBAC4 ( 3 ) in mock - ( D ' ) gA - treated ( D ' ' ) wing discs . Next to maximal projections are shown sum projections of cross - sections along A / P axis . ( E ) of PAC - NAE signal in mock - ( n = 7 ) gA - treated ( n = 9 ) wing discs , shown as fold change from control ( mock - treated ) . ( E ' - E ' ' ) Representative of PAC - NAE uptake in mock - ( E ' ) gA - treated ( E ' ' ) wing discs . GA treatment strongly reduces uptake of PAC - NAE . Error bars indicate ± SD . t - , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . Scale bars = 50 μm .

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D-E'' Dissipation of the plasma membrane potential upon treatment of wing discs with gramicidin A (gA). (D) Quantification of DiBAC4(3) signal intensity in mock-treated (- gA, n=7) and gA-treated (+ gA, n=5) wing discs. (D', D'') Representative images of DiBAC4(3) assay in mock- (D') and gA-treated (D'') wing discs. Next to the maximal projections are shown sum projections of cross-sections along the A/P axis. (E) Quantification of PAC-NAE signal intensity in mock- (n=7) and gA-treated (n=9) wing discs, shown as fold change from control (mock-treated). (E'-E'') Representative images of PAC-NAE uptake in mock- (E') and gA-treated (E'') wing discs. GA treatment strongly reduces the uptake of PAC-NAE. Error bars indicate ± SD. t-test, *p≤0.05, ***p≤0.001. Scale bars= 50 μm.


( a ) WT Pgam5 KO MEFs evaluated by . black arrows indicate mitochondria ; ( b ) after 12 h CCCP treatment ; black arrowheads indicate DMS . Inset shows a DMS with an encapsulated mitochondrion . black arrows indicate mitochondria . Scale bar = 0 . 5 micron ;

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

(a) WT and Pgam5 KO MEFs evaluated by TEM. The black arrows indicate mitochondria; (b) TEM analysis after 12 h CCCP treatment; black arrowheads indicate DMS. Inset shows a DMS with an encapsulated mitochondrion. The black arrows indicate mitochondria. Scale bar=0.5 micron;


( c ) WT KO MEFs were treated with CCCP for 0 , 3 or 6 h fractions were immunoblotted with anti - LC - 3 , anti - p62 or anti - actin as indicated .

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(c) WT and KO MEFs were treated with CCCP for 0, 3 or 6 h and cytosolic fractions were immunoblotted with anti-LC-3, anti-p62 or anti-actin as indicated.


( d ) Similar to c . WT KO MEFs were treated with CCCP for 6 12 h immunoblotted for anti - COXIV , anti - Tomm20 or anti - actin as indicated . For c d , numbers below each lane indicate fractional band density compared with 0 h time point , which has been set at 1 . 0 ;

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(d) Similar to c. WT and KO MEFs were treated with CCCP for 6 and 12 h and immunoblotted for anti-COXIV, anti-Tomm20 or anti-actin as indicated. For c and d, the numbers below each lane indicate the fractional band density compared with the 0 h time point, which has been set at 1.0;


( e ) Confocal photomicrographs of mt - Keima - transduced WT KO MEFs treated with CCCP or DMSO vehicle for 12 h ( six from each group were scanned representative are shown ) ; Intracellular mt - Keima excited at 450 nm was shown in green colour , while red colour indicated excitation by 550 nm in same . Scale bar = 20 μm .

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(e) Confocal photomicrographs of mt-Keima-transduced WT and KO MEFs treated with CCCP or DMSO vehicle for 12 h (six individual cells from each group were scanned and representative cells are shown); Intracellular mt-Keima excited at 450 nm was shown in green colour, while red colour indicated the excitation by 550 nm in the same cell. Scale bar=20 μm.


( f ) Mitochondrial extracts of WT Pgam5 KO MEFs treated with DMSO ( − ) or CCCP ( + ) for 3 h analysed by immunoblot as above . ( Note that mouse PINK1 antibody can only detect full - PINK1 ) . Arrow indicates 63 kDa form of PINK1 .

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(f) Mitochondrial extracts of WT and Pgam5 KO MEFs treated with DMSO (−) or CCCP (+) for 3 h and analysed by immunoblot as above. (Note that mouse PINK1 antibody can only detect the full-length PINK1). Arrow indicates the 63 kDa form of PINK1.


