( A ) representative ECFP / FRET of FAK biosensor in Piezo1 overexpressed HEK before ( left ) after ( right ) shockwave stimulation ( Scale bar , 40 μm ) . Color scale bars in figures are to show ECFP / FRET ratios , with cold hot colors representing low high ratios , respectively . ( B ) time courses of normalized ECFP / FRET ( Mean ± ) of FAK FRET biosensor before after shockwave stimulation in HBSS Ca2 + media with ( green , n = 16 , N = 3 ) or without Piezo1 ( black , n = 10 , N = 3 ) . ( C ) change of FRET of FAK FRET biosensor in HEK with or without Piezo1 after shockwave stimulation in HBSS Ca2 + medium . * P < 0 . 05 from student two - t - .

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

(A) The representative ECFP/FRET ratio images of FAK biosensor in Piezo1 overexpressed HEK cells before (left) and after (right) shockwave stimulation (Scale bar, 40 μm). Color scale bars in the figures are to show the ECFP/FRET ratios, with cold and hot colors representing low and high ratios, respectively. (B) The time courses of normalized ECFP/FRET ratio (Mean ± SEM) of FAK FRET biosensor before and after shockwave stimulation in HBSS Ca2+ media with (green line, n = 16, N = 3) or without Piezo1 (black line, n = 10, N = 3). (C) The percentage change of FRET ratio of the FAK FRET biosensor in HEK cells with or without Piezo1 after shockwave stimulation in HBSS Ca2+ medium. *P < 0.05 from student two-tailed t-test.


( A ) representative ECFP / FRET of FAK biosensor in Piezo1 expressing HEK before after 25 μM Yoda1 stimulation ( Scale bar , 10 μm ) . Color scale bars in figures indicate ECFP / FRET ratios , with cold hot colors representing low high ratios , respectively . ( B ) time courses of normalized FRET ( Mean ± ) of FAK FRET biosensor before after Yoda1 stimulation with ( pink , n = 10 , N = 3 ) or without Piezo1 ( black , n = 11 , N = 3 ) . grey represents DMSO control group ( n = 8 , N = 3 ) .

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

(A) The representative ECFP/FRET ratio images of FAK biosensor in Piezo1 expressing HEK cells before and after 25 μM Yoda1 stimulation (Scale bar, 10 μm). Color scale bars in the figures indicate ECFP/FRET ratios, with cold and hot colors representing low and high ratios, respectively. (B) The time courses of normalized FRET ratio (Mean ± SEM) of FAK FRET biosensor before and after Yoda1 stimulation with (pink line, n = 10, N = 3) or without Piezo1 (black line, n = 11, N = 3). The grey line represents the DMSO control group (n = 8, N = 3).


( C ) representative FRET / ECFP of calcium biosensor in Piezo1 - expressing HEK before after 25 μM Yoda1 stimulation ( Scale bar , 10 μm ) . Color scale bars in figures indicate FRET / ECFP ratios , with cold hot colors representing low high ratios , respectively . ( D ) time courses of normalized FRET ( Mean ± ) of calcium biosensor in Piezo1 - expressing HEK ( purple , n = 9 , N = 3 ) before after 25 μM Yoda1 stimulation in culture medium .

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

(C) The representative FRET/ECFP ratio images of the calcium biosensor in the Piezo1-expressing HEK cells before and after 25 μM Yoda1 stimulation (Scale bar, 10 μm). Color scale bars in the figures indicate FRET/ECFP ratios, with cold and hot colors representing low and high ratios, respectively. (D) The time courses of normalized FRET ratio (Mean ± SEM) of calcium biosensor in the Piezo1-expressing HEK cells (purple line, n = 9, N = 3) before and after 25 μM Yoda1 stimulation in culture medium.


( A ) & ( B ) , time courses ( Mean ± ) ( A ) representative ( Scale bar , 10 μm ) of FRET of SHP2 biosensor ( Y542F ) before after 25 μM or 0 . 5 μM Yoda1 stimulation in with or without Piezo1 overexpression ( red bar , n = 5 , N = 3 ; blue bar , n = 5 , N = 3 ; purple bar , n = 11 , N = 3 ; green bar , n = 5 , N = 3 ) .

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

(A)&(B), The time courses (Mean ± SEM) (A) and representative images (Scale bar, 10 μm) of FRET ratio of SHP2 biosensor (Y542F) before and after 25 μM or 0.5 μM Yoda1 stimulation in cells with or without Piezo1 overexpression (red bar, n = 5, N=3; blue bar, n = 5, N=3; purple bar, n = 11, N=3; green bar, n = 5, N=3).


B ) Expression of 21U sensor ( left ) DAPI ( right ) of gonad arms in indicated genetic backgrounds . Gonads are outlined by a dashed . mCherry signal is represented in pseudo - colours [ LUT fire ( ImageJ ) ] to reflect differences in of signal . Number of analysed with indicated phenotype are given in panel . not showing activation of 21U sensor ( + ) were still silenced , that did not show silenced 21U sensor ( RNAe ) state were expressing weakly , comparable to 21U sensor ( + ) . Scale bar : 25 μm .

O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-TISSUE O O O O O O B-TISSUE 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-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 B-GENEPROD O O O O O O O O O O O

B) Expression of the 21U sensor (left) and DAPI staining (right) of gonad arms in the indicated genetic backgrounds. Gonads are outlined by a dashed line. The mCherry signal is represented in pseudo-colours [LUT fire (ImageJ)] to reflect differences in the intensity of the signal. Number of animals analysed and with indicated phenotype are given in the panel. Animals not showing the activation of the 21U sensor(+) were still silenced, and animals that did not show the silenced 21U sensor(RNAe) state were expressing weakly, comparable to the 21U sensor(+). Scale bar: 25 μm.


C ) Tc1 reversion in different genetic backgrounds . strains tested carried unc - 22 : : Tc1 ( st136 ) allele . Tc1 excision can result in restoration of unc - 22 function , which can be scored visually . Negative control = unc - 22 : : Tc1 ( st136 ) in a wild type background ; positive control = wago - 1 / - 2 / - 3 . Two independent experiments per are represented .

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

C) Tc1 reversion assay in different genetic backgrounds. All the strains tested carried the unc-22::Tc1(st136) allele. Tc1 excision can result in restoration of unc-22 function, which can be scored visually. Negative control = unc-22::Tc1(st136) in a wild type background; positive control = wago-1/-2/-3. Two independent experiments per strain are represented.


D ) Crossing scheme to address re - initiation of silencing of 21U sensor . A mut - 7 mutant expressing 21U sensor is crossed with either a wild type ( left ) , prg - 1 ( middle ) or pid - 2 ( right ) mutant hermaphrodite . Their F1 offspring was scored for expression of 21U sensor by microscopy . Three states of expression were scored , represented in a pie chart . three expression states are exemplified by representative at bottom : OFF ( left ) , FAINT ( right ) or ON ( middle ) . DIC are shown above panels . Gonads are outlined by a dashed . Scale bar : 25 µm .

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

D) Crossing scheme to address the re-initiation of silencing of the 21U sensor. A mut-7 mutant male expressing the 21U sensor is crossed with either a wild type (left), prg-1 (middle) or pid-2 (right) mutant hermaphrodite. Their F1 offspring was scored for expression of the 21U sensor by microscopy. Three states of expression were scored, and represented in a pie chart. The three expression states are exemplified by representative images at the bottom: OFF (left), FAINT (right) or ON (middle). DIC images are shown above the fluorescence panels. Gonads are outlined by a dashed line. Scale bar: 25 µm.


E ) Crossing scheme to address if maternal 21U RNAs are sufficient to re - initiate silencing of 21U sensor . A pid - 1 mutant expressing 21U sensor is crossed with a hermaphrodite , heterozygous for same mutation . their F1 offspring inherit a pool of 21U RNAs from hermaphrodite , but in 50 % of F1 , which is pid - 1 homozygous mutant , no zygotic PID - 1 is present , hence no zygotic 21U RNAs can be made . silencing or expression of 21U sensor in pid - 1 homozygous mutant F1 has been scored by microscopy , depicted in a pie chart . At bottom , a representative image of an carrying a silenced 21U sensor ( lower : mCherry signal ; upper : DAPI ) in pid - 1 mutant offspring . Gonads are outlined by a dashed . Scale bar : 25 μm .

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

E) Crossing scheme to address if maternal 21U RNAs are sufficient to re-initiate the silencing of the 21U sensor. A pid-1 mutant male expressing the 21U sensor is crossed with a hermaphrodite, heterozygous for the same mutation. All their F1 offspring inherit a pool of 21U RNAs from the hermaphrodite, but in 50% of the F1, which is pid-1 homozygous mutant, no zygotic PID-1 is present, hence no zygotic 21U RNAs can be made. The silencing or expression of the 21U sensor in the pid-1 homozygous mutant F1 has been scored by microscopy, and depicted in a pie chart. At the bottom, a representative image of an animal carrying a silenced 21U sensor (lower: mCherry signal; upper: DAPI staining) in pid-1 mutant offspring. Gonads are outlined by a dashed line. Scale bar: 25 μm.


