Figure 3 ( A ) RPE1 cells were transfected with siRNA as indicated , and immunostained for Rab8 ( red ) and γ - tubulin ( green ) in either 10 % FBS medium ( upper panel ) or serum - starvation medium ( lower panel ) . Scale bar , 10 μm . Arrowhead indicates Rab8 localized to the centrioles . ( B ) Quantification of the percentage of cells with Rab8 localized to the centriole Data represent mean ± SD ( n = 3 experiments ) , 200 cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test . ( C ) Cells were transfected with siRNA and immunostained for IFT20 ( red ) and γ - tubulin ( green ) . Scale bar , 10 μm . ( D ) Quantification of the percentage of IFT20 intensity at the centrioles Data represent mean ± SD ( n = 3 experiments ) , 150 cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) RPE1 cells were transfected with siRNA as indicated, and immunostained for Rab8 (red) and γ-tubulin (green) in either 10% FBS medium (upper panel) or serum-starvation medium (lower panel). Scale bar, 10 μm. Arrowhead indicates Rab8 localized to the centrioles.(B) Quantification of the percentage of cells with Rab8 localized to the centriole Data represent mean ± SD (n=3 experiments), 200 cells were scored per condition per experiment, *P < 0.05, Student's t-test.(C) Cells were transfected with siRNA and immunostained for IFT20 (red) and γ-tubulin (green). Scale bar, 10 μm.(D) Quantification of the percentage of IFT20 intensity at the centrioles Data represent mean ± SD (n=3 experiments), 150 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


Figure 4 ( A ) RPE1 cells were transfected with siRNAs as indicated , followed by transfection with wild - type pGFP - Rab8a ( WT - Rab8 ) , constitutively active pGFP - Rab8a ( Q67L ) ( CA - Rab8 ) , or DN Rab8 dominant - negative pGFP Rab8a ( T22N ) , incubated in serum - starved media for 12 h , and immunostained for ARL13B ( Red ) , GFP - Rab8 ( green ) , and DAPI ( blue ) . Scale bar , 10 μm . ( B ) Quantification of the percentage of ciliated cells Data represent mean ± SD ( n = 3 experiments ) , 200 GFP - positive cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test . ( C ) ( Upper panel ) Cells were transfected as indicated and the cell lysates were incubated with purified proteins , including GST or GST - JCF1 ( RBD ) . The amount of GTP - Rab8 bound to GFT - JCF1 ( RBD ) was analyzed by western blot with Rab8 antibody . ( Lower panel ) Intensity of the bands was quantified by Image J software . The amount of GTP - Rab8 were normalized to the control level . Bar graph represents mean ± SD ( n = 3 experiments ) . * P < 0 . 05 , Student ' s t - test . ( D ) Cells were transfected and cell lysates were incubated with purified GST - JCF1 ( RBD ) fusion protein . The amount of GTP - Rab8 bound to GFT - JCF1 ( RBD ) was analyzed by Western blot with Rab8 antibody . ( Lower panel ) Cells were transfected and cell lysates were incubated with purified GST - protein . The amount of GTP - Rab8 bound to GFT - JCF1 ( RBD ) was analyzed by Western blot with Rab8 antibody .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) RPE1 cells were transfected with siRNAs as indicated, followed by transfection with wild-type pGFP-Rab8a (WT-Rab8), constitutively active pGFP-Rab8a (Q67L) (CA-Rab8), or DN Rab8 dominant-negative pGFP Rab8a (T22N), incubated in serum-starved media for 12 h, and immunostained for ARL13B (Red), GFP-Rab8 (green), and DAPI (blue). Scale bar, 10 μm.(B) Quantification of the percentage of ciliated cells Data represent mean ± SD (n=3 experiments), 200 GFP-positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.(C) (Upper panel) Cells were transfected as indicated and the cell lysates were incubated with purified proteins, including GST or GST-JCF1 (RBD). The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by western blot with Rab8 antibody. (Lower panel) Intensity of the bands was quantified by Image J software. The amount of GTP-Rab8 were normalized to the control level. Bar graph represents mean ± SD (n=3 experiments). *P < 0.05, Student's t-test.(D) Cells were transfected and cell lysates were incubated with purified GST-JCF1 (RBD) fusion protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by Western blot with Rab8 antibody. (Lower panel) Cells were transfected and cell lysates were incubated with purified GST- protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by Western blot with Rab8 antibody.


Figure 5 ( A ) RPE1 cells were transfected with siRNAs as indicated , followed by 24 h incubation in serum - starved media . Cells were then and subjected to fractionation , and Western blot for IFT20 , the Golgi marker GM130 , membrane marker UBXD8 , and nuclear marker CREB . ( B ) Efficiency of IFT20 knockdown by western blot . ( C ) Cells were transfected with siRNAs as indicated , followed by incubation in serum - starved media for 24 h , and immunostained for ARL13B ( red ) . Scale bar , 10 μm . The bar graph represents the quantification of the percentage of ciliated cells . Data represent mean ± SD ( n = 3 experiments ) , 250 cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test . ( D ) Cells were transfected and immunostained for Rab8 and γ - tubulin , followed by quantification of the percentage of cells with Rab8 localized to the centriole . Data represent average ( n = 2 experiments ) . ( E ) Cells were transfected with siRNAs as indicated , followed by transfection with Flag - IFT20 , incubation in serum - starvation media for 12 h , and immunostained for ARL13B . Representative fluorescent images of Flag - IFT20 ( green ) , ARL13B ( red ) , and DAPI ( blue ) are shown . Scale bar , 10 μm . ( F ) Quantification of the percentage of ciliated cells with both the Flag - IFT20 and ARL13B localized in the cilium . Data represent mean ± SD ( n = 3 experiments ) , 150 Flag positive cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test . ( G ) Cells were transfected with siRNAs as indicated , followed by further transfection with CA - Rab8 , incubation in serum - starvation media for 12 h , and immunostained for acetylated - tubulin . Representative fluorescent images of GFP - Rab8 Q67L ( green ) , acetylated - tubulin ( red ) , and DNA ( blue ) are shown . Scale bar , 10 μm . ( H ) Quantification of the percentage of GFP - positive ciliated cells ( only those cilia having both GFP - Rab8 and acetylated - tubulin on cilium were considered for quantification ) . Data represent mean ± SD ( n = 3 experiments ) , 200 GFP positive cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) RPE1 cells were transfected with siRNAs as indicated, followed by 24 h incubation in serum-starved media. Cells were then and subjected to fractionation, and Western blot for IFT20, the Golgi marker GM130, membrane marker UBXD8, and nuclear marker CREB.(B) Efficiency of IFT20 knockdown by western blot.(C) Cells were transfected with siRNAs as indicated, followed by incubation in serum-starved media for 24 h, and immunostained for ARL13B (red). Scale bar, 10 μm. The bar graph represents the quantification of the percentage of ciliated cells. Data represent mean ± SD (n=3 experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.(D) Cells were transfected and immunostained for Rab8 and γ-tubulin, followed by quantification of the percentage of cells with Rab8 localized to the centriole. Data represent average (n=2 experiments).(E) Cells were transfected with siRNAs as indicated, followed by transfection with Flag-IFT20, incubation in serum-starvation media for 12 h, and immunostained for ARL13B. Representative fluorescent images of Flag-IFT20 (green), ARL13B (red), and DAPI (blue) are shown. Scale bar, 10 μm. (F) Quantification of the percentage of ciliated cells with both the Flag-IFT20 and ARL13B localized in the cilium. Data represent mean ± SD (n=3 experiments), 150 Flag positive cells were scored per condition per experiment, *P < 0.05, Student's t-test. (G) Cells were transfected with siRNAs as indicated, followed by further transfection with CA-Rab8, incubation in serum-starvation media for 12 h, and immunostained for acetylated-tubulin. Representative fluorescent images of GFP-Rab8 Q67L (green), acetylated-tubulin (red), and DNA (blue) are shown. Scale bar, 10 μm. (H) Quantification of the percentage of GFP -positive ciliated cells (only those cilia having both GFP-Rab8 and acetylated-tubulin on cilium were considered for quantification). Data represent mean ± SD (n = 3 experiments), 200 GFP positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.


Figure 6 ( A ) Efficiency of Rab8a depletion confirmed by Western blot in RPE1 cells . ( B ) RPE1 cells were transfected by siRNAs as indicated , followed by incubation in serum - starvation media for 24 h , and immunostained for ARL13B ( red ) and γ - tubulin ( green ) . Scale bar , 10 μm . ( C ) Quantification of the percentage of ciliated cells Data represent mean ± SD ( n = 3 experiments ) , 250 GFP positive cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test . ( D ) Cells were transfected by siRNa as indicated followed by incubation in serum - starvation media for 24 h , and immunostained for EHD1 ( red ) and γ - tubulin ( green ) . Scale bar , 10 μm . ( E ) Quantification of the percentage of cells with EHD1 in cilium or in the distal end of basal body as shown in ( D ) . Data represent mean ± SD ( n = 3 experiments ) , 150 cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test . ( F ) Cells were transfected by siRNAs as indicated , followed by incubation in serum - starvation media for 24 h , and immunostained for IFT20 ( red ) and γ - tubulin ( green ) . Scale bar , 10 μm . ( G ) Quantification of the percentage of IFT20 fluorescent intensity at the centriole shown in ( F ) . Data represent mean ± SD ( n = 3 experiments ) , 150 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test . ( H , I , J ) Cells were transfected by siRNAs as indicated , followed by transfection with GFP - Rab8 WT , GFP - Rab8 Q67L , or GFP - Rab8 T22N , and further incubated in serum - starvation media for 12 h . Cell lysate was subjected to immunoprecipitation with anti - GFP antibody , followed by Western blot with antibody against GFP .

