Figure 5A - C . The IFT - interaction domain is required for Ccdc108 multiciliogenesis function in Xenopus embryos : ( A ) impaired multiciliogenesis , ( B ) defective apical migration / docking of centrioles , and ( C ) reduced enrichment of apical F - actin . Embryos at stage 27 were fixed and stained with the acetylated tubulin ( A ; Ac - tub ; green ) , Cep164 ( B ; purple ) and Cetn1 ( B ; green ) antibodies or phalloidin ( purple ) . mRNA of a membrane - bound form of GFP was co - injected with each morpholino to indicate targeted cells ( A , B ) . Typical D - SIM images ( A , B ) and confocal images ( C ) are present . Embryos were treated as described in Figure 1C and expressed mGFP or GFP - Ccdc108 mutants . Plots show results from three independent experiments . ( A ) > 70 MCCs from 6 embryos , and ( C ) > 70 MCCs from 6 embryos for each condition . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are presented .

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

Figure 5A-C. The IFT-interaction domain is required for Ccdc108 multiciliogenesis function in Xenopus embryos: (A) impaired multiciliogenesis, (B) defective apical migration/docking of centrioles, and (C) reduced enrichment of apical F-actin. Embryos at stage 27 were fixed and stained with the acetylated tubulin (A; Ac-tub; green), Cep164 (B; purple) and Cetn1 (B; green) antibodies or phalloidin (purple). mRNA of a membrane-bound form of GFP was co-injected with each morpholino to indicate targeted cells (A,B). Typical D-SIM images (A,B) and confocal images (C) are present. Embryos were treated as described in Figure 1C and expressed mGFP or GFP-Ccdc108 mutants. Plots show results from three independent experiments. (A) > 70 MCCs from 6 embryos, and (C) > 70 MCCs from 6 embryos for each condition. Unpaired two-tailed t-test was performed (*** p<0.001; **p<0.01). Mean ± s.d. values are presented.


Figure 6A , ift74 CRISPR mutants display defective cilia formation . Embryos at one cell stage were injected and fixed at stage 27 with two independent sgRNA targeting ift74 . Representative confocal images of the Xenopus epidermis , and plots display defective ciliation . ; 60 MCCs from 6 embryos Cell membranes ( mGFP ) , cilia ( Ac - tub , green ) were labeled with indicated antibodies . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented . B . ift74 CRISPR mutants display defective cilia formation . Embryos at one cell stage were injected and fixed at stage 27 with two independent sgRNA targeting ift74 . Representative 3D - SIM images of the Xenopus epidermis , and plots display defective ciliation . ; 70 MCCs from 6 embryos for each condition . Cell membranes ( mGFP ) , cilia ( Ac - tub , green ) and basal bodies ( Cep164 ) were labeled with indicated antibodies . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented . C . Ift74 depletion affects centriole apical migration in MCCs . Representative 3D - SIM images ( x - y ) and 3D - reconstructions ( x - z ) of MCCs show failure of apical trafficking of basal bodies . Basal bodies ( Cetn1 , green ) and distal appendages ( Cep164 , purple ) were labeled with indicated antibodies . Quantitative plot shows a slight significant reduction in basal bodies by the depletion of Ift74 . > 70 MCCs from 6 embryos for each condition . D . Representative 3D - SIM images and plots reveal that apical expansion of MCCs is slightly affected by Ift74 depletion . > 70 MCCs from 6 embryos for each condition . Embryos were fixed and stained with phalloidin ( purple ) . Cilia ( Ac - tub , gray ) were also presented . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented . E . Representative confocal images and plot show a significant reduction in apical F - actin enrichment upon Ift74 depletion . > 70 MCCs from 6 embryos for each condition . Cell boundaries and apical actin network were marked with mGFP ( green ) and phalloidin ( purple ) . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

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

Figure 6A, ift74 CRISPR mutants display defective cilia formation. Embryos at one cell stage were injected and fixed at stage 27 with two independent sgRNA targeting ift74. Representative confocal images of the Xenopus epidermis, and plots display defective ciliation. ;60 MCCs from 6 embryos Cell membranes (mGFP), cilia (Ac-tub, green) were labeled with indicated antibodies. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.B. ift74 CRISPR mutants display defective cilia formation. Embryos at one cell stage were injected and fixed at stage 27 with two independent sgRNA targeting ift74. Representative 3D-SIM images of the Xenopus epidermis, and plots display defective ciliation. ;70 MCCs from 6 embryos for each condition. Cell membranes (mGFP), cilia (Ac-tub, green) and basal bodies (Cep164) were labeled with indicated antibodies. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.C. Ift74 depletion affects centriole apical migration in MCCs. Representative 3D-SIM images (x-y) and 3D-reconstructions (x-z) of MCCs show failure of apical trafficking of basal bodies. Basal bodies (Cetn1, green) and distal appendages (Cep164, purple) were labeled with indicated antibodies. Quantitative plot shows a slight significant reduction in basal bodies by the depletion of Ift74. >70 MCCs from 6 embryos for each condition. D. Representative 3D-SIM images and plots reveal that apical expansion of MCCs is slightly affected by Ift74 depletion. >70 MCCs from 6 embryos for each condition. Embryos were fixed and stained with phalloidin (purple). Cilia (Ac-tub, gray) were also presented. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.E. Representative confocal images and plot show a significant reduction in apical F-actin enrichment upon Ift74 depletion. >70 MCCs from 6 embryos for each condition. Cell boundaries and apical actin network were marked with mGFP (green) and phalloidin (purple). Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


Figure 7C - E . Ccdc108 depletion affects centriolar distribution of IFT - B proteins during centriole migration in MCCs . Embryos were co - injected with GFP - IFTs ( C : Ift74 ; D : Ift80 ; E : Ift88 ; green ) , Cetn4 - RFP ( purple ) and each morpholino , and imaged at stage 18 . HA tagged Ccdc108 proteins were co - expressed to test for the rescue of phenotypic defects . Xenopus embryos were subjected to immunoblotting to evaluate the protein expression levels . α - tubulin ( α - Tub ) served as loading control . Quantitative plots show that Ccdc108 - depleted MCCs showed a significant decrease of centriolar distribution of IFT - B proteins ( Ift74 , Ift80 and Ift88 ) , and Ccdc108 - IFT interaction deficient mutants are unable to rescue the decreased centriolar accumulation of IFT - B proteins . > 40 MCCs from 6 embryos for each condition . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ) . Mean ± s . d . values are also presented .

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

Figure 7C-E. Ccdc108 depletion affects centriolar distribution of IFT-B proteins during centriole migration in MCCs. Embryos were co-injected with GFP-IFTs (C: Ift74; D: Ift80; E: Ift88; green), Cetn4-RFP (purple) and each morpholino, and imaged at stage 18. HA tagged Ccdc108 proteins were co-expressed to test for the rescue of phenotypic defects. Xenopus embryos were subjected to immunoblotting to evaluate the protein expression levels. α-tubulin (α-Tub) served as loading control. Quantitative plots show that Ccdc108-depleted MCCs showed a significant decrease of centriolar distribution of IFT-B proteins (Ift74, Ift80 and Ift88), and Ccdc108-IFT interaction deficient mutants are unable to rescue the decreased centriolar accumulation of IFT-B proteins. >40 MCCs from 6 embryos for each condition. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are also presented.


Figure 8A , B . Centriolar distribution of ciliary adhesion complex related proteins Fak and Cp110 are not reduced following Ccdc108 depletion . Embryos were co - injected with GFP - Cp110 ( A ) GFP - Fak ( B ) , Cetn4 - RFP ( purple ) and each morpholino , and imaged at stage 18 . > 30 MCCs from 6 embryos for each condition . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented . C , D . Ccdc108 depletion affects centriolar distribution of PCP associated actin cytoskeleton regulators important for centriole migration in MCCs . Embryos were co - injected with GFP - Drg1 ( C ) GFP - RBD ( D ) , Cetn4 - RFP ( purple ) and each morpholino , and imaged at stage 18 . HA tagged Ccdc108 proteins were co - expressed to test for the rescue of phenotypic defects . Xenopus embryos were subjected to immunoblotting to evaluate the protein expression levels . α - tubulin ( α - Tub ) served as loading control . Quantitative plots show that Ccdc108 - depleted MCCs showed a significant decrease of centriolar distribution of Drg1 and RBD , and neither of Ccdc108 - IFT interaction deficient mutants can rescue the centriolar phenotypic defects . > 30 MCCs from 6 embryos for each condition . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

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

Figure 8A, B. Centriolar distribution of ciliary adhesion complex related proteins Fak and Cp110 are not reduced following Ccdc108 depletion. Embryos were co-injected with GFP-Cp110 (A) GFP-Fak (B), Cetn4-RFP (purple) and each morpholino, and imaged at stage 18. >30 MCCs from 6 embryos for each condition. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.C, D. Ccdc108 depletion affects centriolar distribution of PCP associated actin cytoskeleton regulators important for centriole migration in MCCs. Embryos were co-injected with GFP-Drg1 (C) GFP-RBD (D), Cetn4-RFP (purple) and each morpholino, and imaged at stage 18. HA tagged Ccdc108 proteins were co-expressed to test for the rescue of phenotypic defects. Xenopus embryos were subjected to immunoblotting to evaluate the protein expression levels. α-tubulin (α-Tub) served as loading control. Quantitative plots show that Ccdc108-depleted MCCs showed a significant decrease of centriolar distribution of Drg1 and RBD, and neither of Ccdc108-IFT interaction deficient mutants can rescue the centriolar phenotypic defects. >30 MCCs from 6 embryos for each condition. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


Figure 1CSF extract was treated with DMSO , CsA , buffer or His - CnA420 - 508 at the indicated concentrations . Meiotic exit was induced by calcium addition and samples were taken at the indicated time points . Samples were immunoblotted for cyclin B2 . The cyclin B2 membrane was stripped and reprobed for α - tubulin . CSF extract was treated with DMSO or CsA . Both reactions were divided and supplemented with the indicated amounts of calcium or H2O . After 20min , samples were taken and immunoblotted for cyclin B2 for which a low and high exposure is shown . The cyclin B2 membrane was stripped and reprobed for α - tubulin . CSF extract was supplemented with Myc - XErp1 wt IVT at the indicated dilutions . An empty IVT reaction not expressing Myc - XErp1 served as control . All reactions were divided and treated with DMSO or CsA . Calcium was added to all reactions and samples were taken at the indicated time points , treated with λ - phosphatase and immunoblotted for XErp1 , the Myc - tag , cyclin B2 and α - tubulin . Asterisks indicate unspecific bands . CSF extract was supplemented with Myc - XErp1 wt IVT . The reaction was divided and treated with DMSO or CsA . Both reactions were treated with calcium , samples were taken at the indicated time points and as indicated incubated with λ - phosphatase . Samples were immunoblotted for XErp1 and cyclin B2 . The cyclin B2 membrane was stripped and reprobed for α - tubulin . Asterisk indicates unspecific bands .

