A ) Time - lapse experiments of living cells harbouring a non - reparable DSB were carried out after expressing the HO endonuclease in wild - type and cdc14 - 1 mutant cells . Spc110 - RFP was used as SPB reporter and Tub1 - GFP was used as tubulin marker . Three z - planes images every 5 - second intervals over a period of 5 minutes were captured and used to quantify the average SPB track length and velocity using Spc110 - RFP as SPB marker . At least 100 cells were scored . A representative maximum projection image is depicted . Scale bar : 3μm .

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

A) Time-lapse experiments of living cells harbouring a non-reparable DSB were carried out after expressing the HO endonuclease in wild-type and cdc14-1 mutant cells. Spc110-RFP was used as SPB reporter and Tub1-GFP was used as tubulin marker. Three z-planes images every 5-second intervals over a period of 5 minutes were captured and used to quantify the average SPB track length and velocity using Spc110-RFP as SPB marker. At least 100 cells were scored.A representative maximum projection image is depicted. Scale bar: 3μm.


A ) A Western blot is shown to confirm the equal amount of Tub1 - GFP in both wild - type and cdc14 - 1 backgrounds . Coomassie blue staining is shown as loading control .

O O O O O O O O O O O O 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) A Western blot is shown to confirm the equal amount of Tub1-GFP in both wild-type and cdc14-1 backgrounds. Coomassie blue staining is shown as loading control.


B ) The steady state phosphorylation of Spc110 is required to maintain spindle polarity in response to DNA damage . Wild - type , cdc14 - 1 and spc110S36 - 91A cells expressing Spc110 - RFP were scored for their ability to maintain spindle polarity at the axial plane of the cell upon induction of a single DSB induced by the HO endonuclease . A maximum projection of nine z - planes was used to score the cells . At least 100 cells from three independent experiments were scored . Graph represents the percentage ± SD of cells with oriented / miss - oriented SPBs with respect to the axial plane of the cell . P value was calculated using a 2 - tailed unpaired Student t test .

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

B) The steady state phosphorylation of Spc110 is required to maintain spindle polarity in response to DNA damage. Wild-type, cdc14-1 and spc110S36-91A cells expressing Spc110-RFP were scored for their ability to maintain spindle polarity at the axial plane of the cell upon induction of a single DSB induced by the HO endonuclease. A maximum projection of nine z-planes was used to score the cells. At least 100 cells from three independent experiments were scored. Graph represents the percentage ± SD of cells with oriented/miss-oriented SPBs with respect to the axial plane of the cell. P value was calculated using a 2-tailed unpaired Student t test.


C ) The asymmetric loading of dynein into astral MTs emanating from the daughter SPB is affected in cdc14 - 1 and spc110S36 - 91A cells during the DNA damage response . Dynein distribution on astral MTs emerging from each SPB was scored by using Dyn1 - GFP in strains containing Spc110 - RFP . Graph represents the percentage ± SD of cells grouped into three categories depending on the pattern of dynein distribution on astral MTs : Type1 , Dynein is exclusively loaded on astral MTs emerging from the daughter SPB ; Type 2 , Dynein is equally loaded on astral MTs emanating from both SPBs ; Type 3 , Dynein is absent from astral MTs . At least 100 cells from three independent experiments were scored . A representative picture showing the three patterns is included . Scale bar : 3μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The asymmetric loading of dynein into astral MTs emanating from the daughter SPB is affected in cdc14-1 and spc110S36-91A cells during the DNA damage response. Dynein distribution on astral MTs emerging from each SPB was scored by using Dyn1-GFP in strains containing Spc110-RFP. Graph represents the percentage ± SD of cells grouped into three categories depending on the pattern of dynein distribution on astral MTs: Type1, Dynein is exclusively loaded on astral MTs emerging from the daughter SPB; Type 2, Dynein is equally loaded on astral MTs emanating from both SPBs; Type 3, Dynein is absent from astral MTs. At least 100 cells from three independent experiments were scored. A representative picture showing the three patterns is included. Scale bar: 3μm.


A ) Cells treated with nocodazole are affected in DSB - SPB interaction . Cnm67 - CFP and Ddc2 - RFP were used as SPB and DSB markers respectively . Diagram depicts the genomic background used . D1 : inter - SPB distance ; D2 : DSB distance to proximal SPB ; D3 : DSB distance to distal SPB . D1 , D2 and D3 distances after inducing a DSB in the presence of nocodazole or mock dimethyl sulfoxide ( DMSO ) were scored and plotted . Graph represents the mean ± SD of D1 , D2 and D3 distances of three independent experiments . At least 100 cells per experiment were scored using the maximum projection of three z - planes . P values were calculated using a 2 - tailed unpaired Student t test . A representative picture is shown . Scale bar : 3μm .

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

A) Cells treated with nocodazole are affected in DSB-SPB interaction. Cnm67-CFP and Ddc2-RFP were used as SPB and DSB markers respectively. Diagram depicts the genomic background used. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. D1, D2 and D3 distances after inducing a DSB in the presence of nocodazole or mock dimethyl sulfoxide (DMSO) were scored and plotted. Graph represents the mean ± SD of D1, D2 and D3 distances of three independent experiments. At least 100 cells per experiment were scored using the maximum projection of three z-planes. P values were calculated using a 2-tailed unpaired Student t test. A representative picture is shown. Scale bar: 3μm.


B ) Physical analysis of wild - type cells containing the repair pathway choice assay described in Fig 2B . The diagram with the genomic information , the restriction enzymes used and the location of the probe are shown . Cells were synchronized in G1 by using the α - factor pheromone and released into fresh media for 1 hour . Induction of HO expression was attained by adding galactose for 2 hours . After formation of the DSB , glucose was added to repress the HO in the presence of nocodazole or mock DMSO and samples were taken to analyse the kinetics of the repair . FACS analyses are shown .

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

B) Physical analysis of wild-type cells containing the repair pathway choice assay described in Fig 2B. The diagram with the genomic information, the restriction enzymes used and the location of the probe are shown. Cells were synchronized in G1 by using the α-factor pheromone and released into fresh media for 1 hour. Induction of HO expression was attained by adding galactose for 2 hours. After formation of the DSB, glucose was added to repress the HO in the presence of nocodazole or mock DMSO and samples were taken to analyse the kinetics of the repair. FACS analyses are shown.


B ) Physical analysis of wild - type cells containing the repair pathway choice assay described in Fig 2B . The diagram with the genomic information , the restriction enzymes used and the location of the probe are shown . Cells were synchronized in G1 by using the α - factor pheromone and released into fresh media for 1 hour . Induction of HO expression was attained by adding galactose for 2 hours . After formation of the DSB , glucose was added to repress the HO in the presence of nocodazole or mock DMSO and samples were taken to analyse the kinetics of the repair . DNA was extracted , digested with EcoRV , separated on agarose gels and blotted . Both probes for the MATa - distal sequence and the HIS3 gene ( as loading control ) were used . Graphs show the quantification of MATa restoration ( NHEJ ) , mating type switching by gene conversion ( HR ) , and DSB kinetics formation . All data were normalized with the HIS3 gene . Graphs show the mean ± S . D . from three independent experiments . P values were calculated using a 2 - tailed unpaired Student t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Physical analysis of wild-type cells containing the repair pathway choice assay described in Fig 2B. The diagram with the genomic information, the restriction enzymes used and the location of the probe are shown. Cells were synchronized in G1 by using the α-factor pheromone and released into fresh media for 1 hour. Induction of HO expression was attained by adding galactose for 2 hours. After formation of the DSB, glucose was added to repress the HO in the presence of nocodazole or mock DMSO and samples were taken to analyse the kinetics of the repair. DNA was extracted, digested with EcoRV, separated on agarose gels and blotted. Both probes for the MATa-distal sequence and the HIS3 gene (as loading control) were used. Graphs show the quantification of MATa restoration (NHEJ), mating type switching by gene conversion (HR), and DSB kinetics formation. All data were normalized with the HIS3 gene. Graphs show the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test.


A ) Growth sensitivity to MMS and phleomycin of a Cdk phospho - deficient mutant of Spc110 . Ten - fold serial dilutions from overnight cultures of wild - type , spc110S36 - 91A and a cdc14 - 1 cells grown on solid rich media containing mock DMSO , MMS or Phleomycin at 30ºC .

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

A) Growth sensitivity to MMS and phleomycin of a Cdk phospho-deficient mutant of Spc110. Ten-fold serial dilutions from overnight cultures of wild-type, spc110S36-91A and a cdc14-1 cells grown on solid rich media containing mock DMSO, MMS or Phleomycin at 30ºC.


B ) Diagram showing the genomic background and the approximation used to measure DSB - SPB interaction . Spc110 and spc110S36 - 91A were labelled with the RFP in a background containing Ddc2 - GFP as DSB reporter . D1 : inter - SPB distance ; D2 : DSB distance to proximal SPB ; D3 : DSB distance to distal SPB . At least 100 cells from three independent experiments were scored using the maximum projection of nine z - planes . Graph shows the mean ± S . D . P values were calculated using a 2 - tailed unpaired Student t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Diagram showing the genomic background and the approximation used to measure DSB-SPB interaction. Spc110 and spc110S36-91A were labelled with the RFP in a background containing Ddc2-GFP as DSB reporter. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. At least 100 cells from three independent experiments were scored using the maximum projection of nine z-planes. Graph shows the mean ± S.D. P values were calculated using a 2-tailed unpaired Student t test.


C ) Time - lapse experiments of living cells expressing the HO endonuclease in wild - type and spc110S36 - 91A mutants cells . Five z - planes every 5 - second intervals over a period of 5 minutes were captured . Quantifications were made using the average SPB track length and velocity of at least 100 cells . Spc110 and spc110S36 - 91A were labelled with the RFP and used as SPB reporter .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Time-lapse experiments of living cells expressing the HO endonuclease in wild-type and spc110S36-91A mutants cells. Five z-planes every 5-second intervals over a period of 5 minutes were captured. Quantifications were made using the average SPB track length and velocity of at least 100 cells. Spc110 and spc110S36-91A were labelled with the RFP and used as SPB reporter.