( g ) Hela transduced with either nonspecific ( NS ) or PGAM5 shRNA were treated with CCCP for 3 h , mitochondrial fractions were immunoblotted for indicated . Full - ( * 63 kDa ) ( # 54 kDa ) PINK1 are indicated . For f g , numbers below indicate full - PINK1 band density compared with first lane that has been set at 1 .

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(g) Hela cells transduced with either nonspecific (NS) or PGAM5 shRNA viruses were treated with CCCP for 3 h, and mitochondrial fractions were immunoblotted for the proteins indicated. Full-length (*63 kDa) and cleaved (#54 kDa) PINK1 are indicated. For f and g, the numbers below indicate the full-length PINK1 band density compared with the first lane that has been set at 1.0


( h ) Confocal of Hela as in g were transfected with PARKIN - YFP ( green ) plasmid before CCCP treatment , then ( i ) treated with CCCP for 12 h . Mitochondria were stained with anti - Tomm20 ( red ) 4 ' , 6 - diamidino - 2 - phenylindole stained nucleus was shown in blue . Co - localization was indicated by colour . Scale bar = 10 μm . ( j ) of co - localization of Tomm20 ( red ) with parkin ( green ) per by Imaris software ( n = 7 for NS PGAM5 shRNA group separately . Results are presented as mean s . d . values were analysed by student t - ) . blots are representative of three independent experiments . DMSO , dimethylsulphoxide .

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(h) Confocal micrographs of Hela cells as in g were transfected with PARKIN-YFP (green) plasmid before CCCP treatment, then (i) treated with CCCP for 12 h. Mitochondria were stained with anti-Tomm20 (red) 4',6-diamidino-2-phenylindole stained nucleus was shown in blue. Co-localization was indicated by white colour. Scale bar=10 μm. (j) Quantification of the co-localization percentage of Tomm20 (red) with parkin (green) per cell by Imaris software (n=7 for NS and PGAM5 shRNA group separately. Results are presented as mean and s.d. values and were analysed by the student t-test). All the blots and images are representative of three independent experiments. DMSO, dimethylsulphoxide.


( a ) Mitochondria from MEFs from Parl WT ( + / + ) KO ( − / − ) mice were treated with indicated doses of proteinase K ( Prot . K ) for 30 min on ice then immunoblotted for indicated . Full - PGAM5 bands are indicated with arrows .

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(a) Mitochondria from MEFs from Parl WT (+/+) and KO (−/−) mice were treated with the indicated doses of proteinase K (Prot.K) for 30 min on ice and then immunoblotted for the indicated proteins. Full-length and cleaved PGAM5 bands are indicated with arrows.


( b ) Pgam5 WT KO MEFs were transduced with either nonspecific ( NS ) or Parl shRNA either treated ( + ) or not ( − ) with CCCP for 3 h . Mitochondria were then purified analysed by immunoblot for indicated .

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

(b) Pgam5 WT and KO MEFs were transduced with either nonspecific (NS) or Parl shRNA and either treated (+) or not (−) with CCCP for 3 h. Mitochondria were then purified and analysed by immunoblot for the indicated proteins.


( c ) WT PINK1 as well as kinase dead ( KD ) or Parkinson ' s disease - associated PINK1 mutants ( C92F , A168P , H271Q , G309D , L347P , G386S , R464H ) were co - transfected into HEK293T with same amount PGAM5 ( lane 1 - 9 ) or GFP ( lane 10 - 18 ) expression plasmids total lysates were immunoblotting for PINK1 , PGAM5 actin . of WT full - ( 63 kDa ) versus ( 54 kDa ) PINK1 ( * / # ) is shown .

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

(c) WT PINK1 as well as kinase dead (KD) or Parkinson's disease-associated PINK1 mutants (C92F, A168P, H271Q, G309D, L347P, G386S, R464H) were co-transfected into HEK293T cells with the same amount PGAM5 (lane 1-9) or GFP (lane 10-18) expression plasmids and total cell lysates were immunoblotting for PINK1, PGAM5 and actin. The ratio of WT full-length (63 kDa) versus cleaved (54 kDa) PINK1 (*/#) is shown.


( d ) Anti - myc immunoprecipitates ( IP ) of lysates from 293T transfected with combinations of myc - tagged PGAM5 PINK1 - V5 with or without CCCP treatment immunoblotting with anti - PINK1 or anti - myc .

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(d) Anti-myc immunoprecipitates (IP) of lysates from 293T cells transfected with combinations of myc-tagged PGAM5 and PINK1-V5 with or without CCCP treatment and immunoblotting with anti-PINK1 or anti-myc.


