Figure 7A The senescence phenotypes of five - week - old vector control , proSAG12 : : CDF4 and proSAG12 : : CDF4 & 35S : : CAT2 plants , from old to young . Scale bars indicate 1 . 5 cm . B qPCR analysis of the expression of CDF4 and CAT2 in the leaves Four independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t test . qPCR analysis of the expression of CAT2 in the leaves Four independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t test . ( D ) Measurement of chlorophyll in the leaves The third leaves in the rosette were used . Four independent experiments were conducted . Data are presented as the mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t test . ( E ) H2O2 concentrations in the leaves The third leaves in the rosette were used . Four independent experiments were conducted . Data are presented as the mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t test .

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Figure 7A The senescence phenotypes of five-week-old vector control, proSAG12::CDF4 and proSAG12::CDF4 & 35S::CAT2 plants, from old to young. Scale bars indicate 1.5 cm.B qPCR analysis of the expression of CDF4 and CAT2 in the leaves Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t test.qPCR analysis of the expression of CAT2 in the leaves Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t test.(D) Measurement of chlorophyll in the leaves The third leaves in the rosette were used. Four independent experiments were conducted. Data are presented as the mean ± SD, n=4. *p<0.05 by Student's t test.(E) H2O2 concentrations in the leaves The third leaves in the rosette were used. Four independent experiments were conducted. Data are presented as the mean ± SD, n=4. *p<0.05 by Student's t test.


Figure 8 ( A ) Observation of inflorescences of 7 - week - old wild - type ( WT ) Arabidopsis and the CDF4 transgenic plants . The asterisk represents the flower organ attached along the inflorescence . Scale bar indicates 1 . 5 cm . Selected abscission - related gene expression levels in ( B ) WT and proIDA : : CDF4 lines by using qPCR analysis . The expression of these selected genes in the wild - type plant is given as 1 . The relative expression level represents only the level of expression of the gene relative to the wild type . ACTIN2 was used as the internal control . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test . Selected abscission - related gene expression levels in ( C ) WT and proIDA : : CDF4RNAi lines by using qPCR analysis . The expression of these selected genes in the wild - type plant is given as 1 . The relative expression level represents only the level of expression of the gene relative to the wild type . ACTIN2 was used as the internal control . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test . E Observation of GUS staining activity in the proPGAZAT : : GUS and proPGAZAT : : GUS & proIDA : : CDF4 . Scale bar indicates 0 . 5 cm . F ChIP - qPCR analysis of the relative binding of CDF4 to the promoter regions of PGAZAT . An anti - HA monoclonal antibody was used for DNA immunoprecipitation from 6 - week - old pER8 : : CDF4 - HA transgenic plants after estradiol induction . Black columns indicate the enrichment fold changes normalized to ACT2 . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test .

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Figure 8(A) Observation of inflorescences of 7-week-old wild-type (WT) Arabidopsis and the CDF4 transgenic plants. The asterisk represents the flower organ attached along the inflorescence. Scale bar indicates 1.5 cm.Selected abscission-related gene expression levels in (B) WT and proIDA::CDF4 lines by using qPCR analysis. The expression of these selected genes in the wild-type plant is given as 1. The relative expression level represents only the level of expression of the gene relative to the wild type. ACTIN2 was used as the internal control. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.Selected abscission-related gene expression levels in (C) WT and proIDA::CDF4RNAi lines by using qPCR analysis. The expression of these selected genes in the wild-type plant is given as 1. The relative expression level represents only the level of expression of the gene relative to the wild type. ACTIN2 was used as the internal control. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.E Observation of GUS staining activity in the proPGAZAT::GUS and proPGAZAT::GUS & proIDA::CDF4. Scale bar indicates 0.5 cm.F ChIP-qPCR analysis of the relative binding of CDF4 to the promoter regions of PGAZAT. An anti-HA monoclonal antibody was used for DNA immunoprecipitation from 6-week-old pER8::CDF4-HA transgenic plants after estradiol induction. Black columns indicate the enrichment fold changes normalized to ACT2. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


Figure 1 ( A ) CXCL10 assay characteristics . Standard curves of the short , long and total CXCL10Simoa assays are shown . For each curve , limit of detection ( LOD ) defined as blank + 3SD , are shown as horizontal lines . The LOD was 1 . 7pg / ml for long and short CXCL10 assays and 0 . 22pg / ml for total CXCL10 . Standard curves were fitted using the 4 Parameter Logistic non - linear regression model . Samples reporting a signal below the LOD were replaced with 1pg / ml for short and long CXCL10 . Plasma from healthy individuals receiving ( B ) placebo ( n = 9 ) or ( C ) sitagliptin ( n = 27 ) were analyzed by Simoa . Blood samples were collected at screening visit ( SV ) and day 0 ( D0 ) before treatment , at day 3 ( D3 ) , day 14 ( W2 ) and day 28 ( W4 ) under treatment and 5 weeks after treatment interruption ( W9 ) . ( C ) Plasma from chronic HCVpatients receiving sitagliptin ( n = 3 ) were collected before ( D0 ) and weekly during sitagliptin treatment ( W1 W3 ) . Antagonist CXCL103 - 77 ( short CXCL10 , in blue ) , agonist CXCL101 - 77 ( long CXCL10 , in red ) and total CXCL10 ( in black ) levels are shown . Each dot represents a donor and bars are at the median . Grey areas highlight the period under placebo or sitagliptin treatment . Statistical analysis of ( B ) and ( C ) was performed using non parametric Friedman ' s test , ns : non significant , * * : P0 . 01 , * * * * : P0 . 001 . For ( C ) , additional size effect analysis was performed and Cohen ' s d values are reported . No statistical analysis was performed in ( D ) due to sample size , nd : non - determined .

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Figure 1(A) CXCL10 assay characteristics. Standard curves of the short, long and total CXCL10Simoa assays are shown. For each curve, limit of detection (LOD) defined as blank+3SD, are shown as horizontal lines. The LOD was 1.7pg/ml for long and short CXCL10 assays and 0.22pg/ml for total CXCL10. Standard curves were fitted using the 4 Parameter Logistic non-linear regression model. Samples reporting a signal below the LOD were replaced with 1pg/ml for short and long CXCL10.Plasma from healthy individuals receiving (B) placebo (n=9) or (C) sitagliptin (n=27) were analyzed by Simoa. Blood samples were collected at screening visit (SV) and day 0 (D0) before treatment, at day 3 (D3), day 14 (W2) and day 28 (W4) under treatment and 5 weeks after treatment interruption (W9). (C) Plasma from chronic HCVpatients receiving sitagliptin (n=3) were collected before (D0) and weekly during sitagliptin treatment (W1 W3). Antagonist CXCL103-77 (short CXCL10, in blue), agonist CXCL101-77 (long CXCL10, in red) and total CXCL10 (in black) levels are shown. Each dot represents a donor and bars are at the median. Grey areas highlight the period under placebo or sitagliptin treatment. Statistical analysis of (B) and (C) was performed using non parametric Friedman's test, ns: non significant, **: P0.01, ****: P0.001. For (C), additional size effect analysis was performed and Cohen's d values are reported. No statistical analysis was performed in (D) due to sample size, nd: non-determined.


Figure 1C GST pulldown of transiently expressed AdipoR1 ( residues 1 - 375 ) with immobilized GST - P5 or GST - P10 fusion proteins . Pulldown fractions were subjected to western blotting using Flag and GST antibodies .

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Figure 1C GST pulldown of transiently expressed AdipoR1 (residues 1-375) with immobilized GST-P5 or GST-P10 fusion proteins. Pulldown fractions were subjected to western blotting using Flag and GST antibodies.


Figure 2C AdipoR1 siRNA - transfected ORS or DP cells were treated with APN or P5 . The cell lysates were analyzed for p - AMPK . D - H The relative gene expression levels of growth factors in P5 - treated DP cells ( n = 4 ( VEGF , PDGFA ) , 5 ( IGF1 ) , 6 ( HGF , FGF7 ) biological replicates / group )

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Figure 2C AdipoR1 siRNA-transfected ORS or DP cells were treated with APN or P5. The cell lysates were analyzed for p-AMPK.D-H The relative gene expression levels of growth factors in P5-treated DP cells (n = 4 (VEGF, PDGFA), 5 (IGF1), 6 (HGF, FGF7) biological replicates / group)


Figure 3C IF staining ( p - AMPK ) of the HF bulb area . Yellow arrow indicates p - AMPK signal ; scale bar : 100 µm . D IF staining ( Ki - 67 ) of the HF bulb area ; scale bar : 100 µm .

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Figure 3C IF staining (p-AMPK) of the HF bulb area. Yellow arrow indicates p-AMPK signal; scale bar: 100 µm. D IF staining (Ki-67) of the HF bulb area; scale bar: 100 µm.


Figure 4C H & E and IF for versican of skin tissue ; scale bars : 100 µm . G H & E and IF for versican of skin tissue ; scale bars : 100 µm . H Anagen induction scores of vehicle - , or P5 - treated Adipoq - / - mice ( n = 6 tissues / mice , 4 mice / group ) ; Unpaired t test K . H & E and IF for versican of skin tissue ; scale bars : 100 µm . L Anagen induction scores of vehicle - , or P5 - treated Adipor1 - / - mice ( n = 6 tissues / mice , 5 mice / group ) ; Unpaired t test

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Figure 4C H&E and IF for versican of skin tissue; scale bars: 100 µm.G H&E and IF for versican of skin tissue; scale bars: 100 µm. H Anagen induction scores of vehicle-, or P5- treated Adipoq-/- mice (n = 6 tissues / mice, 4 mice / group); Unpaired t test K. H&E and IF for versican of skin tissue; scale bars: 100 µm. L Anagen induction scores of vehicle-, or P5- treated Adipor1-/-mice (n = 6 tissues / mice, 5 mice / group); Unpaired t test


Figure 5Pulldown of Flag - fused AdipoR1 input ( residues 89 - 375 ) with amino acid mutation ( s ) , as indicated , with GST - P5 . The relative pulldown efficiency is expressed as the ratio of the band intensities of bound AdipoR1 to those of AdipoR1 ( residue 89 - 375 ) inputs , as indicated ( n = 3 technical replicates / group ) . F Pulldown of AdipoR1 ( residues 89 - 375 ) with GST - P5 or GST fused to two mutant peptides ( GLYDF and GLYAF ) . The relative pulldown efficiency is expressed as the ratio of the band intensities of bound AdipoR1 to those of AdipoR1 ( residue 89 - 375 ) inputs , as indicated ( n = 3 technical replicates / group ) .