D ) Southern blot of wild - type and spc110S36 - 91A mutant harbouring the inter - chromosomal gene conversion assay portrayed . An overnight culture was induced by adding galactose at 32ºC to express the HO endonuclease . Samples were collected at different time - points , genomic DNA was extracted , digested with EcoRI and analysed by Southern blot . Blots were probed with a MATa - only and actin ( for loading control ) DNA sequences . Graphs show quantification of gene conversion , DSB induction and crossover vs non - crossover ratio . All data were normalized using the actin signal . Graphs represent the mean ± S . D . from three independent experiments . P values were calculated using a 2 - tailed unpaired Student t test . Asterisk denotes an overexposed film to visualize the formation of crossover products .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Southern blot of wild-type and spc110S36-91A mutant harbouring the inter-chromosomal gene conversion assay portrayed. An overnight culture was induced by adding galactose at 32ºC to express the HO endonuclease. Samples were collected at different time-points, genomic DNA was extracted, digested with EcoRI and analysed by Southern blot. Blots were probed with a MATa-only and actin (for loading control) DNA sequences. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.


D ) Southern blot of wild - type and spc110S36 - 91A mutant harbouring the inter - chromosomal gene conversion assay portrayed . An overnight culture was induced by adding galactose at 32ºC to express the HO endonuclease . Samples were collected at different time - points , genomic DNA was extracted , digested with EcoRI and analysed by Southern blot . Blots were probed with a MATa - only and actin ( for loading control ) DNA sequences . FACS profiles for DNA content are included . Graphs show quantification of gene conversion , DSB induction and crossover vs non - crossover ratio . All data were normalized using the actin signal . Graphs represent the mean ± S . D . from three independent experiments . P values were calculated using a 2 - tailed unpaired Student t test . Asterisk denotes an overexposed film to visualize the formation of crossover products .

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

D) Southern blot of wild-type and spc110S36-91A mutant harbouring the inter-chromosomal gene conversion assay portrayed. An overnight culture was induced by adding galactose at 32ºC to express the HO endonuclease. Samples were collected at different time-points, genomic DNA was extracted, digested with EcoRI and analysed by Southern blot. Blots were probed with a MATa-only and actin (for loading control) DNA sequences. FACS profiles for DNA content are included. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.


A ) DNA damage sensitivity to MMS and phleomycin of a Cdk phospho - mimic mutant of Spc110 . Ten - fold serial dilutions from mid - log phase cultures of wild - type , spc110S36 - 91A and spc110S36 - 91D cells grown on solid rich media containing mock DMSO , MMS or Phleomycin at 30ºC .

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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O

A) DNA damage sensitivity to MMS and phleomycin of a Cdk phospho-mimic mutant of Spc110. Ten-fold serial dilutions from mid-log phase cultures of wild-type, spc110S36-91A and spc110S36-91D cells grown on solid rich media containing mock DMSO, MMS or Phleomycin at 30ºC.


B ) Schematic representation of the genomic background and the approximation used to measure DSB - SPB tethering . Wild - type Spc110 and spc110S36 - 91D were labelled with the RFP . Ddc2 was tagged with the GFP to be used as DSB reporter . D1 : inter - SPB distance ; D2 : DSB distance to proximal SPB ; D3 : DSB distance to distal SPB . At least 100 cells from three independent experiments were scored using the maximum projection of nine z - planes . Graph shows the mean ± S . D . P values were calculated using a 2 - tailed unpaired Student t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Schematic representation of the genomic background and the approximation used to measure DSB-SPB tethering. Wild-type Spc110 and spc110S36-91D were labelled with the RFP. Ddc2 was tagged with the GFP to be used as DSB reporter. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. At least 100 cells from three independent experiments were scored using the maximum projection of nine z-planes. Graph shows the mean ± S.D. P values were calculated using a 2-tailed unpaired Student t test.


C ) Time - lapse experiments of living cells after induction of the HO endonuclease in wild - type and spc110S36 - 91D cells . Three z - planes every 10 - second intervals over a period of 5 minutes were captured to measure the average SPBs track length and velocity . Spc110 and spc110S36 - 91D were labelled with the RFP and used to measured SPBs kinetics . At least 100 cells from three independent experiments were scored .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Time-lapse experiments of living cells after induction of the HO endonuclease in wild-type and spc110S36-91D cells. Three z-planes every 10-second intervals over a period of 5 minutes were captured to measure the average SPBs track length and velocity. Spc110 and spc110S36-91D were labelled with the RFP and used to measured SPBs kinetics. At least 100 cells from three independent experiments were scored.


D ) Southern blot of wild - type and spc110S36 - 91D mutant bearing the inter - chromosomal gene conversion assay depicted . Mid - log phase cells were HO - induced at 32ºC by adding galactose to the media . Samples were collected at different time - points and the genomic DNA was extracted . Preps were digested with EcoRI and analysed by Southern blot . Blots were hybridized with MATa - only and actin probes . FACS profiles for DNA content are displayed . Graphs show quantification of gene conversion , DSB induction and crossover vs non - crossover ratio . All data were normalized using the actin signal . Graphs represent the mean ± S . D . from three independent experiments . P values were calculated using a 2 - tailed unpaired Student t test . Asterisk denotes an overexposed film to visualize the formation of crossover products .

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

D) Southern blot of wild-type and spc110S36-91D mutant bearing the inter-chromosomal gene conversion assay depicted. Mid-log phase cells were HO-induced at 32ºC by adding galactose to the media. Samples were collected at different time-points and the genomic DNA was extracted. Preps were digested with EcoRI and analysed by Southern blot. Blots were hybridized with MATa-only and actin probes. FACS profiles for DNA content are displayed. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.


D ) Southern blot of wild - type and spc110S36 - 91D mutant bearing the inter - chromosomal gene conversion assay depicted . Mid - log phase cells were HO - induced at 32ºC by adding galactose to the media . Samples were collected at different time - points and the genomic DNA was extracted . Preps were digested with EcoRI and analysed by Southern blot . Blots were hybridized with MATa - only and actin probes . Graphs show quantification of gene conversion , DSB induction and crossover vs non - crossover ratio . All data were normalized using the actin signal . Graphs represent the mean ± S . D . from three independent experiments . P values were calculated using a 2 - tailed unpaired Student t test . Asterisk denotes an overexposed film to visualize the formation of crossover products .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Southern blot of wild-type and spc110S36-91D mutant bearing the inter-chromosomal gene conversion assay depicted. Mid-log phase cells were HO-induced at 32ºC by adding galactose to the media. Samples were collected at different time-points and the genomic DNA was extracted. Preps were digested with EcoRI and analysed by Southern blot. Blots were hybridized with MATa-only and actin probes. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.


B . Blue native - PAGE loading of protein extracts from nrc2 / 3 / 4 knockout N . benthamiana plants after immunoprecipitation with anti - Flag antibody . Co - migration of Rpi - amr3 - HF and AVRamr3 - V5 are indicated ( * , red ) . Same samples were loaded twice on one blue native - PAGE gel , transferred onto one membrane , and then the membrane was cut into two and immunoblotted separately . GUS - V5 , β - glucuronidase fused with V5 tag . C . NRC2EEE - Myc does not alter association between Rpi - amr3 and AVRamr3 . Samples of Fig 1B were SDS - boiled and loaded on SDS - PAGE . Input samples were taken prior to immunoprecipitation to show expression of all proteins .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Blue native-PAGE loading of protein extracts from nrc2/3/4 knockout N. benthamiana plants after immunoprecipitation with anti-Flag antibody. Co-migration of Rpi-amr3-HF and AVRamr3-V5 are indicated (*, red). Same samples were loaded twice on one blue native-PAGE gel, transferred onto one membrane, and then the membrane was cut into two and immunoblotted separately. GUS-V5, β-glucuronidase fused with V5 tag. C. NRC2EEE-Myc does not alter association between Rpi-amr3 and AVRamr3. Samples of Fig 1B were SDS-boiled and loaded on SDS-PAGE. Input samples were taken prior to immunoprecipitation to show expression of all proteins.


A . NRC2EEE - Myc is oligomerized upon effector - dependent activation of Rpi - amr3 . Protein lysates from Fig 1B , C were loaded on blue native - PAGE . SDS - boiled protein lysate samples serve as control for actual size of NRC2EEE - Myc . Oligomerized NRC2EEE - Myc is indicated ( * , red ) . B . NRC2EEE - Myc oligomerizes upon effector - dependent activation of Rpi - amr1 . Protein lysates from nrc2 / 3 / 4 knockout N . benthamiana plants were loaded on blue native - PAGE . Oligomerized NRC2EEE - Myc is indicated ( * , red ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. NRC2EEE-Myc is oligomerized upon effector-dependent activation of Rpi-amr3. Protein lysates from Fig 1B, C were loaded on blue native-PAGE. SDS-boiled protein lysate samples serve as control for actual size of NRC2EEE-Myc. Oligomerized NRC2EEE-Myc is indicated (*, red). B. NRC2EEE-Myc oligomerizes upon effector-dependent activation of Rpi-amr1. Protein lysates from nrc2/3/4 knockout N. benthamiana plants were loaded on blue native-PAGE. Oligomerized NRC2EEE-Myc is indicated (*, red).


C . Samples from Fig 2B were SDS - boiled and loaded on SDS - PAGE . Protein accumulation of Rpi - amr1 - Flag , NRC2EEE - Myc , AVRamr1 - V5 and AVRamr3 - V5 are shown .

O O O O O O O O O O O O O O O O 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. Samples from Fig 2B were SDS-boiled and loaded on SDS-PAGE. Protein accumulation of Rpi-amr1-Flag, NRC2EEE-Myc, AVRamr1-V5 and AVRamr3-V5 are shown.


B . NRC2EEE and Rpi - amr3 migration in the absence of effector . N . benthamiana nrc2 / 3 / 4 knockout plants were transiently infiltrated with Rpi - amr3 - HF , NRC2EEE - Myc and GUS - V5 followed by 2D - PAGE . NRC2EEE - Myc proteins ( * , red ) and Rpi - amr3 - HF proteins ( * , blue ) is indicated . C . NRC2EEE and Rpi - amr3 migration in the presence of effector . N . benthamiana nrc2 / 3 / 4 knockout plants were transiently infiltrated with Rpi - amr3 - HF , NRC2EEE - Myc and AVRamr3 - V5 followed by 2D - PAGE . NRC2EEE - Myc proteins ( * , red ) and Rpi - amr3 - HF protein ( * , blue ) is indicated .

