D . In vitro ubiquitylation experiment as in B . Blots 1 & 2 : Plk1 or RanGAP1 used as substrates instead of MCAK . Blot 3 : Left : Experiment as in B . Asterisks indicate signals from Plk1 antibody ( same species as anti - Fbxw5 ) . Right : Same reaction except that Cul1 ~ Nedd8 / Rbx1 complexes were replaced by Cul4A ~ Nedd8 / DDB1 / Rbx1 complexes Bottom blots : Same blots as above incubated with anti - Fbxw5 antibodies . Autoubiquitylation of Fbxw5 indicates activity of the E3 ligase complex .

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

D. In vitro ubiquitylation experiment as in B. Blots 1&2: Plk1 or RanGAP1 used as substrates instead of MCAK. Blot 3: Left: Experiment as in B. Asterisks indicate signals from Plk1 antibody (same species as anti-Fbxw5). Right: Same reaction except that Cul1~Nedd8/Rbx1 complexes were replaced by Cul4A~Nedd8/DDB1/Rbx1 complexes Bottom blots: Same blots as above incubated with anti-Fbxw5 antibodies. Autoubiquitylation of Fbxw5 indicates activity of the E3 ligase complex.


G . In vitro ubiquitylation experiment as in B using either wild type , K48R , K63R , KK48 , 63RR or methylated ubiquitin ( Kmethyl , see Fig EV2K ) . Top : Cdc34 used as E2 . Bottom : UbcH5b used as E2 .

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

G. In vitro ubiquitylation experiment as in B using either wild type, K48R, K63R, KK48,63RR or methylated ubiquitin (Kmethyl, see Fig EV2K). Top: Cdc34 used as E2. Bottom: UbcH5b used as E2.


B . Quantification of the MCAK / Uba2 signal ratio normalised to each non - targeting control of 4 independent experiments . Error bars indicate standard deviation , asterisks the p - value of a two - tailed unpaired Student ' s t - test comparing each Fbxw5 - directed siRNA sample with the according non - targeting control ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

B. Quantification of the MCAK/Uba2 signal ratio normalised to each non-targeting control of 4 independent experiments. Error bars indicate standard deviation, asterisks the p-value of a two-tailed unpaired Student's t-test comparing each Fbxw5-directed siRNA sample with the according non-targeting control (*P < 0.05, **P < 0.01, ***P < 0.001).


C . RPE - 1 cells were transfected with the indicated siRNAs ( 72 hours total time ) , split on coverslips 24 hours later and serum - starved for the last 24 hours . Cells were fixed in methanol and analysed via immunofluorescence using the indicated antibodies together with Hoechst staining . Left : Maximum intensity projections ( same for all following microscopy images ) of representative images . Arrows indicate ODF2 signal . Scale bar = 5 µm . Note : Middle panel shows an example image of siFw5 - 3 . Right : Quantification of MCAK signals co - localising with ODF2 . Long black line shows mean intensity and error bars indicate standard error of the mean of 3 independent experiments covering in total more than 280 cells . Asterisks indicate p - value of a Mann Whitney test comparing each sample set with the non - targeting control ( * * P < 0 . 01 , * * * P < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RPE-1 cells were transfected with the indicated siRNAs (72 hours total time), split on coverslips 24 hours later and serum-starved for the last 24 hours. Cells were fixed in methanol and analysed via immunofluorescence using the indicated antibodies together with Hoechst staining. Left: Maximum intensity projections (same for all following microscopy images) of representative images. Arrows indicate ODF2 signal. Scale bar = 5 µm. Note: Middle panel shows an example image of siFw5-3. Right: Quantification of MCAK signals co-localising with ODF2. Long black line shows mean intensity and error bars indicate standard error of the mean of 3 independent experiments covering in total more than 280 cells. Asterisks indicate p-value of a Mann Whitney test comparing each sample set with the non-targeting control (**P < 0.01, ***P < 0.001).


A . Fluorescence - based pulse - chase experiment using a monoclonal RPE - 1 cell line expressing mNG - MCAK under a doxycycline - inducible promoter . Cells were transfected with the indicated siRNAs ( siFw5 - 1 & 3 together ) and split 24 hours later on Ibidi 8 Well Glass Bottom µ - slides while simultaneously inducing mNG - MCAK expression with 6 ng / ml doxycycline ( pulse ) . 24 hours later , doxycycline was washed out and cells were released into serum - free medium . Cells that have just finished or were about to finish mitosis ( distinguishable for example by the midbody signal of mNG - MCAK ( arrow heads ) ) were selected and imaged over 24 hours , taking an image every 20 min ( chase ) . Left : representative images of selected time points . Scale bar = 10 µm . Arrows indicate centrosomal mNG - MCAK signals , arrowheads mNG - MCAK signals at the midbody . Right : Quantification of centrosomal mNG - MCAK signals . Error bars show standard error of the mean of 3 independent experiments with 25 cells in total . P - value of a two - tailed unpaired Students t - test comparing signal intensity of Fbxw5 knockdown over non - targeting control was mostly below 0 . 01 for time points 0 to 8 hours and below 0 . 05 for time points 9h to 20h . Dark and light blue lines indicate the time points at which mNG - MCAK signals are reduced by 50 % compared to time point 0 for siNT and siFbxw5 samples , respectively .

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

A. Fluorescence-based pulse-chase experiment using a monoclonal RPE-1 cell line expressing mNG-MCAK under a doxycycline-inducible promoter. Cells were transfected with the indicated siRNAs (siFw5-1&3 together) and split 24 hours later on Ibidi 8 Well Glass Bottom µ-slides while simultaneously inducing mNG-MCAK expression with 6 ng/ml doxycycline (pulse). 24 hours later, doxycycline was washed out and cells were released into serum-free medium. Cells that have just finished or were about to finish mitosis (distinguishable for example by the midbody signal of mNG-MCAK (arrow heads)) were selected and imaged over 24 hours, taking an image every 20 min (chase). Left: representative images of selected time points. Scale bar = 10 µm. Arrows indicate centrosomal mNG-MCAK signals, arrowheads mNG-MCAK signals at the midbody. Right: Quantification of centrosomal mNG-MCAK signals. Error bars show standard error of the mean of 3 independent experiments with 25 cells in total. P-value of a two-tailed unpaired Students t-test comparing signal intensity of Fbxw5 knockdown over non-targeting control was mostly below 0.01 for time points 0 to 8 hours and below 0.05 for time points 9h to 20h. Dark and light blue lines indicate the time points at which mNG-MCAK signals are reduced by 50% compared to time point 0 for siNT and siFbxw5 samples, respectively.


D . Same samples as in C , except that cells grown on coverslips were fixed in methanol and analysed by immunofluorescence . Left : Representative images . Arrows indicate ODF2 signal . Scale bar = 5 µm . Right : Quantification . Error bars show standard error of the mean of 3 independent experiments , covering in total more than 150 cells . Asterisks indicate the p - value of a Mann Whitney test ( * * * P < 0 . 001 ) .

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D. Same samples as in C, except that cells grown on coverslips were fixed in methanol and analysed by immunofluorescence. Left: Representative images. Arrows indicate ODF2 signal. Scale bar = 5 µm. Right: Quantification. Error bars show standard error of the mean of 3 independent experiments, covering in total more than 150 cells. Asterisks indicate the p-value of a Mann Whitney test (***P < 0.001).


B . CHX chase experiment as in A , except that cells were treated with the indicated siRNAs for 48h and arrested with 200 mM thymidine for 16h , released for 9 hours and again arrested for 16h . Following the 2nd release , 50 µg / ml CHX was added as indicated and samples were analysed as in A . Left : representative blots . Asterisks indicate unspecific bands detected by the Fbxw5 antibody . Right : Quantification of MCAK / Uba2 signal intensity ratios normalised to time point zero . Error bars show standard deviation of 6 independent experiments and asterisks indicate p - value of a two - tailed unpaired Student ' s t - test ( * * P < 0 . 01 ) .

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

B. CHX chase experiment as in A, except that cells were treated with the indicated siRNAs for 48h and arrested with 200 mM thymidine for 16h, released for 9 hours and again arrested for 16h. Following the 2nd release, 50 µg/ml CHX was added as indicated and samples were analysed as in A. Left: representative blots. Asterisks indicate unspecific bands detected by the Fbxw5 antibody. Right: Quantification of MCAK/Uba2 signal intensity ratios normalised to time point zero. Error bars show standard deviation of 6 independent experiments and asterisks indicate p-value of a two-tailed unpaired Student's t-test (**P < 0.01).


C . Extracts of RPE - 1 cells treated with the indicated siRNAs for 72 hours , grown asynchronously or arrested with the indicated compounds for the last 24 hours and treated either with DMSO ( - ) or 10 µM MG - 132 ( + ) for the very last 6 hours . Left : Representative blots . Asterisk indicates an unspecific band detected by the Fbxw5 antibody . Right : Quantification of MCAK / Uba2 signal ratio normalised to non - targeting control with DMSO of 6 independent experiments . Error bars indicate standard deviation , asterisks the p - value of a two - tailed unpaired Student ' s t - test ( * * P < 0 . 01 ) .

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

C. Extracts of RPE-1 cells treated with the indicated siRNAs for 72 hours, grown asynchronously or arrested with the indicated compounds for the last 24 hours and treated either with DMSO (-) or 10 µM MG-132 (+) for the very last 6 hours. Left: Representative blots. Asterisk indicates an unspecific band detected by the Fbxw5 antibody. Right: Quantification of MCAK/Uba2 signal ratio normalised to non-targeting control with DMSO of 6 independent experiments. Error bars indicate standard deviation, asterisks the p-value of a two-tailed unpaired Student's t-test (**P < 0.01).