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Figure 5Pulldown of Flag-fused AdipoR1 input (residues 89-375) with amino acid mutation(s), as indicated, with GST-P5. The relative pulldown efficiency is expressed as the ratio of the band intensities of bound AdipoR1 to those of AdipoR1 (residue 89-375) inputs, as indicated (n = 3 technical replicates / group).F Pulldown of AdipoR1 (residues 89-375) with GST-P5 or GST fused to two mutant peptides (GLYDF and GLYAF). The relative pulldown efficiency is expressed as the ratio of the band intensities of bound AdipoR1 to those of AdipoR1 (residue 89-375) inputs, as indicated (n = 3 technical replicates / group).


Figure 1B Fatty acid composition of B . subtilis WT cells grown in PF medium , and Δbkd grown with MB , IB or depleted for precursor for 90 min ( IBPF ) . For detailed analyses , see Appendix Fig S1B . E Fatty acid composition of E . coli WT and fabA ( Ts ) cells grown at 30 ° C and shifted to different temperatures for 120 min . For detailed analyses , see Appendix Fig S2B and C .

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Figure 1B Fatty acid composition of B. subtilis WT cells grown in PF medium, and Δbkd grown with MB, IB or depleted for precursor for 90 min (IBPF). For detailed analyses, see Appendix Fig S1B.E Fatty acid composition of E. coli WT and fabA(Ts) cells grown at 30°C and shifted to different temperatures for 120 min. For detailed analyses, see Appendix Fig S2B and C.


Figure 2A DPH anisotropy of B . subtilis WT or Δbkd cells supplemented either with MB , IB or depleted for precursor ( IBPF ) for the times indicated . High DPH anisotropy indicates low membrane fluidity . B DPH anisotropy of E . coli WT cells grown steady state at 30 ° C , 37 ° C or shifted from 30 ° C to 37 ° C followed by immediate measurement . In addition , DPH anisotropy of fabA ( Ts ) cells grown steady state at 30 ° C or shifted from 30 ° C to 37 ° C followed by measurement at the times indicated .

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Figure 2A DPH anisotropy of B. subtilis WT or Δbkd cells supplemented either with MB, IB or depleted for precursor (IBPF) for the times indicated. High DPH anisotropy indicates low membrane fluidity. B DPH anisotropy of E. coli WT cells grown steady state at 30°C, 37°C or shifted from 30°C to 37°C followed by immediate measurement. In addition, DPH anisotropy of fabA(Ts) cells grown steady state at 30°C or shifted from 30°C to 37°C followed by measurement at the times indicated. D


Figure 3A Images of B . subtilis WT and Δbkd cells co - labelled with the membrane potential - sensitive dye DISC3 ( 5 ) and the membrane permeability indicator Sytox Green . Membrane properties were assessed for Δbkd cells grown in the presence of MB , IB or washed precursor - free ( IBPF ) for the times indicated . As controls , WT cells were measured in the presence of depolarising antimicrobial peptide gramicidin ABC ( gABC ) or pore - forming lantibiotic Nisin . For cross - correlation between membrane depolarisation and membrane permeabilisation , see Appendix Fig S3A - C . B Quantification of DISC3 ( 5 ) fluorescence for cells ( n = 100 - 142 ) depicted in panel A . Median represented by red line . C Images of E . coli WT and fabA ( Ts ) cells co - labelled with the same indicator dyes as in panel A . Membrane properties were assessed for fabA ( Ts ) at 30 ° C and upon transfer to non - permissive 37 ° C for the times indicated . As controls , WT cells were incubated with the pore - forming antibiotic Polymyxin B ( PolyB ) . For cross - correlation between membrane depolarisation and membrane permeabilisation , see Appendix Fig S3D and E . The integrity of the diffusion barrier function was additionally studied via ONPG permeability in a ΔlacY background ( see Fig EV1 ) . D Quantification of DISC3 ( 5 ) fluorescence for cells ( n = 76 - 141 ) depicted in panel C . Median represented by red line .

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Figure 3A Images of B. subtilis WT and Δbkd cells co-labelled with the membrane potential-sensitive dye DISC3(5) and the membrane permeability indicator Sytox Green. Membrane properties were assessed for Δbkd cells grown in the presence of MB, IB or washed precursor-free (IBPF) for the times indicated. As controls, WT cells were measured in the presence of depolarising antimicrobial peptide gramicidin ABC (gABC) or pore-forming lantibiotic Nisin. For cross-correlation between membrane depolarisation and membrane permeabilisation, see Appendix Fig S3A-C. B Quantification of DISC3(5) fluorescence for cells (n=100-142) depicted in panel A. Median represented by red line. C Images of E. coli WT and fabA(Ts) cells co-labelled with the same indicator dyes as in panel A. Membrane properties were assessed for fabA(Ts) at 30°C and upon transfer to non-permissive 37°C for the times indicated. As controls, WT cells were incubated with the pore-forming antibiotic Polymyxin B (PolyB). For cross-correlation between membrane depolarisation and membrane permeabilisation, see Appendix Fig S3D and E. The integrity of the diffusion barrier function was additionally studied via ONPG permeability in a ΔlacY background (see Fig EV1). D Quantification of DISC3(5) fluorescence for cells (n=76-141) depicted in panel C. Median represented by red line. Dat


Figure 4A Images of B . subtilis Δbkd cells stained with membrane dye FM 5 - 95 and expressing GFP fusions of DNA - binding protein HBsu ( top ) , cell division protein FtsZ ( middle ) or cell elongation protein MreB ( bottom ) . Cells were grown with IB or depleted for precursors for 90 min ( IBPF ) . For further examples and additional time points , see Appendix Fig S4A . B Images of E . coli fabA ( Ts ) cells stained with FM 5 - 95 for the outer membrane and with DNA - intercalating dye DAPI ( top ) , or expressing GFP sandwich ( SW ) fusions to the cell division protein FtsZ ( middle ) and the cell elongation protein MreB ( bottom ) , respectively . Depicted are cells grown at 30 ° C or with a temperature shift to 37 ° C for 120 min . For a more detailed view on the influence of low membrane fluidity on membrane dissociation of RNase E as well as on the cell division machinery , see Figs EV2 , EV3 , EV4 , and S5 . For further examples and additional time points , see Appendix Fig S4B .

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Figure 4A Images of B. subtilis Δbkd cells stained with membrane dye FM 5-95 and expressing GFP fusions of DNA-binding protein HBsu (top), cell division protein FtsZ (middle) or cell elongation protein MreB (bottom). Cells were grown with IB or depleted for precursors for 90 min (IBPF). For further examples and additional time points, see Appendix Fig S4A. B Images of E. coli fabA(Ts) cells stained with FM 5-95 for the outer membrane and with DNA-intercalating dye DAPI (top), or expressing GFP sandwich (SW) fusions to the cell division protein FtsZ (middle) and the cell elongation protein MreB (bottom), respectively. Depicted are cells grown at 30°C or with a temperature shift to 37°C for 120 min. For a more detailed view on the influence of low membrane fluidity on membrane dissociation of RNase E as well as on the cell division machinery, see Figs EV2, EV3, EV4, and S5. For further examples and additional time points, see Appendix Fig S4B. D


Figure 5B Images of B . subtilis Δbkd cells grown with IB or without precursor ( IBPF ) . Cells were stained with membrane fluidity - sensitive dye Laurdan and imaged at 460 nm , 520 nm and as the corresponding colour - coded Laurdan GP map . C Images of E . coli fabA ( Ts ) cells grown at 30 ° C or shifted to non - permissive 37 ° C for 120 min . Cells were stained and imaged as described in panel B . For a more pronounced view on domain formation associated with local differences in membrane fluidity , see Appendix Fig S7 , showing E . coli fabA ( Ts ) grown in LB instead of M9 minimal medium with glucose / casamino acids . D Images of B . subtilis Δbkd cells grown and stained with Laurdan ( imaged at 460 nm ) as in panel B , but additionally expressing WALP23 - mCherry . For corresponding fluorescence intensity correlation between images , see Appendix Fig S8A . Co - localisation of membrane dye FM 5 - 95 and transmembrane peptide WALP23 is shown in Appendix Figs S9 and S8C . E Images of E . coli fabA ( Ts ) cells grown and stained as in panel C , but additionally expressing WALP23 - mScarlet - I . For corresponding fluorescence intensity correlation between images , see Appendix Fig S8B .

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Figure 5B Images of B. subtilis Δbkd cells grown with IB or without precursor (IBPF). Cells were stained with membrane fluidity-sensitive dye Laurdan and imaged at 460 nm, 520 nm and as the corresponding colour-coded Laurdan GP map. C Images of E. coli fabA(Ts) cells grown at 30°C or shifted to non-permissive 37°C for 120 min. Cells were stained and imaged as described in panel B. For a more pronounced view on domain formation associated with local differences in membrane fluidity, see Appendix Fig S7, showing E. coli fabA(Ts) grown in LB instead of M9 minimal medium with glucose/casamino acids. D Images of B. subtilis Δbkd cells grown and stained with Laurdan (imaged at 460 nm) as in panel B, but additionally expressing WALP23-mCherry. For corresponding fluorescence intensity correlation between images, see Appendix Fig S8A. Co-localisation of membrane dye FM 5-95 and transmembrane peptide WALP23 is shown in Appendix Figs S9 and S8C. E Images of E. coli fabA(Ts) cells grown and stained as in panel C, but additionally expressing WALP23-mScarlet-I. For corresponding fluorescence intensity correlation between images, see Appendix Fig S8B. Dat


Figure 6A Time lapse images of E . coli fabA ( Ts ) cells expressing FOF1 a - mNG . For corresponding controls , see Appendix Fig S10 . Cells were grown at 30 ° C and shifted from 30 ° C to non - permissive 37 ° C . See Movie EV2 for full time lapse . E Fatty acid composition of E . coli fabA ( Ts ) cells ( same cell batch as in D ) upon growth at 30 ° C , upon depletion of UFA by incubation at 37 ° C for 120 min and upon recovery by oleate supplementation for additional 120 min . For detailed analyses , see Appendix Fig S11B - D .