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

Figure 1CSF extract was treated with DMSO, CsA, buffer or His-CnA420-508 at the indicated concentrations. Meiotic exit was induced by calcium addition and samples were taken at the indicated time points. Samples were immunoblotted for cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin.CSF extract was treated with DMSO or CsA. Both reactions were divided and supplemented with the indicated amounts of calcium or H2O. After 20min, samples were taken and immunoblotted for cyclin B2 for which a low and high exposure is shown. The cyclin B2 membrane was stripped and reprobed for α-tubulin.CSF extract was supplemented with Myc-XErp1 wt IVT at the indicated dilutions. An empty IVT reaction not expressing Myc-XErp1 served as control. All reactions were divided and treated with DMSO or CsA. Calcium was added to all reactions and samples were taken at the indicated time points, treated with λ-phosphatase and immunoblotted for XErp1, the Myc-tag, cyclin B2 and α-tubulin. Asterisks indicate unspecific bands.CSF extract was supplemented with Myc-XErp1 wt IVT. The reaction was divided and treated with DMSO or CsA. Both reactions were treated with calcium, samples were taken at the indicated time points and as indicated incubated with λ-phosphatase. Samples were immunoblotted for XErp1 and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin. Asterisk indicates unspecific bands.


Figure 2CSF extract was supplemented with Myc - XErp1 DSG - DSA - ZBR - ( S33N S38N S284N S288N C583A ) IVT and as indicated treated with DMSO or CsA . Both reactions were treated with calcium and samples were taken at the indicated time points . Samples were immunoblotted for the Myc - tag , cyclin B2 and α - tubulin . A pseudocoloured representation of the Myc immunoblot is shown in Fig EV2A . Arrow marks the meiotic phosphorylation state of Myc - XErp1 . Asterisk indicates unspecific bands . CSF extract was supplemented with Myc - XErp1 DSG - DSA - ZBR - ( S33N S38N S284N S288N C583A ) IVT that was either wild - type or mutated to alanine at the p90RSK - target sites S335 , T336 and S342 ( Rsk3A ) . Both reactions were divided and treated with DMSO or CsA . Calcium was added and samples were taken at the indicated time points . Samples were immunoblotted for the Myc - tag and cyclin B2 . The cyclin B2 membrane was stripped and reprobed for α - tubulin . Asterisk indicates unspecific bands . CSF extract was treated with Myc - XErp1 CaMKII - ZBR - ( T195A C583A ) IVT . An empty IVT reaction not expressing XErp1 was used as control . The extracts were treated with DMSO or CsA as indicated . Calcium was added , samples were taken at the indicated time points and immunoblotted for cyclin B2 , pSer335 XErp1 and α - tubulin . The pSer335 XErp1 membrane was stripped and reprobed for the Myc - tag . Asterisks indicate unspecific bands . Myc - XErp1 CaMKII - ZBR - ( T195A C583A ) IVT was in vitro phosphorylated by recombinant PKA and isolated by α - Myc immunoprecipitation . The immunoprecipitate was supplemented with calcium and treated with recombinant hs _ CaN and calmodulin as indicated . Samples were taken at the indicated time points and immunoblotted for the catalytic calcineurin subunit CnA and pSer335 XErp1 . The pSer335 XErp1 membrane was stripped and reprobed for the Myc - tag . Asterisk indicates the IgG heavy chain .

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

Figure 2CSF extract was supplemented with Myc-XErp1 DSG- DSA- ZBR- (S33N S38N S284N S288N C583A) IVT and as indicated treated with DMSO or CsA. Both reactions were treated with calcium and samples were taken at the indicated time points. Samples were immunoblotted for the Myc-tag, cyclin B2 and α-tubulin. A pseudocoloured representation of the Myc immunoblot is shown in Fig EV2A. Arrow marks the meiotic phosphorylation state of Myc-XErp1. Asterisk indicates unspecific bands.CSF extract was supplemented with Myc-XErp1 DSG- DSA- ZBR- (S33N S38N S284N S288N C583A) IVT that was either wild-type or mutated to alanine at the p90RSK-target sites S335, T336 and S342 (Rsk3A). Both reactions were divided and treated with DMSO or CsA. Calcium was added and samples were taken at the indicated time points. Samples were immunoblotted for the Myc-tag and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin. Asterisk indicates unspecific bands.CSF extract was treated with Myc-XErp1 CaMKII- ZBR- (T195A C583A) IVT. An empty IVT reaction not expressing XErp1 was used as control. The extracts were treated with DMSO or CsA as indicated. Calcium was added, samples were taken at the indicated time points and immunoblotted for cyclin B2, pSer335 XErp1 and α-tubulin. The pSer335 XErp1 membrane was stripped and reprobed for the Myc-tag. Asterisks indicate unspecific bands.Myc-XErp1 CaMKII- ZBR- (T195A C583A) IVT was in vitro phosphorylated by recombinant PKA and isolated by α-Myc immunoprecipitation. The immunoprecipitate was supplemented with calcium and treated with recombinant hs_CaN and calmodulin as indicated. Samples were taken at the indicated time points and immunoblotted for the catalytic calcineurin subunit CnA and pSer335 XErp1. The pSer335 XErp1 membrane was stripped and reprobed for the Myc-tag. Asterisk indicates the IgG heavy chain.


Figure 3CSF extract was supplemented with mRNA encoding Flag - Cdc20 that was either wild - type or mutated to serine at Thr64 , Thr68 and Thr79 ( 3TS ) . After expression , calcium was added , samples were taken at the indicated time points and resolved by either conventional or Phos - tagTM SDS - PAGE . Samples were immunoblotted for the Flag - tag , Cdc27 and cyclin B2 . CSF extract was supplemented with Myc - XErp1 wt IVT and treated with DMSO or CsA . Calcium was added , samples were taken at the indicated time points , resolved by either conventional or Phos - tagTM SDS - PAGE and immunoblotted for Cdc20 , Cdc27 and cyclin B2 . A control sample was treated with λ - phosphatase . A pseudocoloured representation of the Cdc20 and the Cdc27 immunoblots is shown in Fig . EV4A . The cyclin B2 membrane was stripped and reprobed for α - tubulin . CSF extract was supplemented with Flag - Cdc20 wt IVT coupled to α - Flag beads . Calcium was added and Flag - Cdc20 was isolated by α - Flag immunoprecipitation at the indicated time points . Input and pellet ( IP : Flag ) samples were immunoblotted for the Flag - tag , pThr68 Cdc20 and p150 ( Glued ) . The pThr68 Cdc20 membrane was stripped and reprobed for cyclin B2 . Asterisk in the cyclin B2 immunoblot indicates the IgG heavy chain . Asterisks in other immunoblots indicate unspecific bands . CSF extract was supplemented with Flag - Cdc20 wt IVT . Flag - Cdc20 was re - isolated by α - Flag immunoprecipitation , supplemented with calcium and treated with recombinant hs _ CaN and calmodulin as indicated . Samples were taken at the indicated time points and immunoblotted for the Flag - tag and pThr68 Cdc20 . The pThr68 Cdc20 membrane was stripped and reprobed for the catalytic calcineurin subunit CnA . Asterisk indicates the IgG heavy chain .

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

Figure 3CSF extract was supplemented with mRNA encoding Flag-Cdc20 that was either wild-type or mutated to serine at Thr64, Thr68 and Thr79 (3TS). After expression, calcium was added, samples were taken at the indicated time points and resolved by either conventional or Phos-tagTM SDS-PAGE. Samples were immunoblotted for the Flag-tag, Cdc27 and cyclin B2.CSF extract was supplemented with Myc-XErp1 wt IVT and treated with DMSO or CsA. Calcium was added, samples were taken at the indicated time points, resolved by either conventional or Phos-tagTM SDS-PAGE and immunoblotted for Cdc20, Cdc27 and cyclin B2. A control sample was treated with λ-phosphatase. A pseudocoloured representation of the Cdc20 and the Cdc27 immunoblots is shown in Fig. EV4A. The cyclin B2 membrane was stripped and reprobed for α-tubulin.CSF extract was supplemented with Flag-Cdc20 wt IVT coupled to α-Flag beads. Calcium was added and Flag-Cdc20 was isolated by α-Flag immunoprecipitation at the indicated time points. Input and pellet (IP: Flag) samples were immunoblotted for the Flag-tag, pThr68 Cdc20 and p150(Glued). The pThr68 Cdc20 membrane was stripped and reprobed for cyclin B2. Asterisk in the cyclin B2 immunoblot indicates the IgG heavy chain. Asterisks in other immunoblots indicate unspecific bands.CSF extract was supplemented with Flag-Cdc20 wt IVT. Flag-Cdc20 was re-isolated by α-Flag immunoprecipitation, supplemented with calcium and treated with recombinant hs_CaN and calmodulin as indicated. Samples were taken at the indicated time points and immunoblotted for the Flag-tag and pThr68 Cdc20. The pThr68 Cdc20 membrane was stripped and reprobed for the catalytic calcineurin subunit CnA. Asterisk indicates the IgG heavy chain.