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B. NRC2EEE and Rpi-amr3 migration in the absence of effector. N. benthamiana nrc2/3/4 knockout plants were transiently infiltrated with Rpi-amr3-HF, NRC2EEE-Myc and GUS-V5 followed by 2D-PAGE. NRC2EEE-Myc proteins (*, red) and Rpi-amr3-HF proteins (*, blue) is indicated. C. NRC2EEE and Rpi-amr3 migration in the presence of effector. N. benthamiana nrc2/3/4 knockout plants were transiently infiltrated with Rpi-amr3-HF, NRC2EEE-Myc and AVRamr3 -V5 followed by 2D-PAGE. NRC2EEE-Myc proteins (*, red) and Rpi-amr3-HF protein (*, blue) is indicated.


D . Re - localization of NRC2EEE - Myc upon effector - dependent activation of Rpi - amr1 . Lysates of nrc2 / 3 / 4 knockout plants transiently expressing Rpi - amr1 - Flag , NRC2EEE - Myc with AVRamr3 - V5 or AVRamr1 - V5 were fractionated into total ( T ) , soluble ( S ) , and pellet ( P ) fractions . E . Re - localization of NRC2EEE - Myc upon effector - dependent activation of Rpi - amr3 . Lysates of nrc2 / 3 / 4 knockout plants transiently expressing Rpi - amr3 - HF , NRC2EEE - Myc with AVRamr3 - V5 or AVRamr1 - V5 were fractionated into total ( T ) , soluble ( S ) , and pellet ( P ) fractions .

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D. Re-localization of NRC2EEE-Myc upon effector-dependent activation of Rpi-amr1. Lysates of nrc2/3/4 knockout plants transiently expressing Rpi-amr1-Flag, NRC2EEE-Myc with AVRamr3-V5 or AVRamr1-V5 were fractionated into total (T), soluble (S), and pellet (P) fractions. E. Re-localization of NRC2EEE-Myc upon effector-dependent activation of Rpi-amr3. Lysates of nrc2/3/4 knockout plants transiently expressing Rpi-amr3-HF, NRC2EEE-Myc with AVRamr3-V5 or AVRamr1-V5 were fractionated into total (T), soluble (S), and pellet (P) fractions.


A . P - loop of Rpi - amr3 is required for AVRamr3 - dependent HR in N . benthamiana . Representative leaf phenotype of HR ( hypersensitive response ) in wild - type N . benthamiana is shown . The number of leaves tested and occurrences of HR are indicated in parentheses . B . P - loop of NRC2 is required for AVRamr3 - dependent HR in N . benthamiana nrc2 / 3 / 4 knockout plants . Representative leaf phenotype of HR is shown . The number of leaves tested and occurrences of HR are indicated in parentheses .

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A. P-loop of Rpi-amr3 is required for AVRamr3-dependent HR in N. benthamiana. Representative leaf phenotype of HR (hypersensitive response) in wild-type N. benthamiana is shown. The number of leaves tested and occurrences of HR are indicated in parentheses. B. P-loop of NRC2 is required for AVRamr3-dependent HR in N. benthamiana nrc2/3/4 knockout plants. Representative leaf phenotype of HR is shown. The number of leaves tested and occurrences of HR are indicated in parentheses.


C . NRC2EEE - Myc requires functional P - loop of both Rpi - amr3 and NRC2 for oligomerization . Protein lysates from N . benthamiana nrc2 / 3 / 4 knockout plants expressing wild - type or P - loop mutants of Rpi - amr3 or NRC2EEE were loaded for blue native - PAGE . Membranes were immunoblotted with anti - Myc .

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C. NRC2EEE-Myc requires functional P-loop of both Rpi-amr3 and NRC2 for oligomerization. Protein lysates from N. benthamiana nrc2/3/4 knockout plants expressing wild-type or P-loop mutants of Rpi-amr3 or NRC2EEE were loaded for blue native-PAGE. Membranes were immunoblotted with anti-Myc.


B . Protein lysates expressing NRC4AAA - Myc with Rpi - amr3 - HF or Rpi - amr1 - Flag in the presence and absence of cognate effector were loaded on blue native - PAGE . Proteins were transiently expressed in N . benthamiana nrc2 / 3 / 4 knockout plants . Non - specific bands are indicated with * .

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B. Protein lysates expressing NRC4AAA-Myc with Rpi-amr3-HF or Rpi-amr1-Flag in the presence and absence of cognate effector were loaded on blue native-PAGE. Proteins were transiently expressed in N. benthamiana nrc2/3/4 knockout plants. Non-specific bands are indicated with *.


B . NRC2EEE - Myc oligomerizes in response - dependent manner . Protein lysates from N . benthamiana nrc2 / 3 / 4 knockout transiently expressing NRC2EEE - Myc , Rpi - amr3 - HF and AVRamr3 alleles from P . infestans , P . capsici , and P . parasitica were loaded on blue native - PAGE and blotted for NRC2EEE ( anti - Myc ) . C . Recognition is correlated with interaction and protein complex formation of AVRamr3 with Rpi - amr3 . Protein extracts from Fig 5B were immunoprecipitated with anti - Flag antibody , separated on blue native - PAGE , and AVRamr3 - GFP proteins of P . infestans , P . capsici , and P . parasitica were visualized .

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B. NRC2EEE-Myc oligomerizes in response-dependent manner. Protein lysates from N. benthamiana nrc2/3/4 knockout transiently expressing NRC2EEE-Myc, Rpi-amr3-HF and AVRamr3 alleles from P. infestans, P. capsici, and P. parasitica were loaded on blue native-PAGE and blotted for NRC2EEE (anti-Myc). C. Recognition is correlated with interaction and protein complex formation of AVRamr3 with Rpi-amr3. Protein extracts from Fig 5B were immunoprecipitated with anti-Flag antibody, separated on blue native-PAGE, and AVRamr3-GFP proteins of P. infestans, P. capsici, and P. parasitica were visualized.


( A ) Nissl - stained sagittal brain sections reveal no change in hippocampal morphology between Clcn3unc / unc and Clcn3 + / + mice at P45 or after 20 months . In contrast , the hippocampus was absent ( indicated by asterisk ) in 3 months - old Clcn3 - / - mice ( scale bar : 200 µm ) .

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(A) Nissl-stained sagittal brain sections reveal no change in hippocampal morphology between Clcn3unc/unc and Clcn3+/+ mice at P45 or after 20 months. In contrast, the hippocampus was absent (indicated by asterisk) in 3 months-old Clcn3-/- mice (scale bar: 200 µm).


( B ) Nissl - stained paraffin sections show intact retinal layers in 20 months old Clcn3unc / un mice . Neurodegeneration in Clcn3 - / - mice however , results in a loss of retinal structure already at 11 weeks of age ( scale bar : 100 µm ) . RPE , retinal pigment epithelium ; OS , photoreceptor outer segments ; IS , photoreceptor inner segments ; ONL , outer nuclear layer ; OPL , outer plexiform layer ; INL , inner nuclear layer ; IPL , inner plexiform layer ; GCL , ganglion cell layer .

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(B) Nissl-stained paraffin sections show intact retinal layers in 20 months old Clcn3unc/un mice. Neurodegeneration in Clcn3-/- mice however, results in a loss of retinal structure already at 11 weeks of age (scale bar: 100 µm). RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.


( C ) Immunoblots for ClC - 3 , - 4 and - 5 of membrane fractions of WT ( + / + ) , Clcn3unc / unc ( unc / unc ) , Clcn3unc / + ( unc / + ) and Clcn3 - / - ( - / - ) mice . β - actin , loading control .

(C) Immunoblots for ClC-3, -4 and -5 of membrane fractions of WT (+/+), Clcn3unc/unc (unc/unc), Clcn3unc/+ (unc/+) and Clcn3-/- (-/-) mice. β-actin, loading control.


( D ) Representative Western blot for ClC - 4 of membrane fractions from brain and kidney of WT ( + / + ) , Clcn3 + / - ( + / - ) and Clcn3 - / - ( - / - ) mice . β - actin , loading control . ( E ) Quantification of ClC - 4 immunoblots including those shown in ( C and D ) ( normalized to actin ) . Mean values ± s . e . m . Average from ≥ 5 animals per genotype and at ≥ 2 immunoblots per animal . * * * p < 0 . 0005 , * * p < 0 . 005 , * p < 0 . 05 ( two - tailed unpaired t test ) .

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(D) Representative Western blot for ClC-4 of membrane fractions from brain and kidney of WT (+/+), Clcn3+/- (+/-) and Clcn3-/- (-/-) mice. β-actin, loading control. (E) Quantification of ClC-4 immunoblots including those shown in (C and D) (normalized to actin). Mean values ± s.e.m. Average from ≥5 animals per genotype and at ≥2 immunoblots per animal. *** p < 0.0005 , ** p < 0.005, * p < 0.05 (two-tailed unpaired t test).


( A ) Co - immunoprecipitation reveals a ClC - 3 - ClC - 4 complex . 10 % of solubilized brain membranes of WT and Clcn3unc / unc mice were directly loaded on the gel ( input , or first immunoprecipitated ( IP ) with antibodies against ClC - 3 or ClC - 4 . Western blots were probed for ClC - 3 and ClC - 4 . Equivalent amounts of lysates and precipitates were loaded . * unspecific band / contamination .

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(A) Co-immunoprecipitation reveals a ClC-3-ClC-4 complex. 10% of solubilized brain membranes of WT and Clcn3unc/unc mice were directly loaded on the gel (input, or first immunoprecipitated (IP) with antibodies against ClC-3 or ClC-4. Western blots were probed for ClC-3 and ClC-4. Equivalent amounts of lysates and precipitates were loaded. * unspecific band / contamination.