O O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Efficiency of Rab8a depletion confirmed by Western blot in RPE1 cells.(B) RPE1 cells were transfected by siRNAs as indicated, followed by incubation in serum-starvation media for 24 h, and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm.(C) Quantification of the percentage of ciliated cells Data represent mean ± SD (n = 3 experiments), 250 GFP positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.(D) Cells were transfected by siRNa as indicated followed by incubation in serum-starvation media for 24 h, and immunostained for EHD1 (red) and γ-tubulin (green). Scale bar, 10 μm. (E) Quantification of the percentage of cells with EHD1 in cilium or in the distal end of basal body as shown in (D). Data represent mean ± SD (n=3 experiments), 150 cells were scored per condition per experiment, *P < 0.05, Student's t-test. (F) Cells were transfected by siRNAs as indicated, followed by incubation in serum-starvation media for 24 h, and immunostained for IFT20 (red) and γ-tubulin (green). Scale bar, 10 μm. (G) Quantification of the percentage of IFT20 fluorescent intensity at the centriole shown in (F). Data represent mean ± SD (n=3 experiments), 150 were scored per condition per experiment, *P < 0.05, Student's t-test. (H, I, J) Cells were transfected by siRNAs as indicated, followed by transfection with GFP-Rab8 WT, GFP-Rab8 Q67L, or GFP-Rab8 T22N, and further incubated in serum-starvation media for 12 h. Cell lysate was subjected to immunoprecipitation with anti-GFP antibody, followed by Western blot with antibody against GFP.


Figure 7 ( A ) RPE1 cells were transfected as indicated , followed by incubation in serum - starvation media in the presence or absence of cholesterol / MCD for 24 h , and then immunostained for Rab8 ( red ) and γ - tubulin ( green ) . Scale bar , 10 μm . Representative magnified images are shown from cells labeled with white asterisks . ( B ) Quantification of the percentage of cells with Rab8 localized to the centriole Data represent mean ± SD ( n = 3 experiments ) , 200 cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test . ( C ) ( Upper panel ) Cells were transfected and cell lysate was incubated with purified GST - JCF1 ( RBD ) fusion protein . The amount of GTP - Rab8 bound to GFT - JCF1 ( RBD ) was analyzed by western blot with Rab8 antibody . ( Lower panel ) The intensity of bands was quantified by Image J software . The amount of GTP - Rab8 were normalized to the control level . The bar graph represents mean ± SD ( n = 3 experiments ) . * P < 0 . 05 , Student ' s t - test . ( D ) Cells were transfected and immunostained for IFT20 ( red ) and γ - tubulin ( green ) . Scale bar , 10 μm . ( E ) Quantification of IFT20 intensity at the centriole Data represent mean ± SD ( n = 3 experiments ) , 150 cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) RPE1 cells were transfected as indicated, followed by incubation in serum-starvation media in the presence or absence of cholesterol/MCD for 24 h, and then immunostained for Rab8 (red) and γ-tubulin (green). Scale bar, 10 μm. Representative magnified images are shown from cells labeled with white asterisks.(B) Quantification of the percentage of cells with Rab8 localized to the centriole Data represent mean ± SD (n=3 experiments), 200 cells were scored per condition per experiment, *P < 0.05, Student's t-test.(C) (Upper panel) Cells were transfected and cell lysate was incubated with purified GST-JCF1 (RBD) fusion protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by western blot with Rab8 antibody. (Lower panel) The intensity of bands was quantified by Image J software. The amount of GTP-Rab8 were normalized to the control level. The bar graph represents mean ± SD (n=3 experiments). *P < 0.05, Student's t-test.(D) Cells were transfected and immunostained for IFT20 (red) and γ-tubulin (green). Scale bar, 10 μm.(E) Quantification of IFT20 intensity at the centriole Data represent mean ± SD (n=3 experiments), 150 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


Figure 1 ( D - E ) Measurements of ciliary cAMP levels using cilia - Pink Flamindo . ( D ) Exemplary images are shown for cilia ( cilia - bPAC or cyto - bPAC cells ) expressing the cilia - Pink Flamindo biosensor after light stimulation . Scale bars are indicated ; cAMP levels are color - coded using a high - low look - up table . ( E ) Quantification of data exemplified in ( D ) . The cAMP level is determined as the mean ciliary fluorescence intensity during the first 60 s after light stimulation ( measurement interval 10 s ) ; p value for a Kolmogorov - Smirnov test is indicated . Each data point represents an individual cilium . Bars display median and interquartile range ; cilia - bPAC : 40 cilia ( biological replicates ) from n = 14 independent experiments ; cyto - bPAC : 31 cilia ( biological replicates ) from n = 31 independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(D-E) Measurements of ciliary cAMP levels using cilia-Pink Flamindo. (D) Exemplary images are shown for cilia (cilia-bPAC or cyto-bPAC cells) expressing the cilia-Pink Flamindo biosensor after light stimulation. Scale bars are indicated; cAMP levels are color-coded using a high-low look-up table. (E) Quantification of data exemplified in (D). The cAMP level is determined as the mean ciliary fluorescence intensity during the first 60 s after light stimulation (measurement interval 10 s); p value for a Kolmogorov-Smirnov test is indicated. Each data point represents an individual cilium. Bars display median and interquartile range; cilia-bPAC: 40 cilia (biological replicates) from n = 14 independent experiments; cyto-bPAC: 31 cilia (biological replicates) from n = 31 independent experiments.


Figure 2 ( A ) Wild - type ( WT ) mIMCD - 3 cells cultured in a 3D matrix during continuous exposure to DMSO ( control ) or 10 µM of Forskolin . Exemplary images are shown ( n = 5 biological replicates ) . Quantification of cyst number and area is shown on the right Data are shown as mean ± S . D . , each datapoint corresponds to an independent experiment , p values calculated using an unpaired , two - sided Student ' s t - test are indicated . ( D ) mIMCD - 3 cells stably expressing cilia - bPAC or cyto - bPAC , cultured in a 3D matrix in the dark , during light exposure ( 1 h light / 1 h dark , 9 days , 465 nm , 38 . 8 µW / cm² ) or in the dark and incubated with 10 µM of Forskolin . Exemplary images are shown ( n > 3 ) . ( E ) Quantification of the data exemplified in ( D ) . Data are shown as mean ± S . D . , each datapoint corresponds to an independent experiment ; p values calculated using an unpaired , two - sided Student ' s t - test are indicated .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Wild-type (WT) mIMCD-3 cells cultured in a 3D matrix during continuous exposure to DMSO (control) or 10 µM of Forskolin. Exemplary images are shown (n = 5 biological replicates). Quantification of cyst number and area is shown on the right Data are shown as mean ± S.D., each datapoint corresponds to an independent experiment, p values calculated using an unpaired, two-sided Student's t-test are indicated.(D) mIMCD-3 cells stably expressing cilia-bPAC or cyto-bPAC, cultured in a 3D matrix in the dark, during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²) or in the dark and incubated with 10 µM of Forskolin. Exemplary images are shown (n > 3). (E) Quantification of the data exemplified in (D). Data are shown as mean ± S.D., each datapoint corresponds to an independent experiment; p values calculated using an unpaired, two-sided Student's t-test are indicated.


Figure 3 ( D ) Immunoblotting of lysates from mIMCD - 3 wild - type ( WT ) cells and mIMCD - 3 cells stably expressing cilia - bPAC for S6 ribosomal protein ( S6RP ) phosphorylation at Ser235 / 236 ( pS6RP ) . Levels of total S6RP protein ( S6RP ) and beta - Tubulin have been determined as controls . Cilia - bPAC cells were treated with DMSO ( control ) or with 10 nM of Rapamycin . All cells were stimulated with light ( 1 h light / 1 h dark , 48 h , 465 nm , 38 . 8 µW / cm² ) ( E ) Quantification of the pS6RP / S6RP ratio . Data are shown as mean ± S . D . , n > 3 ; p values were calculated using a paired , two - sided Student ' s t - test . Each data point shows an independent experiment ( G ) mIMCD - 3 cells stably expressing cilia - bPAC , cultured in a 3D matrix in the dark , during light exposure ( 1 h light / 1 h dark , 9 days , 465 nm , 38 . 8 µW / cm² ) and incubation with 10 nM rapamycin . Light exposure started 2 d later than incubation with 10 nM rapamycin . Exemplary images are shown ( n = 3 ) ( H ) Quantification of the data exemplified in ( G ) . Data are shown as mean ± S . D . , each datapoint corresponds to an independent experiment ; p values calculated using a paired , two - sided Student ' s t - test are

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(D) Immunoblotting of lysates from mIMCD-3 wild-type (WT) cells and mIMCD-3 cells stably expressing cilia-bPAC for S6 ribosomal protein (S6RP) phosphorylation at Ser235/236 (pS6RP). Levels of total S6RP protein (S6RP) and beta-Tubulin have been determined as controls. Cilia-bPAC cells were treated with DMSO (control) or with 10 nM of Rapamycin. All cells were stimulated with light (1 h light/1 h dark, 48 h, 465 nm, 38.8 µW/cm²) (E) Quantification of the pS6RP/S6RP ratio. Data are shown as mean ± S.D., n > 3; p values were calculated using a paired, two-sided Student's t-test. Each data point shows an independent experiment(G) mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix in the dark, during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²) and incubation with 10 nM rapamycin. Light exposure started 2 d later than incubation with 10 nM rapamycin. Exemplary images are shown (n = 3) (H) Quantification of the data exemplified in (G). Data are shown as mean ± S.D., each datapoint corresponds to an independent experiment; p values calculated using a paired, two-sided Student's t-test are indicated