Figure 1A Affinity measurements by biolayer interferometry ( BLI ) of bipNbs NM1267 and NM1268 . NM1267 and NM1268 were applied with concentrations ranging from 5 - 0 . 625 nM and 20 ‑ 2 . 5 nM , respectively ( illustrated with gradually lighter shades ) on immobilized RBD derived from SARS - CoV - 2 B . 1 ( RBDB . 1 ) . Global 1 : 1 fits are illustrated as dashed lines . Global 1 : 1 fits are illustrated as dashed lines and binding affinity ( KD ) , association ( kon ) and dissociation constant ( koff ) determined for the individual bipNbs are summarized .

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

Figure 1A Affinity measurements by biolayer interferometry (BLI) of bipNbs NM1267 and NM1268. NM1267 and NM1268 were applied with concentrations ranging from 5-0.625 nM and 20‑2.5 nM, respectively (illustrated with gradually lighter shades) on immobilized RBD derived from SARS-CoV-2 B.1 (RBDB.1). Global 1:1 fits are illustrated as dashed lines. Global 1:1 fits are illustrated as dashed lines and binding affinity (KD), association (kon) and dissociation constant (koff) determined for the individual bipNbs are summarized.


Figure 2A - P Affinity measurements by BLI of bipNb NM1267 and NM1268 on recently identified RBD variants B . 1 . 1 . 7 ( Alpha ) ( A , I ) , B . 1 . 351 ( Beta ) ( B , J ) , P1 ( Gamma ) ( C , K ) , B . 1 . 617 . 2 ( Delta ) ( D , L ) , B . 1 . 429 ( Epsilon ) ( E , M ) , P3 ( Theta ) ( F , N ) , B . 1 . 617 . 1 ( Kappa ) ( G , O ) and A . 23 . 1 ( H , P ) . NM1267 and NM1268 were applied with concentrations ranging from 5 - 0 . 625 nM and 20 ‑ 2 . 5 nM , respectively ( illustrated with gradually lighter shades ) on immobilized RBD variants . Global 1 : 1 fits are illustrated as dashed lines .

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

Figure 2A-P Affinity measurements by BLI of bipNb NM1267 and NM1268 on recently identified RBD variants B.1.1.7 (Alpha) (A, I), B.1.351 (Beta) (B, J), P1 (Gamma) (C, K), B.1.617.2 (Delta) (D, L), B.1.429 (Epsilon) (E, M), P3 (Theta) (F, N), B.1.617.1 (Kappa) (G, O) and A.23.1 (H, P). NM1267 and NM1268 were applied with concentrations ranging from 5-0.625 nM and 20‑2.5 nM, respectively (illustrated with gradually lighter shades) on immobilized RBD variants. Global 1:1 fits are illustrated as dashed lines.


Figure 3A - F Neutralization potency of NM1267 and NM1268 were analyzed in Caco ‑ 2 cells using the SARS - CoV - 2 B . 1 ( A , D ) SARS - CoV - 2 B . 1 . 351 ( Beta ) ( B , E ) and SARS - CoV - 2 B . 1 . 617 . 2 ( Delta ) ( C , F ) . Infection normalized to virus - only infection control is illustrated as percent of infection ( infection [ % ] ) . Data are presented as mean ± SEM of three ( n = 3 ) biological replicates .

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Figure 3A-F Neutralization potency of NM1267 and NM1268 were analyzed in Caco‑2 cells using the SARS-CoV-2 B.1 (A, D) SARS-CoV-2 B.1.351 (Beta) (B, E) and SARS-CoV-2 B.1.617.2 (Delta) (C, F). Infection normalized to virus-only infection control is illustrated as percent of infection (infection [%]). Data are presented as mean ± SEM of three (n = 3) biological replicates.


Figure 4C Hemizygous K18 - hACE2 mice were treated intranasally with 20 µg of NM1251 ( n = 15 ) or NM1267 ( n = 12 ) seven hours prior to infection with 3 * 103 PFU SARS - CoV - 2 B . 1 . survival were monitored for 14 days . , * * * * P < 0 . 0001 , by log - rank testD Nasal swabs were collected from six mice per group ( n = 6 ) at the indicated time points and viral load was determined by plaque - assay . Bars represent mean ± SEM . * * P < 0 . 01 , by unpaired t

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Figure 4C Hemizygous K18-hACE2 mice were treated intranasally with 20 µg of NM1251 (n = 15) or NM1267 (n = 12) seven hours prior to infection with 3*103 PFU SARS-CoV-2 B.1. survival were monitored for 14 days. , ****P < 0.0001, by log-rank testD Nasal swabs were collected from six mice per group (n = 6) at the indicated time points and viral load was determined by plaque-assay. Bars represent mean ± SEM. **P < 0.01, by unpaired t test


Figure 5Histopathological analysis of mice , which were intranasally treated with bipNb NM1267 or control Nb NM1251 and subsequently infected with 3 * 103 PFU SARS - CoV - 2 B . 1 . A - B Serial tissue sections revealed severe inflammation ( H & E ) and numerous widespread SARS - CoV - 2 RNA positive alveolar epithelia cells and macrophages ( in situ hybridization ( ISH ) ) in lungs of infected , control - treated mice . In infected and NM1267 - treated animals no inflammation or only small focal areas with inflammation and a few SARS - CoV - 2 RNA positive cells were observed . Scale bars represent 1 mm and 200 µm , respectively .

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

Figure 5Histopathological analysis of mice, which were intranasally treated with bipNb NM1267 or control Nb NM1251 and subsequently infected with 3*103 PFU SARS-CoV-2 B.1. A-B Serial tissue sections revealed severe inflammation (H&E) and numerous widespread SARS-CoV-2 RNA positive alveolar epithelia cells and macrophages (in situ hybridization (ISH)) in lungs of infected, control-treated mice. In infected and NM1267-treated animals no inflammation or only small focal areas with inflammation and a few SARS-CoV-2 RNA positive cells were observed. Scale bars represent 1 mm and 200 µm, respectively.


Figure 6B , Hemizygous K18 - hACE2 mice were treated intranasally with 20 µg of NM1251 ( n = 8 ) , NM1267 ( n = 9 ) or NM1268 ( n = 9 ) seven hours prior to infection with 3 * 103 PFU SARS - CoV - 2 B . 1 . 617 . 2 ( Delta ) . Weight loss were monitored for 14 days . Dashed line indicates humane endpoint and symbols represent mean ± SEM * * * * P < 0 . 0001 in orange between NM1251 and NM1267 and in purple between NM1251 and NM1268 , * P < 0 . 1 in black between NM1267 and NM1268 , by two - way ANOVA with Sidak ' s multiple comparison testC Hemizygous K18 - hACE2 mice were treated intranasally with 20 µg of NM1251 ( n = 8 ) , NM1267 ( n = 9 ) or NM1268 ( n = 9 ) seven hours prior to infection with 3 * 103 PFU SARS - CoV - 2 B . 1 . 617 . 2 ( Delta ) . survival were monitored for 14 days . * * * P < 0 . 001 in orange between NM1251 and NM1267 and * * * * P < 0 . 0001 in purple between NM1251 and NM1268 , by log - rank

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

Figure 6B, Hemizygous K18-hACE2 mice were treated intranasally with 20 µg of NM1251 (n = 8), NM1267 (n = 9) or NM1268 (n = 9) seven hours prior to infection with 3*103 PFU SARS-CoV-2 B.1.617.2 (Delta). Weight loss were monitored for 14 days. Dashed line indicates humane endpoint and symbols represent mean ± SEM ****P < 0.0001 in orange between NM1251 and NM1267 and in purple between NM1251 and NM1268, *P < 0.1 in black between NM1267 and NM1268, by two-way ANOVA with Sidak's multiple comparison testC Hemizygous K18-hACE2 mice were treated intranasally with 20 µg of NM1251 (n = 8), NM1267 (n = 9) or NM1268 (n = 9) seven hours prior to infection with 3*103 PFU SARS-CoV-2 B.1.617.2 (Delta). survival were monitored for 14 days. ***P < 0.001 in orange between NM1251 and NM1267 and ****P < 0.0001 in purple between NM1251 and NM1268, by log-rank test


figf1 ( a ) Knockdown of dRagA and dRagC decreased dS6K phosphorylation ( Thr 398 ) . Drosophila S2 cells untreated ( lane 2 ) or treated with the indicated RNAi were starved of amino acids for 1 h followed by amino acid stimulation for 30 min Phosphorylation and protein levels of dS6K were determined by immunoblotting with the indicated antibodies . ( b ) dRagA and dRagC are not required for dAkt phosphorylation . Drosophila S2 cells untreated ( lanes 1 - 3 ) or treated with the indicated RNAi were starved of amino acids for 1 h and stimulated with amino acids for 30 min . Phosphorylation and protein levels of dAkt were determined by immunoblotting with appropriate antibodies as indicated . NC , negative control RNAi . ( c ) dRagA and dRagC function in parallel with TSC2 and PTEN . dRagA and / or dRagC RNAi was added to S2 cells in combination with dTSC2 or dPTEN RNAi , as indicated . dTSC2 or dPTEN RNAi treatment increased pdS6K ( Thr 398 ) and this increase was compromised by dRagA or / and dRagC RNAi . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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

figf1(a) Knockdown of dRagA and dRagC decreased dS6K phosphorylation (Thr 398). Drosophila S2 cells untreated (lane 2) or treated with the indicated RNAi were starved of amino acids for 1 h followed by amino acid stimulation for 30 min Phosphorylation and protein levels of dS6K were determined by immunoblotting with the indicated antibodies.(b) dRagA and dRagC are not required for dAkt phosphorylation. Drosophila S2 cells untreated (lanes 1-3) or treated with the indicated RNAi were starved of amino acids for 1 h and stimulated with amino acids for 30 min. Phosphorylation and protein levels of dAkt were determined by immunoblotting with appropriate antibodies as indicated. NC, negative control RNAi.(c) dRagA and dRagC function in parallel with TSC2 and PTEN. dRagA and/or dRagC RNAi was added to S2 cells in combination with dTSC2 or dPTEN RNAi, as indicated. dTSC2 or dPTEN RNAi treatment increased pdS6K (Thr 398) and this increase was compromised by dRagA or/and dRagC RNAi. Full scans of blots are provided in Supplementary Information, Fig. S6.