( e ) Four constructs expressing either : ( 1 ) no PGAM5 ( empty vector ) or ( 2 ) 1 - 98 , ( 3 ) 1 - 110 ( 4 ) 98 - 289 truncated versions of PGAM5 ( left panel ) . Immunoblot of HEK293T expressing PINK1 - GFP myc - tagged PGAM5 truncation mutants as indicated with anti - PINK1 or anti - myc for input as well as anti - GFP IP ( right panel ) .

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(e) Four constructs expressing either: (1) no PGAM5 (empty vector) or (2) amino acids 1-98, (3) 1-110 and (4) 98-289 truncated versions of PGAM5 (left panel). Immunoblot of HEK293T cells expressing PINK1-GFP and myc-tagged PGAM5 truncation mutants as indicated with anti-PINK1 or anti-myc for the input proteins as well as the anti-GFP IP (right panel).


( f ) Co - transfection of GFP , WT PGAM5 , 2RA mutant ( R98A R104A ) or 2HA mutant ( H99A H105A ) with a PINK1 expression plasmid into HEK293T input lysates as well as anti - myc IP were immunoblotted with antibodies against PINK1 , PGAM5 or actin .

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(f) Co-transfection of GFP, WT PGAM5, 2RA mutant (R98A and R104A) or the 2HA mutant (H99A and H105A) with a PINK1 expression plasmid into HEK293T cells and the input cell lysates as well as the anti-myc IP were immunoblotted with antibodies against PINK1, PGAM5 or actin.


WT PINK1 was expressed in HEK293T with or without PGAM5 or a GFP control plasmid after ( g ) DMSO or ( h ) CCCP treatment . were permeabilized with digitonin or Triton X - 100 separately , followed by treatment with indicated amounts of trypsin on ice for 30 min . lysates were immunoblotted for PINK1 , PGAM5 , Tomm20 or HSP60 as indicated . Full - ( * 63 kDa ) ( # 54 kDa ) PINK1 are shown .

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WT PINK1 was expressed in HEK293T cells with or without PGAM5 or a GFP control plasmid after (g) DMSO or (h) CCCP treatment. Cells were permeabilized with digitonin or Triton X-100 separately, followed by treatment with the indicated amounts of trypsin on ice for 30 min. Cell lysates were immunoblotted for PINK1, PGAM5, Tomm20 or HSP60 as indicated. Full-length (*63 kDa) and cleaved (#54 kDa) PINK1 are shown.


( a ) was measured in 5 - month - old 12 - month - old Pgam5 WT KO mice .

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(a) Body weight was measured in 5-month-old and 12-month-old female Pgam5 WT and KO mice.


Behavioral measurements on 12 - month - old WT KO mice ( WT n = 10 , KO n = 8 ) were as follows : ( b ) Open - field including time spent mobile , time spent immobile , whole centre ;

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Behavioral measurements on 12-month-old female WT and KO mice (WT n=10, KO n=8) were as follows: (b) Open-field tests including time spent mobile, time spent immobile, whole area distance and centre area distance;


Behavioral measurements on 12 - month - old WT KO mice ( WT n = 10 , KO n = 8 ) were as follows : ( c ) Gait , gait frequency in steps / ss step angle ; ( d ) rearing , ( e ) akinesia ( f ) ladder climb ( g ) vertical pole . T - turn indicates time mice spent to turn their bodies 180 degrees from head up start position before descending pole . T - down indicates time mice spent to descend to bottom of pole . Graphs show mean s . d . values were analysed by student ' s t - , * P0 . 05 , * * P0 . 01 , * * * P0 . 001 , NS , not significant .

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Behavioral measurements on 12-month-old female WT and KO mice (WT n=10, KO n=8) were as follows: (c) Gait length, gait frequency in steps/ss and step angle; (d) rearing test, (e) akinesia test and (f) ladder climb test and (g) vertical pole test. T-turn indicates the time mice spent to turn their bodies 180 degrees from the head up start position before descending the pole. T-down indicates the time mice spent to descend to the bottom of pole. Graphs show the mean and s.d. values and were analysed by the student's t-test, *P0.05, **P0.01, ***P0.001, NS, not significant.


High - performance liquid chromatography - electrochemical detection quantitation of ( a ) striatal DA its metabolites ( b ) dihydroxyphenylacetic acid ( DOPAC ) ( c ) Homovanillic acid ( HVA ) ( d ) 3MT at three different time points ( 1 , 2 12 - month - old ) in Pgam5 WT or KO mice .