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Figure 6A Time lapse images of E. coli fabA(Ts) cells expressing FOF1 a-mNG. For corresponding controls, see Appendix Fig S10. Cells were grown at 30°C and shifted from 30°C to non-permissive 37°C. See Movie EV2 for full time lapse.E Fatty acid composition of E. coli fabA(Ts) cells (same cell batch as in D) upon growth at 30°C, upon depletion of UFA by incubation at 37°C for 120 min and upon recovery by oleate supplementation for additional 120 min. For detailed analyses, see Appendix Fig S11B-D.


Figure 7A Representative trajectory maps of individual FOF1 a - mNG molecules in E . coli WT and fabA ( Ts ) cells grown at 30 ° C and upon shift to non - permissive 37 ° C for 120 min . See Movie EV3 ( WT ) and Movie EV4 ( fabA ( Ts ) ) for accumulating trajectory maps of FOF1 a - mNG molecules . D Apparent lateral diffusion coefficients ( Dapp ) of FOF1 a - mNG analysed in panel B .

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

Figure 7A Representative trajectory maps of individual FOF1 a-mNG molecules in E. coli WT and fabA(Ts) cells grown at 30°C and upon shift to non-permissive 37°C for 120 min. See Movie EV3 (WT) and Movie EV4 (fabA(Ts)) for accumulating trajectory maps of FOF1 a-mNG molecules.D Apparent lateral diffusion coefficients (Dapp) of FOF1 a-mNG analysed in panel B.


Figure 1 ( A ) Negative stain electron micrograph showing the tendency of S . cerevisiae prApe1 ( sc - prApe1 ) dodecamers to form higher - order assemblies or chains ( black arrowheads ) . ( B ) Negative stain electron micrograph of S . cerevisiae mApe1 ( sc - mApe1 ) showing monodisperse dodecameric particles ( circles ) as well as hexameric particles ( white arrowheads ) . ( C ) Bar plot showing the occurrence of single , double , triple or multiple aggregates of sc - prApe1 in comparison with sc - mApe1 . Insets show single , double , triple or multiple dodadecamers . Data information : n = 3 , 186 particles were counted in the case of sc - prApe1 and n = 3 , 107 particles were counted in the case of sc - mApe1 . Particles were picked from 20 different micrographs . * * * P < 2 . 2x10 - 16 ( Pearson ' s Chi - squared test ) . ( D ) Negative stain electron micrograph of GraFixed sc - mApe1 showing dodecameric particles ( circles ) . ( E ) Class averages of sc - mApe1 dodecamers including views of two - fold and three - fold symmetry . ( F ) Fourier shell correlation of the EM reconstruction of the sc - mApe1 dodecamer indicates a resolution of 24 Å . ( G ) C . thermophilum mApe1 crystal structure fitted into electron microscopy ( EM ) map of sc - mApe1 dodecamer . Each of the 12 chains is depicted by a different color . A black sphere represents the N - terminus where the propeptide would emanate from the dodecamer . From left to right : two - fold axis view from the edge , three - fold axis view from the facet and three - fold axis view from the vertex .

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

Figure 1 (A) Negative stain electron micrograph showing the tendency of S. cerevisiae prApe1 (sc-prApe1) dodecamers to form higher-order assemblies or chains (black arrowheads). (B) Negative stain electron micrograph of S. cerevisiae mApe1 (sc-mApe1) showing monodisperse dodecameric particles (circles) as well as hexameric particles (white arrowheads). (C) Bar plot showing the occurrence of single, double, triple or multiple aggregates of sc-prApe1 in comparison with sc-mApe1. Insets show single, double, triple or multiple dodadecamers. Data information: n=3,186 particles were counted in the case of sc-prApe1 and n=3,107 particles were counted in the case of sc-mApe1. Particles were picked from 20 different micrographs. ***P < 2.2x10-16 (Pearson's Chi-squared test).  (D) Negative stain electron micrograph of GraFixed sc-mApe1 showing dodecameric particles (circles). (E) Class averages of sc-mApe1 dodecamers including views of two-fold and three-fold symmetry. (F) Fourier shell correlation of the EM reconstruction of the sc-mApe1 dodecamer indicates a resolution of 24 Å. (G) C. thermophilum mApe1 crystal structure fitted into electron microscopy (EM) map of sc-mApe1 dodecamer. Each of the 12 chains is depicted by a different color. A black sphere represents the N-terminus where the propeptide would emanate from the dodecamer. From left to right: two-fold axis view from the edge, three-fold axis view from the facet and three-fold axis view from the vertex.


Figure 2 ( A ) Electron micrograph of negatively stained Ams1 with particles and higher - order assemblies ( arrows ) . Class averages exhibit two distinct two - fold views consistent with D2 symmetry . Scale bar 10 nm . ( B ) Electron cryomicrograph of Ams1 with particles highlighted ( circles ) . ( C ) Selected particles ( top row ) are shown with corresponding class averages ( bottom row ) . Scale bar 10 nm . ( D ) Fourier shell correlation indicates a resolution of 6 . 3 Å according to the 0 . 143 criterion . ( E ) Top view of the cryo - EM structure , four Ams1 molecules were segmented and colored ( dark blue , beige , red , light blue ) . ( F ) Molecular architecture of Ams1 monomer . Primary structure , two map segments with fitted pseudo - atomic models are shown . Left . The N - terminal portion was built from distant homology models : a four - helix bundle ( 17 - 27 , 209 - 271 ) in yellow and a jelly - roll fold ( 45 - 203 ) in red . Right . The α - mannosidase core ( 287 - 1083 ) with an α / β barrel ( 287 - 573 ) in blue , a three - helix bundle ( 574 - 671 ) in green , and the β - sandwich domain ( 672 - 1083 ) in purple . ( G ) Density section through the Ams1 monomer with three - helix bundle in the center . ( H ) Complete atomic model of Ams1 with active site indicated as yellow sphere fitted into the cryo - EM map . Corresponding 3D structure in Movie EV1 . ( J ) The β - sandwich domain ( purple ) makes up the transverse interface between chain A and chain B . ( K ) The N - terminal four - helix bundle ( orange ) mediates the longitudinal interface . Asterisk denotes position of W234E mutation between chain A and C / D . ( L ) Negatively stained W234E Ams1 mutant reveals predominantly smaller particles when compared with wild - type tetramers . Representative class averages show a two - lobed structure consistent with reprojections of chain A and B ( dark blue and beige ) dimers depicted in ribbon and surface representation below . Scale bar 10 nm .

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

Figure 2 (A) Electron micrograph of negatively stained Ams1 with particles and higher-order assemblies (arrows). Class averages exhibit two distinct two-fold views consistent with D2 symmetry. Scale bar 10 nm. (B) Electron cryomicrograph of Ams1 with particles highlighted (circles). (C) Selected particles (top row) are shown with corresponding class averages (bottom row). Scale bar 10 nm. (D) Fourier shell correlation indicates a resolution of 6.3 Å according to the 0.143 criterion.  (E) Top view of the cryo-EM structure, four Ams1 molecules were segmented and colored (dark blue, beige, red, light blue).  (F) Molecular architecture of Ams1 monomer. Primary structure, two map segments with fitted pseudo-atomic models are shown. Left. The N-terminal portion was built from distant homology models: a four-helix bundle (17 - 27, 209 - 271) in yellow and a jelly-roll fold (45 - 203) in red. Right. The α-mannosidase core (287 - 1083) with an α/β barrel (287 - 573) in blue, a three-helix bundle (574 - 671) in green, and the β-sandwich domain (672 - 1083) in purple.  (G) Density section through the Ams1 monomer with three-helix bundle in the center.  (H) Complete atomic model of Ams1 with active site indicated as yellow sphere fitted into the cryo-EM map. Corresponding 3D structure in Movie EV1.  (J) The β-sandwich domain (purple) makes up the transverse interface between chain A and chain B.  (K) The N-terminal four-helix bundle (orange) mediates the longitudinal interface. Asterisk denotes position of W234E mutation between chain A and C/D.  (L) Negatively stained W234E Ams1 mutant reveals predominantly smaller particles when compared with wild-type tetramers. Representative class averages show a two-lobed structure consistent with reprojections of chain A and B (dark blue and beige) dimers depicted in ribbon and surface representation below. Scale bar 10 nm.


Figure 3 ( A ) SEC - MALS profile of Atg19 yields a molecular weight estimate of 142 kDa ( measured in triplicate ) . ( B ) Right . In vitro pull - down binding assay and subsequent SDS - PAGE . Amylose beads are used as a bait to demonstrate interaction of purified prApe1 dodecamers with MBP - Atg19 ( lane 1 ) whereas purified mApe1 dodecamers are not capable of interacting with MBP - Atg19 ( lane 3 ) . As controls , MBP does not bind prApe1 and mApe1 ( lanes 2 and 4 ) . Left . SDS - PAGE showing input of the corresponding pull - down assay . ( C ) Co - expression of MBP - Atg19 and prApe1 in Sf21 cells ; SDS - PAGE of eluate from amylose beads . ( D ) Size - exclusion chromatography profile ( Superose 6 column ) of the prApe1 / Atg19 complex after anti MBP - Atg19 purification and cleavage of the MBP tag ( top ) . SDS - PAGE of peak fractions of the gel filtration profile ( bottom ) . Samples were run on two separate gels . ( E ) Negative stain electron micrograph corresponding to the size - exclusion chromatography peak at 10 ml retention volume of ( D ) . Several prApe1 dodecamers possess extra density corresponding to Atg19 and are highlighted in the insets . ( F ) Comparison between class averages of purified prApe1 dodecamers with class averages of prApe1 dodecamers that were co - purified with Atg19 . ( G ) Bar plot of occurrence of single , double , triple and multiple dodecamers from the prApe1 and the prApe1 / Atg19 sample . Data information : n = 3 , 186 particles were counted in the case of prApe1 and n = 3 , 340 particles were counted in the case of prApe1 / Atg19 . Particles were picked from 20 different micrographs . * * * P < 2 . 2x10 - 16 ( Pearson ' s Chi - squared test ) . H ) Epifluorescence microscopy images of the prApe1 - GFP / Atg19 - mCherry / ypt7Δ , prApe1 - GFP / Atg19Δ / ypt7Δ S . cerevisiae cells that were used to analyze the fluorescence intensity of all prApe1 - GFP spots . BF represents bright - field images . I ) Bar plot showing the ratio between the GFP intensity of prApe1 spots in prApe1 - GFP / Atg19 - mCherry / ypt7Δ versus prApe1 - GFP / Atg19Δ / ypt7Δ S . cerevisiae cells . The intensity of Ape1 - GFP was normalized over the average fluorescence intensity of Nuf2 - GFP measured in kinetochores as performed in [ 36 ] . Data information : a total of 54 prApe1 - GFP spots and 80 Nuf2 - GFP spots were analyzed in prApe1 - GFP / Atg19Δ / ypt7Δ cells , whereas 52 prApe1 - GFP spots and 98 Nuf2 - GFP spots were analyzed in prApe1 - GFP / Atg19 - mCherry / ypt7Δ control cells . Error bars indicate standard error of the mean ( SEM ) . * P = 0 . 03 ( Z test ) .