A . Polyclonal RPE - 1 cells expressing mNG - MCAK under a doxycycline - inducible promoter were seeded on coverslips , induced with 20 ng / ml doxycycline and grown for 24 hours . Subsequently , cells were washed twice with PBS and incubated for another 24 hours in serum - free medium without doxycycline . Cells were then fixed in methanol and subjected to IF using the indicated antibodies together with Hoechst staining . Left : Representative images . Scale bar = 10 µm . Right : Quantification of ciliated cells . Error bars show standard deviation of 5 independent experiments covering more than 200 cells and asterisks indicate p - value of a two - tailed unpaired Student ' s t - test ( * * P < 0 . 01 ) .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Polyclonal RPE-1 cells expressing mNG-MCAK under a doxycycline-inducible promoter were seeded on coverslips, induced with 20 ng/ml doxycycline and grown for 24 hours. Subsequently, cells were washed twice with PBS and incubated for another 24 hours in serum-free medium without doxycycline. Cells were then fixed in methanol and subjected to IF using the indicated antibodies together with Hoechst staining. Left: Representative images. Scale bar = 10 µm. Right: Quantification of ciliated cells. Error bars show standard deviation of 5 independent experiments covering more than 200 cells and asterisks indicate p-value of a two-tailed unpaired Student's t-test (**P < 0.01).


C . RPE - 1 cells were treated with the indicated siRNA for 48 hours followed by serum - starvation for another 24 hours . Cells were then fixed in methanol and subjected to IF using the indicated antibodies together with Hoechst staining . Left : Representative images . Note : Left panel shows an example image of siFw5 - 1 . Scale bar = 10 µm . Right : Quantification of ciliated cells . Error bars show standard deviation of 4 independent experiments covering more than 200 cells and asterisks indicate p - value of a two - tailed unpaired Student ' s t - test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RPE-1 cells were treated with the indicated siRNA for 48 hours followed by serum-starvation for another 24 hours. Cells were then fixed in methanol and subjected to IF using the indicated antibodies together with Hoechst staining. Left: Representative images. Note: Left panel shows an example image of siFw5-1. Scale bar = 10 µm. Right: Quantification of ciliated cells. Error bars show standard deviation of 4 independent experiments covering more than 200 cells and asterisks indicate p-value of a two-tailed unpaired Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001).


( B ) The inherent elastic response of a lipid membrane ( POPC ) was studied by enforcing indentations via the isolated SNARE C - termini in the presence or absence of a short linker peptide , or the linker plus a fluorescent protein tag ( FP ) . We define " relative indentation " as the change in C - terminus ( Nyv1 ) - C - terminus ( Vam3 ) distance that occurs when the SNARE complex performs mechanical work . It reflects the depth of the formed well upon squeezing . The slope of the obtained work profile estimates the required force ( thin black line ) . Perforation ( red arrow ) requires a force of 80 pN for the wild - type C - termini . The plateau within the work profile indicates that the membrane gives up its elastic response ( perforation transition ) . C - terminal tags oppose such a perforation transition but do not significantly affect the forces observed within the indentation regime . Furthermore , if the unstructured peptide - spacer would be alpha - helical - and thus much shorter - indentation forces would still be only slightly affected .

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

(B) The inherent elastic response of a lipid membrane (POPC) was studied by enforcing indentations via the isolated SNARE C-termini in the presence or absence of a short linker peptide, or the linker plus a fluorescent protein tag (FP). We define "relative indentation" as the change in C-terminus (Nyv1) - C-terminus (Vam3) distance that occurs when the SNARE complex performs mechanical work. It reflects the depth of the formed well upon squeezing. The slope of the obtained work profile estimates the required force (thin black line). Perforation (red arrow) requires a force of 80 pN for the wild-type C-termini. The plateau within the work profile indicates that the membrane gives up its elastic response (perforation transition). C-terminal tags oppose such a perforation transition but do not significantly affect the forces observed within the indentation regime. Furthermore, if the unstructured peptide-spacer would be alpha-helical - and thus much shorter - indentation forces would still be only slightly affected.


( B ) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS - PAGE and Western blotting .

O 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) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS-PAGE and Western blotting.


( C ) In vivo vacuole morphology was assayed by FM4 - 64 staining . Scale bar : 5 µm . ( D ) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells . Values represent the means and s . d . from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vivo vacuole morphology was assayed by FM4-64 staining. Scale bar: 5 µm. (D) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments.


( E ) Vacuoles were isolated from BJ3505 strains expressing the indicated tagged versions of Vam3 and Nyv1 and Pho8 - EGFP or Pho8 - mCherry . 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy . Arrows indicate examples of fusion products . Scale bar : 5 µm . ( F ) Fusion activity : vacuole fusion was assayed by measuring the percentage of co - localization of the two Pho8 - EGFP and Pho8 - mCherry signals . Means s . d . are shown for at least 100 stained vacuoles from 3 experiments .

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

(E) Vacuoles were isolated from BJ3505 strains expressing the indicated tagged versions of Vam3 and Nyv1 and Pho8-EGFP or Pho8-mCherry. 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy. Arrows indicate examples of fusion products. Scale bar: 5 µm. (F) Fusion activity: vacuole fusion was assayed by measuring the percentage of co-localization of the two Pho8-EGFP and Pho8-mCherry signals. Means s.d. are shown for at least 100 stained vacuoles from 3 experiments.


( A ) Vacuoles were isolated from BJ3505 cells expressing the indicated versions of Vam3 and / or Nyv1 . 10 µg of the organelles were analyzed by SDS - PAGE and Western blotting using antibodies to Vti1 , Vam3 and Nyv1 .

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

(A) Vacuoles were isolated from BJ3505 cells expressing the indicated versions of Vam3 and/or Nyv1. 10 µg of the organelles were analyzed by SDS-PAGE and Western blotting using antibodies to Vti1, Vam3 and Nyv1.


( B ) Localization in vivo . The indicated strains were grown logarithmically in synthetic complete ( SC ) medium . After 16 h at 30 ° C , when the final OD of each culture was about 1 , the cells were observed by confocal fluorescence and differential interference ( DIC ) microscopy . Scale bar : 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Localization in vivo. The indicated strains were grown logarithmically in synthetic complete (SC) medium. After 16 h at 30°C, when the final OD of each culture was about 1, the cells were observed by confocal fluorescence and differential interference (DIC) microscopy. Scale bar: 5 µm.


( A ) In vivo morphology . Cells expressing the indicated SNARE variants were grown in SC medium ( 16 h , 30 ° C ) , stained with FM4 - 64 and their relative morphology was assessed by fluorescence and DIC microscopy . Scale bar : 5 µm . The cells were grouped into three categories according to the number of vacuoles visible per 100 cells . Values represent the means and s . d . from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vivo morphology. Cells expressing the indicated SNARE variants were grown in SC medium (16 h, 30°C), stained with FM4-64 and their relative morphology was assessed by fluorescence and DIC microscopy. Scale bar: 5 µm. The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments.


( B ) and ( C ) SNARE activation on isolated vacuoles . Vacuoles were isolated from a strain co - expressing NYV1 - S9 - EGFP and VAM3 - S9 - mCitrine and from an isogenic wild - type . 150 µg of the organelles were incubated in fusion reactions in the presence or absence of an ATP - regenerating system and recombinant , purified Sec18 / NSF ( rSec18 , 50 µg / ml ) . After 10 min of incubation at 27 ° C , the vacuoles were solubilized and immunoprecipitated with antibodies to Nyv1 . Co - immunoprecipitated proteins analyzed by SDS - PAGE and Western blotting . The histograms provide quantifications of the band intensities as the means and s . d . from three independent experiments .

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

(B) and (C) SNARE activation on isolated vacuoles. Vacuoles were isolated from a strain co-expressing NYV1-S9-EGFP and VAM3-S9-mCitrine and from an isogenic wild-type. 150 µg of the organelles were incubated in fusion reactions in the presence or absence of an ATP-regenerating system and recombinant, purified Sec18/NSF (rSec18, 50 µg/ml). After 10 min of incubation at 27°C, the vacuoles were solubilized and immunoprecipitated with antibodies to Nyv1. Co-immunoprecipitated proteins analyzed by SDS-PAGE and Western blotting. The histograms provide quantifications of the band intensities as the means and s.d. from three independent experiments.


( A ) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS - PAGE and Western blotting .

(A) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS-PAGE and Western blotting.


( B ) Vacuole morphology was assessed for the indicated cells as in Fig . 3C . Scale bar : 5 µm .

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

(B) Vacuole morphology was assessed for the indicated cells as in Fig. 3C. Scale bar: 5 µm.


( C ) Vacuoles were isolated from BJ3505 and DKY6281 cells with SNAREs tagged or deleted as indicated . Note that presence of Nyv1 on only one fusion partner still permits efficient fusion [ 65 ] but ensures that trans - SNARE complexes can only form in one orientation , between the Q - SNARE in DKY6281 and the R - SNARE in BJ3505 . The vacuoles were used in standard fusion reactions and content mixing was measured via the alkaline phosphatase assay . In parallel , identical samples were incubated either incubated on ice , which prevents fusion , or in the presence of 0 . 5 % Triton X - 100 , which allows fusion - independent access of the maturase Pep4 to pro - ALP and controls for the levels of these two reporter enzymes in the samples .