Figure 4 ( A ) mIMCD - 3 cells stably expressing cilia - bPAC , cultured in a 3D matrix in the dark or during light exposure ( 1 h light / 1 h dark , 9 days , 465 nm , 38 . 8 µW / cm² ) and incubated with DMSO ( control ) , 250 µM IBMX , a ubiquitous PDE inhibitor , or 10 µM Rolipram , a PDE4 - specific inhibitor . Exemplary images are shown ( n = 3 ) . Scale bars are indicated . ( B ) Quantification of data exemplified in ( A ) . Data are shown as mean ± S . D . , p values calculated using an unpaired , two - sided Student ' s t - test are indicated . Data points show individual experiments . ( C ) mIMCD - 3 cells stably expressing cyto - bPAC , cultured in a 3D matrix during light exposure ( 1 h light / 1 h dark , 9 days , 465 nm , 38 . 8 µW / cm² , 1 day after pharmacological stimulus ) and incubation with DMSO or 10 µM rolipram . Exemplary images are shown ( n = 4 - 5 ) . Scale bars are indicated . ( D ) Quantification of data exemplified in ( C ) Data are shown as mean ± S . D . , p values calculated using an unpaired Student ' s t - test are indicated . Data points show individual experiments . ( F ) Analysis of Pde4 isoform expression ( long and short isoforms ) in wild - type mIMCD - 3 cells , revealed by immunoblotting against PDE4A , PDE4B , PDE4D , and PDE4D . ( H ) mIMCD - 3 cells stably expressing cilia - bPAC , cultured in a 3D matrix during light exposure ( 1 h light / 1 h dark , 9 days , 465 nm , 38 . 8 µW / cm² , 1 day after pharmacological stimulus ) and incubation with DMSO ( 0 µM ) , 3 µM , or 10 µM of the Pde4 - long - isoform activator MR - L8 . Exemplary images are shown ( n = 3 ) . Scale bars are indicated . ( I ) Quantification of data exemplified in ( H ) . Data are shown as mean ± S . D . , p values calculated using a paired , two - sided Student ' s t - test are indicated . Data have been normalized to the Light , 0 µM MR - L8 condition ( Set to 100 % ) within each experiment . Data points show individual experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix in the dark or during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²) and incubated with DMSO (control), 250 µM IBMX, a ubiquitous PDE inhibitor, or 10 µM Rolipram, a PDE4-specific inhibitor. Exemplary images are shown (n = 3). Scale bars are indicated. (B) Quantification of data exemplified in (A). Data are shown as mean ± S.D., p values calculated using an unpaired, two-sided Student's t-test are indicated. Data points show individual experiments.(C) mIMCD-3 cells stably expressing cyto-bPAC, cultured in a 3D matrix during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², 1 day after pharmacological stimulus) and incubation with DMSO or 10 µM rolipram. Exemplary images are shown (n = 4-5). Scale bars are indicated. (D) Quantification of data exemplified in (C) Data are shown as mean ± S.D., p values calculated using an unpaired Student's t-test are indicated. Data points show individual experiments.(F) Analysis of Pde4 isoform expression (long and short isoforms) in wild-type mIMCD-3 cells, revealed by immunoblotting against PDE4A, PDE4B, PDE4D, and PDE4D.(H) mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², 1 day after pharmacological stimulus) and incubation with DMSO (0 µM), 3 µM, or 10 µM of the Pde4-long-isoform activator MR-L8. Exemplary images are shown (n = 3). Scale bars are indicated. (I) Quantification of data exemplified in (H). Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated. Data have been normalized to the Light, 0 µM MR-L8 condition (Set to 100%) within each experiment. Data points show individual experiments.


Figure 5 ( D ) 3D culture of mIMCD - 3 WT cells continuously exposed to acetone ( control ) or 100 nM PGE2 . Exemplary images are shown ( n = 3 biological replicates ) . ( E ) Quantification of images exemplified in ( D ) . Data are shown as mean ± S . D . , p values calculated using an unpaired , two - sided Student ' s t - test are indicated . Data points show individual biological replicates . ( F ) Quantification of the cyst area in PGE2 - stimulated mIMCD - 3 WT cells in the presence of the PDE4 long isoform - specific activator MR - L8 . Data were normalized to PGE2 - and control - treated cells and are shown as mean ± S . D . p values calculated using a one - sample t - test compared to 100 % are indicated , n = 4 biological replicates . ( G ) 3D culture of mIMCD - 3 WT cells continuously exposed to acetone ( control ) or 100 nM PGE2 , and DMSO ( control ) or 10 µM of the EP4 inhibitor L161 , 982 . PGE2 treatment started 2 d after seeding . Exemplary images are shown ( n = 3 ) . Scale bars are indicated . ( H ) Quantification for images exemplified in ( G ) , including addition of 3 µM AH6809 , an EP2 inhibitor Data are shown as mean ± S . D . , p values calculated using a paired , two - sided Student ' s t - test are indicated ( p value of n / a indicates that the test is not applicable because both conditions showed no variance ) . Data points show individual experiments and have been normalized to addition of 100 nM PGE2 ( set to 100 % ) within each experiment . ( I ) 3D culture of mIMCD - 3 cells stably expressing cilia - bPAC , incubated with DMSO ( control ) or the EP4 - inhibitor L161 , 982 in the dark or during light exposure ( 1 light / 1 h dark , 9 days , 465 nm , 38 . 8 µW / cm² , started 2 d after pre - incubation with DMSO or L161 , 982 ) . Exemplary images are shown ( n = 3 ) . ( J ) Quantification of images exemplified in ( I ) . Data are shown as mean ± S . D . , p values calculated using a paired , two - sided Student ' s t - test are indicated . Data points show individual experiments and have been normalized to the light / DMSO condition ( Set to 100 % ) within each experiment .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(D) 3D culture of mIMCD-3 WT cells continuously exposed to acetone (control) or 100 nM PGE2. Exemplary images are shown (n = 3 biological replicates). (E) Quantification of images exemplified in (D). Data are shown as mean ± S.D., p values calculated using an unpaired, two-sided Student's t-test are indicated. Data points show individual biological replicates. (F) Quantification of the cyst area in PGE2-stimulated mIMCD-3 WT cells in the presence of the PDE4 long isoform-specific activator MR-L8. Data were normalized to PGE2- and control-treated cells and are shown as mean ± S.D. p values calculated using a one-sample t-test compared to 100% are indicated, n = 4 biological replicates.(G) 3D culture of mIMCD-3 WT cells continuously exposed to acetone (control) or 100 nM PGE2, and DMSO (control) or 10 µM of the EP4 inhibitor L161,982. PGE2 treatment started 2 d after seeding. Exemplary images are shown (n = 3). Scale bars are indicated. (H) Quantification for images exemplified in (G), including addition of 3 µM AH6809, an EP2 inhibitor Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated (p value of n/a indicates that the test is not applicable because both conditions showed no variance). Data points show individual experiments and have been normalized to addition of 100 nM PGE2 (set to 100%) within each experiment.(I) 3D culture of mIMCD-3 cells stably expressing cilia-bPAC, incubated with DMSO (control) or the EP4-inhibitor L161,982 in the dark or during light exposure (1 light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², started 2 d after pre-incubation with DMSO or L161,982). Exemplary images are shown (n = 3). (J) Quantification of images exemplified in (I). Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated. Data points show individual experiments and have been normalized to the light/DMSO condition (Set to 100%) within each experiment.


Figure 6 ( F ) 3D culture of mIMCD - 3 cells stably expressing cilia - bPAC , incubated with DMSO ( control ) or 20 µM Celecoxib , a COX - 2 - inhibitor , in the dark or during light exposure ( 1 light / 1 h dark , 9 days , 465 nm , 38 . 8 µW / cm² , started 2 d after pre - incubation with DMSO or Celecoxib ) . Exemplary images are shown ( n = 3 ) . Scale bars are indicated . ( G ) Quantification of images exemplified in ( F ) and from 3D cultures incubated with 1 μM celecoxib . Data are shown as mean ± S . D . , p values calculated using a paired , two - sided Student ' s t - test are indicated . Data points show individual experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(F) 3D culture of mIMCD-3 cells stably expressing cilia-bPAC, incubated with DMSO (control) or 20 µM Celecoxib, a COX-2-inhibitor, in the dark or during light exposure (1 light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², started 2 d after pre-incubation with DMSO or Celecoxib). Exemplary images are shown (n = 3). Scale bars are indicated. (G) Quantification of images exemplified in (F) and from 3D cultures incubated with 1 μM celecoxib. Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated. Data points show individual experiments.


Figure 7 ( A ) Wild - type ( WT ) , cilia - bPAC , or cyto - bPAC ( magenta ) mIMCD - 3 cells were stimulated by light ( for 1 h prior to fixation , 465 nm , 38 . 8 µW / cm² ) , and treated with DMSO ( control ) or 50 µM Ciliobrevin - D , a dynein - inhibitor . Cells were labeled with DAPI ( blue ) to label the DNA , an ARL13B antibody ( white ) to label cilia , and a phospho - specific ( Ser133 ) CREB antibody ( pCREB , green ) . Arrows indicate the direction and the length of the shift of the respective fluorescence channel . The box indicates the position of the magnified view shown on the right of each panel . B ) Quantification of the ciliary pCREB signal in cilia - bPAC and cyto - bPAC cells . Data are shown as mean ± S . D . , n > 3 . Each data point represents an independent experiment and corresponds to the median of > 73 cilia ( biological replicates ) ; p values for an unpaired , two - sided Student ' s t - test with Welch ' s correction are indicated . ( C ) mIMCD - 3 Ift27 - / - cells with or without stable cilia - bPAC expression ( magenta ) were stimulated with light ( for 1 h prior to fixation , 465 nm , 38 . 8 µW / cm² ) . Cells were labeled with DAPI ( blue ) to label the DNA , an ARL13B antibody ( white ) to label cilia , and a phospho - specific ( Ser133 ) CREB antibody ( pCREB , green ) . Arrows indicate the direction and the length of the shift of the respective fluorescence channel . The box indicates the position of the magnified view shown on the right of each panel . ( D ) Quantification of the ciliary pCREB signal . Data are shown as mean ± S . D . , n > 3 . Each data point represents the median of > 90 cilia from the same experiment ; p values are indicated for an unpaired , two - sided Student ' s t - test with Welch ' s correction .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Wild-type (WT), cilia-bPAC, or cyto-bPAC (magenta) mIMCD-3 cells were stimulated by light (for 1 h prior to fixation, 465 nm, 38.8 µW/cm²), and treated with DMSO (control) or 50 µM Ciliobrevin-D, a dynein-inhibitor. Cells were labeled with DAPI (blue) to label the DNA, an ARL13B antibody (white) to label cilia, and a phospho-specific (Ser133) CREB antibody (pCREB, green). Arrows indicate the direction and the length of the shift of the respective fluorescence channel. The box indicates the position of the magnified view shown on the right of each panel. B) Quantification of the ciliary pCREB signal in cilia-bPAC and cyto-bPAC cells. Data are shown as mean ± S.D., n > 3. Each data point represents an independent experiment and corresponds to the median of > 73 cilia (biological replicates); p values for an unpaired, two-sided Student's t-test with Welch's correction are indicated.(C) mIMCD-3 Ift27-/- cells with or without stable cilia-bPAC expression (magenta) were stimulated with light (for 1 h prior to fixation, 465 nm, 38.8 µW/cm²). Cells were labeled with DAPI (blue) to label the DNA, an ARL13B antibody (white) to label cilia, and a phospho-specific (Ser133) CREB antibody (pCREB, green). Arrows indicate the direction and the length of the shift of the respective fluorescence channel. The box indicates the position of the magnified view shown on the right of each panel. (D) Quantification of the ciliary pCREB signal. Data are shown as mean ± S.D., n > 3. Each data point represents the median of > 90 cilia from the same experiment; p values are indicated for an unpaired, two-sided Student's t-test with Welch's correction.