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

Figure 3(A) SEC-MALS profile of Atg19 yields a molecular weight estimate of 142 kDa (measured in triplicate). (B) Right. In vitro pull-down binding assay and subsequent SDS-PAGE. Amylose beads are used as a bait to demonstrate interaction of purified prApe1 dodecamers with MBP-Atg19 (lane 1) whereas purified mApe1 dodecamers are not capable of interacting with MBP-Atg19 (lane 3). As controls, MBP does not bind prApe1 and mApe1 (lanes 2 and 4). Left. SDS-PAGE showing input of the corresponding pull-down assay.  (C) Co-expression of MBP-Atg19 and prApe1 in Sf21 cells; SDS-PAGE of eluate from amylose beads.  (D) Size-exclusion chromatography profile (Superose 6 column) of the prApe1/Atg19 complex after anti MBP-Atg19 purification and cleavage of the MBP tag (top). SDS-PAGE of peak fractions of the gel filtration profile (bottom). Samples were run on two separate gels.  (E) Negative stain electron micrograph corresponding to the size-exclusion chromatography peak at 10 ml retention volume of (D). Several prApe1 dodecamers possess extra density corresponding to Atg19 and are highlighted in the insets.  (F) Comparison between class averages of purified prApe1 dodecamers with class averages of prApe1 dodecamers that were co-purified with Atg19.  (G) Bar plot of occurrence of single, double, triple and multiple dodecamers from the prApe1 and the prApe1/Atg19 sample. Data information: n=3,186 particles were counted in the case of prApe1 and n=3,340 particles were counted in the case of prApe1/Atg19. Particles were picked from 20 different micrographs. ***P < 2.2x10-16 (Pearson's Chi-squared test).  H) Epifluorescence microscopy images of the prApe1-GFP/Atg19-mCherry/ypt7Δ, prApe1-GFP/Atg19Δ/ypt7Δ S. cerevisiae cells that were used to analyze the fluorescence intensity of all prApe1-GFP spots. BF represents bright-field images.  I) Bar plot showing the ratio between the GFP intensity of prApe1 spots in prApe1-GFP/Atg19-mCherry/ypt7Δ versus prApe1-GFP/Atg19Δ/ypt7Δ S. cerevisiae cells. The intensity of Ape1-GFP was normalized over the average fluorescence intensity of Nuf2-GFP measured in kinetochores as performed in [36]. Data information: a total of 54 prApe1-GFP spots and 80 Nuf2-GFP spots were analyzed in prApe1-GFP/Atg19Δ/ypt7Δ cells, whereas 52 prApe1-GFP spots and 98 Nuf2-GFP spots were analyzed in prApe1-GFP/Atg19-mCherry/ypt7Δ control cells. Error bars indicate standard error of the mean (SEM). *P = 0.03 (Z test).


Figure 4 Figure 4 : Quantification of the Ape1 , Ams1 , Atg19 protein abundances in living cells . ( A ) Epifluorescence microscopy images of the prApe1 - GFP / Atg19 - mCherry / ypt7Δ , Ams1 - GFP / Atg19 - mCherry / ypt7Δ , Atg19 - GFP / Ape1 - mCherry / ypt7Δ S . cerevisiae cells that were used to quantify the fluorescence intensity of GFP spots . Only co - localizing GFP and RFP spots were considered . BF represents bright - field images . ( B ) Bar plot showing the number of molecules of prApe1 , Ams1 and Atg19 found in Cvt vesicles in living cells . Data information : data are representative of two independent experiments in which a total of 217 prApe1 - GFP , 181 Ams1 - GFP and 233 Atg19 - GFP spots were analyzed . Error bars indicate standard deviation ( SD ) . * * * P < 0 . 001 ; * P = 0 . 0176 ( Z test ) .

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

Figure 4 Figure 4: Quantification of the Ape1, Ams1, Atg19 protein abundances in living cells. (A) Epifluorescence microscopy images of the prApe1-GFP/Atg19-mCherry/ypt7Δ, Ams1-GFP/Atg19-mCherry/ypt7Δ, Atg19-GFP/Ape1-mCherry/ypt7Δ S. cerevisiae cells that were used to quantify the fluorescence intensity of GFP spots. Only co-localizing GFP and RFP spots were considered. BF represents bright-field images. (B) Bar plot showing the number of molecules of prApe1, Ams1 and Atg19 found in Cvt vesicles in living cells. Data information: data are representative of two independent experiments in which a total of 217 prApe1-GFP, 181 Ams1-GFP and 233 Atg19-GFP spots were analyzed. Error bars indicate standard deviation (SD). ***P < 0.001; *P = 0.0176 (Z test).


Figure 5 ( A ) Fluorescence microscopy image of a resin section of prApe1 - GFP / Atg19 - mCherry / ypt7Δ cells , merge of green ( prApe1 - GFP ) and red ( Atg19 - mcherry ) channels . Tetraspeck beads are detected in the blue channel . ( B ) Electron tomography slice of the boxed area in ( A ) . The outer and inner dashed green circles of 160 and 60 nm radius represent estimated 50 % and 90 % localization accuracy , respectively . Insets show close - up views of a double ( top ) and single ( bottom ) membrane . Corresponding tomogram Movie EV2 . ( C ) Plot of the x and y dimensions of the Cvt vesicles obtained from localization correlation . The average dimensions give rise to spheroids with an average ellipticity of 11 % ( shown in dark grey ) . Both prApe1 - GFP and prApe1 - GFP / Atg19 - mCherry datasets were included . The cases representing autophagosomes were excluded . ( D ) Dense Cvt aggregate that is not surrounded by membrane . Corresponding tomogram Movie EV3 . ( E ) Cvt aggregate that is partially enwrapped by double membrane . Corresponding tomogram Movie EV4 . ( F ) Cvt vesicle fully enclosed by double membrane . Bottom : schematic representation . White arrowheads indicate the double membrane . Corresponding tomogram Movie EV5 .

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

Figure 5 (A) Fluorescence microscopy image of a resin section of prApe1-GFP/Atg19-mCherry/ypt7Δ cells, merge of green (prApe1-GFP) and red (Atg19-mcherry) channels. Tetraspeck beads are detected in the blue channel. (B) Electron tomography slice of the boxed area in (A). The outer and inner dashed green circles of 160 and 60 nm radius represent estimated 50% and 90% localization accuracy, respectively. Insets show close-up views of a double (top) and single (bottom) membrane. Corresponding tomogram Movie EV2. (C) Plot of the x and y dimensions of the Cvt vesicles obtained from localization correlation. The average dimensions give rise to spheroids with an average ellipticity of 11 % (shown in dark grey). Both prApe1-GFP and prApe1-GFP/Atg19-mCherry datasets were included. The cases representing autophagosomes were excluded.  (D) Dense Cvt aggregate that is not surrounded by membrane. Corresponding tomogram Movie EV3. (E) Cvt aggregate that is partially enwrapped by double membrane. Corresponding tomogram Movie EV4. (F) Cvt vesicle fully enclosed by double membrane. Bottom: schematic representation. White arrowheads indicate the double membrane. Corresponding tomogram Movie EV5.


Figure 6 Figure 6 : Model of the Cvt pathway . ( A ) prApe1 the major Cvt cargo forms homo - oligomers as dodecamers that ( B ) assemble into higher - order assemblies mediated by the N - terminal propeptide . ( C ) These assemblies are capable of binding Atg19 that forms a trimer in solution . ( D ) Ams1 , which forms a homo - oligomer as a tetramer , joins the Cvt assembly by binding to Atg19 via a site that is distinct from the Ape1 binding site . ( E ) prApe1 dodecamers , secondary cargo enzymes such as Ams1 tetramers and Atg19 trimers are the main constituents of the Cvt aggregate in the cytosol . ( F ) The Cvt aggregate becomes enwrapped by a double membrane via Atg8 and Atg19 interaction to form ( G ) the Cvt vesicle . Cvt assembly stages in ( E ) , ( F ) and ( G ) are drawn respecting the determined average size of the vesicles , the dimensions and stoichiometry of the major cargo prApe1 and secondary cargo Ams1 found in this article . ( H ) After fusion with the vacuole , prApe1 is subjected to maturation while maintaining the dodecamer organization . ( I ) Ams1 does not undergo a maturation process in the vacuole .

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

Figure 6 Figure 6: Model of the Cvt pathway. (A) prApe1 the major Cvt cargo forms homo-oligomers as dodecamers that (B) assemble into higher-order assemblies mediated by the N-terminal propeptide. (C) These assemblies are capable of binding Atg19 that forms a trimer in solution. (D) Ams1, which forms a homo-oligomer as a tetramer, joins the Cvt assembly by binding to Atg19 via a site that is distinct from the Ape1 binding site. (E) prApe1 dodecamers, secondary cargo enzymes such as Ams1 tetramers and Atg19 trimers are the main constituents of the Cvt aggregate in the cytosol. (F) The Cvt aggregate becomes enwrapped by a double membrane via Atg8 and Atg19 interaction to form (G) the Cvt vesicle. Cvt assembly stages in (E), (F) and (G) are drawn respecting the determined average size of the vesicles, the dimensions and stoichiometry of the major cargo prApe1 and secondary cargo Ams1 found in this article. (H) After fusion with the vacuole, prApe1 is subjected to maturation while maintaining the dodecamer organization. (I) Ams1 does not undergo a maturation process in the vacuole.