figf2 ( a ) Constitutively active RagA and RagB stimulate S6K phosphorylation . 200 ng of each mammalian RagA , RagB , RagC or RagD construct was co - transfected with HA - S6K ( 20 ng ) into HEK293 cells . Their corresponding dominant - negative mutants ( RagAT21N , RagBT54N , RagCS75N , RagDS76N ) and constitutively active mutants ( RagAQ66L , RagBQ99L , RagCQ120L , RagDQ121L ) were also tested . Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies , as indicated . ( b ) RagA has a dominant role over RagC in regulating S6K phosphorylation . Each Rag construct ( 200 ng ) indicated was co - transfected with HA - S6K ( 20 ng ) . The different Rag mutants used in the transfection are indicated at the top of each lane . ( c ) Rag regulates TORC1 but not TORC2 activity . Each indicated Rag construct ( 200 ng ) was co - transfected with either HA - S6K ( 20 ng ) , Myc - 4EBP1 ( 20 ng ) or of GST - Akt ( 100 ng ) . Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies , as indicated . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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

figf2(a) Constitutively active RagA and RagB stimulate S6K phosphorylation. 200 ng of each mammalian RagA, RagB, RagC or RagD construct was co-transfected with HA-S6K (20 ng) into HEK293 cells. Their corresponding dominant-negative mutants (RagAT21N, RagBT54N, RagCS75N, RagDS76N) and constitutively active mutants (RagAQ66L, RagBQ99L, RagCQ120L, RagDQ121L) were also tested. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated.(b) RagA has a dominant role over RagC in regulating S6K phosphorylation. Each Rag construct (200 ng) indicated was co-transfected with HA-S6K (20 ng). The different Rag mutants used in the transfection are indicated at the top of each lane.(c) Rag regulates TORC1 but not TORC2 activity. Each indicated Rag construct (200 ng) was co-transfected with either HA-S6K (20 ng), Myc-4EBP1 (20 ng) or of GST-Akt (100 ng). Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated. Full scans of blots are provided in Supplementary Information, Fig. S6.


figf3 ( a ) RagAQ66L and RagCS75N activate TORC1 in the absence of amino acids . Each Rag construct ( 200 ng ) indicated was co - transfected with HA - S6K ( 20 ng ) into HEK293 cells . Cells were starved of amino acids for 1 h before collection . Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies , as indicated . ( b ) RagAT21N and RagCQ120L block S6K phosphorylation in response to amino acid ( AA ) stimulation . pcDNA3 ( 200 ng , lanes 1 and 2 ) or each indicated Rag construct was co - transfected with HA - S6K into HEK293 cells . Cells were starved of amino acids for 1 h ( - AA ) and either remained in the starvation medium or were stimulated with amino - acid - containing - medium ( + AA ) for 30 min before collection . Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies , as indicated . ( c ) RagAT21N and RagCQ120L suppress insulin - induced stimulation of S6K phosphorylation . pcDNA3 ( 200 ng , lanes 1 and 2 ) or each indicated Rag construct was co - transfected with HA - S6K ( 20 ng ) or GST - Akt ( 100 ng ) into HeLa cells . Cells were serum - starved overnight and stimulated with insulin ( 400 nM ) for 30 min . Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies , as indicated . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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

figf3(a) RagAQ66L and RagCS75N activate TORC1 in the absence of amino acids. Each Rag construct (200 ng) indicated was co-transfected with HA-S6K (20 ng) into HEK293 cells. Cells were starved of amino acids for 1 h before collection. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated.(b) RagAT21N and RagCQ120L block S6K phosphorylation in response to amino acid (AA) stimulation. pcDNA3 (200 ng, lanes 1 and 2) or each indicated Rag construct was co-transfected with HA-S6K into HEK293 cells. Cells were starved of amino acids for 1 h (-AA) and either remained in the starvation medium or were stimulated with amino-acid-containing-medium (+AA) for 30 min before collection. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated.(c) RagAT21N and RagCQ120L suppress insulin-induced stimulation of S6K phosphorylation. pcDNA3 (200 ng, lanes 1 and 2) or each indicated Rag construct was co-transfected with HA-S6K (20 ng) or GST-Akt (100 ng) into HeLa cells. Cells were serum-starved overnight and stimulated with insulin (400 nM) for 30 min. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated. Full scans of blots are provided in Supplementary Information, Fig. S6.


figf4 ( a ) dRagA positively regulates wing compartment size . Wild - type or mutant dRagA transgenes were expressed in posterior compartments with the en - GAL4 driver . The ratios of representative posterior to anterior compartment areas are shown . Posterior compartment area was significantly increased in response to dRagAQ61L expression and decreased in response to dRagAT61N expression . Data are mean ± s . d . , * P = 7 . 32 × 10 − 4 ( n = 7 ) , * * P = 6 . 18 × 10 − 4 ( n = 12 ) , Student ' s two - tailed t - test , ( n is the number of adult wings analysed ) . ( b ) dRagA positively regulates wing cell size . The average area of posterior compartment cells from en - GAL4 UAS - dRagA adult wings is shown . Cells expressing dRagAQ61L are significantly larger and dRagAT61N - expressing cells are smaller than controls . Data are mean ± s . d . , * P = 1 . 15 × 10 − 3 ( n = 7 ) , * * P = 0 . 025 ( n = 12 ) , Student ' s 2 - tailed t - test ( n represents number of adult wings analysed ) . ( c , d ) dRag GTPases positively regulate larval fat body cell size . ( c ) Cell area of clonally - induced dRagA - expressing cells or dRagC homozygous mutant cells relative to neighbouring wild - type control cells is shown . Cell area was determined from phalloidin - stained fixed fat body samples from fed or 48 h starved larvae . Expression of dRagAWT or dRagAQ61L significantly increased relative cell area under starvation but not fed conditions . Cells expressing dRagAT61N and dRagC loss - of - function cells were significantly smaller than control cells only under nutrient replete conditions . Data are means ± s . d . , * P = 2 . 04 × 10 − 3 ( n = 5 ) , * * P = 2 . 94 × 10 − 6 ( n = 14 ) , * * * P = 3 . 79 × 10 − 7 ( n = 14 ) , * * * * P = 1 . 36 × 10 − 5 ( n = 30 ) , Student ' s two - tailed t - test . ( d ) Representative examples of fat body cells with altered dRagA activity . Cells expressing dRagA transgenes are marked by the expression of GFP in the left and middle panels , and dRagC homozygous mutant cells are marked by absence of GFP in the right panel . Scale bar represents 50 μm .

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

figf4(a) dRagA positively regulates wing compartment size. Wild-type or mutant dRagA transgenes were expressed in posterior compartments with the en-GAL4 driver. The ratios of representative posterior to anterior compartment areas are shown. Posterior compartment area was significantly increased in response to dRagAQ61L expression and decreased in response to dRagAT61N expression. Data are mean ± s.d., *P = 7.32 × 10−4 (n = 7), **P = 6.18 × 10−4 (n = 12), Student's two-tailed t-test, (n is the number of adult wings analysed).(b) dRagA positively regulates wing cell size. The average area of posterior compartment cells from en-GAL4 UAS-dRagA adult wings is shown. Cells expressing dRagAQ61L are significantly larger and dRagAT61N-expressing cells are smaller than controls. Data are mean ± s.d., *P = 1.15 × 10−3 (n = 7), **P = 0.025 (n = 12), Student's 2-tailed t-test (n represents number of adult wings analysed).(c, d) dRag GTPases positively regulate larval fat body cell size. (c) Cell area of clonally-induced dRagA-expressing cells or dRagC homozygous mutant cells relative to neighbouring wild-type control cells is shown. Cell area was determined from phalloidin-stained fixed fat body samples from fed or 48 h starved larvae. Expression of dRagAWT or dRagAQ61L significantly increased relative cell area under starvation but not fed conditions. Cells expressing dRagAT61N and dRagC loss-of-function cells were significantly smaller than control cells only under nutrient replete conditions. Data are means ± s.d., *P = 2.04 × 10−3 (n = 5), **P = 2.94 × 10−6 (n = 14), ***P = 3.79 × 10−7 (n = 14), ****P = 1.36 × 10−5 (n = 30), Student's two-tailed t-test. (d) Representative examples of fat body cells with altered dRagA activity. Cells expressing dRagA transgenes are marked by the expression of GFP in the left and middle panels, and dRagC homozygous mutant cells are marked by absence of GFP in the right panel. Scale bar represents 50 μm.