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High-performance liquid chromatography-electrochemical detection quantitation of (a) striatal DA and its metabolites (b) dihydroxyphenylacetic acid (DOPAC) (c) Homovanillic acid (HVA) and (d) 3MT levels at three different time points (1, 2 and 12-month-old) in Pgam5 WT or KO mice.


( e ) Representative of TH - immunohistochemistry from WT KO mice . VTA region is indicated . Stereological quantitation of ( f ) TH ( g ) Nissl - positive neurons in SNpc ( n = 4 mice per group ) . Error bars represent mean ± s . e . m . , n = 4 mice per group . ( two - way of variance , * * * P0 . 01 , * P0 . 05 , NS , not significant ) . mo , month old .

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(e) Representative images of TH-immunohistochemistry from WT and KO mice. The VTA region is indicated. Stereological quantitation of (f) TH and (g) Nissl-positive neurons in the SNpc (n=4 mice per group). Error bars represent the mean±s.e.m., n=4 mice per group. (two-way analysis of variance, ***P0.01, *P0.05, NS, not significant). mo, month old.


18 - month - old Pgam5 WT KO mice received either water as control ( n = 4 ) , or 5 mg kg − 1 L - DOPA 12 . 5 mg kg − 1 Benserazide ( n = 5 ) by intraperitoneal injection . Twenty minutes later , mice were subjected to open - field centre time in seconds ( s ) as well as centre in centimeters ( cm ) were measured as described in Materials Methods . Graphs show mean s . d . values were analysed by student t - , NS , no significance ; * P0 . 05 ; * * P0 . 01 .

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The 18-month-old Pgam5 WT and KO mice received either water as control (n=4), or 5 mg kg−1 L-DOPA and 12.5 mg kg−1 Benserazide (n=5) by intraperitoneal injection. Twenty minutes later, mice were subjected to the open-field test and the centre area time in seconds (s) as well as the centre area distance in centimeters (cm) were measured as described in the Materials and Methods. Graphs show the mean and s.d. values and were analysed by the student t-test, NS, no significance; *P0.05; **P0.01.


( a ) WT dnm1Δ expressing Vph1 - RFP Idp1 - GFP were grown in minimal synthetic dextrose medium transferred to lactate medium at an initial density of OD600 0 . 08 . were then sampled daily analysed by microscopy ( × 100 objective ) during stationary phase mitophagy in lactate medium . Scale bar , 5 μm .

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(a) WT and dnm1Δ cells expressing Vph1-RFP and Idp1-GFP were grown in minimal synthetic dextrose medium and transferred to lactate medium at an initial density of OD600 0.08. The cells were then sampled daily and analysed by fluorescence microscopy (× 100 objective) during stationary phase mitophagy in lactate medium. Scale bar, 5 μm.


( b ) WT , dnm1Δ dnm1Δ mgm1Δ mutants were grown in SL medium as described in Methods , samples ( 10 OD600 units ) were taken at each time point . extracts were prepared equal amounts ( 20 μg ) were subjected to SDS - polyacrylamide gel electrophoresis immunoblotting with anti - GFP antibody .

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(b) WT, dnm1Δ and dnm1Δ mgm1Δ mutants were grown in SL medium as described in Methods, and samples (10 OD600 units) were taken at each time point. Protein extracts were prepared and equal protein amounts (20 μg) were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting with anti-GFP antibody.


( c ) of free GFP at day 4 time point in each of three strains analysed in b , using four independent experiments for each . Error bars indicate s . d . of variance , P0 . 001 ; N = 4 .

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(c) Quantification of the percent free GFP at the day 4 time point in each of the three strains analysed in b, using four independent experiments for each strain. Error bars indicate s.d. analysis of variance, P0.001; N=4.


( c ) Density plot of reproducibility of biological replicates .

(c) Density plot of the reproducibility of biological replicates.


( a ) Ratios from with quantifications from samples were log2 transformed z - score normalized . Columns containing data from different samples were clustered hierarchically rows that contained entries were clustered by k - means . in each k - means cluster were tested for enrichment of GO CC ( indicated in red ) GO BP ( indicated in blue ) terms .

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(a) Ratios from proteins with quantifications from all samples were log2 transformed and z-score normalized. Columns containing data from the different samples were clustered hierarchically and rows that contained protein entries were clustered by k-means. The proteins in each k-means cluster were tested for enrichment of GO CC (indicated in red) and GO BP (indicated in blue) terms.