( B ) FRET experiments show homo - and heteromerization of ClC - 3 and ClC - 4 constructs ( fused to yellow fluorescent protein ( YFP ) and cyan fluorescent protein ( CFP ) ) overexpressed in COS - 7 cells . Co - expressed constructs are indicated . Graph represents energy transfer efficiencies from acceptor photobleaching , depicted as bleach corrected values ( subtraction of ClC - 4CFP alone ) . Mean values ± s . e . m . N = 20 ( ClC - 3YFP / ClC - 4CFP ) ; 33 ( ClC - 3YFP / ClC - 3CFP ) ; 26 ( ClC - 4YFP / ClC - 4CFP ) ; 42 ( ClC - 3YFP / Lamp - 1CFP ) cells . * * * p < 0 . 0005 ( two - tailed unpaired t test )

(B) FRET experiments show homo- and heteromerization of ClC-3 and ClC-4 constructs (fused to yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)) overexpressed in COS-7 cells. Co-expressed constructs are indicated. Graph represents energy transfer efficiencies from acceptor photobleaching, depicted as bleach corrected values (subtraction of ClC-4CFP alone). Mean values ± s.e.m. N = 20 (ClC-3YFP / ClC-4CFP); 33 (ClC-3YFP / ClC-3CFP); 26 (ClC-4YFP / ClC-4CFP); 42 (ClC-3YFP / Lamp-1CFP) cells. *** p < 0.0005 (two-tailed unpaired t test)


( C ) Immunolabelling shows subcellular localization of hClC - 4 ( top , green in merge ) , hClC - 3 ( middle , green in merge ) and hClC - 3unc ( bottom , green in merge ) of transiently transfected COS - 7 cells , in comparison to either PDI or Lamp - 1 ( both red ) as marker for the ER and late endosomes / lysosomes , respectively .

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(C) Immunolabelling shows subcellular localization of hClC-4 (top, green in merge), hClC-3 (middle, green in merge) and hClC-3unc (bottom, green in merge) of transiently transfected COS-7 cells, in comparison to either PDI or Lamp-1 (both red) as marker for the ER and late endosomes/lysosomes, respectively.


( D ) Co - localization of hClC - 3 and hClC - 4 in cytoplasmic vesicles of COS - 7 cells transfected with hClC - 4 cDNA ( hemagglutinin ( HA ) tagged ) together with either hClC - 3 or hClC - 3unc . Immunostaining used antibodies against ClC - 3 and HA tag ( green and red in merge , respectively ) .

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(D) Co-localization of hClC-3 and hClC-4 in cytoplasmic vesicles of COS-7 cells transfected with hClC-4 cDNA (hemagglutinin (HA) tagged) together with either hClC-3 or hClC-3unc. Immunostaining used antibodies against ClC-3 and HA tag (green and red in merge, respectively).


( A ) Clcn3unc / unc / Clcn4 - / - and Clcn3 - / - mice died within 3 - 4 weeks after birth . Approximately 20 % of the animals survived in either line ( Clcn3unc / unc / Clcn4 - / - , n = 136 and Clcn3 - / - , n = 187 ) . All Clcn3 - / - / Clcn4 - / - mice ( n = 4 ) died within 1 - 2 weeks after birth .

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(A) Clcn3unc/unc/Clcn4-/- and Clcn3-/- mice died within 3-4 weeks after birth. Approximately 20% of the animals survived in either line (Clcn3unc/unc/Clcn4-/-, n = 136 and Clcn3-/-, n = 187). All Clcn3-/-/Clcn4-/- mice (n= 4) died within 1-2 weeks after birth.


( B ) Nissl - stained paraffin sections show progressive neuronal cell loss ( arrows ) that begins in hippocampal CA1 region of P14 Clcn3unc / unc / Clcn4 - / - mice and results in a complete loss of the hippocampus at P28 . Neurodegeneration progresses slower in Clcn3 - / - mice Stobrawa et al , 2001 ( ) ( scale bar : 200 µm ) .

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(B) Nissl-stained paraffin sections show progressive neuronal cell loss (arrows) that begins in hippocampal CA1 region of P14 Clcn3unc/unc/Clcn4-/- mice and results in a complete loss of the hippocampus at P28. Neurodegeneration progresses slower in Clcn3-/- mice Stobrawa et al, 2001() (scale bar: 200 µm).


( C ) Semi - thin sections of P28 retinae revealed degeneration of photoreceptor cells in the outer nuclear layer and outer and inner segment of Clcn3unc / unc / Clcn4 - / - , but not of Clcn3unc / unc or Clcn4 - / - mice ( scale bar : 50 µm ) . RPE , retinal pigment epithelium ; OS , photoreceptor outer segments ; IS , photoreceptor inner segments ; ONL , outer nuclear layer ; OPL , outer plexiform layer ; INL , inner nuclear layer ; IPL , inner plexiform layer ; GCL , ganglion cell layer .

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(C) Semi-thin sections of P28 retinae revealed degeneration of photoreceptor cells in the outer nuclear layer and outer and inner segment of Clcn3unc/unc/Clcn4-/-, but not of Clcn3unc/unc or Clcn4-/- mice (scale bar: 50 µm). RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.


( A ) GFP antibodies immunolabel VenusClC - 3 ( green ) in the somata of CA1 pyramidal neurons of Clcn3ven / ven , but not Clcn3 + / + brain sections . sp , stratum pyramidale ( scale bar : 20 µm ) .

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(A) GFP antibodies immunolabel VenusClC-3 (green) in the somata of CA1 pyramidal neurons of Clcn3ven/ven, but not Clcn3+/+ brain sections. sp, stratum pyramidale (scale bar: 20 µm).


( B ) Co - immunolabelling of VenusClC - 3 ( green ) with ClC - 4 ( red ) in the CA3 region of Clcn3ven / ven mice ( scale bar : 20 µm ) . sl , stratum lucidum ; sp , stratum pyramidale .

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(B) Co-immunolabelling of VenusClC-3 (green) with ClC-4 (red) in the CA3 region of Clcn3ven/ven mice (scale bar: 20 µm). sl, stratum lucidum; sp, stratum pyramidale.


( C ) Co - immunostaining of VenusClC - 3 ( green ) with EEA1 ( red , left panel ) , Lamp - 1 ( red , middle panel ) or synaptophysin ( red , right panel ) in either CA1 or CA3 region of Clcn3ven / ven mice . Arrows indicate structures of overlap ( scale bar : 5 µm ) .

(C) Co-immunostaining of VenusClC-3 (green) with EEA1 (red, left panel), Lamp-1 (red, middle panel) or synaptophysin (red, right panel) in either CA1 or CA3 region of Clcn3ven/ven mice. Arrows indicate structures of overlap (scale bar: 5 µm).


( D ) GFP antibody immunolabels VenusClC - 3 ( green ) in neurites of cultured neurons . EEA1 and VGLUT1 are co - stained ( both in red ; left and right panel , respectively ) . The dendritic marker MAP2 is stained in blue ( right panel ) ( scale bars : 10 µm ) . DNA stained with DAPI .

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(D) GFP antibody immunolabels VenusClC-3 (green) in neurites of cultured neurons. EEA1 and VGLUT1 are co-stained (both in red; left and right panel, respectively). The dendritic marker MAP2 is stained in blue (right panel) (scale bars: 10 µm). DNA stained with DAPI.


( E ) Immuno - blot analysis of fractions collected during preparation of SVs from brain of Clcn3ven / ven mice . VenusClC - 3 ( detected with GFP antibody ) co - purified with SV marker protein synaptophysin .

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(E) Immuno-blot analysis of fractions collected during preparation of SVs from brain of Clcn3ven/ven mice. VenusClC-3 (detected with GFP antibody) co-purified with SV marker protein synaptophysin.


( F ) Fluorescence of immobilized Venus - tagged SVs imaged with TIRF microscopy . Scale bar , 2 µm .

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(F) Fluorescence of immobilized Venus-tagged SVs imaged with TIRF microscopy. Scale bar, 2 µm.


( G , H ) Co - localization between the Venus - tagged vesicles with antibodies against VGLUT1 and synaptophysin was analyzed . ( G ) 11 . 4 ± 0 . 6 ( SD ) of VGLUT1 - labeled SVs and 8 . 2 ± 2 ( SD ) of synaptophysin - labeled SVs showed Venus fluorescence ( n = 6 ) . ( H ) 65 ± 5 . 4 ( SD ) and 79 . 4 ± 3 . 8 ( SD ) of Venus - tagged SVs showed co - localization with VGLUT1 and synaptophysin , respectively ( n = 6 ) .

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(G, H) Co-localization between the Venus-tagged vesicles with antibodies against VGLUT1 and synaptophysin was analyzed. (G) 11.4 ± 0.6 (SD) of VGLUT1-labeled SVs and 8.2 ± 2 (SD) of synaptophysin-labeled SVs showed Venus fluorescence (n=6). (H) 65 ± 5.4 (SD) and 79.4 ± 3.8 (SD) of Venus-tagged SVs showed co-localization with VGLUT1 and synaptophysin, respectively (n=6).


( I ) Co - immunolabelling of VenusClC - 3 ( green ) with synaptophysin ( red ) of immobilized SVs imaged with TIRF microscopy . Vesicle in quadrant delimited by dashed line is shown in higher magnification in merge . Scale bar , 2 µm .

(I) Co-immunolabelling of VenusClC-3 (green) with synaptophysin (red) of immobilized SVs imaged with TIRF microscopy. Vesicle in quadrant delimited by dashed line is shown in higher magnification in merge. Scale bar, 2 µm.


( A ) Immunoblots for ClC - 3 , ClC - 4 , VGLUT1 , VGLUT2 , VGAT , GluR4 and GABAA - R - α1 of whole lysates from brain of Clcn3 + / + and Clcn3 - / - mice at 2 , 6 and 12 weeks of age .

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(A) Immunoblots for ClC-3, ClC-4, VGLUT1, VGLUT2, VGAT, GluR4 and GABAA-R-α1 of whole lysates from brain of Clcn3+/+ and Clcn3-/- mice at 2, 6 and 12 weeks of age.


( B ) Acidification of synaptic vesicle LP2 fractions from Clcn3unc / unc and Clcn3 + / + mice at one year of age ( upper panel , n = 2 animals each and ≥ 3 measurements per animal ) , 10 - weeks - old Clcn4 - / - and WT mice ( middle panel , n = 3 animals each and ≥ 3 measurements per animal ) , and 4 - 6 - weeks - old Clcn3unc / unc / Clcn4 - / - and control mice ( lower panel , 6 animals each with ≥ 2 measurements per animal ) . A decrease in fluorescence reflects acidification . The protonophore FCCP dissipated the pH gradient . Mean values ± s . e . m .

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(B) Acidification of synaptic vesicle LP2 fractions from Clcn3unc/unc and Clcn3+/+ mice at one year of age (upper panel, n= 2 animals each and ≥3 measurements per animal), 10-weeks-old Clcn4-/- and WT mice (middle panel, n= 3 animals each and ≥3 measurements per animal), and 4-6-weeks-old Clcn3unc/unc/Clcn4-/- and control mice (lower panel, 6 animals each with ≥2 measurements per animal). A decrease in fluorescence reflects acidification. The protonophore FCCP dissipated the pH gradient. Mean values ± s.e.m.