figf5 ( a ) Rag acts through TORC1 to regulate S6K phosphorylation . HEK293 cells were transfected with constructs as indicated . Co - expression of of mTORKD construct ( 600 ng ) or rapamycin treatment ( rapa , 20 nM , 30 min ) abolished the effect of RagAQ66L and RagCS75N on S6K phosphorylation . The protein level of mTORKD was determined by immunoblotting with anti - mTOR antibody . ( b ) RagA / RagC and TSC1 / TSC2 independently regulate S6K phosphorylation . HEK293 cells were transfected with 200 ng of each Rag and / or ′ constructs as indicated . Amino acid starvation for 1 h ( - AA ) is indicated . Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies , as indicated . ( c ) TSC2 and RagA / B independently affect S6K phosphorylation . HA - S6K ( 20 ng ) was transfected into HeLa cells with or without RNAi against human TSC2 , RagA and RagB as indicated . ( d ) RagAT21N and RagCQ120L do not block Rheb - induced S6K phosphorylation . RagAT21N and RagCQ120L ( 200 ng each ) were transfected into HEK293 cells with or without Rheb construct ( 20 ng ) . S6K was included in the co - transfection . Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies , as indicated . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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figf5(a) Rag acts through TORC1 to regulate S6K phosphorylation. HEK293 cells were transfected with constructs as indicated. Co-expression of of mTORKD construct (600 ng) or rapamycin treatment (rapa, 20 nM, 30 min) abolished the effect of RagAQ66L and RagCS75N on S6K phosphorylation. The protein level of mTORKD was determined by immunoblotting with anti-mTOR antibody.(b) RagA/RagC and TSC1/TSC2 independently regulate S6K phosphorylation. HEK293 cells were transfected with 200 ng of each Rag and/or ′ constructs as indicated. Amino acid starvation for 1 h (-AA) is indicated. Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies, as indicated.(c) TSC2 and RagA/B independently affect S6K phosphorylation. HA-S6K (20 ng) was transfected into HeLa cells with or without RNAi against human TSC2, RagA and RagB as indicated.(d) RagAT21N and RagCQ120L do not block Rheb-induced S6K phosphorylation. RagAT21N and RagCQ120L (200 ng each) were transfected into HEK293 cells with or without Rheb construct (20 ng). S6K was included in the co-transfection. Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies, as indicated. Full scans of blots are provided in Supplementary Information, Fig. S6.


figf6 ( a , b ) dRagC is not required for Rheb - induced cell growth . ( a ) The area of Rheb - overexpressing cells in control or dRagC mutant ( dRagC − / − ) backgrounds under fed conditions , relative to that of neighbouring control cells which were assigned a value of 1 . Overexpression of Rheb led to a significant increase in cell area in both control and dRagC mutant backgrounds . Data are mean ± s . d . , * P = 0 . 034 ( n = 5 ) , * * P = 6 . 1 × 10 − 3 ( n = 5 ) , Student ' s two - tailed t - test ( n represents number of experimental samples ) . ( b ) A representative example of a clone of Rheb - overexpressing cells in a dRagC − / − animal . Rheb transgene - expressing cells are marked by co - expression of GFP . Cell boundaries are labelled by phalloidin staining in red ; nuclei are labelled by DAPI in blue . Scale bar represents 50 μm . ( c , d ) Expression of dRagAQ61L fails to rescue the growth impairment of Rheb mutant cells . ( c ) Relative area of clonally - induced Rheb26 . 2 homozygous mutant cells in a control background and in animals expressing dRagAQ61L throughout the fat body . Clonally induced Rheb26 . 2 homozygous mutant cells were significantly smaller than neighbouring control cells both in wild - type and in dRagAQ61L expressing backgrounds . Data are mean ± s . d . , * P = 2 . 91 × 10 − 4 ( n = 7 ) , * * P = 2 . 59 × 10 − 8 ( n = 5 ) , Student ' s two - tailed t - test ( fed conditions where n represents number of experimental samples ) . ( d ) A representative example of Rheb homozygous mutant cells ( marked by lack of GFP , arrows ) in fat body ubiquitously expressing UAS - dRagAQ61L . GFP - positive control cells in this experiment are a mixture of Rheb + / − and Rheb + / + . Scale bar represents 50 μm .

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figf6(a, b) dRagC is not required for Rheb-induced cell growth. (a) The area of Rheb-overexpressing cells in control or dRagC mutant (dRagC−/−) backgrounds under fed conditions, relative to that of neighbouring control cells which were assigned a value of 1. Overexpression of Rheb led to a significant increase in cell area in both control and dRagC mutant backgrounds. Data are mean ± s.d., *P = 0.034 (n = 5), **P = 6.1 × 10−3 (n = 5), Student's two-tailed t-test (n represents number of experimental samples). (b) A representative example of a clone of Rheb-overexpressing cells in a dRagC−/− animal. Rheb transgene-expressing cells are marked by co-expression of GFP. Cell boundaries are labelled by phalloidin staining in red; nuclei are labelled by DAPI in blue. Scale bar represents 50 μm.(c, d) Expression of dRagAQ61L fails to rescue the growth impairment of Rheb mutant cells. (c) Relative area of clonally-induced Rheb26.2 homozygous mutant cells in a control background and in animals expressing dRagAQ61L throughout the fat body. Clonally induced Rheb26.2 homozygous mutant cells were significantly smaller than neighbouring control cells both in wild-type and in dRagAQ61L expressing backgrounds. Data are mean ±s.d., *P = 2.91 × 10−4 (n = 7), **P = 2.59 × 10−8 (n = 5), Student's two-tailed t-test (fed conditions where n represents number of experimental samples). (d) A representative example of Rheb homozygous mutant cells (marked by lack of GFP, arrows) in fat body ubiquitously expressing UAS-dRagAQ61L. GFP-positive control cells in this experiment are a mixture of Rheb+/− and Rheb+/+. Scale bar represents 50 μm.


figf7 ( a - d ) dRagAQ61L suppresses autophagy . ( a ) Drosophila fat body cells clonally expressing dRagAQ61L ( marked in green by GFP expression ) failed to accumulate autolysosomes ( shown in surrounding control cells by punctate Lysotracker Red staining ) in response to starvation for 4 h . Nuclei are marked in blue by DAPI . ( b - d ) Induction of autophagosomes in response to 4 - h starvation is shown by the punctate pattern of GFP - Atg8a expression in control fat body cells ( b ) , but not in cells expressing dRagAQ61L ( c ) . The average number of GFP - Atg8a - marked autophagosomes per cell in control and dRagAQ61L - expressing clones is shown ( d ) . Data are mean ± s . d . , * P = 2 . 91 × 10 − 6 , Students two - tailed t - test ( n = 33 fat body samples imaged per genotype ) . Scale bars represent 25 μm in each panel . ( e ) RagA regulates LC3 conversion in mammalian cells . Myc - LC3 was co - transfected with RagAQL and RagCSN or RagATN and RagCQL into HEK293 cells as indicated . One day after transfection , cells were cultured in amino - acid - sufficienct medium ( + AA ) or amino - acid - depleted medium ( − AA ) for 4 h before collection . Western blotting for Myc - LC3 and HA - Rag were performed . Autophagic conversion of LC3I into the lipidated LC3II form was blocked by active RagA and stimulated by dominant - negative RagA . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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figf7(a-d) dRagAQ61L suppresses autophagy. (a) Drosophila fat body cells clonally expressing dRagAQ61L (marked in green by GFP expression) failed to accumulate autolysosomes (shown in surrounding control cells by punctate Lysotracker Red staining) in response to starvation for 4 h. Nuclei are marked in blue by DAPI. (b-d) Induction of autophagosomes in response to 4-h starvation is shown by the punctate pattern of GFP-Atg8a expression in control fat body cells (b), but not in cells expressing dRagAQ61L (c). The average number of GFP-Atg8a-marked autophagosomes per cell in control and dRagAQ61L-expressing clones is shown (d). Data are mean ± s.d., *P = 2.91 × 10−6, Students two-tailed t-test (n = 33 fat body samples imaged per genotype). Scale bars represent 25 μm in each panel.(e) RagA regulates LC3 conversion in mammalian cells. Myc-LC3 was co-transfected with RagAQL and RagCSN or RagATN and RagCQL into HEK293 cells as indicated. One day after transfection, cells were cultured in amino-acid-sufficienct medium (+AA) or amino-acid-depleted medium (−AA) for 4 h before collection. Western blotting for Myc-LC3 and HA-Rag were performed. Autophagic conversion of LC3I into the lipidated LC3II form was blocked by active RagA and stimulated by dominant-negative RagA. Full scans of blots are provided in Supplementary Information, Fig. S6.


figf8 ( a , b ) dRagA activation increases sensitivity to starvation . Expression of dRagAQ61L using the fat - body - specific Cg - GAL4 driver significantly decreased survival of adult female flies under starvation ( b ) but not fed ( a ) conditions , relative to controls ( Cg - GAL4 alone ) . Asterisks indicate significant difference , compared with controls . Data are mean ± s . d . , * P 0 . 05 , Students two - tailed t - test ( n = 150 flies / genotype / treatment ) .

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figf8(a, b) dRagA activation increases sensitivity to starvation. Expression of dRagAQ61L using the fat-body-specific Cg-GAL4 driver significantly decreased survival of adult female flies under starvation (b) but not fed (a) conditions, relative to controls (Cg-GAL4 alone). Asterisks indicate significant difference, compared with controls. Data are mean ± s.d., *P 0.05, Students two-tailed t-test (n = 150 flies/genotype/treatment).


Figure 2 ( C ) Asynchronous exponentially growing cells were treated with 0 . 2M HU ( + HU ) , then HU was removed from the medium ( - HU ) . SDS - PAGE of total protein extracts taken at the indicated times were used to detect the kinetic of Rad53 phosphorylation upshift ( Rad53 - P = * * ) . Red and green asterisk indicate the time where mec1 - S1991 phosphomutants show altered Rad53 recovery . A 5 - fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC + / - the indicated dose of HU .