( b ) associated with different compartments were sequentially extracted average of their ratios were compared with averages of remaining .

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(b) Proteins associated with different cellular compartments were sequentially extracted and the average of their ratios were compared with averages of remaining proteins.


( d ) GO BP enrichment of in different clusters compared with remaining .

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(d) GO BP enrichment analysis of proteins in the different clusters compared with the remaining proteins.


A comparison of SILAC - derived abundance as a function of time , for several that either show differential behaviour between WT mutants ( Idh2 , Idp1 Aco2 ) that behave similarly between genetic backgrounds tested ( Hsp78 ) . Shown are mean ratios ( two replicates for mutants , three replicates for WT ) , error bars indicating peptide - relative s . d .

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A comparison of the SILAC-derived abundance as a function of time, for several proteins that either show differential behaviour between WT and the mutants (Idh2, Idp1 and Aco2) and proteins that behave similarly between all the genetic backgrounds tested (Hsp78). Shown are mean protein ratios (two replicates for mutants, three replicates for WT), error bars indicating peptide-based relative s.d.


WT ( a ) , dnm1Δ ( b ) atg1Δ ( c ) atg32Δ ( d ) expressing chromosomally tagged GFP chimeras of Aco2 , Idh2 , Idp1 Hsp78 were incubated in SL medium for 7 days , 10 OD600 units were sampled at day 1 day 7 processed for immunoblotting with anti - GFP antibodies as described in Methods section . Positive control used in c d was from dnm1Δ expressing Idp1 - GFP from a plasmid at 4 - day incubation ( Fig . 1b , lane 4 ) . Release of free GFP in WT is reproducibly variable between these reporters , varies between 0 ( Idh2 ) 60 % ( Aco2 ) . ( e ) Densitometric of release of free GFP ( as % of total signal ) in four independent experiments , comparing results between WT dnm1Δ . Bars denote s . d . ( n = 4 ) .

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WT (a), dnm1Δ (b) and atg1Δ cells (c) and atg32Δ cells (d) expressing chromosomally tagged GFP chimeras of Aco2, Idh2, Idp1 and Hsp78 were incubated in SL medium for 7 days, and 10 OD600 units were sampled at day 1 and day 7 and processed for immunoblotting with anti-GFP antibodies as described in the Methods section. Positive control used in c and d was from dnm1Δ cells expressing Idp1-GFP from a plasmid at 4-day incubation (Fig. 1b, lane 4). Release of free GFP in WT cells is reproducibly variable between these reporters, and varies between 0 (Idh2) and 60% (Aco2). (e) Densitometric quantification of the release of free GFP (as % of total signal) in four independent experiments, comparing the results between WT and dnm1Δ cells. Bars denote s.d. (n=4).


( a ) expressing chromosomally tagged GFP chimeras of Aco2 , Idh2 , Idp1 Hsp78 as well as a plasmid - borne ( pDJB12 ) , mitochondrially targeted RFP ( mtRFP ) were incubated in SL - leucine medium for 3 days imaged daily . Aco2 Idp1 show clear mitophagic targeting ( arrows point at specific examples ) by day 3 , whereas Idh2 Hsp78 show weak or no mitophagy . In addition , Idh2 Hsp78 show different degrees of segregation relative to mtRFP marker , whereas Idp1 - GFP Aco2 - GFP show a near - complete co - localization with mtRFP . Scale bar , 5 μm . ( b ) of frequency of mitophagy from a ; of showing mitophagic profiles in red ( magenta bars ; plasmid - borne mtRFP ) or green ( turquoise bars ; integrated C - terminal GFP chimerae ) was calculated for each GFP chimera . Idp - GFP , red N = 76 , green N = 82 ; Aco2 - GFP , red N = 79 , green N = 82 ; Idh2 - GFP , red N = 72 , green N = 72 ; Hsp78 - GFP , red N = 70 , green N = 70 .

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(a) Cells expressing chromosomally tagged GFP chimeras of Aco2, Idh2, Idp1 and Hsp78 as well as a plasmid-borne (pDJB12), mitochondrially targeted RFP (mtRFP) were incubated in SL-leucine medium for 3 days and imaged daily. Aco2 and Idp1 show clear mitophagic targeting (white arrows point at specific examples) by day 3, whereas Idh2 and Hsp78 show weak or no mitophagy. In addition, Idh2 and Hsp78 show different degrees of segregation relative to the mtRFP marker, whereas Idp1-GFP and Aco2-GFP show a near-complete co-localization with mtRFP. Scale bar, 5 μm. (b) Quantification of the frequency of mitophagy from a; the percentage of cells showing mitophagic profiles in the red (magenta bars; plasmid-borne mtRFP) or green (turquoise bars; integrated C-terminal GFP chimerae) was calculated for each GFP chimera. Idp-GFP, red N=76, green N=82; Aco2-GFP, red N=79, green N=82; Idh2-GFP, red N=72, green N=72; Hsp78-GFP, red N=70, green N=70.