F ) Crossing scheme to role of PID - 2 in re - initiating silencing of 21U sensor , mediated by maternally provided 21U RNAs only . expression of 21U sensor in F1 has been scored by microscopy , depicted in a pie chart . arrow heads indicate many arrayed oocytes , typical of a feminized germline . DIC image of a representative are shown at bottom . Gonads are outlined by a dashed . Scale bar : 25 μm .

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

F) Crossing scheme to test the role of PID-2 in re-initiating the silencing of the 21U sensor, mediated by maternally provided 21U RNAs only. The expression of the 21U sensor in the F1 has been scored by microscopy, and depicted in a pie chart. White arrow heads indicate the many arrayed oocytes, typical of a feminized germline. DIC and fluorescence image of a representative animal are shown at the bottom. Gonads are outlined by a dashed line. Scale bar: 25 μm.


22G RNAs mapping to 21U sensor transgene were identified from small sequencing data , read density was plotted over transgene , which is schematically depicted at bottom . aggregated results of three replicates is shown for each indicated genotype in different panels . shading , in grey , represents standard deviation of read density from three replicates . ( + ) means that sensor was detectably expressed . ( RNAe ) means that sensor was crossed into respective mutant background in an RNAe state ( i . e . its silencing was PRG - 1 - independent ) , its expression remained undetectable by microscopy .

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

The 22G RNAs mapping to the 21U sensor transgene were identified from small RNA sequencing data, and read density was plotted over the transgene, which is schematically depicted at the bottom. The aggregated results of three replicates is shown for each indicated genotype in the different panels. The shading, in grey, represents the standard deviation of the read density from the three replicates. (+) means that the sensor was detectably expressed. (RNAe) means that the sensor was crossed into the respective mutant background in an RNAe state (i.e. its silencing was PRG-1-independent), and its expression remained undetectable by microscopy.


A ) MA - plot of log2 fold changes ( Y - axis ) versus mean of normalized of 22G RNAs ( X - axis ) for pid - 2 ( xf23 ) mutants , compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 fold - change < - 2 .

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

A) MA-plot of log2 fold changes (Y-axis) versus the mean of normalized counts of 22G RNAs (X-axis) for pid-2(xf23) mutants, compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2.


B ) displaying overlap significance between different gene sets genes that either up - or down - regulated in pid - 2 ( xf23 ) mutants . Significance was tested with Fisher ' s exact p - values adjusted with Benjamini - Hochberg Procedure . Colour scheme represents odds of overlaps representing strength of association . N . S . : not significant .

O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O 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) Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in pid-2(xf23) mutants. Significance was tested with Fisher's exact test and p-values adjusted with the Benjamini-Hochberg Procedure. Colour scheme represents the odds ratio of overlaps representing the strength of association. N.S.: not significant.


C - D ) Cumulative 22G coverage along gene of Mutator CSR - 1 targets . Values represent 22G coverage normalized to total coverage of each gene , for wild type ( N2 ) pid - 2 ( xf23 ) mutants . lines represent average of biological replicates , whereas shading represents standard deviation of biological replicates . a . u . : arbitrary units .

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

C-D) Cumulative 22G coverage along the gene body of Mutator and CSR-1 targets. Values represent 22G coverage normalized to the total coverage of each gene, for wild type (N2) and pid-2(xf23) mutants. The lines represent the average of biological replicates, whereas the shading represents the standard deviation of biological replicates. a.u.: arbitrary units.


A ) Volcano plot representing enrichment of interacting with PID - 2 , isolated by immunoprecipitation of eGFP : : PID - 2 , followed by quantitative label - free mass spectrometry . As control , eGFP IPs were performed on extracts from wild - type , non - transgenic . For each IP - MS experiment , quadruplicates were measured analysed . dashed reflects a significance threshold of p - value < 0 . 05 at two - fold enrichment .

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

A) Volcano plot representing the enrichment of proteins interacting with PID-2, isolated by immunoprecipitation of eGFP::PID-2 protein, followed by quantitative label-free mass spectrometry. As control, eGFP IPs were performed on protein extracts from wild-type, non-transgenic animals. For each IP-MS experiment, quadruplicates were measured and analysed. The dashed line reflects a significance threshold of p-value < 0.05 at two-fold enrichment.


Volcano plot representing enrichment of interacting with PID - 2 , isolated by immunoprecipitation of eGFP : : PID - 2 , followed by quantitative label - free mass spectrometry . In this experiment , endogenous PID - 2 was immunoprecipitated , pid - 2 ( xf23 ) mutant extracts were used as control .

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

Volcano plot representing the enrichment of proteins interacting with PID-2, isolated by immunoprecipitation of eGFP::PID-2 protein, followed by quantitative label-free mass spectrometry. In this experiment, the endogenous PID-2 protein was immunoprecipitated, and pid-2(xf23) mutant protein extracts were used as control.


Volcano plot , representing enrichment of interacting with PID - 4 , by immunoprecipitating endogenously tagged PID - 4 . IPs from extracts from wild - type , non - tagged were used as control .

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

Volcano plot , representing the enrichment of proteins interacting with PID-4, by immunoprecipitating endogenously tagged PID-4 protein. IPs from protein extracts from wild-type, non-tagged animals were used as control.


Volcano plot , representing enrichment of interacting with PID - 5 , by immunoprecipitating endogenously tagged PID - 5 . IPs from extracts from wild - type , non - tagged were used as control .

Volcano plot, representing the enrichment of proteins interacting with PID-5, by immunoprecipitating endogenously tagged PID-5 protein. IPs from protein extracts from wild-type, non-tagged animals were used as control.


A ) Expression of 21U sensor in indicated genetic backgrounds . Gonads are outlined by a dashed . mCherry signal is represented in pseudo - colours [ LUT fire ( ImageJ ) ] to reflect differences in of signal . panels on right are DIC of respective . Scale bar : 25 μm .

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

A) Expression of the 21U sensor in the indicated genetic backgrounds. Gonads are outlined by a dashed line. The mCherry signal is represented in pseudo-colours [LUT fire (ImageJ)] to reflect differences in the intensity of the signal. The panels on the right are DIC images of the respective animals. Scale bar: 25 μm.


B ) 22G coverage of 21U sensor ( RNAe ) in indicated genetic backgrounds .

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

B) 22G RNA coverage of the 21U sensor(RNAe) in the indicated genetic backgrounds.


C ) MA - plot of log2 fold changes ( on Y - axis ) versus mean of normalized of 22G RNAs ( on X - axis ) for pid - 4 mutants , compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 fold - change < - 2 .

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

C) MA-plot of log2 fold changes (on the Y-axis) versus the mean of normalized counts of 22G RNAs (on the X-axis) for pid-4 mutants, compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2.


D ) displaying overlap significance between different gene sets genes that either up - or down - regulated in pid - 4 mutants . Colour scheme represents odds of overlaps representing strength of association .

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

D) Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in pid-4 mutants. Colour scheme represents the odds ratio of overlaps representing the strength of association.


MA - plot of log2 fold changes ( on Y - axis ) versus mean of normalized of 22G RNAs ( on X - axis ) , compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 fold - change < - 2 . E ) for pid - 5

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

MA-plot of log2 fold changes (on the Y-axis) versus the mean of normalized counts of 22G RNAs (on the X-axis) , compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2. E) for pid-5 mutant


displaying overlap significance between different gene sets genes that either up - or down - regulated in F ) pid - 5

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

Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in F) pid-5 mutant


MA - plot of log2 fold changes ( on Y - axis ) versus mean of normalized of 22G RNAs ( on X - axis ) compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 fold - change < - 2 . G ) for pid - 4 ; pid - 5 double

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

MA-plot of log2 fold changes (on the Y-axis) versus the mean of normalized counts of 22G RNAs (on the X-axis) compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2. G) for pid-4;pid-5 double mutant


displaying overlap significance between different gene sets genes that either up - or down - regulated in H ) pid - 4 ; pid - 5 double

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

Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in H) pid-4;pid-5 double mutant


I , J ) plots representing fertility over generations for indicated genetic backgrounds at 25oC . Six L2 - L3 larvae for each of indicated backgrounds were - picked to a fresh plate every four or six days , counting as two or three generations , respectively , until no larvae were present on plate to be picked . * : two plates of N2 were contaminated were excluded from .

O O O O O O B-EXP_ASSAY 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 B-TISSUE O O O O O O O O O O O O O O O O O O O O

I, J) Line plots representing fertility over generations for the indicated genetic backgrounds at 25oC. Six L2-L3 larvae for each of the indicated backgrounds were hand-picked to a fresh plate every four or six days, counting as two or three generations, respectively, until no larvae were present on the plate to be picked. *: two plates of N2 were contaminated and were excluded from the assay.


Expression of PID - 2 : : eGFP ( xfSi145 ) ( A ) in perinuclear granules in germline . PGL - 1 : : mTagRFP - T ( A ) mark P granules . indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown for each . Note that most of L4 gonad is in pachytene stage . Arrow heads indicate condensates . Scale bar : 25 µm .