Figure 2 ( B ) Structures of S . cerevisiae Hrr251 - 394 K38R bound to CK1 - 7 ( Chijiwa et al , 1989 ; Xu et al , 1996 ) and C . glabrata Hrr251 - 403 K38R bound to ADP , with domains colored as in ( A ) . Bound PO4 - / SO4 - ions are shown as spheres . See Figure EV2A for additional C . glabrataHrr25 structures , and Figure EV3A for SO4 - ion electron density . ( C ) Overlay of the kinase domain of C . glabrataHrr25 ( Apo form , crystallized with 1 . 2 M PO4 - ) with ratCK1δ crystallized in the presence of tungstate ions ( WO4 ; PDB ID 1CKJ , ( Longenecker et al , 1996 ) ) See Figure EV2B for detailed views of ion binding . ( D ) Stereo view of CK1 - 7 binding to S . cerevisiae Hrr25 . Bound drug is positioned identically to a previous structure of S . pombe Cki1 bound to CK1 - 7 ( Xu et al , 1996 ) . ( E ) Stereo view of ADP binding to C . glabrata Hrr25 ( formate structure ; SO4 - structure is equivalent , but the GxGxxG motif is disordered in that structure ) . All active - site residues shown in panels ( D ) and ( E ) are conserved between S . cerevisiae and C . glabrata Hrr25 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) Structures of S. cerevisiae Hrr251-394 K38R bound to CK1-7 (Chijiwa et al, 1989; Xu et al, 1996) and C. glabrata Hrr251-403 K38R bound to ADP, with domains colored as in (A). Bound PO4-/SO4- ions are shown as spheres. See Figure EV2A for additional C. glabrataHrr25 structures, and Figure EV3A for SO4- ion electron density.(C) Overlay of the kinase domain of C. glabrataHrr25 (Apo form, crystallized with 1.2 M PO4-) with ratCK1δ crystallized in the presence of tungstate ions (WO4; PDB ID 1CKJ, (Longenecker et al, 1996)) See Figure EV2B for detailed views of ion binding.(D) Stereo view of CK1-7 binding to S. cerevisiae Hrr25. Bound drug is positioned identically to a previous structure of S. pombe Cki1 bound to CK1-7 (Xu et al, 1996).(E) Stereo view of ADP binding to C. glabrata Hrr25 (formate structure; SO4- structure is equivalent, but the GxGxxG motif is disordered in that structure). All active-site residues shown in panels (D) and (E) are conserved between S. cerevisiae and C. glabrata Hrr25.


Figure 3 ( A ) Two views of C . glabrata Hrr25 ( Apo form ) with domains colored as in Figure 1 , showing bound PO4 - ions and the previously - identified Mam1 - binding residues His25 and Glu34 ( Petronczki et al , 2006 ) . ( B ) Two views , roughly equivalent to the views in ( A ) , of the Hrr25 central domain , colored as a rainbow according to the schematic at top . Dotted lines in the schematic indicate disordered loops . See Figure EV1A for sequence alignment of this domain .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Two views of C. glabrata Hrr25 (Apo form) with domains colored as in Figure 1, showing bound PO4- ions and the previously-identified Mam1-binding residues His25 and Glu34 (Petronczki et al, 2006).(B) Two views, roughly equivalent to the views in (A), of the Hrr25 central domain, colored as a rainbow according to the schematic at top. Dotted lines in the schematic indicate disordered loops. See Figure EV1A for sequence alignment of this domain.


Figure 4 ( B ) Overall structure of the S . cerevisiae Hrr251 - 394 K38R : Mam187 - 191 complex , colored as in ( A ) . View is equivalent to Figure 2B . ( C ) Close - up view of Mam187 - 191 , colored as in the schematic in ( A ) . ( D ) Close - up of the variant zinc knuckle motif of Mam1 , with zinc anomalous difference electron density shown at 3 σ ( gray ) and 10 σ ( red ) . ( E ) Geometry of zinc binding in Mam1 . Zn - S bonds and the Zn - N bond were restrained to ~ 2 . 3 Å and ~ 2 . 0 Å , respectively , during refinement .

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Figure 4(B) Overall structure of the S. cerevisiae Hrr251-394 K38R:Mam187-191 complex, colored as in (A). View is equivalent to Figure 2B.(C) Close-up view of Mam187-191, colored as in the schematic in (A). (D) Close-up of the variant zinc knuckle motif of Mam1, with zinc anomalous difference electron density shown at 3 σ (gray) and 10 σ (red).(E) Geometry of zinc binding in Mam1. Zn-S bonds and the Zn-N bond were restrained to ~2.3 Å and ~2.0 Å, respectively, during refinement.


Figure 5 ( A ) The Hrr25 : Mam1 complex with Mam1 shown as orange ribbons , and Hrr25 shown as a white surface with Mam1 - binding interface # 1 shown in green and # 2 in magenta . ( B ) Stereo view of interface # 1 , which involves hydrophobic interactions by Mam1 L92 , L97 , L100 , and I104 , and a hydrogen - bond network between Mam1 Q101 / E108 / R131 and Hrr25 residues 12 - 15 . ( C ) Stereo view of interface # 2 . Hrr25 residues H25 and E34 participate in a buried hydrogen - bond network with Mam1 residues R149 and Y158 , and are surrounded by hydrophobic residues on both proteins . For clarity , Mam1 helix 4 , on which R149 , Y153 , and Y158 are located , is not shown . ( D ) Pulldown assay with in vitro - translated Mam187 - 191 ( wild - type and alanine mutants ) and Hrr251 - 394 K38R . Mam1 mutations also disrupt Hrr25 binding when the two are co - expressed in E . coli ( Figure EV6B ) . Bottom : Sporeviability of MAM1 mutant strains ( see Table EV2 for strains ; strains tested are KC549 , KC560 , KC566 , KC552 , KC554 , KC556 , and KC558 ; 46 - 48 tetrads were dissected for each strain ) . " - - " indicates that this mutation was not tested for sporeviability .

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Figure 5(A) The Hrr25:Mam1 complex with Mam1 shown as orange ribbons, and Hrr25 shown as a white surface with Mam1-binding interface #1 shown in green and #2 in magenta.(B) Stereo view of interface #1, which involves hydrophobic interactions by Mam1 L92, L97, L100, and I104, and a hydrogen-bond network between Mam1 Q101/E108/R131 and Hrr25 residues 12-15. (C) Stereo view of interface #2. Hrr25 residues H25 and E34 participate in a buried hydrogen-bond network with Mam1 residues R149 and Y158, and are surrounded by hydrophobic residues on both proteins. For clarity, Mam1 helix 4, on which R149, Y153, and Y158 are located, is not shown.(D) Pulldown assay with in vitro-translated Mam187-191 (wild-type and alanine mutants) and Hrr251-394 K38R. Mam1 mutations also disrupt Hrr25 binding when the two are co-expressed in E. coli (Figure EV6B). Bottom: Sporeviability of MAM1 mutant strains (see Table EV2 for strains; strains tested are KC549, KC560, KC566, KC552, KC554, KC556, and KC558; 46-48 tetrads were dissected for each strain). "--" indicates that this mutation was not tested for sporeviability.


Figure 6 ( A - C ) Ribbon views of the S . cerevisiae Hrr251 - 394 : Mam187 - 191 structure ( form 1 ) , CK1 - 7 bound S . cerevisiae Hrr251 - 394 , and ADP - bound C . glabrata Hrr251 - 403 ( formate condition ) , with Hrr25 colored according to main - chain B - factor from low ( purple ) to cyan ( high ) . For each , coloring is normalized to correspond to the average of the 20 lowest ( purple ) or highest ( cyan ) main - chain B - factors in Hrr25 . Mam1 ( panel A ) is colored in light gray . ( D ) Overall main - chain B - factors for the N - lobe ( residues 1 - 85 ) of four structures of Hrr25 ( both C . glabrata and S . cerevisiae ) and the three crystallographically - unique views of the S . cerevisiae Hrr25 : Mam1 complex . Values are normalized to the average main - chain B - factor for the entire Hrr25 chain ( dotted line at 1 . 0 ) . P - value = 0 . 004 ( student ' s T - test ) . See Figure EV4 for residue - by - residue B - factor plots for each structure .