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Figure 2(C) Asynchronous exponentially growing cells were treated with 0.2M HU (+HU), then HU was removed from the medium (-HU). SDS-PAGE of total protein extracts taken at the indicated times were used to detect the kinetic of Rad53 phosphorylation upshift (Rad53-P=**). Red and green asterisk indicate the time where mec1-S1991 phosphomutants show altered Rad53 recovery. A 5-fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC +/- the indicated dose of HU.


Figure 3Analysis of replication fork progression at the single - molecule level by DNA combing . ( C ) Representative images of DNA fibers . Green : ssDNA , red : BrdU . Scale bar corresponds to 20 kb . ( D ) Graph depict the distribution of BrdU track length . WT 90 min ( n = 427 ) and 180 min ( n = 483 ) , mec1 - S1991A 90 min ( n = 309 ) and 180 min ( n = 477 ) .

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Figure 3Analysis of replication fork progression at the single-molecule level by DNA combing. (C) Representative images of DNA fibers. Green: ssDNA, red: BrdU. Scale bar corresponds to 20 kb. (D) Graph depict the distribution of BrdU track length. WT 90 min (n=427) and 180 min (n=483), mec1-S1991A 90 min (n=309) and 180 min (n=477).


Figure 4 ( A ) tRNA level measured by RT - qPCR in asynchronous ( async . ) culture and after 90 min on HU in the indicated strains . Expression is normalized to ACT1 . The repression is expressed as a ratio of HU treated / asynchronous cells in percent .

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Figure 4(A) tRNA level measured by RT-qPCR in asynchronous (async.) culture and after 90 min on HU in the indicated strains. Expression is normalized to ACT1. The repression is expressed as a ratio of HU treated/asynchronous cells in percent.


Figure 5 ( A - D ) Exponentially growing cells were treated with 0 . 2M HU and total protein extracts were collected at the indicated time points ( in min ) , samples were subjected to SDS - PAGE followed by immunoblotting with Rpb1 and Tubulin antibodies . ( B , D ) Quantitation of total Rpb1 over time was done by normalizing Rpb1 levels to tubulin . ( E , F ) Drop assay on HU showing a 10 - fold dilution series of cells from exponential SC cultures of the indicated strains that were spotted on SC + / - 200mM HU . ( F ) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution .

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Figure 5(A-D) Exponentially growing cells were treated with 0.2M HU and total protein extracts were collected at the indicated time points (in min), samples were subjected to SDS-PAGE followed by immunoblotting with Rpb1 and Tubulin antibodies. (B,D) Quantitation of total Rpb1 over time was done by normalizing Rpb1 levels to tubulin.(E,F) Drop assay on HU showing a 10-fold dilution series of cells from exponential SC cultures of the indicated strains that were spotted on SC +/- 200mM HU. (F) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution.


Figure 6 ( A ) Preparations of chromatin - bound proteins were prepared in triplicate and subjected to SDS - PAGE and immunoblotting with the indicated antibodies in a strain that either does or does not express Rpb3 - TAP Quantification of chromatin - bound Rpb1 - S2P and Rpb1 - S5P is shown on the right . Mcm2 is used as a loading control . ( F , G ) A 10 - fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC + / - 200mM of HU . A high level of HU was used to be able to demonstrate robust suppression of the mec1 - S1991A phenotype . ( G ) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution .

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Figure 6(A) Preparations of chromatin-bound proteins were prepared in triplicate and subjected to SDS-PAGE and immunoblotting with the indicated antibodies in a strain that either does or does not express Rpb3-TAP Quantification of chromatin-bound Rpb1-S2P and Rpb1-S5P is shown on the right. Mcm2 is used as a loading control.(F,G) A 10-fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC +/- 200mM of HU. A high level of HU was used to be able to demonstrate robust suppression of the mec1-S1991A phenotype. (G) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution.


Figure 7 ( B ) Amongst the Mec1 - S1991 - dependent phosphopeptides on HU , factors involved in transcription are highlighted . ( E ) Position relative to the nuclear envelope ( Zone 1 of the lacO - tagged GAL1 - GAL10 locus in the indicated strains grown either 12h on glucose , or 90 min galactose or galactose + 0 . 2M HU . The third panel shows the relative retention at the nuclear periphery ( Zone 1 ) on galactose + HU . The number of cells is > 300 for each condition .

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Figure 7(B) Amongst the Mec1-S1991-dependent phosphopeptides on HU, factors involved in transcription are highlighted.(E) Position relative to the nuclear envelope (Zone 1 of the lacO-tagged GAL1-GAL10 locus in the indicated strains grown either 12h on glucose, or 90 min galactose or galactose + 0.2M HU. The third panel shows the relative retention at the nuclear periphery (Zone 1) on galactose + HU. The number of cells is >300 for each condition.


Figure 8Total Rpb1 levels of exponentially growing cells that were synchronized in G1 with α - factor and released into S phase at 25 ° C . Actin was used as a loading control .

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Figure 8Total Rpb1 levels of exponentially growing cells that were synchronized in G1 with α-factor and released into S phase at 25°C. Actin was used as a loading control.


Figure 1A Ccno mRNA is expressed in the embryonic node at E8 as shown by in situ hybridization . Occasionally , additional expression is observed at the posterior tip of the embryo ( asterisk ) . Double labelling by staining for LacZ expression from the CcnoTA allele ( shown in B ) and in situ hybridization for Dand5 , marking crown cells at the circumference of the node . Ccno expression is restricted to ciliated , ventral pit cells of the node as detailed in the transverse section at the indicated plane . C Whole embryo X ‐ Gal staining of E16 CcnoTA / + and wild ‐ type control embryos and indicated section planes of ( D - F ' ) . D - F ' Histological sections of LacZ ‐ stained CcnoTA / + embryo at E16 reveal Ccno expression in the epithelium of ( D ) plexus choroideus and ependyme , ( E ) snout epithelium , and ( F , F ' ) trachea and bronchi . pc , plexus choroideus ; se , snout epithelium ; tr , trachea ; and br , bronchus . G Co ‐ expression of Ccno and Foxj1 in multiciliated cells of the trachea shown by double staining for X ‐ Gal and immunohistochemistry using a FOXJ1 ‐ specific antibody .

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Figure 1A Ccno mRNA is expressed in the embryonic node at E8 as shown by in situ hybridization. Occasionally, additional expression is observed at the posterior tip of the embryo (asterisk). Double labelling by staining for LacZ expression from the CcnoTA allele (shown in B) and in situ hybridization for Dand5, marking crown cells at the circumference of the node. Ccno expression is restricted to ciliated, ventral pit cells of the node as detailed in the transverse section at the indicated plane.C Whole embryo X‐Gal staining of E16 CcnoTA/+ and wild‐type control embryos and indicated section planes of (D-F').D-F' Histological sections of LacZ‐stained CcnoTA/+ embryo at E16 reveal Ccno expression in the epithelium of (D) plexus choroideus and ependyme, (E) snout epithelium, and (F, F') trachea and bronchi. pc, plexus choroideus; se, snout epithelium; tr, trachea; and br, bronchus.G Co‐expression of Ccno and Foxj1 in multiciliated cells of the trachea shown by double staining for X‐Gal and immunohistochemistry using a FOXJ1‐specific antibody.


Figure 2A Ccno ‐ deficient mice develop severe hydrocephalus resulting in characteristic head deformation at P21 ( arrow ) . B , C Cranial MRI analysis ( B ) shows the drastically enlarged ventricular cavity ( marked by V ) and diminished cerebral cortex tissue , ( C ) also seen when brains are cut in coronal orientation . D , D ' Alcian ‐ blue staining of paranasal cavities reveals mucus congestion along the nasal epithelium ( arrows ) . F , F ' Scanning electron microscopy ( SEM ) of P21 adult trachea shows reduced numbers of cilia of Ccno ‐ deficient MCCs . Remaining cilia are found in the central regions of the cell surface , and cell margins frequently lack the ciliary decoration ( arrows ) . G Transmission electron microscopy ( TEM ) of wild ‐ type and CcnoRA / RA MCCs from adult trachea . Ccno ‐ deficient MCCs show reduced numbers of basal bodies and cilia that correctly docked to the apical cell surface ( arrows ) . Ectopic electron ‐ dense material is found within the cytoplasm of Ccno ‐ deficient MCCs ( arrowheads ) .

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Figure 2A Ccno‐deficient mice develop severe hydrocephalus resulting in characteristic head deformation at P21 (arrow).B, C Cranial MRI analysis (B) shows the drastically enlarged ventricular cavity (marked by V) and diminished cerebral cortex tissue, (C) also seen when brains are cut in coronal orientation.D, D' Alcian‐blue staining of paranasal cavities reveals mucus congestion along the nasal epithelium (arrows).F, F' Scanning electron microscopy (SEM) of P21 adult trachea shows reduced numbers of cilia of Ccno‐deficient MCCs. Remaining cilia are found in the central regions of the cell surface, and cell margins frequently lack the ciliary decoration (arrows).G Transmission electron microscopy (TEM) of wild‐type and CcnoRA/RA MCCs from adult trachea. Ccno‐deficient MCCs show reduced numbers of basal bodies and cilia that correctly docked to the apical cell surface (arrows). Ectopic electron‐dense material is found within the cytoplasm of Ccno‐deficient MCCs (arrowheads).


Figure 3A - H X ‐ Gal ‐ staining of ( A - C ) control and ( D - H ) CcnoTA / + embryonic lungs at indicated stages showing onset of Ccno expression from E13 in the proximal trachea and in the main bronchi . Expression is extending to more distal regions , and from E16 staining is also found in bronchioli . I X ‐ Gal ‐ staining indicating LacZ expression from the CcnoTA / + allele in mTEC cultures after 5 days of differentiation ‐ onset by switching to air - liquid interface ( ALI ) conditions ( ALI d5 ) . J Transcript levels of Ccno and indicated genes with known functions for the generation of multiple cilia during mTEC differentiation . mRNA levels from three independent experiments were measured by qRT - PCR and levels of expression set as 1 on day 0 of ALI cultures . Scales for Ccno and Deup1 are indicated on the left , and for Cep152 , Cep63 and Ift57 on the right side . K Relative mRNA expression levels for indicated genes were measured by qRT - PCR in wild ‐ type and Ccno ‐ deficient mTEC cultures at indicated days after differentiation ‐ onset . Relative values were calculated as in ( J ) relative to day 0 of ALI cultures .

