( 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 ) .

O O O O O O O O O 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 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

(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 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 .

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

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

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

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 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 .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_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 B-MEASURED_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 O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O 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 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 .

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 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

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.


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 .

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

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 O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

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

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

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 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 .

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

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

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 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 .

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

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 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 .

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 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 .

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

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

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

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 O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O O O O O O O O O O O O O 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) 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 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

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

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


Heatmap displaying overlap significance between different gene sets and 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 O O O O O

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 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

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

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


Heatmap displaying overlap significance between different gene sets and 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 O O O O O O O O O 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 .

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

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

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

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

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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 ) 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

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

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

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

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 . 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 .

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

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

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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 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 ) .

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(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 ) 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 .

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(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 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 .

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(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 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 .

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

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

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

(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 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 .

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 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

(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 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 .

O O O B-MEASURED_VAR O O B-CONTROLLED_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 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 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

(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 . 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 .

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

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

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

(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 cell lysates were probed for Ulk1 protein and 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 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 .

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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 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 .

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

(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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 : 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 ) .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 ) .

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(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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 analysis of DNA content and Western blot analysis of cell cycle markers of the 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 and + CDC14 ‐ NLS cells were grown at 35 . 5 ° C for 5 h and sonicated , and differential interference contrast ( DIC ) images 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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 ) , the plasma membrane 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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 cells from ( C ) were plated 6 h following β ‐ estradiol treatment and 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 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 .

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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 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 .

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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 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 .

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

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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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.


B . Typical examples of stage 1 ( day 0 ) , stage 2 ( day 7 ) and stage 3 ( day 12 ) human iPSC - derived NSCs / neurons immunostained for γ - Tubulin and Centrin , marked by arrows . Cells were co - immunostained with Ki67 and DAPI ( day 0 ) or Trim46 and MAP2 ( day 7 and 12 ) to define their neuronal stages . Arrowheads mark the AIS . Scale bar = 10µm in overview , 2µm in zooms .

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B. Typical examples of stage 1 (day 0), stage 2 (day 7) and stage 3 (day 12) human iPSC-derived NSCs/neurons immunostained for γ-Tubulin and Centrin, marked by arrows. Cells were co-immunostained with Ki67 and DAPI (day 0) or Trim46 and MAP2 (day 7 and 12) to define their neuronal stages. Arrowheads mark the AIS. Scale bar = 10µm in overview, 2µm in zooms.


D . 3D STED imaging of αTubulin in stage 1 ( day 0 ) , stage 2 ( day 7 ) and stage 3 ( day 13 ) hiPSC - derived neurons . Scale bar = 5µm in overview , 1µm in zoom .

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D. 3D STED imaging of αTubulin in stage 1 (day 0), stage 2 (day 7) and stage 3 (day 13) hiPSC-derived neurons. Scale bar = 5µm in overview, 1µm in zoom.


F . Typical examples of centrosomes of human iPSC - derived NSCs ( day 0 ) with STED imaging of Trim46 and Centrin immunostaining , and confocal imaging of γ - Tubulin immunostaining . Scale bar = 1µm .

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F. Typical examples of centrosomes of human iPSC-derived NSCs (day 0) with STED imaging of Trim46 and Centrin immunostaining, and confocal imaging of γ-Tubulin immunostaining. Scale bar = 1µm.


G . Typical examples of stage 2 and stage 3 human iPSC - derived neurons immunostained for Trim46 and MAP2 . Inserts represent centrosomes . Zooms on the right represent a non - polarized neurite or a developing axon in stage 2 or stage 3 neurons , respectively . Scale bar = 20µm in overview , 1µm in insert , 10µm in zoom .

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G. Typical examples of stage 2 and stage 3 human iPSC-derived neurons immunostained for Trim46 and MAP2. Inserts represent centrosomes. Zooms on the right represent a non-polarized neurite or a developing axon in stage 2 or stage 3 neurons, respectively. Scale bar = 20µm in overview, 1µm in insert, 10µm in zoom.