Figure 1Detection of native and GFP - fused TgAtg8 . Protein extracts corresponding to 107 tachyzoites from parental RHΔHX and transgenic GFP - TgAtg8 cell lines were separated by SDS - PAGE and analysed by Western blot using anti - TgAtg8 or anti - GFP antibodies . Overexpressed GFP - TgAtg8 and native TgAtg8 are indicated by arrows .

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

Figure 1Detection of native and GFP-fused TgAtg8.Protein extracts corresponding to 107 tachyzoites from parental RHΔHX and transgenic GFP-TgAtg8 cell lines were separated by SDS-PAGE and analysed by Western blot using anti-TgAtg8 or anti-GFP antibodies. Overexpressed GFP-TgAtg8 and native TgAtg8 are indicated by arrows.


Figure 2A . Autophagic vesicles marker GFP - TgAtg8 was found in the cytosol of tachyzoites and occasionally in punctate vesicles in recently egressed parasites . B . Extracellular tachyzoites were put to starve in HBSS medium for increased time periods and the proportions of cells displaying punctate or cytosolic GFP - TgAtg8 signals were assessed . Data are mean from n = 4 independent experiments ± SEM .

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Figure 2A. Autophagic vesicles marker GFP-TgAtg8 was found in the cytosol of tachyzoites and occasionally in punctate vesicles in recently egressed parasites.B. Extracellular tachyzoites were put to starve in HBSS medium for increased time periods and the proportions of cells displaying punctate or cytosolic GFP-TgAtg8 signals were assessed. Data are mean from n = 4 independent experiments ±SEM.


Figure 3A . Extracellular tachyzoites were starved for 8 hours in HBSS medium and observed ( 2 , 3 ) . Unstarved control is shown for comparison ( 1 ) . Several vacuoles ( V ) can be seen in starved parasites , with also cytosol - and organelle - sequestrating membranous structures resembling autophagosomes ( Ap ) , as well as vacuoles containing partially digested material corresponding to autophagic degradative vacuoles ( Av ) . 2 ′ , 3 ′ and 3 " pictures are a magnification of squared regions . Scale bar = 1 µm in initial micrographs ( 1 , 2 , 3 ) and 0 . 2 µm in magnifications . B . Partial co - localisation of GFP - TgAtg8 - labeled autophagosomes and Toxoplasma mitochondrion . Extracellular tachyzoites expressing GFP - TgAtg8 were incubated in complete medium with Mitotracker Red to label the mitochondrion ; partial co - localisation could be observed in some parasites displaying autophagic vesicles . C . Identification of autophagic vesicles in extracellular tachyzoites by immuno - electron microscopy . Extracellular tachyzoites expressing GFP - TgAtg8 were starved for 8 hours in HBSS medium , fixed , cryosectioned and stained with an anti - GFP antibody , then with gold - conjugated secondary antibody and observed . Gold particles can be seen around cytosol - sequestrating membranous structures resembling autophagosomes ( Ap ) , as well as larger vacuoles containing partially digested material corresponding to autophagic degradative vacuoles ( Av ) . Scale bar = 200 nm .

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

Figure 3A. Extracellular tachyzoites were starved for 8 hours in HBSS medium and observed (2, 3). Unstarved control is shown for comparison (1). Several vacuoles (V) can be seen in starved parasites, with also cytosol- and organelle-sequestrating membranous structures resembling autophagosomes (Ap), as well as vacuoles containing partially digested material corresponding to autophagic degradative vacuoles (Av). 2′, 3′ and 3" pictures are a magnification of squared regions. Scale bar = 1 µm in initial micrographs (1, 2, 3) and 0.2 µm in magnifications.B. Partial co-localisation of GFP-TgAtg8-labeled autophagosomes and Toxoplasma mitochondrion. Extracellular tachyzoites expressing GFP-TgAtg8 were incubated in complete medium with Mitotracker Red to label the mitochondrion; partial co-localisation could be observed in some parasites displaying autophagic vesicles.C. Identification of autophagic vesicles in extracellular tachyzoites by immuno-electron microscopy. Extracellular tachyzoites expressing GFP-TgAtg8 were starved for 8 hours in HBSS medium, fixed, cryosectioned and stained with an anti-GFP antibody, then with gold-conjugated secondary antibody and observed. Gold particles can be seen around cytosol-sequestrating membranous structures resembling autophagosomes (Ap), as well as larger vacuoles containing partially digested material corresponding to autophagic degradative vacuoles (Av). Scale bar = 200 nm.


Figure 4A . Protein extracts corresponding to tachyzoites incubated in the same starvation conditions as in Figure 2B were separated by urea SDS - PAGE and analysed with an anti - GFP antibody to detect TgAtg8 and the lipidated TgAtg8 - PE form . GFP - TgAtg8 parasites extracts were analysed with anti GFP antibody ( left ) , while parental cell line was analysed with anti - TgAtg8 antibody ( right ) . Anti - ROP5 was used as a loading control . B . Cell lysates from GFP - TgAtg8 parasites were subjected to a centrifugation at 100 , 000 g to separate the soluble fraction ( high speed supernatant , HSS ) from the membrane fraction ( high speed pellet , HSP ) . The faster migrating form of GFP - TgAtg8 is exclusively present in the membrane fraction as revealed by Western blot analysis after urea SDS - PAGE using anti - GFPC . The HSP fraction described in B . was extracted with various agents and new HSS and HSP fractions were separated and analysed by Western blot after urea SDS - PAGE . Only a treatment with a detergent ( DOC ) resulted in GFP - TgAtg8 solubilisation from the HSP fraction . D . GFP - TgAtg8 - expressing parasites were grown in host cells in the presence of 3H - ethanolamine and then starved in HBSS for 8 hours , still in the presence of the radioactive PE precursor . Tachyzoites were then lysed and GFP - TgAtg8 was immunoprecipitated using anti - TgAtg8 antibody and analysed by urea SDS - PAGE . The immunoprecipitated forms of GFP - TgAtg8 were detected by Western blot using anti - GFP antibody and radioactive ethanolamine was found to be incorporated into immunoprecipitated GFP - TgAtg8 , as detected by fluorography ( 3H - etn ) .

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 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

Figure 4A. Protein extracts corresponding to tachyzoites incubated in the same starvation conditions as in Figure 2B were separated by urea SDS-PAGE and analysed with an anti-GFP antibody to detect TgAtg8 and the lipidated TgAtg8-PE form. GFP-TgAtg8 parasites extracts were analysed with anti GFP antibody (left), while parental cell line was analysed with anti-TgAtg8 antibody (right). Anti-ROP5 was used as a loading control.B. Cell lysates from GFP-TgAtg8 parasites were subjected to a centrifugation at 100,000 g to separate the soluble fraction (high speed supernatant, HSS) from the membrane fraction (high speed pellet, HSP). The faster migrating form of GFP-TgAtg8 is exclusively present in the membrane fraction as revealed by Western blot analysis after urea SDS-PAGE using anti-GFPC. The HSP fraction described in B. was extracted with various agents and new HSS and HSP fractions were separated and analysed by Western blot after urea SDS-PAGE. Only a treatment with a detergent (DOC) resulted in GFP-TgAtg8 solubilisation from the HSP fraction.D. GFP-TgAtg8-expressing parasites were grown in host cells in the presence of 3H-ethanolamine and then starved in HBSS for 8 hours, still in the presence of the radioactive PE precursor. Tachyzoites were then lysed and GFP-TgAtg8 was immunoprecipitated using anti-TgAtg8 antibody and analysed by urea SDS-PAGE. The immunoprecipitated forms of GFP-TgAtg8 were detected by Western blot using anti-GFP antibody and radioactive ethanolamine was found to be incorporated into immunoprecipitated GFP-TgAtg8, as detected by fluorography (3H-etn).


Figure 5A . Fluorescence microscopy analysis of extracellular tachyzoites expressing GFP - TgAtg8 or its glycine mutant version , before or after induction of autophagy for 8 hours in HBSS . Autophagosomes are labelled by GFP - TgAtg8 in control parasites ( arrowheads ) , but not by the glycine mutant version of the protein . B . Extracellular tachyzoites expressing GFP - TgAtg8 or its glycine mutant version were put to starve in HBSS medium for increased lengths of time and the proportions of cells displaying punctate or cytosolic GFP signals were assessed . Data are mean from n = 4 independent experiments ± SEM . C . Protein extracts corresponding to tachyzoites expressing GFP - TgAtg8 and glycine mutant version incubated in HBSS for up to 8 hours , were separated by urea SDS - PAGE and analysed with an anti - GFP antibody to detect GFP - TgAtg8 and lipidated GFP - TgAtg8 - PE forms . Anti - ROP5 was used as a loading control .

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 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

Figure 5A. Fluorescence microscopy analysis of extracellular tachyzoites expressing GFP-TgAtg8 or its glycine mutant version, before or after induction of autophagy for 8 hours in HBSS. Autophagosomes are labelled by GFP-TgAtg8 in control parasites (arrowheads), but not by the glycine mutant version of the protein. B. Extracellular tachyzoites expressing GFP-TgAtg8 or its glycine mutant version were put to starve in HBSS medium for increased lengths of time and the proportions of cells displaying punctate or cytosolic GFP signals were assessed. Data are mean from n = 4 independent experiments ±SEM.C. Protein extracts corresponding to tachyzoites expressing GFP-TgAtg8 and glycine mutant version incubated in HBSS for up to 8 hours, were separated by urea SDS-PAGE and analysed with an anti-GFP antibody to detect GFP-TgAtg8 and lipidated GFP-TgAtg8-PE forms. Anti-ROP5 was used as a loading control.