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Figure 6(A-C) Ribbon views of the S. cerevisiae Hrr251-394:Mam187-191 structure (form 1), CK1-7 bound S. cerevisiae Hrr251-394, and ADP-bound C. glabrata Hrr251-403 (formate condition), with Hrr25 colored according to main-chain B-factor from low (purple) to cyan (high). For each, coloring is normalized to correspond to the average of the 20 lowest (purple) or highest (cyan) main-chain B-factors in Hrr25. Mam1 (panel A) is colored in light gray.(D) Overall main-chain B-factors for the N-lobe (residues 1-85) of four structures of Hrr25 (both C. glabrata and S. cerevisiae) and the three crystallographically-unique views of the S. cerevisiae Hrr25:Mam1 complex. Values are normalized to the average main-chain B-factor for the entire Hrr25 chain (dotted line at 1.0). P-value=0.004 (student's T-test). See Figure EV4 for residue-by-residue B-factor plots for each structure.


Figure 7 ( A ) ADP - Glo ATPase assay showing stimulation of S . cerevisiae Hrr251 - 394 ( blue circles ) , Hrr251 - 394 K38R ( red diamonds ) , Hrr25 full - length : Mam187 - 191 ( black triangles ) , Hrr251 - 394 : Mam187 - 191 ( orange squares ) , and Hrr251 - 394 : Mam187 - 191 R131A ( green triangles ) by bovine casein . See Figure EV6A for SDS - PAGE analysis of purified proteins . ( B ) Stimulation of Hrr251 - 394 , Hrr251 - 394 : Mam187 - 191 , and Hrr251 - 394 : Mam187 - 191 R131A by ATP , measured using an enzyme - coupled ATPase assay . ( C ) ADP - Glo assay showing the effect of added CK1 - 7 on Hrr251 - 394 , Hrr251 - 394 : Mam187 - 191 , and Hrr251 - 394 : Mam187 - 191 R131A . ( D ) Km , Vmax , and IC50 values for tested enzymes . Vmax was calculated from curves in ( A ) at 100 M ATP .

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Figure 7(A) ADP-Glo ATPase assay showing stimulation of S. cerevisiae Hrr251-394 (blue circles), Hrr251-394 K38R (red diamonds), Hrr25 full-length:Mam187-191 (black triangles), Hrr251-394:Mam187-191 (orange squares), and Hrr251-394:Mam187-191 R131A (green triangles) by bovine casein. See Figure EV6A for SDS-PAGE analysis of purified proteins.(B) Stimulation of Hrr251-394, Hrr251-394:Mam187-191, and Hrr251-394:Mam187-191 R131A by ATP, measured using an enzyme-coupled ATPase assay.(C) ADP-Glo assay showing the effect of added CK1-7 on Hrr251-394, Hrr251-394:Mam187-191, and Hrr251-394:Mam187-191 R131A.(D) Km, Vmax, and IC50 values for tested enzymes. Vmax was calculated from curves in (A) at 100 M ATP.


Figure 8 ( A ) Phos - tag gels showing phosphorylation of purified S . cerevisiae Mtw1 complex by Hrr251 - 394 and Hrr251 - 394 : Mam187 - 191 . Asterisks denote N - terminal proteolytic cleavage products of Dsn1 , with the red asterisk denoting a previously - characterized product lacking the first 171 residues ( Hornung et al , 2011 ) .

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Figure 8(A) Phos-tag gels showing phosphorylation of purified S. cerevisiae Mtw1 complex by Hrr251-394 and Hrr251-394:Mam187-191. Asterisks denote N-terminal proteolytic cleavage products of Dsn1, with the red asterisk denoting a previously-characterized product lacking the first 171 residues (Hornung et al, 2011).


figf1 ( a ) Wild - type ( WT ) , atg17Δ , atg7Δ and atg19Δ strains containing RPL25 - GFP were grown to mid log phase , starved in SD - N for 48 hours and examined under a fluorescence microscope ( left panel ) . The arrows indicate vacuolar accumulation of the GFP signal . CVT , cytoplasm - to - vacuole - targeting pathway . After the times indicated , at least 100 cells per sample were scored for the presence of Rpl25p - GFP in the vacuole ( right panel ) . Scale bar , 5 μm . ( b ) Wild - type and atg7Δ strains containing RPL25 - GFP or GFP - ATG8 were grown and starved as in a . Samples were taken at indicated time points and GFP cleavage was analysed by western blotting . ( c ) The levels of endogenous Rps3p ( top ) and Rpl3p ( middle ) were analysed by immunblotting in wild - type and atg7Δ cells . Ponceau staining ( bottom ) controlled for equal loading . Uncropped images of blots are shown in Supplementary Information , Fig . S6 . Starv , starvation . ( d ) Wild - type and pep4Δ cells expressing RPL25 - GFP were analysed as in b ; 0 . 1 mM of PMSF was added to pep4Δ cells at the time of starvation to block all vacuolar proteases completely . The LI - COR imaging system was used to quantify the ratio between cleaved GFP and full - length protein as described in Methods . The last time point of wild - type cells containing RPL25 - GFP was set to 1 arbitrary unit ( a . u . ) . One of two independent experiments is shown . ( e ) Wild - type cells containing either RPL25 - GFP or control GFP ( HOG1 - GFP ) were analysed as in d . The average of four independent experiments is shown , and the standard deviation was less than 10 % ( Supplementary Information , Table S3 ) .

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figf1(a) Wild-type (WT), atg17Δ, atg7Δ and atg19Δ strains containing RPL25-GFP were grown to mid log phase, starved in SD-N for 48 hours and examined under a fluorescence microscope (left panel). The arrows indicate vacuolar accumulation of the GFP signal. CVT, cytoplasm-to-vacuole-targeting pathway. After the times indicated, at least 100 cells per sample were scored for the presence of Rpl25p-GFP in the vacuole (right panel). Scale bar, 5 μm.(b) Wild-type and atg7Δ strains containing RPL25-GFP or GFP-ATG8 were grown and starved as in a. Samples were taken at indicated time points and GFP cleavage was analysed by western blotting.(c) The levels of endogenous Rps3p (top) and Rpl3p (middle) were analysed by immunblotting in wild-type and atg7Δ cells. Ponceau staining (bottom) controlled for equal loading. Uncropped images of blots are shown in Supplementary Information, Fig. S6. Starv, starvation.(d) Wild-type and pep4Δ cells expressing RPL25-GFP were analysed as in b; 0.1 mM of PMSF was added to pep4Δ cells at the time of starvation to block all vacuolar proteases completely. The LI-COR imaging system was used to quantify the ratio between cleaved GFP and full-length protein as described in Methods. The last time point of wild-type cells containing RPL25-GFP was set to 1 arbitrary unit (a.u.). One of two independent experiments is shown.(e) Wild-type cells containing either RPL25-GFP or control GFP (HOG1-GFP) were analysed as in d. The average of four independent experiments is shown, and the standard deviation was less than 10% (Supplementary Information, Table S3).


figf2 ( a ) Wild - type , ubp3Δ or bre5Δ strains containing RPL25 - GFP and either a control plasmid ( pRS315 ) , or plasmids expressing Ubp3p or catalytically inactive Ubp3p - C469A from the endogenous promoter , were grown and starved as described in Fig . 1a . Cells were revealed by phase contrast ( right ) , and the localization of Rpl25p - GFP was analysed by GFP microscopy ( left ) . At least 100 cells per sample were analysed . Scale bar , 5 μm . ( b ) Ribosomes from wild - type and ubp3Δ cells grown as indicated for 16 h under rich or starvation ( Starv ) conditions were enriched through a high - salt cushion ( pellet ) and the levels of endogenous Rpl3p were analysed by immunoblotting . Ponceau staining of the blot revealed the total amount of proteins ( bottom panel ) . Uncropped images of blots are shown in Supplementary Information , Fig . S6 . c ) Wild - type and ubp3Δ strains containing RPL25 - GFP were grown and starved as in Fig . 1a , and GFP cleavage was analysed by western blotting at the times indicated ( hours ) . ( d , e ) GFP cleavage of Rpl25p - GFP was analysed in wild - type , ubp3Δ , atg9Δ and bre5Δ strains containing the indicated plasmids , and quantified at the times indicated as described in Fig . 1d . One of three representative experiments is shown for d . The average of three independent experiments is shown in e , and the statistical analysis showed that the standard deviation was less than 13 % ( Supplementary Information , Table S3 ) .

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figf2(a) Wild-type, ubp3Δ or bre5Δ strains containing RPL25-GFP and either a control plasmid (pRS315), or plasmids expressing Ubp3p or catalytically inactive Ubp3p-C469A from the endogenous promoter, were grown and starved as described in Fig. 1a. Cells were revealed by phase contrast (right), and the localization of Rpl25p-GFP was analysed by GFP microscopy (left). At least 100 cells per sample were analysed. Scale bar, 5 μm.(b) Ribosomes from wild-type and ubp3Δ cells grown as indicated for 16 h under rich or starvation (Starv) conditions were enriched through a high-salt cushion (pellet) and the levels of endogenous Rpl3p were analysed by immunoblotting. Ponceau staining of the blot revealed the total amount of proteins (bottom panel). Uncropped images of blots are shown in Supplementary Information, Fig. S6.c) Wild-type and ubp3Δ strains containing RPL25-GFP were grown and starved as in Fig. 1a, and GFP cleavage was analysed by western blotting at the times indicated (hours).(d, e) GFP cleavage of Rpl25p-GFP was analysed in wild-type, ubp3Δ, atg9Δ and bre5Δ strains containing the indicated plasmids, and quantified at the times indicated as described in Fig. 1d. One of three representative experiments is shown for d. The average of three independent experiments is shown in e, and the statistical analysis showed that the standard deviation was less than 13% (Supplementary Information, Table S3).


figf3 ( a ) The vacuolar localization of GFP - Atg8p was analysed in more than 100 wild - type , ubp3Δ , bre5Δ and atg7Δ cells containing GFP - ATG8 after 10 - 15 h of nitrogen starvation ( right panel ) . Corresponding GFP and phase contrast images of representative cells are shown in the left panel . ( b ) Vacuolar cleavage of GFP - Atg8p was examined at the indicated time points ( hours ) by anti - GFP western blotting . ( c ) Wild - type , ubp3Δ and bre5Δ strains containing APE1 - RFP were analysed by RFP microscopy ( left ) and phase contrast ( right ) 15 h after nitrogen starvation . Scale bars , 5 μm .