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

Expression of PID-2::eGFP(xfSi145) (A) in perinuclear granules in the germline. PGL-1::mTagRFP-T (A) mark P granules. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.


Expression of , PID - 4 : : mTagRFP - T ( B ) in perinuclear granules in germline . DEPS - 1 : : GFP ( B mark P granules . indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown for each . Note that most of L4 gonad is in pachytene stage . Arrow heads indicate condensates . Scale bar : 25 µm .

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

Expression of , PID-4::mTagRFP-T (B) in perinuclear granules in the germline. DEPS-1::GFP (B mark P granules. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.


Expression of PID - 5 : : mTagRFP - T ( C ) in perinuclear granules in germline . DEPS - 1 : : GFP mark P granules . indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown for each . Note that most of L4 gonad is in pachytene stage . Arrow heads indicate condensates . Scale bar : 25 µm .

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

Expression of PID-5::mTagRFP-T (C) in perinuclear granules in the germline. DEPS-1::GFP mark P granules. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.


Expression of PID - 4 : : mTagRFP - T ( D ) together with 3xFLAG : : GFP : : ZNFX - 1 , a Z granule marker . indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown for each . Note that most of L4 gonad is in pachytene stage . Arrow heads indicate condensates . Scale bar : 25 µm .

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

Expression of PID-4::mTagRFP-T (D) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.


Expression of PID - 5 : : mTagRFP - T ( E ) together with 3xFLAG : : GFP : : ZNFX - 1 , a Z granule marker . indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown for each . Note that most of L4 gonad is in pachytene stage . Arrow heads indicate condensates . Scale bar : 25 µm .

Expression of PID-5::mTagRFP-T (E) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.


F ) Box - plots representing ( µm ) between centres of two signals from indicated fusion between each pair of is represented by a dot . Between 4 - 10 different gonads were analyzed for each condition .

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 B-TISSUE O O O O O O

F) Box-plots representing the distance (µm) between the centres of two fluorescent signals from the indicated fusion proteins The distance between each pair of fluorescent proteins is represented by a dot. Between 4-10 different gonads were analyzed for each condition.


Expression of PID - 2 : : eGFP ( xfSi145 ) together with PID - 4 : : mTagRFP - T ( G ) indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown for each . Note that most of L4 gonad is in pachytene stage . Arrow heads indicate condensates . Scale bar = 25

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

Expression of PID-2::eGFP (xfSi145) together with PID-4::mTagRFP-T (G) The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar = 25 µ


Expression of PID - 2 : : eGFP ( xfSi145 ) together with PID - 5 : : mTagRFP - T ( H ) . indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown for each . Note that most of L4 gonad is in pachytene stage . Arrow heads indicate condensates . Scale bar = 25 µm .

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

Expression of PID-2::eGFP (xfSi145) together with PID-5::mTagRFP-T (H). The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar = 25 µm.


A - E ) Expression of 3xFLAG : : GFP : : ZNFX - 1 PGL - 1 : : mTagRFP - T in a wild type ( A ) , pid - 2 ( B ) , pid - 4 ; pid - 5 ( C ) , pid - 4 ( D ) , pid - 5 ( E ) mutant backgrounds . indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown . Arrow heads indicate condensates . Scale bar : 25 µm . F ) of Z / P granules in wild type , pid - 2 , pid - 4 / - 5 , pid - 4 pid - 5 mutant backgrounds . number of granules is indicated in brackets , next to genotype .

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

A-E) Expression of 3xFLAG::GFP::ZNFX-1 and PGL-1::mTagRFP-T in a wild type (A), pid-2 (B), pid-4;pid-5 (C), pid-4 (D), and pid-5 (E) mutant backgrounds. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown. Arrow heads indicate individual condensates. Scale bar: 25 µm. F) Quantification of the ratio Z / P granules in wild type, pid-2, pid-4/-5, pid-4 and pid-5 mutant backgrounds. The number of granules is indicated in brackets, next to the genotype.


Expression of PID - 4 : : mTagRFP - T ( G ) together with 3xFLAG : : GFP : : ZNFX - 1 , a Z granule marker , in a pid - 2 mutant background . indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown for each . Scale bar : 25 µm .

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

Expression of PID-4::mTagRFP-T (G) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker, in a pid-2 mutant background. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Scale bar: 25 µm.


Expression of PID - 4 : : mTagRFP - T H ) together with 3xFLAG : : GFP : : ZNFX - 1 , a Z granule marker , in a pid - 2 mutant background . indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown for each . Scale bar : 25 µm .

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

Expression of PID-4::mTagRFP-T H) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker, in a pid-2 mutant background. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Scale bar: 25 µm.


I ) Box - plots representing ( µm ) between centres of two signals from indicated fusion between each pair of is represented by a dot . Between 4 - 10 different gonads were analyzed for each condition . median is represented by a . interquartile range ( IQR ) , 25th to 75th percentile , is represented by upper lower lines , respectively , whiskers represent first quartile ( down to - 1 . 5 * IQR ) or third quartile ( up to + 1 . 5 * IQR ) . P - values were calculated using a t - ( two - ) .

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 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

I) Box-plots representing the distance (µm) between the centres of two fluorescent signals from the indicated fusion proteins The distance between each pair of fluorescent proteins is represented by a dot. Between 4-10 different gonads were analyzed for each condition. The median is represented by a line. The interquartile range (IQR), 25th to 75th percentile, is represented by the upper and lower lines, respectively, and whiskers represent the first quartile (down to -1.5*IQR) or the third quartile (up to +1.5*IQR). P-values were calculated using a t-test (two-tailed).


( a ) HEK293 were starved of glucose ( 4 h ) as indicated , endogenous Ulk1 was immunoprecipitated an autophosphorylation was performed . were resolved by SDS - PAGE visualized with autoradiography ( top ) or western blotting ( WB ; bottom ) .

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

(a) HEK293 cells were starved of glucose (4 h) as indicated, endogenous Ulk1 was immunoprecipitated and an autophosphorylation assay was performed. Proteins were resolved by SDS-PAGE and visualized with autoradiography (top) or western blotting (WB; bottom).


( b ) were incubated in glucose - free medium ( 4 h ) as indicated lysed . Lysates were incubated with lambda phosphatase ( λ PPase ) as indicated . Endogenous Ulk1 mobility was examined by western blotting .

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

(b) Cells were incubated in glucose-free medium (4 h) as indicated and lysed. Lysates were incubated with lambda phosphatase (λ PPase) as indicated. Endogenous Ulk1 mobility was examined by western blotting.


( c ) HA - Ulk1 was transfected into HEK293 together with wild - type ( WT ) AMPKα1 or a kinase - dead ( DN ) mutant . were starved of glucose ( 4 h ; Glu ) or ( - A . A ) treated with compound C ( 20 μM , C . C ) as indicated . Ulk1 mobility as well as phosphorylation of ACC S6K were determined by western blotting .

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

(c) HA-Ulk1 was transfected into HEK293 cells together with wild-type (WT) AMPKα1 or a kinase-dead (DN) mutant. Cells were starved of glucose (4 h; Glu) or amino acids (-A.A) and treated with compound C (20 μM, C.C) as indicated. Ulk1 mobility as well as phosphorylation levels of ACC and S6K were determined by western blotting.


( d ) HA - Ulk1 were immunopurified from transfected HEK293 , which had undergone glucose starvation ( 4 h ) as indicated . HA - Ulk1 were treated with λ PPase , in vitro kinase assays were performed in presence of GST - ATG13 . were resolved by SDS - PAGE ; phosphorylated were visualized with autoradiography , HA - Ulk1 by western blotting GST - Atg13 by Coomassie .

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

(d) HA-Ulk1 proteins were immunopurified from transfected HEK293 cells, which had undergone glucose starvation (4 h) as indicated. The HA-Ulk1 proteins were treated with λ PPase, and in vitro kinase assays were performed in the presence of GST-ATG13. Proteins were resolved by SDS-PAGE; phosphorylated proteins were visualized with autoradiography , HA-Ulk1 by western blotting and GST-Atg13 by Coomassie staining.


( e ) HA - Ulk1 was immunopurified from transfected HEK293 under glucose - rich media treated with AMPK in presence of cold ATP for 15 min , followed by kinase assays as described in d . In vitro kinase assays were performed in presence of GST - ATG13 . were resolved by SDS - PAGE ; phosphorylated were visualized with autoradiography , HA - Ulk1 by western blotting GST - Atg13 by Coomassie .

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

(e) HA-Ulk1 was immunopurified from transfected HEK293 cells under glucose-rich media and treated with AMPK in the presence of cold ATP for 15 min, followed by kinase assays as described in d. In vitro kinase assays were performed in the presence of GST-ATG13. Proteins were resolved by SDS-PAGE; phosphorylated proteins were visualized with autoradiography, HA-Ulk1 by western blotting and GST-Atg13 by Coomassie staining.