Figure 6A . Intracellular tachyzoites expressing GFP - TgAtg8 were used to invade host HFF and the proportions of cells displaying punctate or cytosolic GFP signals were assessed along time during development . Data are mean from n = 5 independent experiments ± SEM . B . Fluorescence microscopy pictures of GFP - TgAtg8 - expressing tachyzoites during their intracellular development and division . The arrowhead indicates the residual body made of maternal material left after endodyogeny . C . Detection of GFP - TgAtg8 - labelled autophagosomes in dividing parasites . Progression of daughter cells division was followed by expression of an IMC1 - RFP construct .

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Figure 6A. Intracellular tachyzoites expressing GFP-TgAtg8 were used to invade host HFF and the proportions of cells displaying punctate or cytosolic GFP signals were assessed along time during development. Data are mean from n = 5 independent experiments ±SEM.B. Fluorescence microscopy pictures of GFP-TgAtg8-expressing tachyzoites during their intracellular development and division. The arrowhead indicates the residual body made of maternal material left after endodyogeny.C. Detection of GFP-TgAtg8-labelled autophagosomes in dividing parasites. Progression of daughter cells division was followed by expression of an IMC1-RFP construct.


Figure 7A . Schematic representation of the strategy . Recombineered cosmid bearing chloramphenicol acetyl transferase ( CAT ) gene for selection of resistant parasites , flanked by homologous regions from TgAtg3 , was used to replace endogenous genomic locus in the cell line expressing a regulatable extra copy of TgAtg3 ( imyc - sTgAtg3 ) . Mutants obtained after double homologous recombination events were selected with chloramphenicol . PCR amplifications and Southern blot strategy used for subsequent characterisation of the mutant are indicated in red and green , respectively . B . PCR verification of the removal of TgAtg3 in the mutant ( imyc - sTgAtg3 - ΔAtg3 , A ) compared to the parental cell line ( imyc - sTgAtg3 , B ) C . Southern blot verification of the TgAtg3 gene replacement . ∼ 4 µg of genomic DNA from mutant ( A ) and parental ( B ) tachyzoites were digested by HindIII / AvrII , transferred on a nylon membrane and probed with 32P - labeled 5 ′ and 3 ′ DNA probes located as schematised in A .

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Figure 7A. Schematic representation of the strategy. Recombineered cosmid bearing chloramphenicol acetyl transferase (CAT) gene for selection of resistant parasites, flanked by homologous regions from TgAtg3, was used to replace endogenous genomic locus in the cell line expressing a regulatable extra copy of TgAtg3 (imyc-sTgAtg3). Mutants obtained after double homologous recombination events were selected with chloramphenicol. PCR amplifications and Southern blot strategy used for subsequent characterisation of the mutant are indicated in red and green, respectively. B. PCR verification of the removal of TgAtg3 in the mutant (imyc-sTgAtg3-ΔAtg3, A) compared to the parental cell line (imyc-sTgAtg3, B)C. Southern blot verification of the TgAtg3 gene replacement. ∼4 µg of genomic DNA from mutant (A) and parental (B) tachyzoites were digested by HindIII/AvrII, transferred on a nylon membrane and probed with 32P-labeled 5′ and 3′ DNA probes located as schematised in A.


Figure 8A . The vector for the expression of the ATc - regulatable sTgAtg3 extra copy allows myc - epitope tagging of this protein . Anti - myc detection of myc - tagged TgAtg3 protein in intracellular parasites either without treatment with ATc , or with a continuous 2 days treatment with 1 . 5 µg / ml ATc , or after reinvasion following a continuous 4 days treatment . DIC : differential interference contrast . B . Western blot detection of the myc - tagged sTgAtg3 protein ( arrowed ) in parasite lysates corresponding to the induction conditions described in A . Anti - SAG1 antibody was used as a loading control . C . Western blot analysis following urea SDS - PAGE of TgAtg3 - depleted parasites extracts , showing an absence of upregulation of the autophasome - bound TgAtg8 - PE form . Anti - ROP5 was used as a loading control .

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Figure 8A. The vector for the expression of the ATc-regulatable sTgAtg3 extra copy allows myc-epitope tagging of this protein. Anti-myc detection of myc-tagged TgAtg3 protein in intracellular parasites either without treatment with ATc, or with a continuous 2 days treatment with 1.5 µg/ml ATc, or after reinvasion following a continuous 4 days treatment. DIC: differential interference contrast.B. Western blot detection of the myc-tagged sTgAtg3 protein (arrowed) in parasite lysates corresponding to the induction conditions described in A. Anti-SAG1 antibody was used as a loading control.C. Western blot analysis following urea SDS-PAGE of TgAtg3-depleted parasites extracts, showing an absence of upregulation of the autophasome-bound TgAtg8-PE form. Anti-ROP5 was used as a loading control.


Figure 9A . Confluent monolayers of fibroblasts were infected either with extra copy - expressing imyc - sTgAtg3 parental cell line in the presence of ATc ( a ) , imyc - sTgAtg3 - ΔAtg3 mutant cell line without ATc ( b ) , mutant cell line in the presence of ATc ( c ) , or mutant cell line pre - incubated with ATc before start of the experiment and maintained in the presence of ATc . Plaques ( arrowheads ) resulting from the lysis of host cells due to the multiplication of the parasites are only visible when TgAtg3 is still expressed . B . Mean plaque area comparisons between fibroblast layers infected with controls and TgAtg3 - depleted parasites ( a , b , c : legend as in A ) . Plaques observed with the mutant in the presence of ATc were significantly smaller than with control cell lines ( * p < 0 . 005 , Student ' s T test ) . Data are mean from n = 3 independent experiments ± SEM . C . Numbers of parasites per vacuole are significantly lower in TgAtg3 - depleted cell line ( pre - incubated for 4 days with ATc ) compared with controls 24 or 48 hours post invasion . Data are mean from n = 3 independent experiments ± SEM .

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Figure 9A. Confluent monolayers of fibroblasts were infected either with extra copy-expressing imyc-sTgAtg3 parental cell line in the presence of ATc (a), imyc-sTgAtg3-ΔAtg3 mutant cell line without ATc (b), mutant cell line in the presence of ATc (c), or mutant cell line pre-incubated with ATc before start of the experiment and maintained in the presence of ATc. Plaques (arrowheads) resulting from the lysis of host cells due to the multiplication of the parasites are only visible when TgAtg3 is still expressed. B. Mean plaque area comparisons between fibroblast layers infected with controls and TgAtg3-depleted parasites (a, b, c: legend as in A). Plaques observed with the mutant in the presence of ATc were significantly smaller than with control cell lines (* p<0.005, Student's T test). Data are mean from n = 3 independent experiments ±SEM.C. Numbers of parasites per vacuole are significantly lower in TgAtg3-depleted cell line (pre-incubated for 4 days with ATc) compared with controls 24 or 48 hours post invasion. Data are mean from n = 3 independent experiments ±SEM.


Figure 10TgAtg3 - depleted parasites show a defect in mitochondrion morphology . Mitochondrion labelled with specific antibodies ( see results section ) was found as a reduced structure , or even absent , in TgAtg3 - depleted cell lines ( middle series of micrographs , kept for 2 days in the presence of ATc or pre - incubated with ATc for 4 days before invasion , respectively ) . Parasites left to develop for two days in the presence of ATc to progressively extinguish the expression of TgAtg3 showed an accumulation of mitochondrial marker at the residual body . TgAtg3 depletion was verified by detecting myc - tagged regulatable extra - copy with specific antibody . TgAtg3 - expressing cell line imyc - sTgAtg3cultivated in the presence of ATc for 4 days was used as a control for mitochondrial morphology ( bottom ) . DNA was labelled with DAPI .

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

Figure 10TgAtg3-depleted parasites show a defect in mitochondrion morphology.Mitochondrion labelled with specific antibodies (see results section) was found as a reduced structure, or even absent, in TgAtg3-depleted cell lines (middle series of micrographs, kept for 2 days in the presence of ATc or pre-incubated with ATc for 4 days before invasion, respectively). Parasites left to develop for two days in the presence of ATc to progressively extinguish the expression of TgAtg3 showed an accumulation of mitochondrial marker at the residual body. TgAtg3 depletion was verified by detecting myc-tagged regulatable extra-copy with specific antibody. TgAtg3-expressing cell line imyc-sTgAtg3cultivated in the presence of ATc for 4 days was used as a control for mitochondrial morphology (bottom). DNA was labelled with DAPI.


Figure 11Altered mitochondrial ultrastructure in TgAtg3 - depleted tachyzoites observed by electron microscopy . Ultrathin section of a TgAtg3 - depleted ( 3 days of ATc treatment ) tachyzoite showing the dramatic alteration of the mitochondrion , where cristae remnants are still visible ( arrows ) , but which is filled up with membranous profiles ( arrowheads ) , whereas Golgi ( G ) , apicoplast ( A ) , rhoptries ( R ) and dense granule ( D ) look normal .

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Figure 11Altered mitochondrial ultrastructure in TgAtg3-depleted tachyzoites observed by electron microscopy.Ultrathin section of a TgAtg3-depleted (3 days of ATc treatment) tachyzoite showing the dramatic alteration of the mitochondrion, where cristae remnants are still visible (arrows), but which is filled up with membranous profiles (arrowheads), whereas Golgi (G), apicoplast (A), rhoptries (R) and dense granule (D) look normal.


Figure 1About 25 μg of membrane ‐ enriched lysate from the mass spectrometry experiments was immunoblotted with anti ‐ PINK1 antibody . TOMM40 and GAPDH serve as markers for mitochondria and cytoplasm , respectively .

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Figure 1About 25 μg of membrane‐enriched lysate from the mass spectrometry experiments was immunoblotted with anti‐PINK1 antibody. TOMM40 and GAPDH serve as markers for mitochondria and cytoplasm, respectively.