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figf3(a) The vacuolar localization of GFP-Atg8p was analysed in more than 100 wild-type, ubp3Δ, bre5Δ and atg7Δ cells containing GFP-ATG8 after 10-15 h of nitrogen starvation (right panel). Corresponding GFP and phase contrast images of representative cells are shown in the left panel.(b) Vacuolar cleavage of GFP-Atg8p was examined at the indicated time points (hours) by anti-GFP western blotting.(c) Wild-type, ubp3Δ and bre5Δ strains containing APE1-RFP were analysed by RFP microscopy (left) and phase contrast (right) 15 h after nitrogen starvation. Scale bars, 5 μm.


figf4 ( a ) Wild - type , ubp3Δ or bre5Δ strains containing either a control plasmid ( pRS315 ) or plasmids expressing Ubp3p or catalytically inactive Ubp3p - C469A from the endogenous promoter were spotted in serial dilutions onto YPD plates with ( left ) or without ( right ) 5 nM rapamycin . The plates were photographed after 2 days at 30 ° C . ( b ) Induction of a GCN4 - LacZ reporter was measured 0 , 2 and 4 h after amino - acid starvation , and is plotted as Miller units . ( c ) Polysome profiles of exponentially growing wild - type and ubp3Δ cells were determined in rich medium or 3 h after nitrogen starvation . The arrows mark the 60S peak . ( d ) Wild - type , atg7Δ and ubp3Δ cells containing either RPL25 - GFP , control GFP ( HOG1 - GFP ) or RPS2 - GFP were analysed as in Fig . 1c . One of three representative experiments is shown . ( e ) Ribosomes and associated proteins were immunopurified from starved wild - type and ubp3Δ cells containing ( RPL25 - GFP ) and ( 9 × MYC - UBI ) as indicated . The GFP immunoprecipitates ( IP ; left ) and total extracts ( TCA , right ) were analysed by immunoblotting with anti - Myc antibodies to reveal ubiquitinated proteins ( top ) . Ub , ubiquitin . Bottom : immunoblotting with anti - GFP antibodies controls for the presence of Rpl25p - GFP , and anti - Rpl1p antibodies confirm equal loading of the extract . The asterisk marks protein G on the beads recognized by the secondary antibody .

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figf4(a) Wild-type, ubp3Δ or bre5Δ strains containing either a control plasmid (pRS315) or plasmids expressing Ubp3p or catalytically inactive Ubp3p-C469A from the endogenous promoter were spotted in serial dilutions onto YPD plates with (left) or without (right) 5 nM rapamycin. The plates were photographed after 2 days at 30 °C.(b) Induction of a GCN4-LacZ reporter was measured 0, 2 and 4 h after amino-acid starvation, and is plotted as Miller units.(c) Polysome profiles of exponentially growing wild-type and ubp3Δ cells were determined in rich medium or 3 h after nitrogen starvation. The arrows mark the 60S peak.(d) Wild-type, atg7Δ and ubp3Δ cells containing either RPL25-GFP, control GFP (HOG1-GFP) or RPS2-GFP were analysed as in Fig. 1c. One of three representative experiments is shown.(e) Ribosomes and associated proteins were immunopurified from starved wild-type and ubp3Δ cells containing (RPL25-GFP) and (9 × MYC-UBI) as indicated. The GFP immunoprecipitates (IP; left) and total extracts (TCA, right) were analysed by immunoblotting with anti-Myc antibodies to reveal ubiquitinated proteins (top). Ub, ubiquitin. Bottom: immunoblotting with anti-GFP antibodies controls for the presence of Rpl25p-GFP, and anti-Rpl1p antibodies confirm equal loading of the extract. The asterisk marks protein G on the beads recognized by the secondary antibody.


Figure 1A , Representative images of DIV 3 + 2 cortical neurons transfected with FlucSM - EGFP ( green ) and subjected to the indicated treatments : 5 µM MG - 132 for 4 hours ; 10 nM Bafilomycin A1 ( BafA1 ) for 24 hours ; 0 . 5 µM 17 - AAG for 4 hours ; heat shock at 43 ° C for 30 min . Nuclei were labeled with DAPI ( blue ) . Insets show higher magnification of the areas outlined by the boxes . D , DIV 3 + 2 cortical neurons co - transfected with FlucWT - EGFP ( green ) and HTT - Q97 - mCherry ( magenta ) and treated with 1 mM 4 - PBA ( lower row ) or vehicle control ( upper row ) from DIV 3 . Cells were stained for MAP2 ( grey ) as a neuronal marker , nuclei were labeled with DAPI ( blue ) . Arrowhead points to Fluc - EGFP foci . Corresponding cultures transfected with control HTT are shown in Fig . EV1D . E , Quantification of the fraction of double - transfected cells showing Fluc - EGFP foci . N = 4 biological replicates . Two - way ANOVA with Tukey ' s multiple comparisons test . ANOVA : HTT , * * p = 0 . 0046 ; 4 - PBA , n . s . ; HTT x 4 - PBA , n . s . Significant pairwise comparisons are indicated on the graph .

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Figure 1A, Representative images of DIV 3+2 cortical neurons transfected with FlucSM-EGFP (green) and subjected to the indicated treatments: 5 µM MG-132 for 4 hours; 10 nM Bafilomycin A1 (BafA1) for 24 hours; 0.5 µM 17-AAG for 4 hours; heat shock at 43°C for 30 min. Nuclei were labeled with DAPI (blue). Insets show higher magnification of the areas outlined by the boxes.D, DIV 3+2 cortical neurons co-transfected with FlucWT-EGFP (green) and HTT-Q97-mCherry (magenta) and treated with 1 mM 4-PBA (lower row) or vehicle control (upper row) from DIV 3. Cells were stained for MAP2 (grey) as a neuronal marker, nuclei were labeled with DAPI (blue). Arrowhead points to Fluc-EGFP foci. Corresponding cultures transfected with control HTT are shown in Fig. EV1D. E, Quantification of the fraction of double-transfected cells showing Fluc-EGFP foci. N=4 biological replicates. Two-way ANOVA with Tukey's multiple comparisons test. ANOVA: HTT, **p=0.0046; 4-PBA, n.s.; HTT x 4-PBA, n.s. Significant pairwise comparisons are indicated on the graph. D


Figure 2C , Left , Western blot of acute brain slice lysates from Fluc - EGFP mice and non - transgenic littermates . The slices were treated with 1 mM 4 - PBA or vehicle control , and subjected to heat shock at 43 ° C for 15 min , as indicated above the blot . Middle , quantification of specific luciferase activity of Fluc - EGFP upon indicated treatments , normalized to vehicle - treated slices kept at 37 ° C . Right , quantification of Fluc - EGFP protein levels in the indicated conditions . N = 3 mice . Colored asterisks indicate comparisons to the corresponding vehicle - treated 37 ° C control group ( one - sample t - test ) , black asterisk indicates comparison between 4 - PBA and vehicle - treated heat shock groups ( two - tailed t - test ) . D , Representative Western blot of hippocampal lysates from Fluc - EGFP mice at the indicated ages . Short bracket with an arrow indicates the high molecular weight species observed in older mice . Long bracket indicates the part of the lane that was used for Fluc protein quantification ( see Materials and Methods ) . Several lanes on the blot between 6 - month - old and 12 - month - old samples were digitally removed .

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Figure 2C, Left, Western blot of acute brain slice lysates from Fluc-EGFP mice and non-transgenic littermates. The slices were treated with 1 mM 4-PBA or vehicle control, and subjected to heat shock at 43°C for 15 min, as indicated above the blot. Middle, quantification of specific luciferase activity of Fluc-EGFP upon indicated treatments, normalized to vehicle-treated slices kept at 37°C. Right, quantification of Fluc-EGFP protein levels in the indicated conditions. N=3 mice. Colored asterisks indicate comparisons to the corresponding vehicle-treated 37°C control group (one-sample t-test), black asterisk indicates comparison between 4-PBA and vehicle-treated heat shock groups (two-tailed t-test).D, Representative Western blot of hippocampal lysates from Fluc-EGFP mice at the indicated ages. Short bracket with an arrow indicates the high molecular weight species observed in older mice. Long bracket indicates the part of the lane that was used for Fluc protein quantification (see Materials and Methods). Several lanes on the blot between 6-month-old and 12-month-old samples were digitally removed.


Figure 3A , Cortical sections from rTg4510 : Fluc - EGFP mice ( right ) and control littermates ( left ) stained for total human tau ( HT7 , magenta ) and p - tau ( AT8 , yellow ) . Fluc - EGFP was detected by EGFP fluorescence ( green ) , nuclei were labeled with DAPI ( blue ) . Images on the right show higher magnification of the areas indicated by the boxes , with neuronal cell bodies outlined . Note the presence of EGFP foci ( arrowheads ) in the majority of neurons in rTg4510 : Fluc - EGFP mice . Occasional foci of lower relative fluorescence intensity can be observed in control mice ( arrow ) . D , Quantification of the fraction of cells with EGFP foci among the cells with and without tau expression and p - tau pathology . N = 4 rTg4510 : Fluc - EGFP mice , 3 WT : Fluc - EGFP mice , 3 CaMKIIα - tTA : Fluc - EGFP mice and 3 tetO - tauP301L : Fluc - EGFP mice . WT : Fluc - EGFP , CaMKIIα - tTA : Fluc - EGFP and tetO - tauP301L : Fluc - EGFP littermates did not differ from each other and were pooled together as controls . One - way ANOVA with Tukey ' s multiple comparisons test . ANOVA : * * * * p < 0 . 0001 . Pairwise comparisons between groups are indicated on the graph .