WT dnm1Δ expressing integrated Hsp78 - GFP plasmid - borne mtRFP were grown on lactate medium for 3 days . were imaged daily were analysed using ImageJ as described in Methods . Graphs illustrate distribution of pixels associated with specific values of RFP GFP channel intensities , respectively . Diagonal concentrations of dots indicate spatial correlation between signals . coloured photographic panels at bottom illustrate increased correlation of two channels at 3 - day time point in dnm1Δ mutant , relative to WT . Scale bar , 5 μm .

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WT and dnm1Δ cells expressing integrated Hsp78-GFP and plasmid-borne mtRFP were grown on lactate medium for 3 days. Cells were imaged daily and the images were analysed using ImageJ as described in Methods. Graphs illustrate the distribution of pixels associated with specific values of RFP and GFP channel intensities, respectively. Diagonal concentrations of dots indicate spatial correlation between the signals. The coloured photographic panels at the bottom illustrate the increased intensity correlation of the two channels at the 3-day time point in the dnm1Δ mutant, relative to the WT cells. Scale bar, 5 μm.


B , C Serial transplantations of PR + / + PR − / − mammary epithelia . ( B ) stereo microscopy of third ‐ generation mammary outgrowths derived from 8 ‐ week ‐ old PR + / + ; EGFP PR − / − ; EGFP donor mice . ( C ) Table summarizing 3 independent serial transplant experiments with PR + / + ; EGFP PR − / − ; EGFP . Each engrafted is represented by a micrograph ; black sectors represent of fat pad filled by engrafted epithelium . Scale bar : 200 μm .

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B, C Serial transplantations of PR+/+ and PR−/− mammary epithelia. (B) Fluorescence stereo microscopy of third‐generation mammary outgrowths derived from 8‐week‐old PR+/+; EGFP and PR−/−; EGFP donor mice. (C) Table summarizing 3 independent serial transplant experiments with PR+/+; EGFP and PR−/−; EGFP. Each engrafted gland is represented by a micrograph; black sectors represent area of fat pad filled by engrafted epithelium. Scale bar: 200 μm.


D , E Serial transplantation of RANKL + / + RANKL − / − mammary epithelia . ( D ) stereo microscopy of third ‐ generation mammary outgrowths derived from 5 ‐ week ‐ old RANKL + / + ; EGFP RANKL − / − ; EGFP donor mice . Insets : higher magnification showing side branches present in WT control ( arrowheads ) absent from RANKL − / − ; EGFP epithelium . ( E ) Table summarizing three independent serial transplant experiments with RANKL + / + ; EGFP RANKL − / − ; EGFP donor mice . Scale bar : 200 μm .

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D, E Serial transplantation of RANKL+/+ and RANKL−/− mammary epithelia. (D) Fluorescence stereo microscopy of third‐generation mammary outgrowths derived from 5‐week‐old RANKL+/+; EGFP and RANKL−/−; EGFP donor mice. Insets: higher magnification showing side branches present in the WT control (arrowheads) absent from RANKL−/−; EGFP epithelium. (E) Table summarizing three independent serial transplant experiments with RANKL+/+; EGFP and RANKL−/−; EGFP donor mice. Scale bar: 200 μm.


F , G Serial transplantation of Wnt4 + / + Wnt4 − / − mammary epithelia . ( F ) stereo microscopy of third ‐ generation mammary outgrowths derived from mammary buds of E12 . 5 E13 . 5 Wnt4 + / + ; EGFP Wnt4 − / − ; EGFP . Scale bar : 200 μm . ( G ) Table summarizing three independent serial transplant experiments with Wnt4 + / + ; EGFP donor mice . Scale bar : 200 μm .

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F, G Serial transplantation of Wnt4+/+ and Wnt4−/− mammary epithelia. (F) Fluorescence stereo microscopy of third‐generation mammary outgrowths derived from mammary buds of E12.5 and E13.5 Wnt4+/+; EGFP and Wnt4−/−; EGFP embryos. Scale bar: 200 μm. (G) Table summarizing three independent serial transplant experiments with Wnt4+/+; EGFP donor mice. Scale bar: 200 μm.