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

(C) Vacuoles were isolated from BJ3505 and DKY6281 cells with SNAREs tagged or deleted as indicated. Note that presence of Nyv1 on only one fusion partner still permits efficient fusion [65] but ensures that trans-SNARE complexes can only form in one orientation, between the Q-SNARE in DKY6281 and the R-SNARE in BJ3505. The vacuoles were used in standard fusion reactions and content mixing was measured via the alkaline phosphatase assay. In parallel, identical samples were incubated either incubated on ice, which prevents fusion, or in the presence of 0.5% Triton X-100, which allows fusion-independent access of the maturase Pep4 to pro-ALP and controls for the levels of these two reporter enzymes in the samples.


( D ) Using strains from C , lipid and content mixing ( left and right panel , respectively ) were performed in parallel in the presence or absence of ATP . Anti - Vam3 antibody ( 3 µg ) was added to some reactions in order to inhibit trans - SNARE pairing and fusion . Means and the s . d . are shown for three independent experiments .

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(D) Using strains from C, lipid and content mixing (left and right panel, respectively) were performed in parallel in the presence or absence of ATP. Anti-Vam3 antibody (3 µg) was added to some reactions in order to inhibit trans-SNARE pairing and fusion. Means and the s.d. are shown for three independent experiments.


Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig . 3A but the spacer was extended ( S34 ) by an additional 25 - amino acid sequence ( SGGGGSGGGGSGGGGSGGGGAAAGG ) to the previous short spacer . ( A ) Protein levels on isolated vacuoles from these strains were compared as in Fig . 3B .

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

Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig. 3A but the spacer was extended (S34) by an additional 25-amino acid sequence (SGGGGSGGGGSGGGGSGGGGAAAGG) to the previous short spacer. (A) Protein levels on isolated vacuoles from these strains were compared as in Fig. 3B.


Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig . 3A but the spacer was extended ( S34 ) by an additional 25 - amino acid sequence ( SGGGGSGGGGSGGGGSGGGGAAAGG ) to the previous short spacer . ( B ) Vacuole morphology was assessed as in Fig . 3C . Scale bar : 5 µm . ( C ) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells . Values represent the means and s . d . from three independent experiments .

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

Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig. 3A but the spacer was extended (S34) by an additional 25-amino acid sequence (SGGGGSGGGGSGGGGSGGGGAAAGG) to the previous short spacer. (B) Vacuole morphology was assessed as in Fig. 3C. Scale bar: 5 µm. (C) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments.


Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig . 3A but the spacer was extended ( S34 ) by an additional 25 - amino acid sequence ( SGGGGSGGGGSGGGGSGGGGAAAGG ) to the previous short spacer . ( D ) Fusion activity : vacuoles were isolated from BJ3505 strains expressing the indicated versions of Vam3 and Nyv1 and Pho8 - mCFP or Pho8 - mCherry . 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy . Arrows indicate examples of fusion products . Means s . d . are shown for at least 100 stained vacuoles from 3 experiments . Scale bar : 5 µm . ( E ) Vacuole fusion was assayed by measuring the percentage of co - localisation of the two Pho8 - mCFP and Pho8 - mCherry signals .

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

Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig. 3A but the spacer was extended (S34) by an additional 25-amino acid sequence (SGGGGSGGGGSGGGGSGGGGAAAGG) to the previous short spacer. (D) Fusion activity: vacuoles were isolated from BJ3505 strains expressing the indicated versions of Vam3 and Nyv1 and Pho8-mCFP or Pho8-mCherry. 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy. Arrows indicate examples of fusion products. Means s.d. are shown for at least 100 stained vacuoles from 3 experiments. Scale bar: 5 µm. (E) Vacuole fusion was assayed by measuring the percentage of co-localisation of the two Pho8-mCFP and Pho8-mCherry signals.


( A ) Yeast strain expressing a plasmid with Vph1 - GFP was grown in SC - URA medium for 16 h at 30 ° C . The mobility of Vph1 - GFP in the absence ( upper panels ) or presence ( lower panel ) of CPZ ( 150 µM ) was assessed by FRAP . ( B ) The histogram shows the GFP signals in the bleached area during the FRAP procedure . ( C ) The half - time is shown . Means and s . d . are shown for 20 cells of 3 independent experiments .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Yeast strain expressing a plasmid with Vph1-GFP was grown in SC-URA medium for 16 h at 30°C. The mobility of Vph1-GFP in the absence (upper panels) or presence (lower panel) of CPZ (150 µM) was assessed by FRAP. (B) The histogram shows the GFP signals in the bleached area during the FRAP procedure. (C) The half-time is shown. Means and s.d. are shown for 20 cells of 3 independent experiments.


( D ) Yeast cells were grown in SC medium for 16 h at 30 ° C . After staining with FM4 - 64 , cells were treated with CPZ ( 150 µM ) . Vacuole morphology was assessed by confocal microscopy before and after 5 and 20 min of CPZ addition . Scale bar : 5 µm .

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(D) Yeast cells were grown in SC medium for 16 h at 30°C. After staining with FM4-64, cells were treated with CPZ (150 µM). Vacuole morphology was assessed by confocal microscopy before and after 5 and 20 min of CPZ addition. Scale bar: 5 µm.


( E ) Fusion activity . Vacuoles were isolated from BJ cells carrying Nyv1 - S9 - EGFP and DKY cells deleted for Nyv1 and carrying Vam3 - S9 - mCitrine or from wild - type cells and incubated in standard fusion reactions in the presence or absence of chlorpromazine ( CPZ , 150 µM ) and anti - Vam3 ( 3 µg ) . Content mixing was assayed via alkaline phosphatase activity . Means and s . d . are shown for three independent experiments .

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

(E) Fusion activity. Vacuoles were isolated from BJ cells carrying Nyv1-S9-EGFP and DKY cells deleted for Nyv1 and carrying Vam3-S9-mCitrine or from wild-type cells and incubated in standard fusion reactions in the presence or absence of chlorpromazine (CPZ, 150 µM) and anti-Vam3 (3 µg). Content mixing was assayed via alkaline phosphatase activity. Means and s.d. are shown for three independent experiments.


( A ) OCR of wt and gas - 1 mutant nematodes ( normalized to nematode size as given by the COPAS Biosort ( Time of Flight - TOF ) ) . One representative curve shown ( mean ± SEM ; n = 3 ) * * * * p value < 0 . 0001 ; Kruskal - Wallis Test , Dunn ' s post hoc correction .

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(A) OCR of wt and gas-1 mutant nematodes (normalized to nematode size as given by the COPAS Biosort (Time of Flight -TOF)). One representative curve shown (mean ± SEM; n=3) ****p value< 0.0001; Kruskal-Wallis Test, Dunn's post hoc correction.


Representative curves show the lifespan of wt and gas - 1 ( fc21 ) nematodes compared to ( B ) age - 1 and age - 1 ; gas - 1 mutants animals . Average median lifespans ± SEM from all replicates indicated beneath representative curves .

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

Representative curves show the lifespan of wt and gas-1(fc21) nematodes compared to (B) age-1 and age-1; gas-1 mutants animals. Average median lifespans ± SEM from all replicates indicated beneath representative curves.


Representative curves show the lifespan of wt and gas - 1 ( fc21 ) nematodes compared to ( C ) daf - 16 and daf - 16 ; age - 1 ; gas - 1 mutant animals . Average median lifespans ± SEM from all replicates indicated beneath representative curves .

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

Representative curves show the lifespan of wt and gas-1(fc21) nematodes compared to (C) daf-16 and daf-16; age-1; gas-1 mutant animals. Average median lifespans ± SEM from all replicates indicated beneath representative curves.


( D ) Volcano plot of differentially phosphorylated proteins in gas - 1 ( fc21 ) mutants compared to wt . Cutoffs : 20 % FDR and | log2 ( foldchange ) | > 0 . 345 ( in red ) .

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(D) Volcano plot of differentially phosphorylated proteins in gas-1(fc21) mutants compared to wt. Cutoffs: 20% FDR and |log2(foldchange)| >0.345 (in red).


( F ) Lifespan analysis of mev - 1 ( kn1 ) nematodes compared to wt , age - 1 ( hx546 ) and age - 1 ( hx546 ) ; mev - 1 ( kn1 ) animals . ( G )

(F) Lifespan analysis of mev-1(kn1) nematodes compared to wt, age-1(hx546) and age-1(hx546); mev-1(kn1) animals. (G)


( G ) ATP measurements in wt , age - 1 , gas - 1 and age - 1 ; gas - 1 mutant nematodes ( normalized to wt ) , n = 4 . Error bars : Mean ± SEM , * p value < 0 . 05 , * * * p value = 0 . 001 , Kruskal - Wallis Test , Dunn ' s post hoc correction .

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

(G) ATP measurements in wt, age-1, gas-1 and age-1; gas-1 mutant nematodes (normalized to wt), n=4. Error bars: Mean ± SEM, *p value <0.05, ***p value =0.001, Kruskal-Wallis Test, Dunn's post hoc correction.


( H ) OCR of gas - 1 and age - 1 ; gas - 1 mutant nematodes ( normalized to TOF ) . Left panel : One representative curve ( Mean ± SEM ) . Right panel : Basal respiration in wt , gas - 1 and age - 1 ; gas - 1 animals , pooled from 3 biological replicates , * * * * p value < 0 . 0001 , ns = non - significant , Kruskal - Wallis Test , Dunn ' s post hoc correction .

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

(H) OCR of gas-1 and age-1; gas-1 mutant nematodes (normalized to TOF). Left panel: One representative curve (Mean ± SEM). Right panel: Basal respiration in wt, gas-1 and age-1; gas-1 animals, pooled from 3 biological replicates, ****p value <0.0001, ns= non-significant, Kruskal-Wallis Test, Dunn's post hoc correction.