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Figure 3A-H X‐Gal‐staining of (A-C) control and (D-H) CcnoTA/+ embryonic lungs at indicated stages showing onset of Ccno expression from E13 in the proximal trachea and in the main bronchi. Expression is extending to more distal regions, and from E16 staining is also found in bronchioli.I X‐Gal‐staining indicating LacZ expression from the CcnoTA/+ allele in mTEC cultures after 5 days of differentiation‐onset by switching to air-liquid interface (ALI) conditions (ALI d5).J Transcript levels of Ccno and indicated genes with known functions for the generation of multiple cilia during mTEC differentiation. mRNA levels from three independent experiments were measured by qRT-PCR and levels of expression set as 1 on day 0 of ALI cultures. Scales for Ccno and Deup1 are indicated on the left, and for Cep152, Cep63 and Ift57 on the right side.K Relative mRNA expression levels for indicated genes were measured by qRT-PCR in wild‐type and Ccno‐deficient mTEC cultures at indicated days after differentiation‐onset. Relative values were calculated as in (J) relative to day 0 of ALI cultures.


Figure 4A - B ' SEM of tracheae from ( A , A ' ) wild ‐ type and ( B , B ' ) CcnoRA / RA embryos at E17 . In comparison to wild ‐ type MCCs , Ccno ‐ deficient MCCs almost completely lack formation of multiple cilia and only single , or doublet ( arrow ) cilia are observed at E17 . C - E The lack of multiple cilia at E17 stages is reflected by the almost complete absence of acetylated α ‐ tubulin staining as marker for axonemes in ( D ) Ccno ‐ deficient MCCs of E17 embryonic trachea as quantified in ( E ) . FOV , field of view . F - I TEM of MCCs from E17 embryonic tracheae shows deuterosomes with forming procentrioles in ( F , F ' , H ) Ccno ‐ proficient and ( G , G ' , I ) Ccno ‐ deficient MCCs . Asterisks in ( F , G ) indicate microvilli that extend from the apical cell surface . ( F ' , G ' ) Deuterosomes in Ccno ‐ deficient MCCs are significantly enlarged ( identical size bars in F ' and G ' ) and show irregular morphology , which is different to the annular shape of wild ‐ type deuterosomes as seen in ( F ' , H ) . Procentrioles ( arrowheads in F ' and I ) that are found at deuterosomes of ( I ) Ccno ‐ deficient MCCs were recurrently found being shorter and appeared less structured in comparison to ( F ' , H ) wild ‐ type cells . J The average length of procentrioles was quantified for wild ‐ type and Ccno ‐ deficient MCCs of E17 tracheae ( 49 procentrioles in n = 3 independent samples for wild ‐ types and 47 procentrioles in n = 3 independent samples for Ccno ‐ deficient MCCs ) . K The diameter of deuterosomes ( 41 deuterosomes in n = 3 independent samples for wild ‐ types ; 38 deuterosomes in n = 5 independent samples for Ccno ‐ deficient MCCs ) was measured in wild ‐ type and Ccno ‐ deficient MCCs of E17 tracheae .

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

Figure 4A-B' SEM of tracheae from (A, A') wild‐type and (B, B') CcnoRA/RA embryos at E17. In comparison to wild‐type MCCs, Ccno‐deficient MCCs almost completely lack formation of multiple cilia and only single, or doublet (arrow) cilia are observed at E17.C-E The lack of multiple cilia at E17 stages is reflected by the almost complete absence of acetylated α‐tubulin staining as marker for axonemes in (D) Ccno‐deficient MCCs of E17 embryonic trachea as quantified in (E). FOV, field of view.F-I TEM of MCCs from E17 embryonic tracheae shows deuterosomes with forming procentrioles in (F, F', H) Ccno‐proficient and (G, G', I) Ccno‐deficient MCCs. Asterisks in (F, G) indicate microvilli that extend from the apical cell surface. (F', G') Deuterosomes in Ccno‐deficient MCCs are significantly enlarged (identical size bars in F' and G') and show irregular morphology, which is different to the annular shape of wild‐type deuterosomes as seen in (F', H). Procentrioles (arrowheads in F' and I) that are found at deuterosomes of (I) Ccno‐deficient MCCs were recurrently found being shorter and appeared less structured in comparison to (F', H) wild‐type cells.J The average length of procentrioles was quantified for wild‐type and Ccno‐deficient MCCs of E17 tracheae (49 procentrioles in n = 3 independent samples for wild‐types and 47 procentrioles in n = 3 independent samples for Ccno‐deficient MCCs).K The diameter of deuterosomes (41 deuterosomes in n = 3 independent samples for wild‐types; 38 deuterosomes in n = 5 independent samples for Ccno‐deficient MCCs) was measured in wild‐type and Ccno‐deficient MCCs of E17 tracheae.


Figure 5Double IF analysis of mTEC cultures at early stage of MCC differentiation and centriole amplification ( ALI day 3 ) using antibodies for the deuterosome protein DEUP1 and the early centriole marker SAS ‐ 6 . Deuterosome numbers are decreased and deuterosome size enlarged in Ccno ‐ deficient MCCs as quantified in ( C ) . Deuterosome ‐ dependent centriole biogenesis is severely reduced in Ccno ‐ deficient MCCs as shown by reduced staining for the early centriole marker SAS ‐ 6 . IFstaining for DEUP1 reveals a further size increase of deuterosomes of Ccno ‐ deficient MCCs at later stages of centriole biogenesis ( ALI day 7 ) . Deuterosome number and size were quantified in ( C ) . IF using a Centrin antibody shows globally reduced presence of centrioles at ALI day 7 , which stay localized below the level of deuterosomes and fail to dock to the apical cellmembrane in Ccno ‐ deficient MCCs as seen in z ‐ projection . IFstaining for DEUP1 in ( A , B ) was analysed by Imaris 7 . 7 . 2 software to calculate average deuterosome number of wild ‐ type ( n = 17 ) and Ccno ‐ deficient ( n = 19 ) MCCs at ALI day 3 , and wild ‐ type ( n = 12 ) and Ccno ‐ deficient ( n = 14 ) MCCs at ALI day 7 . Deuterosome size was measured using the spot measurement tool in n = 10 MCCs for both genotypes at ALI day 3 , and in wild ‐ type ( n = 12 ) and Ccno ‐ deficient ( n = 14 ) MCCs at ALI day 7 .

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

Figure 5Double IF analysis of mTEC cultures at early stage of MCC differentiation and centriole amplification (ALI day 3) using antibodies for the deuterosome protein DEUP1 and the early centriole marker SAS‐6. Deuterosome numbers are decreased and deuterosome size enlarged in Ccno‐deficient MCCs as quantified in (C). Deuterosome‐dependent centriole biogenesis is severely reduced in Ccno‐deficient MCCs as shown by reduced staining for the early centriole marker SAS‐6.IFstaining for DEUP1 reveals a further size increase of deuterosomes of Ccno‐deficient MCCs at later stages of centriole biogenesis (ALI day 7). Deuterosome number and size were quantified in (C). IF using a Centrin antibody shows globally reduced presence of centrioles at ALI day 7, which stay localized below the level of deuterosomes and fail to dock to the apical cellmembrane in Ccno‐deficient MCCs as seen in z‐projection.IFstaining for DEUP1 in (A, B) was analysed by Imaris 7.7.2 software to calculate average deuterosome number of wild‐type (n = 17) and Ccno‐deficient (n = 19) MCCs at ALI day 3, and wild‐type (n = 12) and Ccno‐deficient (n = 14) MCCs at ALI day 7. Deuterosome size was measured using the spot measurement tool in n = 10 MCCs for both genotypes at ALI day 3, and in wild‐type (n = 12) and Ccno‐deficient (n = 14) MCCs at ALI day 7.


Figure 6A , B IF analysis of mTEC cultures at ALI day 7 using antibodies against the centriole marker Centrin and CEP164 , which marks maturing centrioles . At ALI day 7 , centrioles of wild ‐ type MCCs are decorated with CEP164 and are mostly localized to the apical cellmembrane . The fewer centrioles of Ccno ‐ deficient MCCs do not colocalize with CEP164 and fail to reach the apical cell surface as seen in z ‐ projection . Lack of CEP164 indicates immature centrioles in Ccno ‐ deficient MCCs . ( B ) Total centriole numbers per MCC were counted in n = 11 MCCs for both genotypes using Imaris 7 . 7 . 2 software . C , D Double IFstaining using Centrin ‐ and CP110 ‐ specific antibodies shows an almost complete absence of CP110 from Centrin ‐ positive centrioles in the majority of wild ‐ type MCCs at day 7 of ALI cultures . Most Ccno ‐ deficient MCCs exhibit pronounced CP110 staining that colocalizes with Centrin as seen in z ‐ projection . ( D ) CP110 ‐ positive MCCs were counted in wild ‐ type and Ccno ‐ deficient MTEC cultures and the percentages of CP110 ‐ positive MCCs from total MCCs calculated .