( f ) AMPK wild - type ( WT ) α1 / α2 double knockout ( DKO ) MEFs were incubated with or without glucose ( 4 h ) . Endogenous Ulk1 was immunoprecipitated autophosphorylation was measured ( mean ± s . d . , n = 3 ) . Autophosphorylation activity was normalized to Ulk1 level ; relative activity is calculated by normalization to Ulk1 activity from AMPK wild - type MEFs in glucose - rich conditions .

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

(f) AMPK wild-type (WT) and α1/α2 double knockout (DKO) MEFs were incubated with or without glucose (4 h). Endogenous Ulk1 was immunoprecipitated and autophosphorylation was measured (mean ± s.d., n = 3). Autophosphorylation activity was normalized to Ulk1 protein level; relative activity is calculated by normalization to Ulk1 activity from AMPK wild-type MEFs in glucose-rich conditions.


( g ) HA - Ulk1 was transfected into HEK293 together with vector ( Vec ) or an AMPKα1 kinase - dead mutant ( DN ) . were starved of glucose ( - Glu ) or ( - A . A ) , or treated with 50 nM rapamycin ( Rapa ) for 3 h before lysis . Left : autophosphorylation activity was assessed normalized as in f ( mean ± s . d . , n = 3 ) . Right : fold induction in Ulk1 autophosphorylation , compared with Ulk1 autophosphorylation from under nutrient - rich conditions . Uncropped of blots are shown in Supplementary Fig . S5 .

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

(g) HA-Ulk1 was transfected into HEK293 cells together with vector (Vec) or an AMPKα1 kinase-dead mutant (DN). The cells were starved of glucose (-Glu) or amino acids (-A.A), or treated with 50 nM rapamycin (Rapa) for 3 h before lysis. Left: autophosphorylation activity was assessed and normalized as in f (mean ± s.d., n = 3). Right: fold induction in Ulk1 autophosphorylation, compared with Ulk1 autophosphorylation from cells under nutrient-rich conditions. Uncropped images of blots are shown in Supplementary Fig. S5.


( a ) AMPK phosphorylates Ulk1 S / T domain in vitro . Top : schematic representation of Ulk1 domain structure deletion constructs used to map phosphorylation sites . mouse Ulk1 consists of an N - terminal kinase domain ( KD ; 1 - 278 ) , serine / threonine - rich domain ( S / T domain , 279 - 828 ) , C - terminal domain ( CTD , 829 - 1051 ) . Bottom : indicated Flag - Ulk1 deletion mutants were immunopurified from transfected HEK293 were used for in vitro AMPK as a substrate . Phosphorylation was examined by 32P - autoradiogram level was determined by western blot .

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

(a) AMPK phosphorylates the Ulk1 S/T domain in vitro. Top: schematic representation of Ulk1 domain structure and deletion constructs used to map phosphorylation sites. The mouse Ulk1 protein consists of an N-terminal kinase domain (KD; 1-278), serine/threonine-rich domain (S/T domain, 279-828), and C-terminal domain (CTD, 829-1051). Bottom: the indicated Flag-Ulk1 deletion mutants were immunopurified from transfected HEK293 cells and were used for in vitro AMPK assay as a substrate. Phosphorylation was examined by 32P-autoradiogram and protein level was determined by western blot.


( b ) Determination of AMPK phosphorylation sites in Ulk1 . indicated recombinant GST - Ulk1 mutants were expressed purified from Escherichia coli , used as substrates for in vitro phosphorylation by AMPK . Deletion analyses indicated that two Ulk1 fragments in S / T domain , 279 - 425 769 - 782 , were highly phosphorylated by AMPK in vitro . Mutation of Ser 317 abolished majority of phosphorylation in Ulk1 fragment 279 - 425 . Within fragment 769 - 782 , mutations of five serine residues ( Ser 774 , Ser 777 , Ser 778 , Ser 779 Ser 780 ) to alanine , denoted as ( 769 - 782 ) 5SA , completely abolished phosphorylation by AMPK . Reconstitution of Ser 777 in this mutation background , ( 769 - 782 ) 4SA - S777 , but not any of other four residues , restored phosphorylation by AMPK . GST GST - TSC2F ( TSC2 fragment 1300 - 1367 containing AMPK phosphorylation site at Ser 1345 ) were used as negative positive controls for AMPK reaction , respectively . Phosphorylation was determined by 32P - autoradiograph were detected by Coomassie .

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

(b) Determination of AMPK phosphorylation sites in Ulk1. The indicated recombinant GST-Ulk1 mutants were expressed and purified from Escherichia coli, and used as substrates for in vitro phosphorylation by AMPK. Deletion analyses indicated that two Ulk1 fragments in the S/T domain, 279-425 and 769-782, were highly phosphorylated by AMPK in vitro. Mutation of Ser 317 abolished the majority of phosphorylation in the Ulk1 fragment 279-425. Within the fragment 769-782, mutations of five serine residues (Ser 774, Ser 777, Ser 778, Ser 779 and Ser 780) to alanine, denoted as (769-782) 5SA, completely abolished the phosphorylation by AMPK. Reconstitution of Ser 777 in this mutation background, (769-782) 4SA-S777, but not any of the other four residues, restored the phosphorylation by AMPK. GST and GST-TSC2F (TSC2 fragment 1300-1367 containing AMPK phosphorylation site at Ser 1345) were used as negative and positive controls for AMPK reaction, respectively. Phosphorylation was determined by 32P-autoradiograph and the protein levels were detected by Coomassie staining.


( c ) Ser 317 / Ser 777 are required for glucose - starvation induced Ulk1 phosphorylation in vivo . HA - Ulk1 mutants were transfected into HEK293 . were starved for glucose for 4 h as indicated . HA - Ulk1 was immunoprecipitated examined by western blot for mobility .

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

(c) Ser 317/Ser 777 are required for glucose-starvation induced Ulk1 phosphorylation in vivo. HA-Ulk1 and mutants were transfected into HEK293 cells. Cells were starved for glucose for 4 h as indicated. HA-Ulk1 was immunoprecipitated and examined by western blot for mobility.


( d ) Phosphorylation of Ulk1 Ser 317 Ser 777 are induced by AMPK . Wild - type HA - Ulk1 or S317 / 777A mutant were co - transfected with AMPK into HEK293 as indicated . HA - Ulk1 was immunoprecipitated ( IP ) phosphorylation of Ser 317 Ser 777 were determined by western blotting . Uncropped of blots are shown in Supplementary Fig . S5 .

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(d) Phosphorylation of Ulk1 Ser 317 and Ser 777 are induced by AMPK. Wild-type HA-Ulk1 or S317/777A mutant were co-transfected with AMPK into HEK293 cells as indicated. HA-Ulk1 was immunoprecipitated (IP) and phosphorylation of Ser 317 and Ser 777 were determined by western blotting. Uncropped images of blots are shown in Supplementary Fig. S5.


( a ) AMPK wild - type or DKO MEFs were starved of glucose ( 4 h ) as indicated . Total lysates were probed for Ulk1 phosphorylation .

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(a) AMPK wild-type or DKO MEFs were starved of glucose (4 h) as indicated. Total cell lysates were probed for Ulk1 protein and phosphorylation.


( b ) Time course of Ulk1 Ser 317 Ser 777 phosphorylation in response to glucose starvation / re - addition . MEFs were starved of glucose ( - Glu ) for indicated times . After 3 h starvation , culture was switched to glucose - containing ( 25 mM ) medium samples were harvested ( Re - Glu ) . In parallel , were treated with - acid - free ( - A . A ) medium or 50 nM rapamycin ( Rapa ) for 3 h .

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(b) Time course of Ulk1 Ser 317 and Ser 777 phosphorylation in response to glucose starvation/re-addition. MEFs were starved of glucose (-Glu) for the indicated times. After 3 h starvation, the culture was switched to glucose-containing (25 mM) medium and samples were harvested (Re-Glu). In parallel, cells were treated with amino-acid-free (-A.A) medium or 50 nM rapamycin (Rapa) for 3 h.


( c ) Phosphorylation of Ulk1 Ser 317 Ser 777 correlates with AMPK activity . MEFs were starved of glucose ( 4 h ) as indicated in presence or absence of 20 μM compound C ( C . C ) . In parallel , were treated with 2 mM Metformin ( Met , 2 h ) in glucose - rich medium . Phosphorylation of ACC S79 was tested as a positive control for AMPK activation .

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(c) Phosphorylation of Ulk1 Ser 317 and Ser 777 correlates with AMPK activity. MEFs were starved of glucose (4 h) as indicated in the presence or absence of 20 μM compound C (C.C). In parallel, cells were treated with 2 mM Metformin (Met, 2 h) in glucose-rich medium. Phosphorylation of ACC S79 was tested as a positive control for AMPK activation.


( d ) Ulk1 is highly phosphorylated at Ser 317 Ser 777 by glucose starvation in vivo . To determine Ulk1 phosphorylation level in vivo , immunopurified HA - Ulk1 was phosphorylated by AMPK in vitro ( 100 % represents full phosphorylation of Ulk1 by AMPK ) . In vitro phosphorylated HA - Ulk1 was diluted as indicated , was immunoblotted along with immunoprecipitated HA - Ulk1 from grown in either glucose - rich ( + Glu ) or glucose - free ( - Glu , 4 h ) medium . density of bands was then quantified . By this measurement , approximately 50 % of Ulk1 isolated from glucose - starved was phosphorylated on Ser 317 Ser 777 .