( I ) STED images of representative mitochondrial morphology in muscle cells of L4 mutant worms . Scale bar : 2 μm . ( J ) Left panel : Percentage of FRAP . Each point : mean recovery of ~ 30 bleached regions ( 2 regions per animal , 15 animals per condition ) ± SEM . Right panel : values of the maximum ( total ) recovery per strain ± SEM , * * * p value < 0 . 001 , * * * * p value < 0 . 0001 , one - way ANOVA , Tukey ' s post - hoc correction .

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

(I) STED images of representative mitochondrial morphology in muscle cells of L4 mutant worms. Scale bar: 2 μm. (J) Left panel: Percentage of FRAP. Each point: mean recovery of ~30 bleached regions (2 regions per animal, 15 animals per condition) ± SEM. Right panel: values of the maximum (total) recovery per strain ± SEM, ***p value <0.001, **** p value <0.0001, one-way ANOVA, Tukey's post-hoc correction.


( K ) Representative lifespan of age - 1 ; gas - 1 nematodes fed with empty vector ( EV ) and fzo - 1 RNAi bacteria . Average median lifespans ± SEM from all replicates indicated beneath representative curves . For all panels : wt = wild type , a = age - 1 , g = gas - 1 , a ; g = age - 1 ; gas - 1 .

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(K) Representative lifespan of age-1; gas-1 nematodes fed with empty vector (EV) and fzo-1 RNAi bacteria. Average median lifespans ± SEM from all replicates indicated beneath representative curves. For all panels: wt= wild type, a= age-1, g= gas-1, a; g= age-1; gas-1.


( A ) Left panel : Venn Diagram of the number of genes significantly dysregulated in age - 1 ; gas - 1 versus wt ( green ) and gas - 1 versus wt ( red ) . Right panel : Scatter - plots of the expression of genes significantly downregulated ( left ) or upregulated ( right ) in gas - 1 and age - 1 ; gas - 1 mutants compared to wt . In black : expression levels of genes that are significantly up - or down - regulated in gas - 1 mutants compared to wt . In red : expression levels of the same genes in age - 1 ; gas - 1 mutants compared to wt .

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

(A) Left panel: Venn Diagram of the number of genes significantly dysregulated in age-1; gas-1 versus wt (green) and gas-1 versus wt (red). Right panel: Scatter-plots of the expression of genes significantly downregulated (left) or upregulated (right) in gas-1 and age-1; gas-1 mutants compared to wt. In black: expression levels of genes that are significantly up- or down-regulated in gas-1 mutants compared to wt. In red: expression levels of the same genes in age-1; gas-1 mutants compared to wt.


( C ) Representative lifespans of the indicated strains with average median lifespans ± SEM from all replicates indicated beneath representative

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

(C) Representative lifespans of the indicated strains with average median lifespans ± SEM from all replicates indicated beneath representative curves


( D ) Volcano plot of TMT / iTRAQ - based quantitative proteomic analysis . In red , proteins significantly dysregulated in age - 1 ; gas - 1 compared to gas - 1 ( FDR < 20 % ; | log2 ( fold change ) | > 0 . 47 ) . Dashed lines indicate cutoffs . Proteins involved in carbohydrate ( green ) and lipid ( blue ) metabolism are labeled .

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(D) Volcano plot of TMT/iTRAQ-based quantitative proteomic analysis. In red, proteins significantly dysregulated in age-1; gas-1 compared to gas-1 (FDR< 20%; |log2 (fold change)| >0.47). Dashed lines indicate cutoffs. Proteins involved in carbohydrate (green) and lipid (blue) metabolism are labeled.


( H ) Left panel : Glucose quantification in wt , gas - 1 and age - 1 ; gas - 1 nematode lysates with NMR , n = 7 , ns = non - significant , Mann Whitney U test . Right panel : Quantification of total carbohydrates in the indicated strains with phenol - sulfuric assay . Error bars : mean ± SEM , 2 biological replicates ( 5 technical replicates each ) , normalized to wt and number of animals used . * * p value < 0 . 01 , ns = non - significant , Mann - Whitney U test .

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

(H) Left panel: Glucose quantification in wt, gas-1 and age-1; gas-1 nematode lysates with NMR, n=7, ns= non-significant, Mann Whitney U test. Right panel: Quantification of total carbohydrates in the indicated strains with phenol-sulfuric assay. Error bars: mean ± SEM, 2 biological replicates (5 technical replicates each), normalized to wt and number of animals used. **p value < 0.01, ns= non-significant, Mann-Whitney U test.


( C ) Immunoblot analysis of phospho - AAK - 2 in wt , age - 1 , gas - 1 and age - 1 ; gas - 1 mutant nematodes ( actin loading control ) . Numbers correspond to band densitometries across 3 biological replicates .

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(C) Immunoblot analysis of phospho-AAK-2 in wt, age-1, gas-1 and age-1; gas-1 mutant nematodes (actin loading control). Numbers correspond to band densitometries across 3 biological replicates.


( D ) Representative lifespan of age - 1 ; gas - 1 double mutant and age - 1 ; aak - 2 ; gas - 1 triple mutant nematodes with average median lifespans ± SEM from all replicates indicated beneath representative curves .

(D) Representative lifespan of age-1; gas-1 double mutant and age-1; aak-2; gas-1 triple mutant nematodes with average median lifespans ± SEM from all replicates indicated beneath representative curves.


( E ) Lifespan assay of gas - 1 mutant animals compared to gas - 1 ; AAK - 2 overexpressing ( O / E ) nematodes and their wt counterparts . Inset : immunoblot analysis of phospho - AAK - 2 in gas - 1 ( g ) and gas - 1 ; AAK - 2 O / E ( g ; a O / E ) animals ( actin loading control ) , showing the overexpressed protein ( lower band ) and the endogenous one ( upper band ) .

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(E) Lifespan assay of gas-1 mutant animals compared to gas-1; AAK-2 overexpressing (O/E) nematodes and their wt counterparts. Inset: immunoblot analysis of phospho-AAK-2 in gas-1 (g) and gas-1; AAK-2 O/E (g; a O/E) animals (actin loading control), showing the overexpressed protein (lower band) and the endogenous one (upper band).


( F ) Left panel : Immunoblot analysis of phospho - KIN - 1 substrates in young adult nematodes . Numbers correspond to band densitometries across 2 biological replicates . Right panel : immunoblot analysis of phospho - KIN - 1 substrates in age - 1 ; gas - 1 animals grown on bacteria carrying an empty vector ( EV ) or expressing kin - 1 RNAi . Two different kin - 1 RNAi clones ( g1 , b5 ) were used in this experiment . Numbers correspond to band densitometries of the presented immunoblot . Dashed rectangles : band corresponding to downregulated KIN - 1 phospho - substrate .

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

(F) Left panel: Immunoblot analysis of phospho-KIN-1 substrates in young adult nematodes. Numbers correspond to band densitometries across 2 biological replicates. Right panel: immunoblot analysis of phospho-KIN-1 substrates in age-1; gas-1 animals grown on bacteria carrying an empty vector (EV) or expressing kin-1 RNAi. Two different kin-1 RNAi clones (g1, b5) were used in this experiment. Numbers correspond to band densitometries of the presented immunoblot. Dashed rectangles: band corresponding to downregulated KIN-1 phospho-substrate.


( G ) Representative lifespan assay of wt and age - 1 ; gas - 1 nematodes fed with empty vector ( EV ) and kin - 1 RNAi bacteria with average median lifespans ± SEM from all replicates indicated beneath representative curves .

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(G) Representative lifespan assay of wt and age-1; gas-1 nematodes fed with empty vector (EV) and kin-1 RNAi bacteria with average median lifespans ± SEM from all replicates indicated beneath representative curves.


ATP measurements in : ( H ) age - 1 ( hx546 ) ; gas - 1 ( fc21 ) ( a ; g ) and age - 1 ( hx546 ) ; aak - 2 ( ok524 ) ; gas - 1 ( fc21 ) ( a ; aa ; g ) mutant nematodes ;

ATP measurements in: (H) age-1(hx546); gas-1(fc21) (a; g) and age-1(hx546); aak-2(ok524); gas-1(fc21) (a; aa; g) mutant nematodes;


ATP measurements in : ( I ) age - 1 ; gas - 1 ( EV ) ( a ; g ( EV ) ) and age - 1 ; gas - 1 ; kin - 1 ( RNAi ) ( a ; g ; kin - 1 ( RNAi ) ) . Error bars : Mean ± SEM , n = 4 , * p value < 0 . 05 , Mann Whitney U test .

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ATP measurements in: (I) age-1; gas-1 (EV) (a; g (EV)) and age-1; gas-1; kin-1 (RNAi) (a; g; kin-1(RNAi)). Error bars: Mean ± SEM, n=4, *p value <0.05, Mann Whitney U test.


( J ) Left panel : curves represent FRAP in age - 1 ; gas - 1 animals grown on RNAi from larval L1 ( or from egg for fzo - 1 RNAi ) to L4 stage . Each point : mean recovery of bleached regions ( 2 - 5 regions per animal , 10 - 20 animals per condition ) ± SEM . Right panel : Maximum ( total ) recovery per condition are shown , ± SEM . * p value < 0 . 05 , * * p value < 0 . 01 , * * * * p value < 0 . 0001 , Kruskal - Wallis test , Dunn ' s post - hoc correction .