Figure 3A - CConfirmation by mass spectrometry that Rab8A ( A ) , Rab8B ( B ) and Rab13 ( C ) Ser111 is phosphorylated upon PINK1 activation after CCCP treatment . Flp ‐ In T ‐ Rex HEK293 cells expressing empty ‐ FLAG , WT PINK1 ‐ FLAG and KI ( D384A ) PINK1 ‐ FLAG were transfected either with HA ‐ Rab8A ( A ) , HA ‐ Rab8B ( B ) or HA ‐ Rab13 ( C ) induced with doxycycline and stimulated with 10 μM of CCCP for 3 h . Whole ‐ cell lysates ( 10 mg ) were immunoprecipitated with anti ‐ HA agarose , resolved by SDS - PAGE and stained with colloidal Coomassie blue ( second panel ) . Coomassie ‐ stained bands migrating with expected molecular mass of HA ‐ Rabs were excised , in ‐ gel digested with trypsin and subjected to high ‐ performance liquid chromatography with LC ‐ MS / MS on an LTQ ‐ Orbitrap mass spectrometer . Upper panel shows the extracted ion chromatogram ( XIC ) analysis of Ser111 ‐ containing phosphopeptides ( 8A , NIEEHApSADVEK ; 8B , NIEEHApSSDVER ; 13 , SIKENApSAGVER ) with the combined signal intensity of the 2 + and 3 + forms of the peptide indicated on the y ‐ axis . Note that the Ser111 phosphopeptide was only detected in samples from WT PINK1 ‐ FLAG ‐ expressing cells following CCCP stimulation . DCharacterisation of Rab8A , Rab8B and Rab13 phospho ‐ Ser111 antibodies . Flp ‐ In T ‐ Rex HEK293 cells expressing empty ‐ FLAG , WT PINK1 ‐ FLAG and KI ( D384A ) PINK1 ‐ FLAG were transfected with either WT or Ser111Ala ‐ mutant ( S111A ) HA ‐ Rab8A , HA ‐ Rab8B or HA ‐ Rab13 , induced with doxycycline and stimulated with 10 μM of CCCP for 3 h . Whole ‐ cell lysates ( 0 . 25 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with Rab8A , Rab8B or Rab13 phospho ‐ Ser111 antibodies . Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls .

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 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

Figure 3A-CConfirmation by mass spectrometry that Rab8A (A), Rab8B (B) and Rab13 (C) Ser111 is phosphorylated upon PINK1 activation after CCCP treatment. Flp‐In T‐Rex HEK293 cells expressing empty‐FLAG, WT PINK1‐FLAG and KI (D384A) PINK1‐FLAG were transfected either with HA‐Rab8A (A), HA‐Rab8B (B) or HA‐Rab13 (C) induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole‐cell lysates (10 mg) were immunoprecipitated with anti‐HA agarose, resolved by SDS-PAGE and stained with colloidal Coomassie blue (second panel). Coomassie‐stained bands migrating with expected molecular mass of HA‐Rabs were excised, in‐gel digested with trypsin and subjected to high‐performance liquid chromatography with LC‐MS/MS on an LTQ‐Orbitrap mass spectrometer. Upper panel shows the extracted ion chromatogram (XIC) analysis of Ser111‐containing phosphopeptides (8A, NIEEHApSADVEK; 8B, NIEEHApSSDVER; 13, SIKENApSAGVER) with the combined signal intensity of the 2+ and 3+ forms of the peptide indicated on the y‐axis. Note that the Ser111 phosphopeptide was only detected in samples from WT PINK1‐FLAG‐expressing cells following CCCP stimulation.DCharacterisation of Rab8A, Rab8B and Rab13 phospho‐Ser111 antibodies. Flp‐In T‐Rex HEK293 cells expressing empty‐FLAG, WT PINK1‐FLAG and KI (D384A) PINK1‐FLAG were transfected with either WT or Ser111Ala‐mutant (S111A) HA‐Rab8A, HA‐Rab8B or HA‐Rab13, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole‐cell lysates (0.25 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rab8A, Rab8B or Rab13 phospho‐Ser111 antibodies. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls.


Figure 4PINK1 is essential for CCCP ‐ mediated Rab8A Ser111 phosphorylation . WT and PINK1 KO HeLa cells were transfected with either WT or Ser111Ala ( S111A ) mutant constructs of HA ‐ Rab8A , HA ‐ Rab8B or HA ‐ Rab13 . Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1 ‐ 3xFLAG or KI ( D384A ) PINK1 ‐ 3xFLAG as indicated . After transfection for at least 24 h , cells were treated with DMSO as a vehicle control or CCCP for 20 h . Whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with Rab8A , Rab8B or Rab13 phospho ‐ Ser111 antibody . Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls . For the lower panel , whole ‐ cell lysates ( 30 μg ) were immunoblotted with total PINK1 antibody to confirm PINK1 expression and with GAPDH as loading controls . Endogenous Rab8A Ser111 phosphorylation is PINK1 dependent . WT and PINK1 KO HeLa cells were treated with DMSO as a vehicle control or CCCP for 20 h . Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1 ‐ 3xFLAG or KI ( D384A ) PINK1 ‐ 3xFLAG as indicated for at least 24 h before CCCP treatment . Whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ Rab8A pre ‐ bound with protein A agarose followed by immunoblot with Rab8A phospho ‐ Ser111 antibody . Part of the immunoprecipitates was used to immunoblot with anti ‐ total Rab8A antibody as loading controls .

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Figure 4PINK1 is essential for CCCP‐mediated Rab8A Ser111 phosphorylation. WT and PINK1 KO HeLa cells were transfected with either WT or Ser111Ala (S111A) mutant constructs of HA‐Rab8A, HA‐Rab8B or HA‐Rab13. Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1‐3xFLAG or KI (D384A) PINK1‐3xFLAG as indicated. After transfection for at least 24 h, cells were treated with DMSO as a vehicle control or CCCP for 20 h. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rab8A, Rab8B or Rab13 phospho‐Ser111 antibody. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls. For the lower panel, whole‐cell lysates (30 μg) were immunoblotted with total PINK1 antibody to confirm PINK1 expression and with GAPDH as loading controls.Endogenous Rab8A Ser111 phosphorylation is PINK1 dependent. WT and PINK1 KO HeLa cells were treated with DMSO as a vehicle control or CCCP for 20 h. Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1‐3xFLAG or KI (D384A) PINK1‐3xFLAG as indicated for at least 24 h before CCCP treatment. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A pre‐bound with protein A agarose followed by immunoblot with Rab8A phospho‐Ser111 antibody. Part of the immunoprecipitates was used to immunoblot with anti‐total Rab8A antibody as loading controls.


Figure 5Absence of Rab8A Ser111 phosphorylation in human mutant PINK1 patient fibroblasts . Primary skinfibroblasts were derived from a patient with homozygous PINK1 Q456X mutation or unaffected control . Cells were incubated with DMSO or CCCP for 20 h , and whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho ‐ Ser111 antibody . Lysates ( 1 mg ) were also subjected to immunoprecipitation with polyclonal anti ‐ PINK1 antibody and immunoblotted with monoclonal PINK1 antibody . Equal loading of protein extracts was confirmed by GAPDH . Absence of Rab8A Ser111 phosphorylation in PINK1 knockout MEFs . MEFs were derived from PINK1 knockout embryos or wild ‐ type controls ( see Materials and Methods ) . Cells were incubated with DMSO or CCCP for 20 h , and whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho ‐ Ser111 antibody . Lysates ( 1 mg ) were also subjected to immunoprecipitation with a polyclonal anti ‐ mouse ‐ specific PINK1 antibody and immunoblotted with a different anti ‐ mouse ‐ specific PINK1 antibody . Equal loading of protein extracts was confirmed by GAPDH .

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Figure 5Absence of Rab8A Ser111 phosphorylation in human mutant PINK1 patient fibroblasts. Primary skinfibroblasts were derived from a patient with homozygous PINK1 Q456X mutation or unaffected control. Cells were incubated with DMSO or CCCP for 20 h, and whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho‐Ser111 antibody. Lysates (1 mg) were also subjected to immunoprecipitation with polyclonal anti‐PINK1 antibody and immunoblotted with monoclonal PINK1 antibody. Equal loading of protein extracts was confirmed by GAPDH.Absence of Rab8A Ser111 phosphorylation in PINK1 knockout MEFs. MEFs were derived from PINK1 knockout embryos or wild‐type controls (see Materials and Methods). Cells were incubated with DMSO or CCCP for 20 h, and whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho‐Ser111 antibody. Lysates (1 mg) were also subjected to immunoprecipitation with a polyclonal anti‐mouse‐specific PINK1 antibody and immunoblotted with a different anti‐mouse‐specific PINK1 antibody. Equal loading of protein extracts was confirmed by GAPDH.


Figure 6Rab8A is not required for the activation of Parkin E3 ligase activity at mitochondria in response to PINK1 activation by CCCPWild ‐ type ( WT ) or Rab8A knockout ( KO ) HeLa cells were transfected with WT or Cys431Phe ( C341F ) ‐ mutant Parkin . After transfection for 24 h , cells were treated with DMSO as a vehicle control or 10 μM CCCP for 6 h . Mitochondrial enriched extracts ( mitochondrial lysate ) were incubated with ubiquitin ‐ binding resins derived from his ‐ halo ‐ ubiquilin1 UBA domain tetramer ( UBAUBQLN1 ) . Captured ubiquitylated proteins were subject to immunoblotting with anti ‐ CISD1 and anti ‐ mitofusin 2 antibodies . Mitochondrial lysate and total lysate were also subjected to immunoblotting with indicated antibodies for loading and protein expression controls . Phospho ‐ Ser111 Rab8A was detected after Rab8A immunoprecipitation from 200 μg of mitochondrial lysate with anti ‐ Rab8A antibody .

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Figure 6Rab8A is not required for the activation of Parkin E3 ligase activity at mitochondria in response to PINK1 activation by CCCPWild‐type (WT) or Rab8A knockout (KO) HeLa cells were transfected with WT or Cys431Phe (C341F)‐mutant Parkin. After transfection for 24 h, cells were treated with DMSO as a vehicle control or 10 μM CCCP for 6 h. Mitochondrial enriched extracts (mitochondrial lysate) were incubated with ubiquitin‐binding resins derived from his‐halo‐ubiquilin1 UBA domain tetramer (UBAUBQLN1). Captured ubiquitylated proteins were subject to immunoblotting with anti‐CISD1 and anti‐mitofusin 2 antibodies. Mitochondrial lysate and total lysate were also subjected to immunoblotting with indicated antibodies for loading and protein expression controls. Phospho‐Ser111 Rab8A was detected after Rab8A immunoprecipitation from 200 μg of mitochondrial lysate with anti‐Rab8A antibody.