( C ) Quantification of ATP - induced acidification of LP2 fractions derived from 2 , 6 , and 10 - 12 weeks - old Clcn3 - / - compared to wild - type mice . Acidification measured by acridine orange fluorescence in the presence of 60 mM KCl . At least 2 animals in at least 2 independent experiments were pooled . Each measurement was performed at least 3 times . Mean ± s . e . m . is shown . * p < 0 . 05 , * * * p < 0 . 0005 ( two - tailed unpaired t test ) .

(C) Quantification of ATP-induced acidification of LP2 fractions derived from 2, 6, and 10-12 weeks-old Clcn3-/- compared to wild-type mice. Acidification measured by acridine orange fluorescence in the presence of 60 mM KCl. At least 2 animals in at least 2 independent experiments were pooled. Each measurement was performed at least 3 times. Mean ± s.e.m. is shown.* p < 0.05, *** p < 0.0005 (two-tailed unpaired t test).


( D ) Immunoblot revealed strong reduction of VGLUT1 levels in the homogenate ( H ) and LP2 fraction of Clcn3unc / unc / Clcn4 - / - mice .

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(D) Immunoblot revealed strong reduction of VGLUT1 levels in the homogenate (H) and LP2 fraction of Clcn3unc/unc/Clcn4-/- mice.


( E ) Endosomal pH of Clcn3 + / + and Clcn3 - / - primary hippocampal neurons determined with a pH - sensitive transferrin conjugate . N = 5 independent cell lines with at least 2 live cell dishes per cell line with about 10 images each and ≥ 10 Tfn - positive compartments per image and genotype were analysed . Mean ± s . e . m . is shown .

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(E) Endosomal pH of Clcn3+/+ and Clcn3-/- primary hippocampal neurons determined with a pH-sensitive transferrin conjugate. N= 5 independent cell lines with at least 2 live cell dishes per cell line with about 10 images each and ≥10 Tfn-positive compartments per image and genotype were analysed. Mean ± s.e.m. is shown.


Miniature inhibitory postsynaptic currents ( mIPSCs ) measured in CA1 pyramidal neurons in different mouse models at P14 - P16 . ( A ) example mIPSCs of WT ( left ) and Clcn3 - / - neurons .

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

Miniature inhibitory postsynaptic currents (mIPSCs) measured in CA1 pyramidal neurons in different mouse models at P14-P16. (A) example mIPSCs of WT (left) and Clcn3-/- neurons.


Mean values ± s . e . m . and cumulative histogram of amplitude ( top ) and frequency / interevent - interval ( bottom ) of mIPSCs in different mouse - models as indicated . Several cells of 3 mice of each genotype were analyzed . Compared genotypes were siblings . No difference in amplitude or frequency / interevent - interval distribution of mIPSCs was found in ( B ) Clcn3 - / - and Clcn3 + / + ( Clcn3 + / + : 16 cells , Clcn3 - / - : 15 cells ; mean amplitude : p > 0 . 8 , t test ; mean frequency : p > 0 . 4 , MW - test ) , in ( C ) Clcn3unc / unc and Clcn3 + / + ( Clcn3 + / + : 21 cells , Clcn3unc / unc : 13 cells ; mean amplitude : p > 0 . 7 ; mean frequency : p = 0 . 3 , t test ) , and in ( D ) Clcn4 - / - and Clcn3 + / + ( Clcn3 + / + : 18 cells , Clcn4 - / - : 18 cells ; mean amplitude : p > 0 . 3 , t - test ; mean frequency : p = 0 . 6 , MW - test ) .

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

Mean values ± s.e.m. and cumulative histogram of amplitude (top) and frequency/interevent-interval (bottom) of mIPSCs in different mouse-models as indicated . Several cells of 3 mice of each genotype were analyzed. Compared genotypes were siblings. No difference in amplitude or frequency/ interevent-interval distribution of mIPSCs was found in (B) Clcn3-/- and Clcn3+/+ (Clcn3+/+: 16 cells, Clcn3-/-: 15 cells; mean amplitude: p > 0.8, t test; mean frequency: p > 0.4, MW-test), in (C) Clcn3unc/unc and Clcn3+/+ (Clcn3+/+: 21 cells, Clcn3unc/unc: 13 cells; mean amplitude: p > 0.7; mean frequency: p = 0.3, t test), and in (D) Clcn4-/- and Clcn3+/+ (Clcn3+/+: 18 cells, Clcn4-/-: 18 cells; mean amplitude: p > 0.3, t-test; mean frequency: p = 0.6, MW-test).


( E ) No difference in mean amplitude ( p > 0 . 5 , t test ) , in cumulative amplitude and mean frequency of mIPSCs ( p > 0 . 1 , t test ) was found in Clcn3unc / unc / Clcn4 - / - and Clcn3 + / + / Clcn4 - / - . The interevent - interval distribution was shifted to the right , indicating a reduced frequency of mIPSCs in Clcn3unc / unc / Clcn4 - / - ( KS - test , p < 0 , 05 ) ( Clcn3 + / + / Clcn4 - / - : 16 cells , Clcn3unc / unc / Clcn4 - / - : 12 cells ) .

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

(E) No difference in mean amplitude (p > 0.5, t test), in cumulative amplitude and mean frequency of mIPSCs (p > 0.1, t test) was found in Clcn3unc/unc/Clcn4-/- and Clcn3+/+/Clcn4-/-. The interevent-interval distribution was shifted to the right, indicating a reduced frequency of mIPSCs in Clcn3unc/unc/Clcn4-/- (KS-test, p < 0,05) (Clcn3+/+/Clcn4-/-: 16 cells, Clcn3unc/unc/Clcn4-/-: 12 cells).


( F , G ) miniature excitatory postsynaptic currents ( mEPSCs ) . ( F ) example traces of mEPSCs in WT and Clcn3 - / - neurons ( G ) No significant difference between Clcn3unc / unc / Clcn4 - / - and Clcn3 + / + / Clcn4 - / - mEPSCs in mean amplitudes ( p > 0 . 9 , t test ) or in the distribution of amplitudes ( Clcn4 - / - : 12 cells , 2 mice ; Clcn3unc / unc / Clcn4 - / - : 8 cells , 2 mice ) .

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

(F,G) miniature excitatory postsynaptic currents (mEPSCs). (F) example traces of mEPSCs in WT and Clcn3-/- neurons (G) No significant difference between Clcn3unc/unc/Clcn4-/- and Clcn3+/+/Clcn4-/- mEPSCs in mean amplitudes (p > 0.9, t test) or in the distribution of amplitudes (Clcn4-/-: 12 cells, 2 mice; Clcn3unc/unc/Clcn4-/-: 8 cells, 2 mice).


( H ) Primary hippocampal neurons co - transfected with ClC - 3 and synaptobrevin ( syb2 ) , both luminally tagged with pHluorin or the pH - sensitive mOrange Ramirez et al , 2012 ( ) , respectively , were stimulated by 200 action potentials ( APs ) at 20 Hz ( indicated by bar ) . Vesicle exocytosis was monitored by fluorescence increase upon exposure to extracellular neutral pH . Note that only Syb2 and not ClC - 3 shows signs of exocytosis . Traces were corrected for photobleaching . The initial decay of the pHluorin signal is probably due to the high light exposure , which forces the fluorophores into a transient dark - state Dean et al , 2011 ( ) . Averaged fluorescence traces ( n = 10 ) , error bars , s . e . m .

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

(H) Primary hippocampal neurons co-transfected with ClC-3 and synaptobrevin (syb2), both luminally tagged with pHluorin or the pH-sensitive mOrange Ramirez et al, 2012(), respectively, were stimulated by 200 action potentials (APs) at 20 Hz (indicated by bar). Vesicle exocytosis was monitored by fluorescence increase upon exposure to extracellular neutral pH. Note that only Syb2 and not ClC-3 shows signs of exocytosis. Traces were corrected for photobleaching. The initial decay of the pHluorin signal is probably due to the high light exposure, which forces the fluorophores into a transient dark-state Dean et al, 2011(). Averaged fluorescence traces (n=10), error bars, s.e.m.


( C ) day 20 ± 2 cerebral organoids derived from controls , patients and EML1 - heKOs stained for the neuronal marker MAPT and the adherens junction marker NCAD , counterstained with DAPI . Ectopic neural rosettes and neuronal heterotopia are highlighted with dotted yellow lines . ( D ) Quantification of VZ areas with ectopic neural rosettes ( 3 batches , 3 organoids analyzed per batch , significance based on Kruskal - Wallis - test , P = 0 . 0001 , Dunn ' s post hoc test for multiple comparisons performed to define statistical differences between genotypes , single data points are presented colored by batch ) . ( E ) Quantification of heterotopic , disorganized or organized cortical areas ( 3 batches , 3 organoids per batch , significance based on Kruskal - Wallis - test , P = 0 . 0001 for " organized " and " heterotopic " ; no significant difference for " disorganized " , Dunn ' s post hoc test performed for multiple comparisons to define statistical differences between genotypes ) . Data information : * marks statistical significance in relation to Control 1 , # in relation to Control 2 . P - values : * * * / # # # < 0 . 001 . VZ : ventricular zone ; Data in graphs are represented as means ± SD . Scale bars : 50µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) day 20±2 cerebral organoids derived from controls, patients and EML1-heKOs stained for the neuronal marker MAPT and the adherens junction marker NCAD, counterstained with DAPI. Ectopic neural rosettes and neuronal heterotopia are highlighted with dotted yellow lines. (D) Quantification of VZ areas with ectopic neural rosettes (3 batches, 3 organoids analyzed per batch, significance based on Kruskal-Wallis-test, P=0.0001, Dunn's post hoc test for multiple comparisons performed to define statistical differences between genotypes, single data points are presented colored by batch). (E) Quantification of heterotopic, disorganized or organized cortical areas (3 batches, 3 organoids per batch, significance based on Kruskal-Wallis-test, P=0.0001 for "organized" and "heterotopic"; no significant difference for "disorganized", Dunn's post hoc test performed for multiple comparisons to define statistical differences between genotypes). Data information: * marks statistical significance in relation to Control 1, # in relation to Control 2. P-values: ***/###<0.001. VZ: ventricular zone; Data in graphs are represented as means ± SD. Scale bars: 50µm.