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(J) Left panel: curves represent FRAP in age-1; gas-1 animals grown on RNAi from larval L1 (or from egg for fzo-1 RNAi) to L4 stage. Each point: mean recovery of bleached regions (2-5 regions per animal, 10-20 animals per condition) ± SEM. Right panel: Maximum (total) recovery per condition are shown, ± SEM. *p value< 0.05, **p value< 0.01, ****p value< 0.0001, Kruskal-Wallis test, Dunn's post-hoc correction.


A ) Volcano plot of lipidomic profiling in gas - 1 versus age - 1 ; gas - 1 mutants . Red stars : significantly altered lipids in age - 1 ; gas - 1 versus gas - 1 ( 5 % FDR ; n = 5 ) .

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A) Volcano plot of lipidomic profiling in gas-1 versus age-1; gas-1 mutants. Red stars: significantly altered lipids in age-1; gas-1 versus gas-1 (5% FDR; n=5).


B ) NMR - 1D targeted metabolic profiling in age - 1 ; gas - 1 mutants ( normalized to gas - 1 ) . Dashed lines indicate fold change cutoff ( | log2 ( fold change ) | < 1 . 23 ) . Red bars : not significant , Light blue bars : FDR < 10 % , | log2 ( fold change ) | < 1 . 23 , Dark blue bars : FDR < 10 % , | log2 ( fold change ) | > 1 . 23 . Error bar : mean + SEM , n = 7 .

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B) NMR-1D targeted metabolic profiling in age-1; gas-1 mutants (normalized to gas-1). Dashed lines indicate fold change cutoff (|log2(fold change)| <1.23). Red bars: not significant, Light blue bars: FDR<10%, |log2(fold change)| <1.23, Dark blue bars: FDR <10%, |log2(fold change)| >1.23. Error bar: mean + SEM, n=7.


D ) Left panel : qRT - PCR of xdh - 1 ( normalized to wt ) . Error bars : Mean ± SEM , n = 6 . * * * * p value < 0 . 0001 , * p value < 0 . 05 , ns = non - significant , Kruskal - Wallis with Dunn ' s post hoc test . Right : xanthine oxidase activity levels normalized to animal number . Error bars : mean ± SEM , n = 2 , ns = non - significant , Mann Whitney U test .

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D) Left panel: qRT-PCR of xdh-1 (normalized to wt). Error bars: Mean ± SEM, n=6. ****p value< 0.0001, *p value< 0.05, ns= non-significant, Kruskal-Wallis with Dunn's post hoc test. Right: xanthine oxidase activity levels normalized to animal number. Error bars: mean ± SEM, n=2, ns= non-significant, Mann Whitney U test.


F ) Immunoblot analysis of phospho - Akt ( S473 ) , pan Akt , phospho - PRAS40 ( T246 ) and total PRAS40 in WT and NDUFA9 KO HAP1 cells treated with 0 . 2 % DMSO ( - ) or AMP ( 2 μM ) , ENPF ( 2 μM ) and PTX ( 36 μM ) ( + ) for 24 h ( actin loading control ) .

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F) Immunoblot analysis of phospho-Akt (S473), pan Akt, phospho-PRAS40 (T246) and total PRAS40 in WT and NDUFA9 KO HAP1 cells treated with 0.2% DMSO (-) or AMP (2 μM), ENPF (2 μM) and PTX (36 μM) (+) for 24 h (actin loading control).


( G ) Immunoblot analysis of phospho - PKA substrates in WT and NDUFA9 KO HAP1 cells treated as indicated ( actin loading control ) . For all immunoblot panels : numbers correspond to band densitometries across 2 - 3 biological replicates .

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(G) Immunoblot analysis of phospho-PKA substrates in WT and NDUFA9 KO HAP1 cells treated as indicated (actin loading control). For all immunoblot panels: numbers correspond to band densitometries across 2-3 biological replicates.


( A ) Effect of methylxanthines on wt lifespan . Average median lifespans ± SEM from all replicates indicated beneath representative curve .

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(A) Effect of methylxanthines on wt lifespan. Average median lifespans ± SEM from all replicates indicated beneath representative curve.


( B ) Locomotory activity of 10 - day - old gas - 1 mutant nematodes treated with vehicle ( DMSO ) or methylxanthines . Values represent arbitrary units from 3 biological replicates with 4 technical replicates each , 35 worms per technical replicate . Error bars : mean ± SEM , * * p value < 0 . 01 , * * * p value < 0 . 001 , one - way ANOVA , Dunnett ' s post - hoc correction .

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(B) Locomotory activity of 10-day-old gas-1 mutant nematodes treated with vehicle (DMSO) or methylxanthines. Values represent arbitrary units from 3 biological replicates with 4 technical replicates each, 35 worms per technical replicate. Error bars: mean ± SEM, **p value< 0.01, ***p value< 0.001, one-way ANOVA, Dunnett's post-hoc correction.


( C ) Effect of methylxanthines on the survival of gas - 1 mutants . Average median lifespans ± SEM from all replicates indicated beneath representative curves .

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(C) Effect of methylxanthines on the survival of gas-1 mutants. Average median lifespans ± SEM from all replicates indicated beneath representative curves.


( D ) Pharyngeal pumps / minute in young adult gas - 1 mutant nematodes treated with vehicle or methylxanthines . Error bars : mean ± SEM , n = 18 . * * * * p value < 0 . 0001 , one - way ANOVA , Dunnett ' s post - hoc correction .

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(D) Pharyngeal pumps/minute in young adult gas-1 mutant nematodes treated with vehicle or methylxanthines. Error bars: mean ± SEM, n=18. ****p value< 0.0001, one-way ANOVA, Dunnett's post-hoc correction.


( E - F ) Immunoblot analysis of ( E ) KIN - 1 phospho - substrates in young adult gas - 1 mutant nematodes treated with vehicle ( DMSO ) or methylxanthines ( actin loading control ) .

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(E-F) Immunoblot analysis of (E) KIN-1 phospho-substrates in young adult gas-1 mutant nematodes treated with vehicle (DMSO) or methylxanthines (actin loading control).


( E - F ) Immunoblot analysis of ( F ) phospho - AAK - 2 in young adult gas - 1 mutant nematodes treated with vehicle ( DMSO ) or methylxanthines ( actin loading control ) .

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(E-F) Immunoblot analysis of (F) phospho-AAK-2 in young adult gas-1 mutant nematodes treated with vehicle (DMSO) or methylxanthines (actin loading control).


( G ) Left panel : FRAP in vehicle or PTX - treated gas - 1 animals . Each point : mean recovery of 14 bleached regions ( 2 regions per animal , 7 animals per condition ) ± SEM . Right panel : maximum ( total ) recovery per condition ± SEM . * * p value < 0 . 01 unpaired t test . For all immunoblot panels : numbers correspond to band densitometries across biological replicates and for all lifespan curves : the legend shows Mean median lifespan ± SEM across all biological replicates .

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(G) Left panel: FRAP in vehicle or PTX-treated gas-1 animals. Each point: mean recovery of 14 bleached regions (2 regions per animal, 7 animals per condition) ± SEM. Right panel: maximum (total) recovery per condition ± SEM. **p value< 0.01 unpaired t test. For all immunoblot panels: numbers correspond to band densitometries across biological replicates and for all lifespan curves: the legend shows Mean median lifespan ± SEM across all biological replicates.


A . Root tip of Arabidopsis with distinct growth zones marked : meristematic ( MZ , green ) , transition ( TZ , blue ) , elongation ( EZ , pink ) and differentiation ( DZ , yellow ) . Double arrow indicates the first expanded cortex cell considered as a start point of the TZ , which encompasses epidermal cells smaller than 45 µm . Epidermal cells reaching a length of more than 45 µm prior termination of the elongation are in the EZ . Scale bar 25 µm .

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A. Root tip of Arabidopsis with distinct growth zones marked: meristematic (MZ, green), transition (TZ, blue), elongation (EZ, pink) and differentiation (DZ, yellow). Double arrow indicates the first expanded cortex cell considered as a start point of the TZ, which encompasses epidermal cells smaller than 45 µm. Epidermal cells reaching a length of more than 45 µm prior termination of the elongation are in the EZ. Scale bar 25 µm.


B . CMTs visualized by the MAP4 - GFP reporter ( left ) and scheme of the CMT orientations ( right ) in epidermal cells of distinct growth zones ( TZ , EZ and DZ ) . Individual cells monitored at two time points ( 0 and 60 min ) . Dark blue arrows mark the cell expansion direction . Scale bar 10 µm .

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B. CMTs visualized by the MAP4-GFP reporter (left) and scheme of the CMT orientations (right) in epidermal cells of distinct growth zones (TZ, EZ and DZ). Individual cells monitored at two time points (0 and 60 min). Dark blue arrows mark the cell expansion direction. Scale bar 10 µm.


D . Analysis of the CMT plus - end growth with the EB1b - GFP reporter . CMT plus - end growth rates ( μm / min ) measured by tracking the EB1b - GFP marker for 20 - 30 min in epidermal root cells of three growth zones . In the boxplots , the center lines show medians ; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software ; whiskers span minimum to maximum values , individual data points are represented by dots . * * P < 0 . 01 , * * * * P < 0 . 0001by the Student ' s t - test . The number of CMT plus - end events ( EB1b - GFP ) tracked per cell was 20 - 100 / min , of which the growth rate average is represented as a single dot ; 3 - 5 cells per root growth zone , 3 - 5 roots analyzed per biological replicate in 3 independent experiments . On the right , single trajectories of EB1b - GFP signal tracked over 60 sec in root epidermal cells at different growth zones . Scale bar 0 . 5 µm .