Figure 7In vitro phosphorylation analysis of Rab8A by PINK1 . WT or S111A ‐ mutant Rab8A ( 1 . 2 μg ) was incubated in the presence of MBP ‐ fused WT or KI ( D359A ) TcPINK1 ( 1 . 1 μg ) and Mg2 + ‐ [ γ ‐ 32P ] ATP for the indicated time . Samples were subjected to SDS - PAGE , and proteins were detected by colloidal Coomassie blue staining ( lower panel ) . The [ γ ‐ 32P ] incorporation to substrate was detected by autoradiography ( upper panel ) . Cerenkov counting was used to calculate the stoichiometry of substrate phosphorylation as mol of [ γ ‐ 32P ] incorporation / mol of substrate . Ubiquitin was used as a positive control of the TcPINK1 substrate .

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Figure 7In vitro phosphorylation analysis of Rab8A by PINK1. WT or S111A‐mutant Rab8A (1.2 μg) was incubated in the presence of MBP‐fused WT or KI (D359A) TcPINK1 (1.1 μg) and Mg2+‐[γ‐32P] ATP for the indicated time. Samples were subjected to SDS-PAGE, and proteins were detected by colloidal Coomassie blue staining (lower panel). The [γ‐32P] incorporation to substrate was detected by autoradiography (upper panel). Cerenkov counting was used to calculate the stoichiometry of substrate phosphorylation as mol of [γ‐32P] incorporation/mol of substrate. Ubiquitin was used as a positive control of the TcPINK1 substrate.


Figure 8Time ‐ course comparison of PINK1 ‐ mediated Rab8A , Rab8B and Rab13 Ser111 phosphorylation vs . Parkin Ser65 phosphorylation . Flp ‐ In T ‐ Rex HEK293 cells expressing WT PINK1 ‐ FLAG were transfected with either WT or Ser111Ala ‐ ( S111A ) mutant HA ‐ Rab8A , HA ‐ Rab8B or HA ‐ Rab13 , induced with doxycycline and stimulated with CCCP for the indicated time . In parallel , Flp ‐ In T ‐ Rex HEK293 cells expressing WT PINK1 ‐ FLAG were transfected with either WT or Ser65 Ala ( S65A ) ‐ mutant Parkin . Whole ‐ cell lysates ( 0 . 25 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with indicated phospho ‐ Ser111 antibodies . Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls . For the lower panel , whole ‐ cell lysates ( 30 μg ) were immunoblotted with indicated antibodies . Time ‐ course comparison of endogenous PINK1 ‐ mediated Rab8A , Rab8B and Rab13 Ser111 phosphorylation vs . Parkin Ser65 phosphorylation . HeLa cells were transfected with either WT or S111A ‐ mutant HA ‐ Rab8A , HA ‐ Rab8B or HA ‐ Rab13 for at least 24 h before CCCP treatment for the indicated time . Whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with indicated phospho ‐ Ser111 antibodies . In parallel , HeLa cells were transfected with either WT or S65A ‐ mutant Parkin and whole ‐ cell lysates ( 30 μg ) were immunoblotted with indicated antibodies .

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Figure 8Time‐course comparison of PINK1‐mediated Rab8A, Rab8B and Rab13 Ser111 phosphorylation vs. Parkin Ser65 phosphorylation. Flp‐In T‐Rex HEK293 cells expressing WT PINK1‐FLAG were transfected with either WT or Ser111Ala‐(S111A) mutant HA‐Rab8A, HA‐Rab8B or HA‐Rab13, induced with doxycycline and stimulated with CCCP for the indicated time. In parallel, Flp‐In T‐Rex HEK293 cells expressing WT PINK1‐FLAG were transfected with either WT or Ser65 Ala (S65A)‐mutant Parkin. Whole‐cell lysates (0.25 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with indicated phospho‐Ser111 antibodies. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls. For the lower panel, whole‐cell lysates (30 μg) were immunoblotted with indicated antibodies.Time‐course comparison of endogenous PINK1‐mediated Rab8A, Rab8B and Rab13 Ser111 phosphorylation vs. Parkin Ser65 phosphorylation. HeLa cells were transfected with either WT or S111A‐mutant HA‐Rab8A, HA‐Rab8B or HA‐Rab13 for at least 24 h before CCCP treatment for the indicated time. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with indicated phospho‐Ser111 antibodies. In parallel, HeLa cells were transfected with either WT or S65A‐mutant Parkin and whole‐cell lysates (30 μg) were immunoblotted with indicated antibodies.


Figure 9Rabin8 ‐ catalysed mant ‐ GDP release from mant ‐ GDP loaded WT , S111A and S111E mutants of Rab8A . The Rab proteins ( 1 μM ) were incubated with 100 μM GDP in buffer ( 20 mM HEPES pH 7 . 5 , 50 mM NaCl , 1 mM MgCl2 , 2 mM DTE ) , and the reaction was started by the addition of 0 . 5 μM Rabin8 . The decrease in mant fluorescence was used as a measure of mantGDP release . Rab8A Ser111 phosphorylation impairs Rabin8 interaction in cells . Rab8A KO HeLa cells were transfected with wild ‐ type ( WT ) , S111E or S111A HA ‐ Rab8A . Whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with Rabin8 or anti ‐ HA antibody . Lysates were immunoblotted with Rabin8 or anti ‐ HA antibody to confirm equivalent expression of Rabin8 and WT and mutant HA ‐ Rab8A in extracts .

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Figure 9Rabin8‐catalysed mant‐GDP release from mant‐GDP loaded WT, S111A and S111E mutants of Rab8A. The Rab proteins (1 μM) were incubated with 100 μM GDP in buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 1 mM MgCl2, 2 mM DTE), and the reaction was started by the addition of 0.5 μM Rabin8. The decrease in mant fluorescence was used as a measure of mantGDP release.Rab8A Ser111 phosphorylation impairs Rabin8 interaction in cells. Rab8A KO HeLa cells were transfected with wild‐type (WT), S111E or S111A HA‐Rab8A. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rabin8 or anti‐HA antibody. Lysates were immunoblotted with Rabin8 or anti‐HA antibody to confirm equivalent expression of Rabin8 and WT and mutant HA‐Rab8A in extracts.


Figure 1VGLL4 protein expression levels in 6 murine tumor cell lines . Total cell lysates were analyzed by western blotVGLL4 protein levels were reduced in Vgll4 depleted cells . Total cell lysates of Vgll4 - knockdown LLC and MB49 cells were analyzed by western blotControl or shVgll4 LLC cells were transplanted into C57BL / 6 mice , and tumor - growth kinetics was measured at the indicated times . n = 12 tumors for each groupQuantification of tumor weights 21 days after transplantation of control or shVgll4 LLC cells into C57BL / 6 mice from ( C ) Control or shVgll4 LLC cells were transplanted into nude mice , and tumor - growth kinetics was measured at the indicated times . n = 8 tumors for each group . ( F ) Quantification of tumor weights 15 days after transplantation of control or shVgll4 LLC cells into nude mice from ( E ) . n = 8 tumors for each group . n . s . p > 0 . 05 , two - tailed Student " s t - test . The solid line represents the average weightControl or shVgll4 MB49 cells were transplanted into C57BL / 6 mice , and tumor - growth kinetics was measured at the indicated times . n = 9 tumors for each group Quantification of tumor weights 30 days after transplantation of control or shVgll4 MB49 cells into C57BL / 6 miceControl or shVgll4 MB49 cells were transplanted into nude mice , and tumor - growth kinetics was measured at the indicated times . n = 8 tumors for each group Quantification of tumor weights 15 days after transplantation of control or shVgll4 MB49 cells into nude mice from ( I ) . n = 8 tumors for each groupImmunofluorescent staining of CD3 , CD8 and CD45 ( red ) counterstained with DAPI for DNA ( blue ) using control or shVgll4 LLC tumors from C57BL / 6 mice . Statistical analysis of the percentage of CD3 , CD8 and CD45 positive cells in the tumors is shown in the right panel respectively . n = 3 tumors for each groupQuantification of tumor weights 30 days after transplantation of shVgll4 MB49 cells into C57BL / 6 mice receiving anti - CD4 alone , anti - CD8 alone , or both anti - CD4 and anti - CD8 blocking antibodies . n = 20 tumors for each

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Figure 1VGLL4 protein expression levels in 6 murine tumor cell lines. Total cell lysates were analyzed by western blotVGLL4 protein levels were reduced in Vgll4 depleted cells. Total cell lysates of Vgll4-knockdown LLC and MB49 cells were analyzed by western blotControl or shVgll4 LLC cells were transplanted into C57BL/6 mice, and tumor-growth kinetics was measured at the indicated times. n=12 tumors for each groupQuantification of tumor weights 21 days after transplantation of control or shVgll4 LLC cells into C57BL/6 mice from (C)Control or shVgll4 LLC cells were transplanted into nude mice, and tumor-growth kinetics was measured at the indicated times. n=8 tumors for each group. (F) Quantification of tumor weights 15 days after transplantation of control or shVgll4 LLC cells into nude mice from (E). n=8 tumors for each group. n.s. p>0.05, two-tailed Student"s t-test. The solid line represents the average weightControl or shVgll4 MB49 cells were transplanted into C57BL/6 mice, and tumor-growth kinetics was measured at the indicated times. n=9 tumors for each group Quantification of tumor weights 30 days after transplantation of control or shVgll4 MB49 cells into C57BL/6 miceControl or shVgll4 MB49 cells were transplanted into nude mice, and tumor-growth kinetics was measured at the indicated times. n=8 tumors for each group Quantification of tumor weights 15 days after transplantation of control or shVgll4 MB49 cells into nude mice from (I). n=8 tumors for each groupImmunofluorescent staining of CD3, CD8 and CD45 (red) counterstained with DAPI for DNA (blue) using control or shVgll4 LLC tumors from C57BL/6 mice. Statistical analysis of the percentage of CD3, CD8 and CD45 positive cells in the tumors is shown in the right panel respectively. n=3 tumors for each groupQuantification of tumor weights 30 days after transplantation of shVgll4 MB49 cells into C57BL/6 mice receiving anti-CD4 alone, anti-CD8 alone, or both anti-CD4 and anti-CD8 blocking antibodies. n=20 tumors for each group