H Box plot showing difference between of reconstitution between WT MT grafts in each transplant generation . P values were determined by Mann - Whitney U ‐ .

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H Box plot showing the difference between percentage of reconstitution between WT and MT contralateral grafts in each transplant generation. P values were determined by Mann-Whitney U‐test.


A Epifluorescence stereo microscopy of inguinal mammary from a 5 ‐ day ‐ old Wnt4 : : Cre ; mT / mG ( n = 7 ) . Scale bars : 0 . 5 mm 0 . 1 mm ( inset ) .

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A Epifluorescence stereo microscopy of inguinal mammary gland from a 5‐day‐old Wnt4::Cre; mT/mG female (n = 7). Scale bars: 0.5 mm and 0.1 mm (inset).


B , C Histology sections of mammary from a 5 ‐ day ‐ old ( B ; n = 7 ) a 10 ‐ day ‐ old ( C ; n = 5 ) Wnt4 : : Cre ; mT / mG stained by double immunofluorescence microscopy for EGFP ( green ) PR ( magenta , not detected ) , counterstained with DAPI ( blue ) . Scale bar : 50 mm .

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B, C Histology sections of mammary glands from a 5‐day‐old (B; n = 7) and a 10‐day‐old (C; n = 5) Wnt4::Cre; mT/mG female stained by double immunofluorescence microscopy for EGFP (green) and PR (magenta, not detected), counterstained with DAPI (blue). Scale bar: 50 mm.


D - F EGFP ( green ) PR ( magenta ) co ‐ immunofluorescence microscopy counterstained with DAPI ( blue ) on histology sections from mT / mG ; Wnt4 : : Cre mammary at different developmental stages . ( D ) TEB of a 4 ‐ week ‐ old ( n = 4 ) ; scale bar : 30 μm . ( E ) of an 8 ‐ week ‐ old ( n = 3 ) ; scale bar : 100 μm ; inset , scale bar : 20 μm . ( F ) of a at day 10 . 5 of pregnancy ( n = 3 ) ; scale bar : 150 μm ; inset , scale bar : 30 μm .

O O O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O B-GENEPROD B-TISSUE 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F EGFP (green) and PR (magenta) co‐immunofluorescence microscopy counterstained with DAPI (blue) on histology sections from mT/mG; Wnt4::Cre mammary glands at different developmental stages. (D) TEB of a 4‐week‐old female (n = 4); scale bar: 30 μm. (E) Ducts of an 8‐week‐old female (n = 3); scale bar: 100 μm; inset, scale bar: 20 μm. (F) Duct of a female at day 10.5 of pregnancy (n = 3); scale bar: 150 μm; inset, scale bar: 30 μm.


G - I EGFP ( green ) , PR ( magenta ) , p63 ( ) triple co ‐ immunofluorescence microscopy counterstained with DAPI ( blue ) on histological sections from mT / mG ; Wnt4 : : Cre mammary at different developmental stages . ( G ) of 5 ‐ day ‐ old ( n = 3 ) . ( H ) TEB of a 4 ‐ week ‐ old ( n = 3 ) . ( I ) of an 8 ‐ week ‐ old ( n = 3 ) . Scale bars : 30 μm .

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

G-I EGFP (green), PR (magenta), and p63 (white) triple co‐immunofluorescence microscopy counterstained with DAPI (blue) on histological sections from mT/mG; Wnt4::Cre mammary glands at different developmental stages. (G) Ducts of 5‐day‐old female (n = 3). (H) TEB of a 4‐week‐old female (n = 3). (I) Duct of an 8‐week‐old female (n = 3). Scale bars: 30 μm.


J - O Epifluorescence stereo microscopy of mammary harvested from 15 ‐ day ‐ old Wnt4 : : GFP either WT ( n = 18 ) ( J , M ) , ERα − / − ( n = 4 ) ( K , N ) , or PR − / − ( n = 3 ) ( L , O ) . dTomato expression ( J - L ) ; EGFP expression ( M - O ) is not abrogated in ERα − / − nor PR − / − epithelia . Arrowheads mark main originating from nipple . Scale bar : 50 μm .

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

J-O Epifluorescence stereo microscopy of mammary glands harvested from 15‐day‐old Wnt4::GFP females either WT (n = 18) (J, M), ERα−/− (n = 4) (K, N), or PR−/− (n = 3) (L, O). dTomato expression (J-L); EGFP expression (M-O) is not abrogated in ERα−/− nor PR−/− epithelia. Arrowheads mark the main duct originating from the nipple. Scale bar: 50 μm.