( A ) Hybrid organoids generated from controls mixed with control - EGFP + or EML1 - heKO - EGFP + cells ( day 20 ± 2 ) . Yellow dotted lines indicate VZ areas . ( B ) Quantification of control - EGFP + or EML1 - heKO - EGFP + cells within or at the basal side of VZ areas ( 3 batches , 3 organoids each , significance based on Mann Whitney test , * * * P = 0 . 0001 for " EGFP + cells at the basal side of VZ areas " ) . Data information : Data in graphs are represented as means ± SD . Scale bars : ( A )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Hybrid organoids generated from controls mixed with control-EGFP+ or EML1-heKO-EGFP+ cells (day 20±2). Yellow dotted lines indicate VZ areas. (B) Quantification of control-EGFP+ or EML1-heKO-EGFP+ cells within or at the basal side of VZ areas (3 batches, 3 organoids each, significance based on Mann Whitney test, ***P= 0.0001 for "EGFP+ cells at the basal side of VZ areas"). Data information: Data in graphs are represented as means ± SD. Scale bars: (A) 50µm


( C ) Representative images and scheme of cleavage plan orientation . Upper panel : cells immunostained for pVim ( red ) and TPX2 ( green ) , counterstained with DAPI , white dotted line indicates cleavage plane orientation , yellow dotted line VZ - surface . ( D ) Quantification of horizontal , vertical and oblique plane of cell divisions in control and EML1 - deficient organoids ( day 20 ± 2 ; 3 batches , 3 organoids each ) . Data information : Scale bars :

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images and scheme of cleavage plan orientation. Upper panel: cells immunostained for pVim (red) and TPX2 (green), counterstained with DAPI, white dotted line indicates cleavage plane orientation, yellow dotted line VZ-surface. (D) Quantification of horizontal, vertical and oblique plane of cell divisions in control and EML1-deficient organoids (day 20±2; 3 batches, 3 organoids each). Data information: Scale bars: 5µm


( F ) Electron microscopy of primary cilia derived from control or EML1 - deficient cortical progenitors in DMSO - control or following EpothiloneD ( EpoD ) exposure ( blue arrow : basal body , yellow arrow : appendages , red arrow : ciliary pockets ) . Data information : Scale bars : 0 , 5µm .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O 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) Electron microscopy of primary cilia derived from control or EML1-deficient cortical progenitors in DMSO-control or following EpothiloneD (EpoD) exposure (blue arrow: basal body, yellow arrow: appendages, red arrow: ciliary pockets). Data information: Scale bars: 0,5µm.


( E ) Control and EML1 - heKO - derived cerebral organoid stained for COL1A2 and MEIS2 . VZ areas are encircled , squares indicate areas enlarged in F . ( F ) Control and EML1 - heKO - derived cerebral organoid stained for COL1A2 , MEIS2 and MAPT . ( G ) Representative images showing EGFP - control and EGFP - EML1 - heKO hybrid cerebral organoid stained for EGFP , FAM107A and NCAD . Yellow dotted lines indicate morphology of EGFP + cells ( 4 independent batches each , 3 organoids per batch , at least 30 ectopic rosettes in total ) . Data information : Scale bars : ( E ) 50µm ; ( F ) 10µm ; ( G ) 50µm , enlarged 10µm .

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

(E) Control and EML1-heKO-derived cerebral organoid stained for COL1A2 and MEIS2. VZ areas are encircled, squares indicate areas enlarged in F. (F) Control and EML1-heKO-derived cerebral organoid stained for COL1A2, MEIS2 and MAPT. (G) Representative images showing EGFP-control and EGFP-EML1-heKO hybrid cerebral organoid stained for EGFP, FAM107A and NCAD. Yellow dotted lines indicate morphology of EGFP+ cells (4 independent batches each, 3 organoids per batch, at least 30 ectopic rosettes in total). Data information: Scale bars: (E) 50µm; (F) 10µm; (G) 50µm, enlarged 10µm.


( D , E ) Immunofluorescence staining for ( D ) p - VIM and YAP1 or ( E ) Pax6 and YAP1 in control and EML1 - heKO - derived organoids ( day 20 ± 2 , squares indicate areas enlarged in adjacent part of the panel respectively ) . ( F ) Quantification of basally located pVim + cells with nuclear YAP1 signal per VZ area in control and EML1 - heKO - derived organoids ( 3 batches , 3 organoids analyzed per batch ) . ( G ) Quantification of PAX6 + cells with nuclear YAP1 signal per VZ area in control and EML1 - heKO - derived organoids ( 3 batches , 3 organoids analyzed per batch ) . Data information : Data in graphs are represented as means ± SD . Significance based on Kruskal - Wallis - test Dunn ' s post hoc test for multiple comparisons were performed to define statistical differences between genotypes . P - values : * * * < 0 . 001 , * * < 0 . 01 , * < 0 . 05 . Scale bars : ( D and E ) 50µm , enlarged

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

(D, E) Immunofluorescence staining for (D) p-VIM and YAP1 or (E) Pax6 and YAP1 in control and EML1-heKO-derived organoids (day 20±2, squares indicate areas enlarged in adjacent part of the panel respectively). (F) Quantification of basally located pVim+ cells with nuclear YAP1 signal per VZ area in control and EML1-heKO-derived organoids (3 batches, 3 organoids analyzed per batch). (G) Quantification of PAX6+ cells with nuclear YAP1 signal per VZ area in control and EML1-heKO-derived organoids (3 batches, 3 organoids analyzed per batch). Data information: Data in graphs are represented as means ± SD. Significance based on Kruskal-Wallis-test Dunn's post hoc test for multiple comparisons were performed to define statistical differences between genotypes. P-values: ***<0.001, **<0.01, *<0.05. Scale bars: (D and E) 50µm, enlarged 10µm


( H ) Immunofluorescence staining for p - Vim in EML1 - heKO and control derived organoids in DMSO , Verteporfin or Fluvastatin treated conditions , counterstained with DAPI . Dotted line indicates VZ areas . ( I ) Quantification of p - VIM + cells located at the basal side of VZ areas in control or EML1 - heKO - derived organoids under DMSO , Verteporfin or Fluvastatin treated condition ( 3 batches , 3 organoids analyzed per batch ) . Data information : Data in graphs are represented as means ± SD . Significance based on two - way ANOVA ( on log10 normalized data ) Tukey post hoc test for multiple comparisons were performed to define statistical differences between genotypes . P - values : * * * < 0 . 001 , * * < 0 . 01 , * < 0 . 05 . Scale bars : ( H )

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

(H) Immunofluorescence staining for p-Vim in EML1-heKO and control derived organoids in DMSO, Verteporfin or Fluvastatin treated conditions, counterstained with DAPI. Dotted line indicates VZ areas. (I) Quantification of p-VIM+ cells located at the basal side of VZ areas in control or EML1-heKO-derived organoids under DMSO, Verteporfin or Fluvastatin treated condition (3 batches, 3 organoids analyzed per batch). Data information: Data in graphs are represented as means ± SD. Significance based on two-way ANOVA (on log10 normalized data) Tukey post hoc test for multiple comparisons were performed to define statistical differences between genotypes. P-values: ***<0.001, **<0.01, *<0.05. Scale bars: (H) 50µm


A . Binding of ubiquitin chains to immobilized GST - fusion proteins . Indicated proteins were purified from E . coli and incubated with equal amounts of preformed fluorescently labeled ubiquitin chains , which were either K48 - or K63 - linked . The fraction of ubiquitin chains interacting with the bait protein was analyzed by SDS ‑ PAGE and fluorescence scan . Total fluorescence was quantified per lane and normalized to the first lane ( rel . fluo . ) . Asterisk ( * ) indicates lane from the same gel that was cropped and moved .

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

A. Binding of ubiquitin chains to immobilized GST-fusion proteins. Indicated proteins were purified from E. coli and incubated with equal amounts of preformed fluorescently labeled ubiquitin chains, which were either K48- or K63-linked. The fraction of ubiquitin chains interacting with the bait protein was analyzed by SDS‑PAGE and fluorescence scan. Total fluorescence was quantified per lane and normalized to the first lane (rel. fluo.). Asterisk (*) indicates lane from the same gel that was cropped and moved.


B . At indicated time points , samples were taken from the reaction mix and analyzed by SDS ‑ PAGE and fluorescence scan . Product fluorescence over total fluorescence per lane was quantified ( rel . fluo . ) . D . Initial reaction rates were determined for a panel of acceptor ubiquitin molecules in reactions with either full ‑ length Ubc1 or Ubc1 - ΔUBA ( aa1 ‑ 150 ) . The binding of the UBA domain to individual ubiquitin molecules was disrupted by introducing an R42A amino acid exchange in ubiquitin , while acceptor sites were blocked by introducing the K48R substitution . Reactions were performed in triplicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR 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. At indicated time points, samples were taken from the reaction mix and analyzed by SDS‑PAGE and fluorescence scan. Product fluorescence over total fluorescence per lane was quantified (rel. fluo.). D. Initial reaction rates were determined for a panel of acceptor ubiquitin molecules in reactions with either full‑length Ubc1 or Ubc1-ΔUBA (aa1‑150). The binding of the UBA domain to individual ubiquitin molecules was disrupted by introducing an R42A amino acid exchange in ubiquitin, while acceptor sites were blocked by introducing the K48R substitution. Reactions were performed in triplicate.


b . L143E and / or L147E amino acid substitutions were introduced into Ubc1 to disrupt the hydrophobic UBC / UBA domain interface . Single turnover ubiquitination experiments were performed with these Ubc1 variants in presence of K63 ‑ Ub2 with Ub ( K48R ) at the distal position or monoubiquitin as acceptor . Experiment and initial rate

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

b. L143E and/or L147E amino acid substitutions were introduced into Ubc1 to disrupt the hydrophobic UBC/UBA domain interface. Single turnover ubiquitination experiments were performed with these Ubc1 variants in presence of K63‑Ub2 with Ub(K48R) at the distal position or monoubiquitin as acceptor. Experiment and initial rate determination


c . Ubc1 variants were generated with deletions in the linker region ( aa152 ‑ 167 ) , which connects the UBA domain and the UBC domain . Single turnover ubiquitination reaction mixes were incubated for 5 min with K63 - Ub2 or for 20 min with monoubiquitin as acceptor . Final product formation was analyzed by SDS - PAGE and fluorescence scan . Product fluorescence over total fluorescence ( rel . fluo . ) was quantified .