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D. Analysis of the CMT plus-end growth with the EB1b-GFP reporter. CMT plus-end growth rates (μm/min) measured by tracking the EB1b-GFP marker for 20-30 min in epidermal root cells of three growth zones. In the boxplots, the center lines show medians; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software; whiskers span minimum to maximum values, individual data points are represented by dots. ** P < 0.01, **** P < 0.0001by the Student's t-test. The number of CMT plus-end events (EB1b-GFP) tracked per cell was 20-100/min, of which the growth rate average is represented as a single dot; 3-5 cells per root growth zone, 3-5 roots analyzed per biological replicate in 3 independent experiments. On the right, single trajectories of EB1b-GFP signal tracked over 60 sec in root epidermal cells at different growth zones. Scale bar 0.5 µm.


E . Z - stack maximum image projection of the EB1b - GFP plus - end reporter tracked over 30 sec , in epidermal cells at different growth zones . Scale bars 10 µm .

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E. Z-stack maximum image projection of the EB1b-GFP plus-end reporter tracked over 30 sec, in epidermal cells at different growth zones. Scale bars 10 µm.


F . CMTs visualized by MAP4 - GFP in root epidermal cells at the TZ , the EZ and the DZ prior ( 0 ) and 60 min after treatment with 1 µm oryzalin . Scale bar 10 µm .

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F. CMTs visualized by MAP4-GFP in root epidermal cells at the TZ, the EZ and the DZ prior (0) and 60 min after treatment with 1 µm oryzalin. Scale bar 10 µm.


G . Quantification of the MAP4 - GFP CMTs reporter signal in wild - type root epidermal cells at the TZ , the EZ and the DZ zone treated with mock ( DMSO , white box ) , oryzalin ( 1 µM , light grey box ) and cytokinin ( CK ; 10 µM 6 - benzylaminopurine , BAP ) and oryzalin ( 1 µM ) ( dark grey box ) . For double treatments , roots pretreated for 60 min with CK prior to transfer to medium supplemented with both compounds . Boxplots represent ratio between mean fluorescence intensity ( arbitrary units ) measured in epidermal cells at 60 and 0 min .

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G. Quantification of the MAP4-GFP CMTs reporter signal in wild-type root epidermal cells at the TZ, the EZ and the DZ zone treated with mock (DMSO, white box), oryzalin (1 µM, light grey box) and cytokinin (CK; 10 µM 6-benzylaminopurine, BAP) and oryzalin (1 µM) (dark grey box). For double treatments, roots pretreated for 60 min with CK prior to transfer to medium supplemented with both compounds. Boxplots represent ratio between mean fluorescence intensity (arbitrary units) measured in epidermal cells at 60 and 0 min.


A . Arabidopsis root expressing the dual reporter TCSn : : dndT - tNOS DR5v2 : 3nGFP that is sensitive to cytokinin ( CK ) and auxin . CK ( red ) , auxin ( green ) and overlay of both signals detected in nuclei of cells at the root tip ( top , scale bar 50 µm ) . Green , pink , and blue arrowheads point at epidermal cells located in the meristematic ( MZ ) , transition ( TZ ) and elongation ( EZ ) zones , respectively . Magnification of the TZ shown ( bottom , scale bar 10 µm ) .

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A. Arabidopsis root expressing the dual reporter TCSn::dndT-tNOS DR5v2:3nGFP that is sensitive to cytokinin (CK) and auxin. CK (red), auxin (green) and overlay of both signals detected in nuclei of cells at the root tip (top, scale bar 50 µm). Green, pink, and blue arrowheads point at epidermal cells located in the meristematic (MZ), transition (TZ) and elongation (EZ) zones, respectively. Magnification of the TZ shown (bottom, scale bar 10 µm).


B . Relative fluorescence intensity of TCSn : : ntdT : tNOS signal ( red ) and DR5v2 : 3nGFP signal ( green ) measured in epidermal cells along the longitudinal root growth axis . Cell number 1 corresponds to a meristematic cell placed at position - 4 before the beginning of TZ ( as marked by green arrow at 2A ) . Mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 by Student ' s t - test , n = 18 roots .

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B. Relative fluorescence intensity of TCSn::ntdT:tNOS signal (red) and DR5v2:3nGFP signal (green) measured in epidermal cells along the longitudinal root growth axis. Cell number 1 corresponds to a meristematic cell placed at position -4 before the beginning of TZ (as marked by green arrow at 2A). Mean ± s.d., * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by Student's t-test, n= 18 roots.


C - F . CMTs visualized with MAP4 - GFP in root epidermal cells of the TZ and the EZ at time point 0 and 60 min after CK ( C ) , CK and oryzalin ( D ) , auxin ( E ) and CK and auxin ( F ) treatments , and in DZ cells at time point 0 and 60 min after auxin treatment ( E ) . As CK and auxin sources 10 µM BAP and 0 . 1 µM NAA were used , respectively , and 1 µM oryzalin . For the double ( CK / Oryzalin and CK / Auxin ) treatments , roots were pretreated with CK for 60 min prior to transfer to medium supplemented with both compounds . Scale bar 10 µm .

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C-F. CMTs visualized with MAP4-GFP in root epidermal cells of the TZ and the EZ at time point 0 and 60 min after CK (C), CK and oryzalin (D), auxin (E) and CK and auxin (F) treatments, and in DZ cells at time point 0 and 60 min after auxin treatment (E). As CK and auxin sources 10 µM BAP and 0.1 µM NAA were used, respectively, and 1 µM oryzalin. For the double (CK/Oryzalin and CK/Auxin) treatments, roots were pretreated with CK for 60 min prior to transfer to medium supplemented with both compounds. Scale bar 10 µm.


H . Analysis of the CMT plus - end growth with the EB1b - GFP reporter . Z - stack maximum image projections of EB1b - GFP tracked over 30 sec in epidermal cells without ( mock , DMSO ) and with CK , auxin or CK and auxin treatment ( 60 min ) . Scale bars 10 µm . I . CMT plus - end growth rates ( µm / min ) measured by tracking of EB1b - GFP reporter over 20 - 30 min in epidermal cells of different growth zones treated for 60 min with mock ( DMSO ) , or media supplemented with CK ( 30 or 60 min ) , auxin and CK plus auxin . In the boxplots , the center lines show the medians ; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software ; whiskers span minimum to maximum values , individual data points are represented by dots . The number of CMT plus - end events ( EB1b - GFP ) tracked per cell was 20 - 100 / min , of which the growth rate average is represented as a single dot . Statistical significance evaluated by two - way ANOVA ( n = 3 - 5 cells per root growth zone with 3 - 5 roots per replicate in 3 independent experiments . On the right , single trajectories of EB1b - GFP signal tracked over 60 sec in root epidermal cells at different growth zones and treatment conditions . Scale bar 0 . 5 µm . Hormone concentrations and treatment conditions ( H , I ) were as described ( C , E and F ) .

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H. Analysis of the CMT plus-end growth with the EB1b-GFP reporter. Z-stack maximum image projections of EB1b-GFP tracked over 30 sec in epidermal cells without (mock, DMSO) and with CK, auxin or CK and auxin treatment (60 min). Scale bars 10 µm. I. CMT plus-end growth rates (µm/min) measured by tracking of EB1b-GFP reporter over 20-30 min in epidermal cells of different growth zones treated for 60 min with mock (DMSO), or media supplemented with CK (30 or 60 min), auxin and CK plus auxin. In the boxplots, the center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software; whiskers span minimum to maximum values, individual data points are represented by dots. The number of CMT plus-end events (EB1b-GFP) tracked per cell was 20-100/min, of which the growth rate average is represented as a single dot. Statistical significance evaluated by two-way ANOVA (n = 3-5 cells per root growth zone with 3-5 roots per replicate in 3 independent experiments. On the right, single trajectories of EB1b-GFP signal tracked over 60 sec in root epidermal cells at different growth zones and treatment conditions. Scale bar 0.5 µm. Hormone concentrations and treatment conditions (H, I) were as described (C, E and F).


H . Analysis of the CMT plus - end growth with the EB1b - GFP reporter . Z - stack maximum image projections of EB1b - GFP tracked over 30 sec in epidermal cells without ( mock , DMSO ) and with CK , auxin or CK and auxin treatment ( 60 min ) . Scale bars 10 µm .

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H. Analysis of the CMT plus-end growth with the EB1b-GFP reporter. Z-stack maximum image projections of EB1b-GFP tracked over 30 sec in epidermal cells without (mock, DMSO) and with CK, auxin or CK and auxin treatment (60 min). Scale bars 10 µm.


A - D . CMTs visualized with MAP4 - GFP in root epidermal cells of the cre1 - 12 mutant at the transition ( TZ ) and the elongation ( EZ ) zones at time point 0 and 60 min after mock ( DMSO ) ( A ) , cytokinin ( CK ) ( B ) , auxin ( C ) and CK plus auxin ( D ) treatments . As CK and auxin sources 10 µM BAP and 0 . 1 µM NAA were used , respectively . For the double CK plus auxin treatment roots were pretreated for 60 min with CK , whereafter they were transferred to medium supplemented with both compounds . Scale bar 10 µm .

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A-D. CMTs visualized with MAP4-GFP in root epidermal cells of the cre1-12 mutant at the transition (TZ) and the elongation (EZ) zones at time point 0 and 60 min after mock (DMSO) (A), cytokinin (CK) (B), auxin (C) and CK plus auxin (D) treatments. As CK and auxin sources 10 µM BAP and 0.1 µM NAA were used, respectively. For the double CK plus auxin treatment roots were pretreated for 60 min with CK, whereafter they were transferred to medium supplemented with both compounds. Scale bar 10 µm.