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

Figure 6A, B IF analysis of mTEC cultures at ALI day 7 using antibodies against the centriole marker Centrin and CEP164, which marks maturing centrioles. At ALI day 7, centrioles of wild‐type MCCs are decorated with CEP164 and are mostly localized to the apical cellmembrane. The fewer centrioles of Ccno‐deficient MCCs do not colocalize with CEP164 and fail to reach the apical cell surface as seen in z‐projection. Lack of CEP164 indicates immature centrioles in Ccno‐deficient MCCs. (B) Total centriole numbers per MCC were counted in n = 11 MCCs for both genotypes using Imaris 7.7.2 software.C, D Double IFstaining using Centrin‐ and CP110‐specific antibodies shows an almost complete absence of CP110 from Centrin‐positive centrioles in the majority of wild‐type MCCs at day 7 of ALI cultures. Most Ccno‐deficient MCCs exhibit pronounced CP110 staining that colocalizes with Centrin as seen in z‐projection. (D) CP110‐positive MCCs were counted in wild‐type and Ccno‐deficient MTEC cultures and the percentages of CP110‐positive MCCs from total MCCs calculated.


Figure 2Luciferase sequestration as an assay for in vitro uptake . ( A ) Luciferase cofractionating with vacuoles is proteinase K protected . In vitro uptake reactions of three times the standard volume were performed ( 60 min , 27 ° C ) in the presence or absence of the ATP - regenerating system . The vacuoles were reisolated , washed , resuspended in 150 mM KCl in PS buffer to a concentration of 0 . 23 mg / ml , and split into aliquots . After addition of 1 % Triton X - 100 or buffer , the mixture was digested with the indicated concentrations of proteinase K ( 15 min , 0 ° C ) . Proteinase treatment was stopped by adding one volume of 0 . 5 mM PMSF in 150 mM KCl in PS buffer . Then , luciferase activity was assayed . ( B ) Luciferase can be released from the vacuolar lumen by freeze - thaw treatment . An uptake reaction was performed ( 60 min , 27 ° C ) . The vacuoles were reisolated and washed as described in the Materials and Methods section . The final pellet was resuspended in 90 μl PS buffer with 150 mM KCl and split into two aliquots . One was frozen at − 80 ° C and thawed again slowly , whereas the other remained on ice . The samples were centrifuged ( 15 min , 145 , 000 g , 4 ° C ) . The supernatants ( Sup ) were recovered and the pellets were resuspended in an equal volume of PS with 150 mM KCl . Luciferase activity in both fractions was determined . The reaction tubes for this experiment had been coated with BSA ( 0 . 5 mg / ml , 15 min at room temperature ) to reduce unspecific binding of luciferase to the surface .

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

Figure 2Luciferase sequestration as an assay for in vitro uptake. (A) Luciferase cofractionating with vacuoles is proteinase K protected. In vitro uptake reactions of three times the standard volume were performed (60 min, 27°C) in the presence or absence of the ATP-regenerating system. The vacuoles were reisolated, washed, resuspended in 150 mM KCl in PS buffer to a concentration of 0.23 mg/ml, and split into aliquots. After addition of 1% Triton X-100 or buffer, the mixture was digested with the indicated concentrations of proteinase K (15 min, 0°C). Proteinase treatment was stopped by adding one volume of 0.5 mM PMSF in 150 mM KCl in PS buffer. Then, luciferase activity was assayed.(B) Luciferase can be released from the vacuolar lumen by freeze-thaw treatment. An uptake reaction was performed (60 min, 27°C). The vacuoles were reisolated and washed as described in the Materials and Methods section. The final pellet was resuspended in 90 μl PS buffer with 150 mM KCl and split into two aliquots. One was frozen at −80°C and thawed again slowly, whereas the other remained on ice. The samples were centrifuged (15 min, 145,000 g, 4°C). The supernatants (Sup)were recovered and the pellets were resuspended in an equal volume of PS with 150 mM KCl. Luciferase activity in both fractions was determined. The reaction tubes for this experiment had been coated with BSA (0.5 mg/ml, 15 min at room temperature) to reduce unspecific binding of luciferase to the surface.


Figure 3time course and temperature dependence of uptake . ( A ) Standard uptake reactions were performed in the presence or absence of the ATP - regenerating system . After the indicated times at 27 ° C , the vacuoles were reisolated , washed , treated with protease , and luciferaseuptake activity was assayed . ( B ) A standard uptake reaction was performed . After 70 min of incubation , the vacuoles were reisolated , washed , and treated with protease . The vacuoles were reisolated again , resuspended in reaction buffer without luciferase , and incubated for the indicated times at 27 ° C . Aliquots were withdrawn to assay the total luciferase remaining . As a control , luciferase was incubated in reaction buffer for the same periods of time and assayed for activity .

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

Figure 3time course and temperature dependence of uptake. (A) Standard uptake reactions were performed in the presence or absence of the ATP-regenerating system. After the indicated times at 27°C, the vacuoles were reisolated, washed, treated with protease, and luciferaseuptake activity was assayed.(B) A standard uptake reaction was performed. After 70 min of incubation, the vacuoles were reisolated, washed, and treated with protease. The vacuoles were reisolated again, resuspended in reaction buffer without luciferase, and incubated for the indicated times at 27°C. Aliquots were withdrawn to assay the total luciferase remaining. As a control, luciferase was incubated in reaction buffer for the same periods of time and assayed for activity.


Figure 4Requirements for in vitro uptake . Standard uptake reactions were performed ( 60 min ) ( A ) at different temperatures , or in the presence of different concentrations of ( B ) cytosol , ( C ) ATP , or ( D ) MgCl2 . Luciferase uptake was determined as described in the legend to Fig . 3 A . In B , titration curves for four independent cytosol preparations are shown . The maximal uptake signal of each titration curve was set at 100 % . In D , the sample drawn as 0 mM MgCl2 contained 10 mM EDTA to chelate free magnesium in the reaction .

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Figure 4Requirements for in vitro uptake. Standard uptake reactions were performed (60 min) (A) at different temperatures, or in the presence of different concentrations of (B) cytosol, (C) ATP, or (D) MgCl2. Luciferase uptake was determined as described in the legend to Fig. 3 A. In B, titration curves for four independent cytosol preparations are shown. The maximal uptake signal of each titration curve was set at 100%. In D, the sample drawn as 0 mM MgCl2 contained 10 mM EDTA to chelate free magnesium in the reaction.


Figure 6In vitro uptake is independent of components involved in homotypic vacuolar fusion . ( A ) Standard uptake reactions were performed ( 60 min , 27 ° C ) in the presence of the indicated reagents using a mixture of vacuoles from strains DBY5734 - 16 ( Δpep4 ) and DBY5734 - 19 ( Δpho8 ) . Therefore , homotypic vacuolar fusion and luciferase uptake could be assayed simultaneously from the same sample . Four independent experiments were averaged . Error bars indicate SD . For averaging , uptake and fusion activities of the control sample without inhibitor were set at 100 % . Inhibitors were used at the following concentrations : anti - Sec17p , 1 μM ; anti - Sec18p , 1 μM ; Gdi1p , 2 . 5 μM ; anti - Vam3p , 1 μM ; anti - Vam7p , 1 μM ; and anti - Nyv1p , 1 μM . ( B ) Vacuoles and cytosols were prepared from sec18 - 1 and from corresponding wild - type ( Wt ) cells grown and spheroplasted at 23 ° C . The isolated vacuoles and the cytosols were exposed to heat treatment ( 10 min , 37 ° C ) . Standard uptake reactions were performed with vacuoles and cytosols in the indicated combinations . Control reactions were performed in the absence of ATP . Five independent experiments were averaged ; the sample with vacuoles and cytosol from wild - type cells was used as the 100 % reference . Error bars indicate SD . Mut , mutant .

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

Figure 6In vitro uptake is independent of components involved in homotypic vacuolar fusion. (A) Standard uptake reactions were performed (60 min, 27°C) in the presence of the indicated reagents using a mixture of vacuoles from strains DBY5734-16 (Δpep4) and DBY5734-19 (Δpho8). Therefore, homotypic vacuolar fusion and luciferase uptake could be assayed simultaneously from the same sample. Four independent experiments were averaged. Error bars indicate SD. For averaging, uptake and fusion activities of the control sample without inhibitor were set at 100%. Inhibitors were used at the following concentrations: anti-Sec17p, 1 μM; anti-Sec18p, 1 μM; Gdi1p, 2.5 μM; anti-Vam3p, 1 μM; anti-Vam7p, 1 μM; and anti-Nyv1p, 1 μM.(B) Vacuoles and cytosols were prepared from sec18-1 and from corresponding wild-type (Wt) cells grown and spheroplasted at 23°C. The isolated vacuoles and the cytosols were exposed to heat treatment (10 min, 37°C). Standard uptake reactions were performed with vacuoles and cytosols in the indicated combinations. Control reactions were performed in the absence of ATP. Five independent experiments were averaged; the sample with vacuoles and cytosol from wild-type cells was used as the 100% reference. Error bars indicate SD. Mut, mutant.


Figure 7In vitro uptake depends on GTP hydrolysis and on the V - ATPase . ( A ) Standard uptake reactions were performed in the presence of different concentrations of GTPγS . Luciferase uptake was assayed as in the legend to Fig . 3 A . Four independent experiments were averaged , using the sample without inhibitor as the 100 % reference . Error bars indicate SD . GTPγS did not influence luciferase activity by itself ( not shown ) . ( B ) Standard uptake reactions were performed in the presence of FCCP ( CMA , 20 μM ) or concanamycin A ( 20 μM ) , or in the absence of ATP .

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Figure 7In vitro uptake depends on GTP hydrolysis and on the V-ATPase. (A) Standard uptake reactions were performed in the presence of different concentrations of GTPγS. Luciferase uptake was assayed as in the legend to Fig. 3 A. Four independent experiments were averaged, using the sample without inhibitor as the 100% reference. Error bars indicate SD. GTPγS did not influence luciferase activity by itself (not shown).(B) Standard uptake reactions were performed in the presence of FCCP (CMA, 20 μM) or concanamycin A (20 μM), or in the absence of ATP.