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

c. Ubc1 variants were generated with deletions in the linker region (aa152‑167), which connects the UBA domain and the UBC domain. Single turnover ubiquitination reaction mixes were incubated for 5 min with K63-Ub2 or for 20 min with monoubiquitin as acceptor. Final product formation was analyzed by SDS-PAGE and fluorescence scan. Product fluorescence over total fluorescence (rel. fluo.) was quantified.


d . C - terminally blocked M1 - linked diubiquitin with or without a K48R amino acid substitution at the distal or proximal position were employed in single turnover experiments with full - length Ubc1 or Ubc1 - ΔUBA . Reactions with acceptor K63 - Ub2 were carried out controls . Initial rate

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. C-terminally blocked M1-linked diubiquitin with or without a K48R amino acid substitution at the distal or proximal position were employed in single turnover experiments with full-length Ubc1 or Ubc1-ΔUBA. Reactions with acceptor K63-Ub2 were carried out controls. Initial rate determination


e . CSPs of I44 were plotted over UBA domain concentration . f . KD values for binding of the UBA domain were determined from results in ( E ) assuming two independent but different binding sites .

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

e. CSPs of I44 were plotted over UBA domain concentration. f. KD values for binding of the UBA domain were determined from results in (E) assuming two independent but different binding sites.


a . The activity of Ubc1 was compared with the K48 - linked chain building enzyme Ubc7 / Cue1 in single turnover ubiquitination experiments in presence of either monoubiquitin , K48 ‑ Ub2 or K63 - Ub2 . Initial rates were

O O O O 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

a. The activity of Ubc1 was compared with the K48-linked chain building enzyme Ubc7/Cue1 in single turnover ubiquitination experiments in presence of either monoubiquitin, K48‑Ub2 or K63-Ub2. Initial rates were determined


b . The K63 - linked chain building enzyme Ubc13 with its cofactor Uev1a or the Ubc1 homologue Ube2K ( h . sapiens ) were employed in single turnover ubiquitination experiments in presence of K63 - Ub2 with Ub ( K48R ) at the proximal or distal position or in presence of K48 ‑ Ub2 . Initial rate determination c . Experiment as in ( B ) but with the Ubc1 homologue ubc - 20 ( c . elegans ) .

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

b. The K63-linked chain building enzyme Ubc13 with its cofactor Uev1a or the Ubc1 homologue Ube2K (h. sapiens) were employed in single turnover ubiquitination experiments in presence of K63-Ub2 with Ub(K48R) at the proximal or distal position or in presence of K48‑Ub2. Initial rate determination c. Experiment as in (B) but with the Ubc1 homologue ubc-20 (c. elegans).


D . Alexa488 - labeled monoubiquitin and C - terminally capped K63 - Ub4 were used in a 4 : 1 molar ratio for in vitro ubiquitination reactions with full ‑ length Ubc1 or Ubc1 - ΔUBA . Where indicated , Alexa488 - labeled monoubiquitin was methylated prior to the reaction ( met - Ub ) to block de novo chain assembly . Reactions were incubated overnight , quenched and half the reaction mix from each condition was treated with the K63 specific deubiquitinating enzyme AMSH ( lanes 4 - 6 ) . Samples were analyzed by SDS - PAGE and fluorescence scan .

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D. Alexa488-labeled monoubiquitin and C-terminally capped K63-Ub4 were used in a 4:1 molar ratio for in vitro ubiquitination reactions with full‑length Ubc1 or Ubc1-ΔUBA. Where indicated, Alexa488-labeled monoubiquitin was methylated prior to the reaction (met-Ub) to block de novo chain assembly. Reactions were incubated overnight, quenched and half the reaction mix from each condition was treated with the K63 specific deubiquitinating enzyme AMSH (lanes 4-6). Samples were analyzed by SDS-PAGE and fluorescence scan.


F . Ubc1 - EA was genomically integrated into various yeast strains . Exponentially growing cells of the indicated genotype were spotted onto YPD plates in serial 10 ‑ fold dilutions . Where noted cells were incubated at elevated temperature ( 37 ° C ) or in presence of hydroxyurea ( HU ) to induce DNA replication stress .

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F. Ubc1-EA was genomically integrated into various yeast strains. Exponentially growing cells of the indicated genotype were spotted onto YPD plates in serial 10‑fold dilutions. Where noted cells were incubated at elevated temperature (37 °C) or in presence of hydroxyurea (HU) to induce DNA replication stress.


A . 293T cells were co - transfected with CMV - renilla , 8X - TBS ( eight repeats of the TEAD - binding - sequence ) luciferase reporter , Flag - YAP , HA - TEAD2 and wild - type ( WT ) or catalytically inactive ( CS ) USP9X - V5 . One day after transfection , luciferase activity was measured and normalized with respect to renilla activity . The value for Flag - YAP / HA - TEAD - transfected cells ( 2nd column ) was adjusted to 1 . Shown below is a representative Western blot showing that the expression levels were comparable between samples ( n = 4 ) . Error bars indicate the S . E . M . ( * p < 0 . 05 , * * p < 0 . 01 ; paired Student ' s t - test ) .

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A. 293T cells were co-transfected with CMV-renilla, 8X-TBS (eight repeats of the TEAD-binding-sequence) luciferase reporter, Flag-YAP, HA-TEAD2 and wild-type (WT) or catalytically inactive (CS) USP9X-V5. One day after transfection, luciferase activity was measured and normalized with respect to renilla activity. The value for Flag-YAP/HA-TEAD-transfected cells (2nd column) was adjusted to 1. Shown below is a representative Western blot showing that the expression levels were comparable between samples (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01; paired Student's t-test).


B . 293T cells were transfected with control or USP9X - targeting siRNAs . One day after siRNA transfection , the cells were co - transfected with CMV - renilla , 8X - TBS luciferase , Flag - YAP and HA - TEAD2 . Reporter activity was quantified as described in ( A ) ( n = 4 ) . Error bars indicate the S . E . M . ( * * * p < 0 . 001 ; paired Student ' s t - test ) .

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B. 293T cells were transfected with control or USP9X-targeting siRNAs. One day after siRNA transfection, the cells were co-transfected with CMV-renilla, 8X-TBS luciferase, Flag-YAP and HA-TEAD2. Reporter activity was quantified as described in (A) (n=4). Error bars indicate the S.E.M. (***p < 0.001; paired Student's t-test).


C . RPE or MCF10A cells were transfected with the indicated siRNAs for 24 hours and then re - seeded to either sparse or dense culture conditions . At 48 hours after siRNA transfection , the cells were harvested and cell extracts were analyzed by Western blotting for the indicated proteins . L . E . , long exposure , S . E . , short exposure .

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C. RPE or MCF10A cells were transfected with the indicated siRNAs for 24 hours and then re-seeded to either sparse or dense culture conditions. At 48 hours after siRNA transfection, the cells were harvested and cell extracts were analyzed by Western blotting for the indicated proteins. L.E., long exposure, S.E., short exposure.


D . Cells were treated as described in ( C ) , the indicated mRNAs were analyzed with RT - qPCR , and the results were normalized with respect to the β - actin mRNA ( n = 4 ) . Error bars indicate the S . E . M . ( * p < 0 . 05 , * * p < 0 . 01 ; paired Student ' s t - test ) .

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D. Cells were treated as described in (C), the indicated mRNAs were analyzed with RT-qPCR, and the results were normalized with respect to the β-actin mRNA (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01; paired Student's t-test).


E . RPE cells were transfected with the indicated siRNAs for 24 hours , and then co - transfected with CMV - renilla and 8X - TBS luciferase . One day after the latter transfection , the cells were re - seeded to either sparse or dense conditions , and reporter activity was measured at 24 hours after re - seeding ( n = 3 ) . Error bars indicate the S . E . M . ( * * p < 0 . 01 ; paired Student ' s t - test ) .

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E. RPE cells were transfected with the indicated siRNAs for 24 hours, and then co-transfected with CMV-renilla and 8X-TBS luciferase. One day after the latter transfection, the cells were re-seeded to either sparse or dense conditions, and reporter activity was measured at 24 hours after re-seeding (n=3). Error bars indicate the S.E.M. (**p < 0.01; paired Student's t-test).


A . Representative anti - YAP immunofluorescence images of sparsely cultured RPE cells transfected with the indicated siRNAs . Green , YAP . Blue , DAPI . Scale bar , 10 μm .

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A. Representative anti-YAP immunofluorescence images of sparsely cultured RPE cells transfected with the indicated siRNAs. Green, YAP. Blue, DAPI. Scale bar, 10 μm.


B . The nuclear YAP intensities of the cells shown in ( A ) were quantified using the Image J software . At least 70 cells from five or six random fields were analyzed . Quantified values were normalized by adjusting the average of " siControl Dense cells " to 1 . We performed two independent experiments and obtained similar results . Data from one representative experiment are presented . The red bar indicates the average value . S , Sparse . D , Dense ( * * * p < 0 . 001 ; paired Student ' s t - test ) .

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B. The nuclear YAP intensities of the cells shown in (A) were quantified using the Image J software. At least 70 cells from five or six random fields were analyzed. Quantified values were normalized by adjusting the average of "siControl Dense cells" to 1. We performed two independent experiments and obtained similar results. Data from one representative experiment are presented. The red bar indicates the average value. S, Sparse. D, Dense (***p < 0.001; paired Student's t-test).


C . Sparsely cultured RPE or MCF10A cells were transfected with the indicated siRNAs , fractionated into cytosolic and nuclear extracts , and analyzed by Western blotting for the indicated proteins .

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C. Sparsely cultured RPE or MCF10A cells were transfected with the indicated siRNAs, fractionated into cytosolic and nuclear extracts, and analyzed by Western blotting for the indicated proteins.


D . Representative anti - YAP immunofluorescence images of densely cultured RPE cells transfected with the indicated plasmids . Green , GFP or V5 . Red , YAP . Blue , DAPI . Scale bar , 10 μm .

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D. Representative anti-YAP immunofluorescence images of densely cultured RPE cells transfected with the indicated plasmids. Green, GFP or V5. Red, YAP. Blue, DAPI. Scale bar, 10 μm.


E . The nuclear YAP intensities of transfected cells in ( D ) were quantified using the Image J software . At least 70 cells from five to six random fields were analyzed . Quantified values were normalized by adjusting the average of " EGFP transfected cells " to 1 . Similar results were obtained from two independent experiments . Data from one representative experiment are presented . The red bar indicates the average value . ( * * * p < 0 . 001 ; paired Student ' s t - test ) .