F , G . Analysis of the CMT plus - end growth with the EB1b - GFP reporter in cre1 - 12 . F . Z - stack maximum image projection of EB1b - GFP tracked for 30 seconds ( on the left ) and single trajectories of EB1b - GFP signal tracked over 60 sec ( on the right ) in root epidermal cells of the EZ in the wild type and cre1 - 12 treated with CK for 60 min . Scale bars 10 µm and 0 . 5 µm , respectively . G . CMT plus - end growth rates ( µm / min ) quantified from the EB1b - GFP trajectories tracked during 5 min in epidermal cells of the EZ treated with mock ( DMSO ) or CK . In the boxplots , the center lines show the medians ; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software ; whiskers span minimum to maximum values , individual data points are represented by dots . ( ns = non - significant , * * * P < 0 . 001 by Student ' s t - test , n = 7 - 9 cells with 7 roots per condition in 3 independent experiments ) . Concentrations and treatment conditions ( F , G ) were as for ( A , B ) .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G. Analysis of the CMT plus-end growth with the EB1b-GFP reporter in cre1-12. F. Z-stack maximum image projection of EB1b-GFP tracked for 30 seconds (on the left) and single trajectories of EB1b-GFP signal tracked over 60 sec (on the right) in root epidermal cells of the EZ in the wild type and cre1-12 treated with CK for 60 min. Scale bars 10 µm and 0.5 µm, respectively. G. CMT plus-end growth rates (µm/min) quantified from the EB1b-GFP trajectories tracked during 5 min in epidermal cells of the EZ treated with mock (DMSO) or CK. In the boxplots, the center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software; whiskers span minimum to maximum values, individual data points are represented by dots. (ns = non-significant, *** P < 0.001 by Student's t-test, n= 7-9 cells with 7 roots per condition in 3 independent experiments). Concentrations and treatment conditions (F, G) were as for (A, B).


H , I . CMTs visualized with MAP4 - GFP in root epidermal cells of the cre1 - 12 mutant . Cells at the TZ , the EZ and the DZ were monitored at time point 0 and 60 min after treatment with oryzalin ( 1 µM ) ( H ) , and CK plus oryzalin ( I ) . As CK source 10 µM BAP was used and 1 µM oryzalin . For double ( CK and oryzalin ) treatment roots were pretreated for 60 min with CK and then transferred to medium supplemented with both compounds . Scale bar 10 µm . J . Quantification of the MAP4 - GFP CMTs reporter signal in cre1 - 12 root epidermal cells at different root zones treated with mock ( DMSO , white box ) , oryzalin ( 1 µM , light grey box ) and CK ( 10 µM BAP ) plus oryzalin ( 1 µM ) ( dark grey box ) . For double treatments , roots pretreated for 60 min with CK prior to transfer to medium supplemented with both compounds . Boxplots represent ratio between mean fluorescence intensity ( arbitrary units ) measured in epidermal cells at 60 and 0 min . The center lines show the medians and the box limits indicate the 25th and 75th percentiles ; whiskers span the minimal to maximal values , individual data points are represented by dots . Ratio close to 1 ( segmented line ) corresponds to the unchanged MAP - GFP signal for 60 min . ( * P < 0 . 05 , * * * * P < 0 . 0001 by Student ' s t - test , n = 3 - 10 cells per root growth zone with 5 - 8 roots per condition in 4 independent replicates ) .

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

H, I. CMTs visualized with MAP4-GFP in root epidermal cells of the cre1-12 mutant. Cells at the TZ, the EZ and the DZ were monitored at time point 0 and 60 min after treatment with oryzalin (1 µM) (H), and CK plus oryzalin (I). As CK source 10 µM BAP was used and 1 µM oryzalin. For double (CK and oryzalin) treatment roots were pretreated for 60 min with CK and then transferred to medium supplemented with both compounds. Scale bar 10 µm. J. Quantification of the MAP4-GFP CMTs reporter signal in cre1-12 root epidermal cells at different root zones treated with mock (DMSO, white box), oryzalin (1 µM, light grey box) and CK (10 µM BAP) plus oryzalin (1 µM) (dark grey box). For double treatments, roots pretreated for 60 min with CK prior to transfer to medium supplemented with both compounds. Boxplots represent ratio between mean fluorescence intensity (arbitrary units) measured in epidermal cells at 60 and 0 min. The center lines show the medians and the box limits indicate the 25th and 75th percentiles; whiskers span the minimal to maximal values, individual data points are represented by dots. Ratio close to 1 (segmented line) corresponds to the unchanged MAP-GFP signal for 60 min. (* P < 0.05, **** P < 0.0001 by Student's t-test, n= 3-10 cells per root growth zone with 5-8 roots per condition in 4 independent replicates).


H , I . CMTs visualized with MAP4 - GFP in root epidermal cells of the cre1 - 12 mutant . Cells at the TZ , the EZ and the DZ were monitored at time point 0 and 60 min after treatment with oryzalin ( 1 µM ) ( H ) , and CK plus oryzalin ( I ) . As CK source 10 µM BAP was used and 1 µM oryzalin . For double ( CK and oryzalin ) treatment roots were pretreated for 60 min with CK and then transferred to medium supplemented with both compounds . Scale bar 10 µm .

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

H, I. CMTs visualized with MAP4-GFP in root epidermal cells of the cre1-12 mutant. Cells at the TZ, the EZ and the DZ were monitored at time point 0 and 60 min after treatment with oryzalin (1 µM) (H), and CK plus oryzalin (I). As CK source 10 µM BAP was used and 1 µM oryzalin. For double (CK and oryzalin) treatment roots were pretreated for 60 min with CK and then transferred to medium supplemented with both compounds. Scale bar 10 µm.


A . Relative root growth of wild - type ( Col - 0 ) ( grey lines ) and 35S : : ARR1 ∆ DDK - GR ( purple lines ) seedlings grown on mock ( DMSO ; continuous line ) or with dexamethasone ( DEX 10 µM ; dashed line ) supplemented medium . Five days old seedlings ( Day 0 ) were monitored for 3 days . Mean ± s . d . ; * * P < 0 . 01 , * * * * P < 0 . 0001 by Student ' s t - test , referred to wild - type - mock . n = 12 - 14 roots .

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A. Relative root growth of wild-type (Col-0) (grey lines) and 35S::ARR1∆DDK-GR (purple lines) seedlings grown on mock (DMSO; continuous line) or with dexamethasone (DEX 10 µM; dashed line) supplemented medium. Five days old seedlings (Day 0) were monitored for 3 days. Mean ± s.d.; ** P < 0.01, **** P < 0.0001 by Student's t-test, referred to wild-type - mock. n = 12-14 roots.


B . Immunostaining of α - tubulin in epidermal cells of the transition ( TZ ) and the elongation ( EZ ) zone of 35S : : ARR1 ∆ DDK - GR roots after non - , 3h or 16h of treatment with dexamethasone ( DEX 10 µM ) . Scale bar 10 µm .

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B. Immunostaining of α-tubulin in epidermal cells of the transition (TZ) and the elongation (EZ) zone of 35S::ARR1∆DDK-GR roots after non-, 3h or 16h of treatment with dexamethasone (DEX 10 µM). Scale bar 10 µm.


D , E . Analysis of root sensitivity to oryzalin in wild - type ( Col - 0 ) and 35S : : ARR1 ∆ DDK - GR . Seedlings were grown for 5 days on mock ( Murashige and Skoog ) medium and then transferred to medium supplemented with 1 µM oryzalin , CK with oryzalin ( 10 µM BAP and 1 µM oryzalin ) , with or without DEX 10 µM , for 3 days . For the double CK and oryzalin treatment , seedlings were pretreated with 10 µM BAP for 60 min prior to transfer to medium supplemented with both compounds . Representative images ; white arrowheads indicate root length at day of transfer . Scale bar 1 mm ( D ) . Quantifications calculated as percentage ( % ) of root tips exhibiting swelling ( dark grey bars ) , and weak and strong resistance to oryzalin ( grey and white bars , respectively ) . On the right representative images of root phenotype categories . n = 10 - 25 roots per treatment were evaluated . ( E ) .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Analysis of root sensitivity to oryzalin in wild-type (Col-0) and 35S::ARR1∆DDK-GR. Seedlings were grown for 5 days on mock (Murashige and Skoog) medium and then transferred to medium supplemented with 1 µM oryzalin, CK with oryzalin (10 µM BAP and 1 µM oryzalin), with or without DEX 10 µM, for 3 days. For the double CK and oryzalin treatment, seedlings were pretreated with 10 µM BAP for 60 min prior to transfer to medium supplemented with both compounds. Representative images; white arrowheads indicate root length at day of transfer. Scale bar 1 mm (D). Quantifications calculated as percentage (%) of root tips exhibiting swelling (dark grey bars), and weak and strong resistance to oryzalin (grey and white bars, respectively). On the right representative images of root phenotype categories. n=10-25 roots per treatment were evaluated. (E).


F . Relative root growth of wild - type ( Col - 0 ) ( grey lines ) and 35S : : ARR1 ∆ DDK - GR ( purple lines ) seedlings grown on oryzalin 1µM ( continuous line ) or on oryzalin 1µM and DEX 10 µM ( dashed line ) supplemented media monitored for 3 days . Day 0 , day of transfer . Mean ± s . d . ; n = 12 - 14 roots .

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F. Relative root growth of wild-type (Col-0) (grey lines) and 35S::ARR1∆DDK-GR (purple lines) seedlings grown on oryzalin 1µM (continuous line) or on oryzalin 1µM and DEX 10 µM (dashed line) supplemented media monitored for 3 days. Day 0, day of transfer. Mean ± s.d.; n = 12-14 roots.