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(d) Ulk1 is highly phosphorylated at Ser 317 and Ser 777 by glucose starvation in vivo. To determine the Ulk1 phosphorylation level in vivo, immunopurified HA-Ulk1 protein was phosphorylated by AMPK in vitro (100% represents full phosphorylation of Ulk1 by AMPK). In vitro phosphorylated HA-Ulk1 was diluted as indicated, and was immunoblotted along with the immunoprecipitated HA-Ulk1 from cells grown in either glucose-rich (+ Glu) or glucose-free (- Glu, 4 h) medium. The density of the bands was then quantified. By this measurement, approximately 50% of Ulk1 isolated from glucose-starved cells was phosphorylated on Ser 317 and Ser 777.


( e ) indicated HA - Ulk1 were immunopurified from transfected HEK293 grown in high - glucose medium , then incubated with AMPK in presence of cold ATP for 15 minin vitro . After reaction , AMPK was removed by extensive washing , resulting Ulk1 immuno - complexes were assayed for kinase activity in presence of 32P - ATP .

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(e) The indicated HA-Ulk1 proteins were immunopurified from transfected HEK293 cells grown in high-glucose medium, and then incubated with AMPK in the presence of cold ATP for 15 minin vitro. After the reaction, AMPK was removed by extensive washing, the resulting Ulk1 immuno-complexes were assayed for kinase activity in the presence of 32P-ATP.


( f ) HA - Ulk1 ( wild type or S317 / 777A mutant ) were immunoprecipitated from transfected HEK293 , which were incubated with or without glucose ( 4 h ) before lysis . An in vitro kinase reaction was performed in presence of GST - ATG13 FIP200 . Uncropped of blots are shown in Supplementary Fig . S5 .

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(f) HA-Ulk1 proteins (wild type or S317/777A mutant) were immunoprecipitated from the transfected HEK293 cells, which were incubated with or without glucose (4 h) before lysis. An in vitro kinase reaction was performed in the presence of GST-ATG13 and FIP200. Uncropped images of blots are shown in Supplementary Fig. S5.


( a ) AMPK interacts with Ulk1 . HEK293 were transfected with various Flag - Ulk1 deletion mutants together with AMPK α / β / γ , Atg13 FIP200 . Flag - Ulk1 ( indicated by arrows ) was immunoprecipitated co - immunoprecipitation of AMPK α / β / γ , Atg13 FIP200 were examined by western blots .

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(a) AMPK interacts with Ulk1. HEK293 cells were transfected with the various Flag-Ulk1 deletion mutants together with AMPK α/β/γ, Atg13 and FIP200. Flag-Ulk1 protein (indicated by white arrows) was immunoprecipitated and co-immunoprecipitation of AMPK α/β/γ, Atg13 and FIP200 were examined by western blots.


( b ) Deletion of Ulk1 regions responsible for AMPK interaction . indicated Flag - Ulk1 truncation mutants were immunoprecipitated from transfected HEK293 co - expressing AMPK ( α / β / γ ) . Co - immunoprecipitation of AMPK subunits was determined by western blots .

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(b) Deletion analysis of Ulk1 regions responsible for AMPK interaction. The indicated Flag-Ulk1 truncation mutants were immunoprecipitated from transfected HEK293 cells co-expressing AMPK complex (α/β/γ). Co-immunoprecipitation of AMPK subunits was determined by western blots.


( c ) Rheb inhibits Ulk1 - AMPK interaction . HA - AMPKα , Flag - Ulk1 Myc - Rheb were co - transfected into HEK293 as indicated . were treated with or without rapamycin ( 50 nM Rapa ) for 1 h before lysis . Flag - Ulk1 was immunoprecipitated co - immunoprecipitates of AMPKα were determined by western blot .

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(c) Rheb inhibits the Ulk1-AMPK interaction. HA-AMPKα, Flag-Ulk1 and Myc-Rheb were co-transfected into HEK293 cells as indicated. Cells were treated with or without rapamycin (50 nM Rapa) for 1 h before lysis. Flag-Ulk1 was immunoprecipitated and co-immunoprecipitates of AMPKα were determined by western blot.


( d ) Rapamycin treatment enhances interaction of endogenous Ulk1 AMPK . Endogenous Ulk1 were immunoprecipitated from either Ulk1 or AMPK wild - type knockout ( single - knockout ; KO or double - knockout ; DKO ) MEFs . Treatment with 50 nM rapamycin for 1 h is indicated ( Rapa ) . Co - immunoprecipitation of endogenous AMPKα was determined by western blot . arrow indicates AMPKα .

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(d) Rapamycin treatment enhances the interaction of endogenous Ulk1 and AMPK. Endogenous Ulk1 proteins were immunoprecipitated from either Ulk1 or AMPK wild-type and knockout (single-knockout; KO or double-knockout; DKO) MEFs. Treatment with 50 nM rapamycin for 1 h is indicated (Rapa). Co-immunoprecipitation of endogenous AMPKα protein was determined by western blot. The arrow indicates AMPKα protein.


( e ) Phosphorylation by mTORC1 inhibits ability of Ulk1 to bind AMPK in vitro . CBP / SBP - Ulk1 was purified from transfected HEK293 by streptavidin beads Ulk1 - bead was incubated with mTORC1 , which was prepared by Raptor immunoprecipitation , in presence of cold ATP , as indicated . resulting Ulk1 was incubated with lysates containing AMPK , then extensively washed . Ulk1 associated AMPKα were detected by western blot . Uncropped of blots are shown in Supplementary Fig . S5 .

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(e) Phosphorylation by mTORC1 inhibits the ability of Ulk1 to bind AMPK in vitro. CBP/SBP-Ulk1 was purified from transfected HEK293 cells by streptavidin beads and the Ulk1-bead complex was incubated with mTORC1, which was prepared by Raptor immunoprecipitation, in the presence of cold ATP, as indicated. The resulting Ulk1 complex was incubated with the cell lysates containing AMPK, then extensively washed. The Ulk1 and associated AMPKα were detected by western blot. Uncropped images of blots are shown in Supplementary Fig. S5.


( a ) mTORC1 phosphorylates Ulk1 S / T domain . Ulk1 deletion mutants were prepared from transfected HEK293 used for in vitro mTORC1 . Phosphorylation was examined by 32P - autoradiogram ( top ) level was determined by western blot ( bottom ) .

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(a) mTORC1 phosphorylates the Ulk1 S/T domain. Ulk1 deletion mutants were prepared from the transfected HEK293 cells and used for in vitro mTORC1 assay. Phosphorylation was examined by 32P-autoradiogram (top) and protein level was determined by western blot (bottom).


( b ) Ser 757 is phosphorylated by mTORC1 . Left : indicated recombinant GST - mUlk1 mutants were purified from E . coli used for in vitro mTORC1 as substrates . Deletion analyses isolated fragment ( 753 - 771 ) as a target for mTORC1 . Ulk1 ( 753 - 771 ) fragment contains five conserved serine / threonine residues , Thr 754 , Ser 757 , Ser 760 , Thr 763 Thr 770 . Right : mutation of Ser 757 abolished Ulk1 phosphorylation by mTORC1 in vitro . GST was used as negative control for mTORC1 phosphorylation reaction . Phosphorylation was determined by 32P - autoradiograph ( top ) , whereas were detected by Coomassie ( bottom ) .

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(b) Ser 757 is phosphorylated by mTORC1. Left: the indicated recombinant GST-mUlk1 mutants were purified from E. coli and used for in vitro mTORC1 assay as substrates. Deletion analyses isolated the fragment (753-771) as a target for mTORC1. The Ulk1 (753-771) fragment contains five conserved serine/threonine residues, Thr 754, Ser 757, Ser 760, Thr 763 and Thr 770. Right: mutation of Ser 757 abolished Ulk1 phosphorylation by mTORC1 in vitro. GST was used as negative control for mTORC1 phosphorylation reaction. Phosphorylation was determined by 32P-autoradiograph (top), whereas protein levels were detected by Coomassie staining (bottom).


( c ) Rheb increases Ulk1 Ser 757 phosphorylation . HA - Ulk1 wild type S757A mutant were immunoprecipitated from transfected HEK293 . Co - transfection with Rheb rapamycin ( Rapa ) treatment are indicated . Ulk1 Ser 757 phosphorylation was determined by western blot .

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(c) Rheb increases Ulk1 Ser 757 phosphorylation. HA-Ulk1 wild type and the S757A mutant were immunoprecipitated from transfected HEK293 cells. Co-transfection with Rheb and rapamycin (Rapa) treatment are indicated. Ulk1 Ser 757 phosphorylation was determined by western blot.