E. The nuclear YAP intensities of transfected cells in (D) were quantified using the Image J software. At least 70 cells from five to six random fields were analyzed. Quantified values were normalized by adjusting the average of "EGFP transfected cells" to 1. Similar results were obtained from two independent experiments. Data from one representative experiment are presented. The red bar indicates the average value. (***p < 0.001; paired Student's t-test).


F . Densely cultured stable RPE clones integrated with empty vector ( Vec ) , USP9X - V5 WT or CS mutant were fractionated into cytosolic and nuclear extracts , and analyzed by Western blotting for the indicated proteins .

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F. Densely cultured stable RPE clones integrated with empty vector (Vec), USP9X-V5 WT or CS mutant were fractionated into cytosolic and nuclear extracts, and analyzed by Western blotting for the indicated proteins.


G . Densely cultured RPE cells transduced with CRISPR - SAM targeting USP9X were fractionated into cytosolic and nuclear extracts , and analyzed by Western blotting for the indicated proteins .

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G. Densely cultured RPE cells transduced with CRISPR-SAM targeting USP9X were fractionated into cytosolic and nuclear extracts, and analyzed by Western blotting for the indicated proteins.


A . RPE cells were analyzed by Western blotting for the indicated proteins .

A. RPE cells were analyzed by Western blotting for the indicated proteins.


B . Indicated stable RPE clones in densely cultured conditions were analyzed by Western blotting for the indicated proteins .

B. Indicated stable RPE clones in densely cultured conditions were analyzed by Western blotting for the indicated proteins.


C . Densely cultured RPE cells transduced with CRISPR - SAM targeting USP9X were analyzed by Western blotting for the indicated proteins .

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C. Densely cultured RPE cells transduced with CRISPR-SAM targeting USP9X were analyzed by Western blotting for the indicated proteins.


D . RPE or MCF10A cells expressing the control or USP9X shRNA were transfected with the LATS1 / 2 siRNA as indicated . Cells were re - seeded to low density and harvested after one day .

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D. RPE or MCF10A cells expressing the control or USP9X shRNA were transfected with the LATS1/2 siRNA as indicated. Cells were re-seeded to low density and harvested after one day.


E . RPE cells transduced with Flag - YAP WT or Flag - YAP 5SA were transfected with the indicated siRNAs . Cells were re - seeded to low density and harvested after one day .

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E. RPE cells transduced with Flag-YAP WT or Flag-YAP 5SA were transfected with the indicated siRNAs. Cells were re-seeded to low density and harvested after one day.


F . RT - qPCR of CTGF mRNAs in the cells described in ( E ) ( n = 3 ) . Error bars indicate the S . E . M . ( * p < 0 . 05 , paired Student ' s t - test ) .

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F. RT-qPCR of CTGF mRNAs in the cells described in (E) (n=3). Error bars indicate the S.E.M. (*p< 0.05, paired Student's t-test).


A . RPE or MCF10A cells were transfected with the indicated combinations of siRNAs and re - seeded to low density , and cell extracts were analyzed by Western blotting for the indicated proteins .

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A. RPE or MCF10A cells were transfected with the indicated combinations of siRNAs and re-seeded to low density, and cell extracts were analyzed by Western blotting for the indicated proteins.


B . The cells described in ( A ) were analyzed by RT - qPCR for the indicated target genes ( n = 4 ) . Error bars indicate the S . E . M . ( * p < 0 . 05 , * * p < 0 . 01 ; paired Student ' s t - test ) .

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B. The cells described in (A) were analyzed by RT-qPCR for the indicated target genes (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01; paired Student's t-test).


C . Sparsely cultured RPE cells transfected with the indicated siRNAs were immunoprecipitated with an anti - AMOTL2 antibody and analyzed by Western blotting for the indicated proteins . WCL , whole cell lysates .

C. Sparsely cultured RPE cells transfected with the indicated siRNAs were immunoprecipitated with an anti-AMOTL2 antibody and analyzed by Western blotting for the indicated proteins. WCL, whole cell lysates.


A . 293T cells were transfected with the indicated siRNAs , cultured for 24 hours , and then transfected with the indicated DNAs . 48 hours after siRNA transfection , the cells were harvested and ubiquitination of Flag - AMOTL2 was examined . C , control siRNA .

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A. 293T cells were transfected with the indicated siRNAs, cultured for 24 hours, and then transfected with the indicated DNAs. 48 hours after siRNA transfection, the cells were harvested and ubiquitination of Flag-AMOTL2 was examined. C, control siRNA.


B . 293T cells were transfected with the indicated combinations of DNAs , cultured for 24 hours , and subjected to in vivo ubiquitination assays .

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B. 293T cells were transfected with the indicated combinations of DNAs, cultured for 24 hours, and subjected to in vivo ubiquitination assays.


C . RPE cells transduced with vector ( control ) or Flag - AMOTL2 were seeded under sparse ( S ) or dense ( D ) conditions , and an in vivo ubiquitination assay was performed .

C. RPE cells transduced with vector (control) or Flag-AMOTL2 were seeded under sparse (S) or dense (D) conditions, and an in vivo ubiquitination assay was performed.


D . RPE cells transduced with Flag - AMOTL2 were transfected with a 1 : 1 mixture of the two USP9X siRNAs . One day after siRNA transfection , the cells were re - seeded to the sparse condition and an in vivo ubiquitination assay was performed after one day .

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D. RPE cells transduced with Flag-AMOTL2 were transfected with a 1:1 mixture of the two USP9X siRNAs. One day after siRNA transfection, the cells were re-seeded to the sparse condition and an in vivo ubiquitination assay was performed after one day.


E . 293T cells were transfected with the indicated combinations of DNAs , and a co - immunoprecipitation assay was performed .

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E. 293T cells were transfected with the indicated combinations of DNAs, and a co-immunoprecipitation assay was performed.


F . Sparsely cultured RPE or MCF10A cells were immunoprecipitated with an anti - AMOTL2 antibody .

F. Sparsely cultured RPE or MCF10A cells were immunoprecipitated with an anti-AMOTL2 antibody.


A . Flag - AMOTL2 constructs were designed to express regions spanning from the N - terminus to the coiled - coil domain ( AA 1 - 306 ) , the coiled - coil domain ( AA 307 - 581 ) , and the C - terminal portion ( AA 582 - 781 ) . These constructs were co - transfected into 293T cells with Myc - Ub , and an in vivo ubiquitination assay was conducted . * , non - specific band .

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A. Flag-AMOTL2 constructs were designed to express regions spanning from the N-terminus to the coiled-coil domain (AA 1-306), the coiled-coil domain (AA 307-581), and the C-terminal portion (AA 582-781). These constructs were co-transfected into 293T cells with Myc-Ub, and an in vivo ubiquitination assay was conducted. *, non-specific band.


B . The SBP - AMOTL2 coiled - coil domain was expressed in 293T cells , subjected to tandem affinity purification , fractionated by SDS - PAGE and stained with Coomassie blue . Bands corresponding to the AMOTL2 coiled - coil domain were excised and analyzed by mass spectrometry for identification of ubiquitinated sites ( K - G - G linkages ) . Shown are MS / MS spectra recoded using an LTQ - Orbitrap mass spectrometer for the doubly charged peptide , IEK * LESEIQR ( MH + = 1358 . 72 , z = 2 + ; upper ) , and the triply charged peptide , LASK * TQEAQAGSQDMVAK ( MH + = 1992 . 96 , z = 3 + ; lower ) . These peptides contain the K347 and K408 ubiquitination sites , respectively ( marked by asterisks ) . Fragmented ions are annotated according to the nomenclature for peptide fragmentation in mass spectrometry .

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B. The SBP-AMOTL2 coiled-coil domain was expressed in 293T cells, subjected to tandem affinity purification, fractionated by SDS-PAGE and stained with Coomassie blue. Bands corresponding to the AMOTL2 coiled-coil domain were excised and analyzed by mass spectrometry for identification of ubiquitinated sites (K-G-G linkages). Shown are MS/MS spectra recoded using an LTQ-Orbitrap mass spectrometer for the doubly charged peptide, IEK*LESEIQR (MH+ = 1358.72, z = 2+; upper), and the triply charged peptide, LASK*TQEAQAGSQDMVAK (MH+ = 1992.96, z = 3+; lower). These peptides contain the K347 and K408 ubiquitination sites, respectively (marked by asterisks). Fragmented ions are annotated according to the nomenclature for peptide fragmentation in mass spectrometry.


C . The indicated Flag - AMOTL2 WT or mutant proteins were subjected to an in vivo ubiquitination assay in 293T cells .

C. The indicated Flag-AMOTL2 WT or mutant proteins were subjected to an in vivo ubiquitination assay in 293T cells.


D . RPE or MCF10A cells were transduced with Flag - AMOTL2 WT or Flag - AMOTL2 K347 / 408R , and immunoprecipitated with an anti - Flag antibody . Immunoprecipitated and WCL samples were analyzed by Western blotting for the indicated proteins .

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D. RPE or MCF10A cells were transduced with Flag-AMOTL2 WT or Flag-AMOTL2 K347/408R, and immunoprecipitated with an anti-Flag antibody. Immunoprecipitated and WCL samples were analyzed by Western blotting for the indicated proteins.


E . RPE or MCF10A cells were transduced with Flag - AMOTL2 WT or Flag - AMOTL2 Y213A , and processed as ( D ) .

E. RPE or MCF10A cells were transduced with Flag-AMOTL2 WT or Flag-AMOTL2 Y213A, and processed as (D).


A . MCF10A cells expressing shRNA against AMOTL2 were complemented with shRNA - resistant Flag - AMOTL2 WT or the K347 / 408R mutant . The indicated cells were harvested and analyzed by Western blotting for the indicated proteins .

A. MCF10A cells expressing shRNA against AMOTL2 were complemented with shRNA-resistant Flag-AMOTL2 WT or the K347/408R mutant. The indicated cells were harvested and analyzed by Western blotting for the indicated proteins.


B . mRNAs isolated from cells in ( A ) were analyzed by RT - qPCR against indicated EMT marker genes ( n = 4 ) . Error bars indicate the S . E . M . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; paired Student ' s t - test ) .

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B. mRNAs isolated from cells in (A) were analyzed by RT-qPCR against indicated EMT marker genes (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01, ***p < 0.001; paired Student's t-test).