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Figure 3A, Cortical sections from rTg4510:Fluc-EGFP mice (right) and control littermates (left) stained for total human tau (HT7, magenta) and p-tau (AT8, yellow). Fluc-EGFP was detected by EGFP fluorescence (green), nuclei were labeled with DAPI (blue). Images on the right show higher magnification of the areas indicated by the boxes, with neuronal cell bodies outlined. Note the presence of EGFP foci (arrowheads) in the majority of neurons in rTg4510:Fluc-EGFP mice. Occasional foci of lower relative fluorescence intensity can be observed in control mice (arrow).D, Quantification of the fraction of cells with EGFP foci among the cells with and without tau expression and p-tau pathology. N=4 rTg4510:Fluc-EGFP mice, 3 WT:Fluc-EGFP mice, 3 CaMKIIα-tTA:Fluc-EGFP mice and 3 tetO-tauP301L:Fluc-EGFP mice. WT:Fluc-EGFP, CaMKIIα-tTA:Fluc-EGFP and tetO-tauP301L:Fluc-EGFP littermates did not differ from each other and were pooled together as controls. One-way ANOVA with Tukey's multiple comparisons test. ANOVA: ****p < 0.0001. Pairwise comparisons between groups are indicated on the graph.


Figure 4A , Cortical ( upper row ) and striatal ( lower row ) sections from 12 - week - old R6 / 2 : Fluc - EGFP mice ( right ) and control WT : Fluc - EGFP littermates ( left ) stained for aggregated mHTT ( EM48 , magenta ) . Fluc - EGFP was detected by EGFP fluorescence ( green ) , nuclei were counterstained with DAPI ( blue ) . Images on the right show higher magnification of the areas indicated by the boxes . Continuous and stippled lines mark cell bodies and nuclei , respectively . Luciferase assay in R6 / 2 mice . C , Fluc - EGFP protein quantity in the indicated brain regions of R6 / 2 : Fluc - EGFP mice measured by Western blot . Values are normalized to WT : Fluc - EGFP littermates . N = 5 mice of each genotype . Colored asterisks in B and C indicate significant comparisons to the respective brain regions of control littermates ( one - sample t - test ) .

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Figure 4A, Cortical (upper row) and striatal (lower row) sections from 12-week-old R6/2:Fluc-EGFP mice (right) and control WT:Fluc-EGFP littermates (left) stained for aggregated mHTT (EM48, magenta). Fluc-EGFP was detected by EGFP fluorescence (green), nuclei were counterstained with DAPI (blue). Images on the right show higher magnification of the areas indicated by the boxes. Continuous and stippled lines mark cell bodies and nuclei, respectively.Luciferase assay in R6/2 mice. C, Fluc-EGFP protein quantity in the indicated brain regions of R6/2:Fluc-EGFP mice measured by Western blot. Values are normalized to WT:Fluc-EGFP littermates. N=5 mice of each genotype. Colored asterisks in B and C indicate significant comparisons to the respective brain regions of control littermates (one-sample t-test).


Figure 5A , B , Primary cortical neurons transfected with cyt - Fluc - EGFP ( A ) or nuc - Fluc - EGFP ( B ) ( green ) , in combination with nuc - mHTT ( upper rows ) or cyt - mHTT ( lower rows ) ( magenta ) . Cells were fixed at DIV 3 + 2 and stained for the neuronal marker MAP2 ( grey ) . Nuclei were labeled with DAPI ( blue ) . Arrowheads point to Fluc - EGFP foci . Dashed lines mark the nuclei . Schemes on the right summarize the cellular distribution of the respective Fluc - EGFP ( green ) and mHTT ( magenta ) proteins . Corresponding cultures transfected with control HTT are shown in Appendix Fig . S3A .

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Figure 5A, B, Primary cortical neurons transfected with cyt-Fluc-EGFP (A) or nuc-Fluc-EGFP (B) (green), in combination with nuc-mHTT (upper rows) or cyt-mHTT (lower rows) (magenta). Cells were fixed at DIV 3+2 and stained for the neuronal marker MAP2 (grey). Nuclei were labeled with DAPI (blue). Arrowheads point to Fluc-EGFP foci. Dashed lines mark the nuclei. Schemes on the right summarize the cellular distribution of the respective Fluc-EGFP (green) and mHTT (magenta) proteins. Corresponding cultures transfected with control HTT are shown in Appendix Fig. S3A.


Figure 1A HeLa cells were transfected with constructs for a non - targeting shRNA ( NT ) , USP17 shRNA1 , or USP17 shRNA2 . Cells were stained using an anti - LAMP1 antibody ( green ) and the nuclei counterstained with DAPI ( blue ) . Lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line . B HeLa and MDA - MB - 231 cells were transfected as in A and the distribution of LAMP1 positive vesicles was plotted as vesicle relative position ( see C ) . CD HeLa cells transfected as in A , in conjunction with a construct coding GFP tagged CD63 . The lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line . E HeLa cells transfected as in A , in conjunction with constructs for either GFP tagged LAMP1 or CD63 . The distribution of GFP positive vesicles plotted as vesicle relative position .

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Figure 1A HeLa cells were transfected with constructs for a non-targeting shRNA (NT), USP17 shRNA1, or USP17 shRNA2. Cells were stained using an anti-LAMP1 antibody (green) and the nuclei counterstained with DAPI (blue). Lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line. B HeLa and MDA-MB-231 cells were transfected as in A and the distribution of LAMP1 positive vesicles was plotted as vesicle relative position (see C). CD HeLa cells transfected as in A, in conjunction with a construct coding GFP tagged CD63. The lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line. E HeLa cells transfected as in A, in conjunction with constructs for either GFP tagged LAMP1 or CD63. The distribution of GFP positive vesicles plotted as vesicle relative position. F


Figure 2A HeLa cells were transfected with constructs for a non - targeting ( NT ) shRNA , USP17 shRNA1 or USP17 shRNA2 . 48 hours post transfection the cells were either serum starved ( upper panels ) , or treated with serum free medium containing 100ug / ml EGF ( lower panels ) for 16 hours prior to staining for LAMP1 ( green ) and DAPI ( blue ) . Right hand panels are enlarged images of the indicated area in left panels and the cell membrane is marked by dotted line . Scale bar represents 25μm . B The distribution of at least 300 LAMP1 positive vesicles from a number of cells ( n ) from a series of confocal images across three separate experiments was plotted as vesicle relative position ( mean value is red bar ) . Error bars represent standard error , ns indicates not significant , and * * * * indicates a p - values less than 0 . 0001 . One way ANOVA used to determine if statistically significant differences between groups .

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Figure 2A HeLa cells were transfected with constructs for a non-targeting (NT) shRNA, USP17 shRNA1 or USP17 shRNA2. 48 hours post transfection the cells were either serum starved (upper panels), or treated with serum free medium containing 100ug/ml EGF (lower panels) for 16 hours prior to staining for LAMP1 (green) and DAPI (blue). Right hand panels are enlarged images of the indicated area in left panels and the cell membrane is marked by dotted line. Scale bar represents 25μm. B The distribution of at least 300 LAMP1 positive vesicles from a number of cells (n) from a series of confocal images across three separate experiments was plotted as vesicle relative position (mean value is red bar). Error bars represent standard error, ns indicates not significant, and **** indicates a p-values less than 0.0001. One way ANOVA used to determine if statistically significant differences between groups.


Figure 3A 48 hours post transfection the cells were washed with Ca2 + and Mg2 + free ice cold HBSS . The different cell populations were split into three aliquots . One aliquot was placed into fresh medium ( Left panels ) . SLO was added to other two aliquots and then the toxin - containing medium was removed and the aliquots were either placed into Ca2 + and Mg2 + free HBSS ( Middle panels ) , or EBSS containing Ca2 + and Mg2 + ( Right panels ) prior to staining with propidium iodide and analysis by flow cytometry .

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Figure 3A 48 hours post transfection the cells were washed with Ca2+ and Mg2+ free ice cold HBSS. The different cell populations were split into three aliquots. One aliquot was placed into fresh medium (Left panels). SLO was added to other two aliquots and then the toxin-containing medium was removed and the aliquots were either placed into Ca2+ and Mg2+ free HBSS (Middle panels), or EBSS containing Ca2+ and Mg2+ (Right panels) prior to staining with propidium iodide and analysis by flow cytometry.


Figure 4A HeLa cells were transfected with negative control siRNA and RNF26 siRNA as well as constructs coding a non - targeting ( NT ) shRNA , USP17 shRNA1 or USP17 shRNA2 , as indicated . 48 hours post transfection cells were stained for LAMP - 1 ( green ) and DAPI ( blue ) . Lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line . Scale bar represents 25μm . C HeLa cells were transfected with expression constructs for HA - tagged p62 and FLAG - tagged ubiquitin as well as empty vector , USP17 or USP17CS . After 48hrs cell lysates were prepared and HA tagged proteins were pulled down using anti - HA agarose . Pull downs , and lysates , were immunoblotted with anti - HA , anti - FLAG , and anti - USP17 antibodies , as indicated , to confirm the presence of ubiquitinated p62 .

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Figure 4A HeLa cells were transfected with negative control siRNA and RNF26 siRNA as well as constructs coding a non-targeting (NT) shRNA, USP17 shRNA1 or USP17 shRNA2, as indicated. 48 hours post transfection cells were stained for LAMP-1 (green) and DAPI (blue). Lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line. Scale bar represents 25μm.C HeLa cells were transfected with expression constructs for HA-tagged p62 and FLAG-tagged ubiquitin as well as empty vector, USP17 or USP17CS. After 48hrs cell lysates were prepared and HA tagged proteins were pulled down using anti-HA agarose. Pull downs, and lysates, were immunoblotted with anti-HA, anti-FLAG, and anti-USP17 antibodies, as indicated, to confirm the presence of ubiquitinated p62.