( d ) Rheb induces a mobility shift in wild - type Ulk1 , but not Ulk1S757A mutant . HA - Ulk1 was transfected with or without Rheb into HEK293 . HA - Ulk1 was immunoprecipitated from under nutrient - rich medium Ulk1 mobility was examined by western blot .

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(d) Rheb induces a mobility shift in wild-type Ulk1, but not the Ulk1S757A mutant. HA-Ulk1 was transfected with or without Rheb into HEK293 cells. HA-Ulk1 was immunoprecipitated from the cells under nutrient-rich medium and Ulk1 mobility was examined by western blot.


( e ) Endogenous Ulk1 Ser 757 phosphorylation is elevated in Tsc1 − / − Tsc1 . Tsc1 + / + ( WT ) Tsc1 − / − ( KO ) MEFs were starved of glucose ( 4 h ) , or treated with 50 nM rapamycin ( Rapa , 1 h ) . Ser 757 phosphorylation of endogenous Ulk1 was detected by a phospho - Ulk1 Ser 757 antibody . Uncropped of blots are shown in Supplementary Fig . S5 .

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(e) Endogenous Ulk1 Ser 757 phosphorylation is elevated in Tsc1−/− Tsc1. Tsc1+/+ (WT) and Tsc1−/− (KO) MEFs were starved of glucose (4 h), or treated with 50 nM rapamycin (Rapa, 1 h). Ser 757 phosphorylation of endogenous Ulk1 was detected by a phospho-Ulk1 Ser 757 antibody. Uncropped images of blots are shown in Supplementary Fig. S5.


( a ) Ulk1 Ser 757 is required for mTORC1 to regulate interaction of Ulk1 with AMPK in vivo . CBP / SBP tagged Ulk1 ( wild type or S757C ) was co - transfected with HA - AMPKα Rheb into HEK293 as indicated . Ulk1 was purified by streptavidin beads co - precipitatedHA - AMPKα was examined by western blot ( Rapa , 50 nM rapamycin treatment for 1 h before lysis ) .

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

(a) Ulk1 Ser 757 is required for mTORC1 to regulate the interaction of Ulk1 with AMPK in vivo. CBP/SBP tagged Ulk1 (wild type or S757C) was co-transfected with HA-AMPKα and Rheb into HEK293 cells as indicated. Ulk1 was purified by streptavidin beads and the co-precipitatedHA- AMPKα was examined by western blot (Rapa, 50 nM rapamycin treatment for 1 h before cell lysis).


( b ) Ulk1 Ser 757 is required for rapamycin to enhance Ulk1 - AMPK interaction in vitro . CBP / SBP Ulk1 ( wild type or S757C ) were prepared from transfected HEK293 , which were pre - incubated with 50 nM rapamycin ( Rapa , 1h ) as indicated . Ulk1 were purified by streptavidin beads resulting Ulk1 - bead was incubated with purified AMPKα / β / γ . AMPKα in in vitro pulldown assays were examined by western blot using AMPKα antibody . L . E . ; long exposure .

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(b) Ulk1 Ser 757 is required for rapamycin to enhance the Ulk1-AMPK interaction in vitro. CBP/SBP Ulk1 proteins (wild type or S757C) were prepared from transfected HEK293 cells, which were pre-incubated with 50 nM rapamycin (Rapa, 1h) as indicated. The Ulk1 proteins were purified by streptavidin beads and the resulting Ulk1-bead was incubated with the bacterial purified AMPKα/β/γ complex. AMPKα protein levels in the in vitro pulldown assays were examined by western blot using AMPKα antibody. L.E.; long exposure.


( c ) Phosphorylation of AMPK sites Ser 317 Ser 777 in Ulk1 are decreased in Tsc1 − / − Tsc1 . Tsc1 + / + ( WT ) Tsc1 − / − ( KO ) MEFs were starved of glucose ( 4 h ) , or treated with 50 nM rapamycin ( Rapa , 1 h ) . Ser 317 Ser 777 phosphorylation of endogenous Ulk1 was examined by western blotting with antibodies against Ulk1 phosphorylated at Ser 317 or Ser 777 .

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(c) Phosphorylation of AMPK sites Ser 317 and Ser 777 in Ulk1 are decreased in Tsc1−/− Tsc1. Tsc1+/+ (WT) and Tsc1−/− (KO) MEFs were starved of glucose (4 h), or treated with 50 nM rapamycin (Rapa, 1 h). Ser 317 and Ser 777 phosphorylation of endogenous Ulk1 was examined by western blotting with antibodies against Ulk1 phosphorylated at Ser 317 or Ser 777.


( d ) Rheb suppresses Ulk1 Ser 317 Ser 777 phosphorylation in a manner dependent on mTORC1 . HA - Ulk1 , AMPKα kinase - dead mutant ( DN ) , Myc - Rheb were co - transfected into HEK293 as indicated . were incubated with glucose - free medium ( - Glu , glucose ) , in which either 20 μM compound C ( C . C . ) or 50 nM Rapamycin ( Rapa ) was added . Total lysates were probed with antibodies against Ulk1 phosphorylated at Ser 317 , Ser 777 , Ser 757 , HA , as indicated .

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(d) Rheb suppresses Ulk1 Ser 317 and Ser 777 phosphorylation in a manner dependent on mTORC1. HA-Ulk1, AMPKα kinase-dead mutant (DN), and Myc-Rheb were co-transfected into HEK293 cells as indicated. The cells were incubated with glucose-free medium (-Glu, glucose), in which either 20 μM compound C (C.C.) or 50 nM Rapamycin (Rapa) was added. Total cell lysates were probed with antibodies against Ulk1 phosphorylated at Ser 317, Ser 777, Ser 757, and HA, as indicated.


( e ) Rheb inhibits glucose starvation - induced Ulk1 activation . HA - Ulk1 Myc - Rheb was transfected into HEK293 , which were incubated with glucose - free ( - Glu ) , - acid - free ( - A . A ) medium , or 50 nM rapamycin ( Rapa ) for 4 h before lysis . HA - Ulk1 was immunoprecipitated kinase assays were performed . Ulk1 activity was measured by 32P - autoradiogram level of HA - Ulk1 GST - Atg13 used in was determined by western blot by Coomassie , respectively . Uncropped of blots are shown in Supplementary Fig . S5 .

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(e) Rheb inhibits glucose starvation-induced Ulk1 activation. HA-Ulk1 and Myc-Rheb was transfected into HEK293 cells, which were incubated with glucose-free (-Glu), amino-acid-free (-A.A) medium, or 50 nM rapamycin (Rapa) for 4 h before lysis. HA-Ulk1 was immunoprecipitated and kinase assays were performed. Ulk1 activity was measured by 32P-autoradiogram and the protein level of HA-Ulk1 and GST-Atg13 used in the assay was determined by western blot and by Coomassie staining, respectively. Uncropped images of blots are shown in Supplementary Fig. S5.


( a ) Ser 317 / Ser 777 is required for Ulk1 to protect from glucose starvation . Viability ( 24 h , mean ± s . d . , n = 4 ; top ) PARP cleavage ( 8 h ; western blot , middle ; , n = 2 , bottom ) was examined in Ulk1 + / + ( WT ) , Ulk1 − / − ( KO ) , Ulk1 − / − re - expressing wild - type Ulk1 ( KO - WT ) , Ulk1 − / − re - expressing Ulk1 S317 / 777A mutant ( KO - S317 / 777A ) MEFs . Arrows in western blots indicate non - PARP .

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(a) Ser 317/Ser 777 is required for Ulk1 to protect cells from glucose starvation. Viability (24 h, mean ± s.d., n = 4; top) and PARP cleavage (8 h; western blot, middle; quantification, n = 2, bottom) was examined in Ulk1+/+ (WT), Ulk1−/− (KO), Ulk1−/− re-expressing wild-type Ulk1 (KO-WT), and Ulk1−/− re-expressing Ulk1 S317/777A mutant (KO-S317/777A) MEFs. Arrows in western blots indicate non-cleaved and cleaved PARP.


( b ) Ulk1 S317 / 777A mutant is compromised in LC3 lipidation in response to glucose starvation . ULK1MEFs were cultured in MEFs - free medium for indicated times . LC3 - II level was determined by western blotting LC3 - II accumulation was normalized by α - tubulin quantified ( bottom , n = 3 , mean ± s . d . ) . A representative western blot was shown . LC3 antibody used in this experiment seemed to preferentially recognise lipid - modified form of LC3 - II , which migrated faster on gel .

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(b) The Ulk1 S317/777A mutant is compromised in LC3 lipidation in response to glucose starvation. ULK1MEFs were cultured in MEFs-free medium for the indicated times. LC3-II level was determined by western blotting and the LC3-II accumulation was normalized by α-tubulin and quantified (bottom, n = 3, mean ± s.d.). A representative western blot was shown. The LC3 antibody used in this experiment seemed to preferentially recognise the lipid-modified form of LC3-II, which migrated faster on the gel.


( c ) Ulk1 S317 / 777A mutant is defective in autophagosome formation . indicated MEFs were starved of glucose ( 4 h ) formation of GFP - LC3 - positive autophagosomes was examined by confocal microscopy . GFP - LC3 ; green DAPI ; blue . Scale bar , 20 μm .