A . Time lapse imaging of Arabidopsis root expressing the plasma membrane marker ( PM ) pUB10 : : EYFP - NPSN12 ( wave line W131Y ) ( Geldner et al , 2009 ) . Root growth recorded for 2 min and overlay of root tip images at time point 0 ( magenta ) and 2 min ( cyan ) used for the calculation of the root growth rate ( RGR ) and the cell elongation rate ( CER ) . Scale bar 25 µm . B . RGR of wild type ( grey bars ) and cre1 - 12 ( blue bars ) treated for 1 and 4 hours with mock ( DMSO ) , CK ( 10 µM BAP ) or auxin ( 0 . 1 µM NAA ) . Mean ± s . d . , ( * P < 0 . 05and * * * * P < 0 . 0001 by the Student ' s t - test , n = 5 - 8 roots per condition in 3 independent replicates ) .

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A. Time lapse imaging of Arabidopsis root expressing the plasma membrane marker (PM) pUB10::EYFP-NPSN12 (wave line W131Y)(Geldner et al, 2009). Root growth recorded for 2 min and overlay of root tip images at time point 0 (magenta) and 2 min (cyan) used for the calculation of the root growth rate (RGR) and the cell elongation rate (CER). Scale bar 25 µm. B. RGR of wild type (grey bars) and cre1-12 (blue bars) treated for 1 and 4 hours with mock (DMSO), CK (10 µM BAP) or auxin (0.1 µM NAA). Mean ± s.d., (* P < 0.05and **** P < 0.0001 by the Student's t-test, n= 5-8 roots per condition in 3 independent replicates).


A . Time lapse imaging of Arabidopsis root expressing the plasma membrane marker ( PM ) pUB10 : : EYFP - NPSN12 ( wave line W131Y ) ( Geldner et al , 2009 ) . Root growth recorded for 2 min and overlay of root tip images at time point 0 ( magenta ) and 2 min ( cyan ) used for the calculation of the root growth rate ( RGR ) and the cell elongation rate ( CER ) . Scale bar 25 µm .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Time lapse imaging of Arabidopsis root expressing the plasma membrane marker (PM) pUB10::EYFP-NPSN12 (wave line W131Y)(Geldner et al, 2009). Root growth recorded for 2 min and overlay of root tip images at time point 0 (magenta) and 2 min (cyan) used for the calculation of the root growth rate (RGR) and the cell elongation rate (CER). Scale bar 25 µm.


C - E . Analysis of CK and auxin effects on CER of epidermal cells . Epidermal cells of the TZ and the EZ in the wild type and cre1 - 12 with PMs visualized with the YFP - NPSN12 marker . Overlay of images of epidermal cells at time 0 ( magenta ) and 2 min ( cyan ) is presented . Scale bar 10 µm ( C ) . CER ( µm / min ) of root epidermal cells of the TZ ( D ) and the EZ ( E ) of the wild type ( grey bars ) and cre1 - 12 ( blue bars ) mutant . Hormone concentrations and conditions of treatment ( D , E ) were as described for ( C ) . Mean ± s . d . , ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 by Student ' s t - test , n = 15 - 20 cells per root growth zone in 3 - 6 roots per condition , in 3 independent ) .

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C-E. Analysis of CK and auxin effects on CER of epidermal cells. Epidermal cells of the TZ and the EZ in the wild type and cre1-12 with PMs visualized with the YFP-NPSN12 marker. Overlay of images of epidermal cells at time 0 (magenta) and 2 min (cyan) is presented. Scale bar 10 µm (C). CER (µm/min) of root epidermal cells of the TZ (D) and the EZ (E) of the wild type (grey bars) and cre1-12 (blue bars) mutant. Hormone concentrations and conditions of treatment (D, E) were as described for (C). Mean ± s.d., (* P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 by Student's t-test, n=15-20 cells per root growth zone in 3-6 roots per condition, in 3 independent).


A . MT plus - end growth rates ( µm / min ) in leukocytes expressing the EB3 - mCherry plus - end marker before ( dark grey ) and after ( light grey ) treatment with mock ( DMSO ) , CK ( 10 µM BAP or 10 µM trans - zeatin ) and auxin ( 0 . 1 µM NAA ) . MT plus - end growth rates were monitored for 10 min before and for 10 min after each treatment . The number of MT plus - end events ( EB3 - mCherry ) tracked per cell was 100 - 200 / min . Mean ± s . d . , ( * * * P < 0 . 001 by Student ' s t - test , n = 1 - 3 cells per condition in 4 independent replicate ) .

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A. MT plus-end growth rates (µm/min) in leukocytes expressing the EB3-mCherry plus-end marker before (dark grey) and after (light grey) treatment with mock (DMSO), CK (10 µM BAP or 10 µM trans-zeatin) and auxin (0.1 µM NAA). MT plus-end growth rates were monitored for 10 min before and for 10 min after each treatment. The number of MT plus-end events (EB3-mCherry) tracked per cell was 100-200/min. Mean ± s.d., (*** P < 0.001 by Student's t-test, n = 1-3 cells per condition in 4 independent replicate).


A . MT plus - end growth rates ( µm / min ) in leukocytes expressing the EB3 -

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A. MT plus-end growth rates (µm/min) in leukocytes expressing the EB3-mCherry


C - E . Immunostaining of α - tubulin ( green ) in leukocytes mounted in media containing DAPI ( blue , nuclear marker ) and quantification of immunolabeled MTs . Cells were not or treated with 300 nM nocodazole ( N ) for 10 min ( C ) . Leukocytes pretreated for 30 min with mock ( DMSO ) , CK ( 10 µM BAP or 10 µM trans - zeatin ) , adenine ( 10 µM ) or auxin ( 10 µM NAA ) prior to a 10 - min treatment with 300 nM nocodazol ( D ) . Scale bar 50 µm ( C , D ) . E . Relative fluorescence intensity of MTs in leukocytes treated with nocodazole , or nocodazole and hormones as described for ( D ) compared to untreated ( black bar ) cells . Mean ± s . d . , ( P < 0 . 05 by two - way Anova , n = 18 - 33 cells per condition in 3 independent replicates ) .

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C-E. Immunostaining of α-tubulin (green) in leukocytes mounted in media containing DAPI (blue, nuclear marker) and quantification of immunolabeled MTs. Cells were not or treated with 300 nM nocodazole (N) for 10 min (C). Leukocytes pretreated for 30 min with mock (DMSO), CK (10 µM BAP or 10 µM trans-zeatin), adenine (10 µM) or auxin (10 µM NAA) prior to a 10-min treatment with 300 nM nocodazol (D). Scale bar 50 µm (C, D). E. Relative fluorescence intensity of MTs in leukocytes treated with nocodazole, or nocodazole and hormones as described for (D) compared to untreated (black bar) cells. Mean ± s.d., (P < 0.05 by two-way Anova, n = 18-33 cells per condition in 3 independent replicates).


( A ) Immunofluorescence labeling of FTSJ1 - Flag ( red ) in HEK293T cells . The nucleus was stained by DAPI ( blue ) . Scale bar , 20 μm .

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(A) Immunofluorescence labeling of FTSJ1-Flag (red) in HEK293T cells. The nucleus was stained by DAPI (blue). Scale bar, 20 μm.


( B ) Subcellular localization of FTSJ1 . Cytoplasmic ( C ) and nuclear ( N ) fractions were separated from HEK293T cells expressing FTSJ1 - Flag . α - Tubulin and Lamin A / C were used as indicators of the cytoplasmic and nuclear fractions , respectively .

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(B) Subcellular localization of FTSJ1. Cytoplasmic (C) and nuclear (N) fractions were separated from HEK293T cells expressing FTSJ1-Flag. α-Tubulin and Lamin A/C were used as indicators of the cytoplasmic and nuclear fractions, respectively.


( C ) Co - IP assays of the interaction between FTSJ1 and WDR6 in co - transfected HEK293T cells . Anti - HA IP ( top ) and anti - Flag IP ( middle ) were immunoblotted by anti - HA and anti - Flag antibodies , respectively . Bottom : cell lysates immunoblotted by anti - HA or anti - Flag antibodies , with GAPDH serving as a loading control .

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(C) Co-IP assays of the interaction between FTSJ1 and WDR6 in co-transfected HEK293T cells. Anti-HA IP (top) and anti-Flag IP (middle) were immunoblotted by anti-HA and anti-Flag antibodies, respectively. Bottom: cell lysates immunoblotted by anti-HA or anti-Flag antibodies, with GAPDH serving as a loading control.


( D ) Co - IP assays of the interaction between FTSJ1 and WDR6 with RNase A addition .

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(D) Co-IP assays of the interaction between FTSJ1 and WDR6 with RNase A addition.


B , C ) Co - IP assays of the interaction between FTSJ1 - Flag or truncated FTSJ1 mutants and WDR6 - HA in co - transfected HEK293T cells . Anti - Flag IP ( top ) were respectively immunoblotted by anti - HA and anti - Flag antibodies . Bottom : cell lysates immunoblotted by anti - HA or anti - Flag antibodies , with GAPDH serving as a loading control .

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B, C) Co-IP assays of the interaction between FTSJ1-Flag or truncated FTSJ1 mutants and WDR6-HA in co-transfected HEK293T cells. Anti-Flag IP (top) were respectively immunoblotted by anti-HA and anti-Flag antibodies. Bottom: cell lysates immunoblotted by anti-HA or anti-Flag antibodies, with GAPDH serving as a loading control.