Figure 5A HeLa cells were transfected with constructs for EEA1 - GFP and RAB5 - GFP in addition to non - targeting scrambled ( NT ) shRNA , USP17 shRNA1 or USP17 shRNA2 . 72 hours post transfection the cells were stained with DAPI . The scale bar represents 25μm . B The distribution of at least 400 EEA1 or RAB5 positive vesicles from a number of cells ( n ) from a series of confocal images across three separate experiments was plotted as vesicle relative position ( mean value is red bar ) . Error bars represent standard error and * * * * indicates a p - values less than 0 . 0001 . One way ANOVA used to determine if statistically significant differences between groups .

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Figure 5A HeLa cells were transfected with constructs for EEA1-GFP and RAB5-GFP in addition to non-targeting scrambled (NT) shRNA, USP17 shRNA1 or USP17 shRNA2. 72 hours post transfection the cells were stained with DAPI. The scale bar represents 25μm. B The distribution of at least 400 EEA1 or RAB5 positive vesicles from a number of cells (n) from a series of confocal images across three separate experiments was plotted as vesicle relative position (mean value is red bar). Error bars represent standard error and **** indicates a p-values less than 0.0001. One way ANOVA used to determine if statistically significant differences between groups. E


Figure 1 ( A ) Using denaturing immunoprecipitation ( IP ) , Munc18 - 1 was isolated from cell lysate of HEK293T cells expressing M18WT , M18Y145F or M18Y473F together with or without n - Src . Munc18 - 1 was immunoblotted for tyrosine phosphorylation using the 4G10 antibody ( αPtyr ) , after which the blot was stripped and re - blotted for Munc18 - 1 . Total lysate ( TL ) is shown as loading control . N - Src was tagged with a flag - tag for visualization . ( B ) Same as ( A ) but using lysate from HEK293T cells expressing M18WT or M18Y473F together with or without n - Src , c - Src or Fyn . The first two lanes are IPs performed using empty beads to control for non - specific binding of Munc18 or kinases to the beads . ( F ) Lipid mixing was monitored by the increase of Atto 488 fluorescence for 12 minutes . Data were normalized to the maximum fluorescence after liposome lysis by detergent . Error bars = s . e . m . , N = 3 . ( G ) Same as ( E - F ) but the reactions were preincubated on ice for 5 minutes in the presence of Complexin II ( CpxII 600 pmol ) . Error bars = s . e . m . , N = 5 . ( I ) Lipid mixing was monitored by the increase of Atto 488 fluorescence for 60 minutes . Data were normalized to the maximum fluorescence after liposome lysis by detergent . Error bars = s . e . m . , N = 3 .

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Figure 1(A) Using denaturing immunoprecipitation (IP), Munc18-1 was isolated from cell lysate of HEK293T cells expressing M18WT, M18Y145F or M18Y473F together with or without n-Src. Munc18-1 was immunoblotted for tyrosine phosphorylation using the 4G10 antibody (αPtyr), after which the blot was stripped and re-blotted for Munc18-1. Total lysate (TL) is shown as loading control. N-Src was tagged with a flag-tag for visualization.(B) Same as (A) but using lysate from HEK293T cells expressing M18WT or M18Y473F together with or without n-Src, c-Src or Fyn. The first two lanes are IPs performed using empty beads to control for non-specific binding of Munc18 or kinases to the beads.(F) Lipid mixing was monitored by the increase of Atto 488 fluorescence for 12 minutes. Data were normalized to the maximum fluorescence after liposome lysis by detergent. Error bars = s.e.m., N = 3.(G) Same as (E-F) but the reactions were preincubated on ice for 5 minutes in the presence of Complexin II (CpxII 600 pmol). Error bars = s.e.m., N =5.(I) Lipid mixing was monitored by the increase of Atto 488 fluorescence for 60 minutes. Data were normalized to the maximum fluorescence after liposome lysis by detergent. Error bars = s.e.m., N =3.


Figure 2Autaptic munc18 - 1 null hippocampal neurons were rescued with a phosphomimetic Munc18 - 1 mutant , Y473D or wild - type Munc18 - 1 as control and assessed on morphology and synaptic transmission using confocal microscopy ( A ) Typical confocal images from neurons stained for MAP2 , Synaptobrevin / VAMP2 and Munc18 - 1 . Autaptic munc18 - 1 null hippocampal neurons were rescued with a phosphomimetic Munc18 - 1 mutant , Y473D or wild - type Munc18 - 1 as control and assessed on morphology and synaptic transmission using confocal microscopy ( B ) Average synaptic Munc18 - 1 intensity ( M18WT : 389 ± 85 a . u . , n = 21 ; M18Y473D : 261 ± 49 a . u . , n = 20 , t - test with Welch correction , p = 0 . 2000 ) , somatic Munc18 - 1 intensity ( M18WT : 328 ± 49 a . u . , n = 21 ; M18Y473D : 188 ± 21 a . u . , n = 20 , t - test with Welch correction , p = 0 . 0138 ) , synapse number ( M18WT : 129 ± 14 , n = 21 ; M18Y473D : 159 ± 14 , n = 20 , t - test , p = 0 . 1203 ) and dendrite length ( M18WT : 0 . 433 ± 0 . 028 , n = 21 ; M18Y473D : 0 . 454 ± 0 . 037 , n = 20 , t - test , p = 0 . 6613 ) . Autaptic munc18 - 1 null hippocampal neurons were rescued with a phosphomimetic Munc18 - 1 mutant , Y473D or wild - type Munc18 - 1 as control and assessed on morphology and synaptic transmission using whole - cell patch clamp electrophysiology ( C ) Left : typical examples of evoked release upon action potential stimulation . Autaptic munc18 - 1 null hippocampal neurons were rescued with a phosphomimetic Munc18 - 1 mutant , Y473D or wild - type Munc18 - 1 as control and assessed on morphology and synaptic transmission using whole - cell patch clamp electrophysiology ( D ) Left : typical responses to hyperosmotic sucrose application ( 500 mM , 3 . 5 sec . ) used to assess the RRP . Average RRP charge ( M18WT : 1 . 68 ± 0 . 64 nC , n = 13 ; M18Y473D : 0 . 08 ± 0 . 02 nC , n = 10 , t - test with Welch correction , p = 0 . 0274 ) . Autaptic munc18 - 1 null hippocampal neurons were rescued with a phosphomimetic Munc18 - 1 mutant , Y473D or wild - type Munc18 - 1 as control and assessed on morphology and synaptic transmission using whole - cell patch clamp electrophysiology ( E ) Average Pves ( EPSC charge / RRP charge ) ( M18WT : 6 . 75 ± 0 . 77 % , n = 13 ; M18Y473D : 0 . 24 ± 0 . 20 % , n = 10 ; Mann - Whitney U test , p < 0 . 0001 ) . Autaptic munc18 - 1 null hippocampal neurons were rescued with a phosphomimetic Munc18 - 1 mutant , Y473D or wild - type Munc18 - 1 as control and assessed on morphology and synaptic transmission using whole - cell patch clamp electrophysiology ( F ) Left : example traces of spontaneous release of single SVs ( mEPSCs ) . Average mEPSC frequency ( M18WT : 20 . 18 ± 5 . 18 Hz , n = 15 ; M18Y473D : 0 . 28 ± 0 . 13 Hz , n = 17 , Mann - Whitney U test , p < 0 . 0001 ) , mEPSC amplitude ( M18WT : 26 . 0 ± 1 . 7 pA , n = 15 ; M18Y473D : 27 . 8 ± 2 . 2 pA , n = 17 , t - test , p = 0 . 5372 ) and mEPSC decay time ( M18WT : 1 . 98 ± 0 . 10 ms , n = 15 ; M18Y473D : 2 . 84 ± 0 . 41 ms , n = 17 , t - test with Welch correction , p = 0 . 0549 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using confocal microscopy (A) Typical confocal images from neurons stained for MAP2, Synaptobrevin/VAMP2 and Munc18-1.Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using confocal microscopy (B) Average synaptic Munc18-1 intensity (M18WT: 389 ± 85 a.u., n=21; M18Y473D: 261 ± 49 a.u., n=20, t-test with Welch correction, p = 0.2000), somatic Munc18-1 intensity (M18WT: 328 ± 49 a.u., n=21; M18Y473D: 188 ± 21 a.u., n=20, t-test with Welch correction, p = 0.0138), synapse number (M18WT: 129 ± 14, n=21; M18Y473D: 159 ± 14, n=20, t-test, p = 0.1203) and dendrite length (M18WT: 0.433 ± 0.028, n=21; M18Y473D: 0.454 ± 0.037, n=20, t-test, p = 0.6613).Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using whole-cell patch clamp electrophysiology(C) Left: typical examples of evoked release upon action potential stimulation.Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using whole-cell patch clamp electrophysiology (D) Left: typical responses to hyperosmotic sucrose application (500 mM, 3.5 sec.) used to assess the RRP. Average RRP charge (M18WT: 1.68 ± 0.64 nC, n=13; M18Y473D: 0.08 ± 0.02 nC, n=10, t-test with Welch correction, p = 0.0274).Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using whole-cell patch clamp electrophysiology (E) Average Pves (EPSC charge / RRP charge) (M18WT: 6.75 ± 0.77%, n=13; M18Y473D: 0.24 ± 0.20%, n=10; Mann-Whitney U test, p < 0.0001).Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using whole-cell patch clamp electrophysiology (F) Left: example traces of spontaneous release of single SVs (mEPSCs). Average mEPSC frequency (M18WT: 20.18 ± 5.18 Hz, n=15; M18Y473D: 0.28 ± 0.13 Hz, n=17, Mann-Whitney U test, p < 0.0001), mEPSC amplitude (M18WT: 26.0 ± 1.7 pA, n=15; M18Y473D: 27.8 ± 2.2 pA, n=17, t-test, p = 0.5372) and mEPSC decay time (M18WT: 1.98 ± 0.10 ms, n=15; M18Y473D: 2.84 ± 0.41 ms, n=17, t-test with Welch correction, p = 0.0549).