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(c) The Ulk1 S317/777A mutant is defective in autophagosome formation. The indicated MEFs were starved of glucose (4 h) and the formation of GFP-LC3-positive autophagosomes was examined by confocal microscopy. GFP-LC3; green and DAPI; blue. Scale bar, 20 μm.


( d ) Autophagy vacuole by electron microscopy . Low - magnification of Ulk1 − / − ( KO , upper left panel ) , Ulk1 − / − reconstituted with wild - type Ulk1 ( KO - WT , two middle panels with accompanying higher magnification ) , Ulk1 − / − reconstituted with Ulk1 S317 / 777A ( KO - S317 / 777A , lower left panel ) are shown . High - magnification of autophagosomes from KO - WT are shown in upper right lower right panels . Autophagosome / autolysosome - like structures indicated by arrowheads on lower - magnification arrows in higher - magnification . Scale bars ; lower - magnification , 1 μm ; higher - magnification , 200 nm .

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(d) Autophagy vacuole analysis by electron microscopy. Low-magnification images of Ulk1−/− (KO, upper left panel), Ulk1−/− reconstituted with wild-type Ulk1 (KO-WT, two middle panels with accompanying higher magnification images), and Ulk1−/− reconstituted with Ulk1 S317/777A (KO-S317/777A, lower left panel) are shown. High-magnification images of autophagosomes from KO-WT are shown in upper right and lower right panels. Autophagosome/autolysosome-like structures indicated by arrowheads on the lower-magnification images and arrows in higher-magnification images. Scale bars; lower-magnification, 1 μm; higher-magnification, 200 nm.


A cdc14 ‐ 3 ‐ sensitive reconstituted with CDC14 or CDC14 ‐ NLS were released from a G1 arrest at restrictive stained for Cdc14 spindle ( Tub1 ) once had reached late anaphase .

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A cdc14‐3 temperature‐sensitive cells reconstituted with CDC14 or CDC14‐NLS were released from a G1 arrest at the restrictive temperature and stained for Cdc14 and the spindle (Tub1) once cells had reached late anaphase.


B Wild ‐ type , cdc14 ‐ 3 , + CDC14 + CDC14 ‐ NLS were released from an α ‐ factor ‐ induced G1 arrest re ‐ arrested in next G1 . Samples were harvested at indicated time points stained with DAPI for nucleolar Nop1 ( n > 100 ) . of examples of without ( top ) with ( bottom ) rDNA segregation defects at 80 min after release are shown together with over time .

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B Wild‐type, cdc14‐3, +CDC14 and +CDC14‐NLS cells were released from an α‐factor‐induced G1 arrest and re‐arrested in the next G1. Samples were harvested at the indicated time points and stained with DAPI and for the nucleolar protein Nop1 (n > 100). Photographs of examples of cells without (top) and with (bottom) rDNA segregation defects at 80 min after release are shown together with quantification over time.


C from experiment in ( B ) were stained for spindle ( n > 100 ) . without ( top ) with ( bottom ) anaphase spindles at 100 min after release are shown together with their over time .

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C Cells from the experiment in (B) were stained for the spindle (n > 100). Cells without (top) and with (bottom) anaphase spindles at 100 min after release are shown together with their quantification over time.


D FACS of content Western blot of cycle markers of samples from ( B ) . Tub1 served as a loading control .

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D FACS analysis of DNA content and Western blot analysis of cell cycle markers of the samples from (B). Tub1 served as a loading control.


A + CDC14 + CDC14 ‐ NLS were grown at 35 . 5 ° C for 5 h sonicated , differential interference contrast ( DIC ) were acquired .

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A +CDC14 and +CDC14‐NLS cells were grown at 35.5°C for 5 h and sonicated, and differential interference contrast (DIC) images were acquired.


B from a synchronous culture ( as in Fig B ) were treated with zymolyase to remove wall . fraction of remaining budded is shown over time ( n = 100 ) .

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B Cells from a synchronous culture (as in Fig B) were treated with zymolyase to remove the cell wall. The fraction of remaining budded cells is shown over time (n = 100).


C expressing plasma marker GFP ‐ Spo2051 - 91 , exemplifying indicated bud neck stages of cytokinesis . D Bud neck of synchronized GFP ‐ Spo2051 - 91 ‐ expressing + CDC14 or + CDC14 ‐ NLS during progression of a cycle ( n > 75 ) .

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C Cells expressing the plasma membrane marker GFP‐Spo2051-91, exemplifying the indicated bud neck phenotypes and stages of cytokinesis.D Bud neck phenotypes of synchronized GFP‐Spo2051-91‐expressing +CDC14 or +CDC14‐NLS cells during the progression of a cell cycle (n > 75).


E + CDC14 or + CDC14 ‐ NLS expressing Spc42 ‐ YFP GFP fusions with indicated cytokinetic were synchronized in G1 using α ‐ factor . Upon release at 35 . 5 ° C , were imaged every 2 min time from anaphase onset to indicated events was recorded . Error bars represent SD ; Student ' s t ‐ was applied to analyze differences between indicated conditions ; * * P ≤ 0 . 01 ; * * * * P ≤ 0 . 0001 . For representative image sequences , see Supplementary Fig S2C .

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E +CDC14 or +CDC14‐NLS cells expressing Spc42‐YFP and GFP fusions with the indicated cytokinetic protein were synchronized in G1 using α‐factor. Upon release at 35.5°C, cells were imaged every 2 min and the time from anaphase onset to the indicated events was recorded. Error bars represent SD; Student's t‐test was applied to analyze differences between the indicated conditions; **P ≤ 0.01; ****P ≤ 0.0001. For representative image sequences, see Supplementary Fig S2C.


A MET3pr ‐ CDC20cdc15 ‐ as1 were arrested in metaphase by Cdc20 depletion subsequently treated with 1NM ‐ PP1 to inhibit Cdc15 ‐ as1 . were released from metaphase arrest by Cdc20 re ‐ induction ( left graph ) , or Cdc14 / or Sic1 expression were induced from GAL1 promoter while remained arrested ( right graph ) . fraction of large ‐ budded after spheroplastation ( n = 100 ) was quantified . Representative 120 min after galactose addition are shown . Scale bar , 10 μm .

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A MET3pr‐CDC20cdc15‐as1 cells were arrested in metaphase by Cdc20 depletion and subsequently treated with 1NM‐PP1 to inhibit Cdc15‐as1. Cells were released from the metaphase arrest by Cdc20 re‐induction (left graph), or Cdc14 and/or Sic1 expression were induced from the GAL1 promoter while cells remained arrested (right graph). The fraction of large‐budded cells after spheroplastation (n = 100) was quantified. Representative photographs 120 min after galactose addition are shown. Scale bar, 10 μm.


B As ( A ) , plasma marker GFP ‐ Spo2051 - 91 was visualized following Cdc14 induction ( n = 100 ) .

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B As (A), the plasma membrane marker GFP‐Spo2051-91 was visualized following Cdc14 induction (n = 100).


C Samples from experiment in ( B ) were processed for Western blot of indicated cycle . Arrowheads indicate endogenous exogenous forms of Cdc14 . Tub1 served as a loading control . A schematic of downstream processing for phosphoproteome is shown on right .

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C Samples from the experiment in (B) were processed for Western blot analysis of the indicated cell cycle proteins. Arrowheads indicate endogenous and exogenous forms of Cdc14. Tub1 served as a loading control. A schematic of downstream processing for the phosphoproteome analysis is shown on the right.


A Phospho ‐ peptide abundance during Cdc14 ‐ induced mitotic exit . Examples of phospho ‐ peptides that disappear with early ( red ) , intermediate ( orange ) late ( green ) timing or that remain stable ( black ) are shown . This color coding is used throughout panels of this figure . B Numbers of unique peptides corresponding that do or do not contain a minimal Cdk consensus motif are shown , categorized according to timing of disappearance as described in ( A ) . C stability data for corresponding to disappearing phospho ‐ peptides ( blue ) for other ( black ) . D fractions of peptides that contain a minimal Cdk consensus motif ( [ S / T ] ‐ P ) within timing categories shown in ( B ) were calculated . P ‐ value of enrichment of each category relative to combined stable / category ( gray ) are shown . E Similar to ( D ) , enrichment for reported physical Cdc14 interactors is assessed within [ S / T ] ‐ P ‐ containing phospho ‐ peptides . F Peptides that contain indicated Cdk consensus motifs that have disappeared over time are represented as a fraction of phospho ‐ peptides containing that motif .

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A Phospho‐peptide abundance during Cdc14‐induced mitotic exit. Examples of phospho‐peptides that disappear with early (red), intermediate (orange) and late (green) timing or that remain stable (black) are shown. This color coding is used throughout all panels of this figure.B Numbers of unique peptides and corresponding proteins that do or do not contain a minimal Cdk consensus motif are shown, categorized according to timing of disappearance as described in (A).C Protein stability data for proteins corresponding to disappearing phospho‐peptides (blue) and for all other proteins (black).D The fractions of peptides that contain a minimal Cdk consensus motif ([S/T]‐P) within the timing categories shown in (B) were calculated. The ratio and P‐value of enrichment of each category relative to the combined stable/rest category (gray) are shown.E Similar to (D), the enrichment for reported physical Cdc14 interactors is assessed within [S/T]‐P‐containing phospho‐peptides.F Peptides that contain the indicated Cdk consensus sequence motifs and that have disappeared over time are represented as a fraction of all phospho‐peptides containing that motif.