Q Bar plots showing relative PR Wnt4 mRNA expression normalized to CK18 mRNA in mammary organoids from 5 pubertal ( 6 old ) 3 adult ( 11 old ) mice exposed for 6 h to vehicle ( C ) , 17β ‐ estradiol ( 20 nmol ) ( E2 ) , or R5020 ( 20 nmol ) ( P ) . Bars represent mean ± SD of 3 independent experiments .

O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-ORGANISM O O O 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 B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

Q Bar plots showing relative PR and Wnt4 mRNA expression normalized to CK18 mRNA in mammary organoids from 5 pubertal (6 weeks old) and 3 adult (11 weeks old) mice exposed for 6 h to vehicle (C), 17β‐estradiol (20 nmol) (E2), or R5020 (20 nmol) (P). Bars represent the mean ± SD of 3 independent experiments.


R - X Epifluorescence stereo microscopy of mammary that were engrafted with Wnt4 : : GFP epithelium from 8 ‐ week ‐ old , either PRWT ( R , T , V , X ) or PR − / − ( S , U , W ) . dTomato expression ( R , S ) ; EGFP expression ( T , U ) double epifluorescence ( V , W , X ) on engrafted 3 after surgery when recipients were 6 old . Representative result from three independent experiments . Arrowheads point to TEBs ( V , X ) or to origin of growth ( W ) . Scale bar ( R - W ) : 5 mm , ( X ) : 1 mm .

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

R-X Epifluorescence stereo microscopy of contralateral mammary glands that were engrafted with Wnt4::GFP epithelium from 8‐week‐old females, either PRWT (R, T, V, X) or PR−/− (S, U, W). dTomato expression (R, S); EGFP expression (T, U) double epifluorescence (V, W, X) on contralateral engrafted glands 3 weeks after surgery when recipients were 6 weeks old. Representative result from three independent experiments. Arrowheads point to TEBs (V, X) or to origin of growth (W). Scale bar (R-W): 5 mm, (X): 1 mm.


A - D Representative epifluorescence stereo microscopy of inguinal mammary from a 10 ‐ day ‐ old mT / mG ( A , C ) MMTV : : Cre ; mT / mG ( B , D ) ( n = 7 ) . Scale bar : 1 mm .

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

A-D Representative epifluorescence stereo microscopy of inguinal mammary glands from a 10‐day‐old mT/mG (A, C) and MMTV::Cre; mT/mG (B, D) female (n = 7). Scale bar: 1 mm.


E Higher magnification of inguinal mammary from a 10 ‐ day ‐ old MMTV : : Cre ; mT / mG . Scale bar : 0 . 2 mm . F Immunofluorescence microscopy for EGFP ( green ) on a mammary section from a 10 ‐ day ‐ old MMTV : : Cre ; mT / mG counterstained with DAPI ( blue ) ( n = 7 ) . Scale bar : 30 μm .

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

E Higher magnification of inguinal mammary gland from a 10‐day‐old MMTV::Cre; mT/mG female. Scale bar: 0.2 mm.F Immunofluorescence microscopy for EGFP (green) on a mammary gland section from a 10‐day‐old MMTV::Cre; mT/mG female counterstained with DAPI (blue) (n = 7). Scale bar: 30 μm.


G Epifluorescence stereo microscopy of mammary from 10 ‐ day ‐ old either MMTV : : Cre ; mT / mG Wnt4fl / + or MMTV : : Cre ; mT / mG ; Wnt4fl / fl . Scale bar : 1 mm . Arrowhead marks main originating from nipple . H Bar plot showing of branching points in prepubertal Cre + ; Wnt4fl / fl ( n = 12 ) relative to Cre + ; Wnt4fl / + ( n = 10 ) . Two ‐ , paired Student ' s t ‐ was used to calculate statistical significance .

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

G Epifluorescence stereo microscopy of mammary glands from 10‐day‐old littermates either MMTV::Cre; mT/mG Wnt4fl/+ or MMTV::Cre; mT/mG; Wnt4fl/fl. Scale bar: 1 mm. Arrowhead marks the main duct originating from the nipple.H Bar plot showing ratio of branching points in prepubertal Cre+;Wnt4fl/fl (n = 12) relative to Cre+;Wnt4fl/+ littermates (n = 10). Two‐tailed, paired Student's t‐test was used to calculate statistical significance.