G Unique phospho ‐ peptides of Spa2 , as an example , are categorized on their dephosphorylation timing ( graph ) . Enrichment of clustered timing for covered by phospho ‐ peptides was compared to random for Cdk or non ‐ Cdk consensus site ‐ containing peptides ( table ) . Fisher ' s exact were used to determine significance .

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G Unique phospho‐peptides of Spa2, as an example, are categorized based on their dephosphorylation timing (graph). Enrichment of clustered timing for all proteins covered by phospho‐peptides was compared to random for Cdk or non‐Cdk consensus site‐containing peptides (table). Fisher's exact tests were used to determine significance.


B Western blot of expressing conditional Clb2m ( + ) or Clb2mΔCdk ( Δ ) fusions to indicated , before ( − ) 5 h after ( + ) β ‐ estradiol treatment . Tub1 served as a loading control . c , control .

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B Western blot analysis of cells expressing conditional Clb2m (+) or Clb2mΔCdk (Δ) fusions to the indicated proteins, before (−) and 5 h after (+) β‐estradiol treatment. Tub1 served as a loading control. c, control cells.


C FACS of content after conditional Clb2m or Clb2mΔCdk fusion to indicated . of fraction of with > 2C content is shown in bar graph for two independent biological replicates , with red dots representing bars representing average values . Bnr1 fusions were included as controls for cytokinetic defects . Student ' s t ‐ was applied to compare matched Clb2m versus Clb2mΔCdk fusion strains . ‐ P > 0 . 05 ; * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 .

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C FACS analysis of DNA content after conditional Clb2m or Clb2mΔCdk fusion to the indicated proteins. Quantification of the fraction of cells with > 2C DNA content is shown in the bar graph for two independent biological replicates, with red dots representing individual and bars representing average values. Bnr1 fusions were included as controls for cytokinetic defects. Student's t‐test was applied to compare matched Clb2m versus Clb2mΔCdk fusion strains. ‐P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.


D Serial dilutions of from ( C ) were plated 6 h following β ‐ estradiol treatment grown for 1 day at 36 ° C .

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D Serial dilutions of cells from (C) were plated 6 h following β‐estradiol treatment and grown for 1 day at 36°C.


E of chains containing a common cytoplasm was scored 6 h after β ‐ estradiol treatment using GFP ‐ Spo2051 - 91 plasma marker ( n > 150 ) . c , control ; scale bars , 10 μm .

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E The length of cell chains containing a common cytoplasm was scored 6 h after β‐estradiol treatment using the GFP‐Spo2051-91 plasma membrane marker (n > 150). c, control cells; scale bars, 10 μm.


A Genes corresponding to indicated were replaced by wild ‐ type ( wt ) or Cdk phospho ‐ site mutant ( xA ) sequences then conditionally fused to Clb2m or Clb2mΔCdk . Cytokinetic defects were scored by FACS of content as in Fig C . values from two independent clones ( red dots ) as well as their average ( bars ) are shown . Student ' s t ‐ was applied to compare matched wt xA strains . c , control .

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A Genes corresponding to the indicated proteins were replaced by wild‐type (wt) or Cdk phospho‐site mutant (xA) sequences and then conditionally fused to Clb2m or Clb2mΔCdk. Cytokinetic defects were scored by FACS analysis of DNA content as in Fig C. The individual values from two independent clones (red dots) as well as their average (bars) are shown. Student's t‐test was applied to compare matched wt and xA strains. c, control cells.


B chain formation in samples from ( A ) . fraction of with > 2 bodies for each clone are represented with red dots their averages by black bar ( n > 150 ) . Significance is determined by Student ' s t ‐ for indicated conditions . Scale bars , 5 μm .

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B Cell chain formation in the samples from (A). The fraction of cells with > 2 cell bodies for each clone are represented with red dots and their averages by the black bar (n > 150). Significance is determined by Student's t‐tests for the indicated conditions. Scale bars, 5 μm.


C indicated strains were spotted in serial dilutions onto auxin ‐ containing plates grown for 2 days .

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C The indicated strains were spotted in serial dilutions onto auxin‐containing plates and grown for 2 days.


D Auxin was added to cultures of indicated strains , after 6 h , were sonicated number of attached bodies was counted ( n > 200 ) . Significance was calculated using a Wilcoxon rank ‐ sum .

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D Auxin was added to cultures of the indicated strains, and after 6 h, cells were sonicated and the number of attached cell bodies was counted (n > 200). Significance was calculated using a Wilcoxon rank‐sum test.


E fraction of in asynchronously proliferating cultures of indicated strains with a content of > 2C was determined by FACS ( n = 3 ) . Error bars represent SD .

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E The fraction of cells in asynchronously proliferating cultures of the indicated strains with a DNA content of > 2C was determined by FACS analysis (n = 3). Error bars represent SD.


A Cyk3 ‐ GFP signal at GFP relative to onset of actomyosin ring contraction ( t = 0 ) . mean from at least 20 are shown .

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A Cyk3‐GFP signal intensity at the GFP relative to the onset of actomyosin ring contraction (t = 0). The mean and SEM from at least 20 cells are shown.


B Inn1 ‐ Clb2m , Inn1 ‐ Clb2mΔCdk control before Clb2m fusion were arrested in G1 using α ‐ factor , released harvested 90 - 100 min later at time of cytokinesis . Indirect immunofluorescence microscopy was used to determine Inn1bud neck spindle status . Representative of full spindles partially fully disassembled spindles are shown . nr , non ‐ recombined . Scale bars , 5 μm .

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B Inn1‐Clb2m, Inn1‐Clb2mΔCdk and control cells before Clb2m fusion were arrested in G1 using α‐factor, released and harvested 90-100 min later at the time of cytokinesis. Indirect immunofluorescence microscopy was used to determine Inn1bud neck staining and the spindle status. Representative images of full spindles and partially and fully disassembled spindles are shown. nr, non‐recombined cells. Scale bars, 5 μm.


C time from anaphase onset until GFP appearance of Inn1 was determined by filming synchronized Inn1 ‐ Clb2m ‐ GFP Inn1 ‐ Clb2mΔCdk ‐ GFP containing Spc42 ‐ YFP ‐ labeled spindle pole bodies . Error bars represent SD .

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C The time from anaphase onset until the GFP appearance of Inn1 was determined by filming synchronized Inn1‐Clb2m‐GFP and Inn1‐Clb2mΔCdk‐GFP cells containing Spc42‐YFP‐labeled spindle pole bodies. Error bars represent SD.


D As in ( C ) , now persistence of a full actomyosin ring was measured in expressing Inn1 ‐ Clb2m , Inn1 ‐ Clb2mΔCdk or their respective non ‐ phosphorylatable mutants together with Myo1 ‐ GFP Spc42 ‐ YFP . Error bars represent SD .

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D As in (C), now the persistence of a full actomyosin ring was measured in cells expressing Inn1‐Clb2m, Inn1‐Clb2mΔCdk or their respective non‐phosphorylatable mutants together with Myo1‐GFP and Spc42‐YFP. Error bars represent SD.


E Montage of medial view projections of actomyosin rings in Inn1 ‐ Clb2m Inn1 ‐ Clb2mΔCdk . Frames are shown in 2 ‐ min intervals . F Actomyosin ring contraction from at least 21 filmed as in ( E ) were blindly scored as having a normal ( " constriction " ) or aberrant ( " disintegration " ) cytokinetic phenotype .

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E Montage of medial view projections of actomyosin rings in Inn1‐Clb2m and Inn1‐Clb2mΔCdk cells. Frames are shown in 2‐min intervals.F Actomyosin ring contraction phenotypes from at least 21 cells filmed as in (E) were blindly scored as having a normal ("constriction") or aberrant ("disintegration") cytokinetic phenotype.


G Cytokinetic defect viability of Inn1 ‐ Clb2m or Inn1 ‐ Clb2mΔCdk in absence or presence of Chs2V377I . In bar graph , red dots represent values of duplicate biological replicates with bars representing average . Significance is calculated using Student ' s t ‐ . 10 ‐ fold spot dilution assays are represented , together with Western blotting for Inn1 before after its fusion to Clb2m . Tub1 served as a loading control .

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G Cytokinetic defect and cell viability of Inn1‐Clb2m or Inn1‐Clb2mΔCdk cells in the absence or presence of Chs2V377I. In the bar graph, red dots represent values of duplicate biological replicates with bars representing the average. Significance is calculated using Student's t‐test. 10‐fold spot dilution assays are represented, together with the Western blotting analysis for Inn1 before and after its fusion to Clb2m. Tub1 served as a loading control.