( A ) Overview of a reconstructed superresolution image of GFP - Bax overexpressing HCT116Bax / Bak - / - cells stained with AF647 - anti - GFP - nanobodies Image was acquired on fixed cells 7 h after apoptosis induction with STS . Dotted line shows the cell shape ( see Fig EV3 ) . Scale bar , 5 µm . ( B ) Gallery of typical GFP - Bax WT non - random structures during apoptosis on HCT116Bax / Bak - / - cells . Scale bars , 100 nm .

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(A) Overview of a reconstructed superresolution image of GFP-Bax overexpressing HCT116Bax/Bak -/- cells stained with AF647-anti-GFP-nanobodies Image was acquired on fixed cells 7 h after apoptosis induction with STS. Dotted line shows the cell shape (see Fig EV3). Scale bar, 5 µm.(B) Gallery of typical GFP-Bax WT non-random structures during apoptosis on HCT116Bax/Bak -/- cells. Scale bars, 100 nm.


( C ) Comparison of confocal ( left ) and STED ( right ) images of GFP - Bax overexpressing HeLa cells 2h after STS treatment . STED reveals a notable increase in resolution . Scale bars , 500 nm . ( D ) Representative GFP - Bax non - random structures found with STED ( lines , arcs , rings , double lines ) . Scale bars , 100 nm .

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(C) Comparison of confocal (left) and STED (right) images of GFP-Bax overexpressing HeLa cells 2h after STS treatment. STED reveals a notable increase in resolution. Scale bars, 500 nm.(D) Representative GFP-Bax non-random structures found with STED (lines, arcs, rings, double lines). Scale bars, 100 nm.


( A ) Representative TEM pictures of HeLa wild - type cells . Bottom panels show healthy mitochondria . Scale bars , 500nm ( overview ) and 200 nm ( zooms ) . ( B ) Representative TEM pictures of apoptotic HeLa wild - type cells 4h after STS treatment ( 1µM ) . Black arrowheads point to defects on the mitochondrial outer membrane . Scale bars , 500nm ( overview ) and 200 nm ( zooms ) ( C ) Representative TEM pictures of apoptotic HeLaGFP - Bax transfected cells 4h after STS treatment ( 1µM ) . Black arrowheads point to defects on the mitochondrial outer membrane . Scale bars , 500nm ( overview ) and 200 nm ( zooms ) .

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(A) Representative TEM pictures of HeLa wild-type cells. Bottom panels show healthy mitochondria. Scale bars, 500nm (overview) and 200 nm (zooms).(B) Representative TEM pictures of apoptotic HeLa wild-type cells 4h after STS treatment (1µM). Black arrowheads point to defects on the mitochondrial outer membrane. Scale bars, 500nm (overview) and 200 nm (zooms)(C) Representative TEM pictures of apoptotic HeLaGFP-Bax transfected cells 4h after STS treatment (1µM). Black arrowheads point to defects on the mitochondrial outer membrane. Scale bars, 500nm (overview) and 200 nm (zooms).


( A ) Left panel shows the individual channels and overlaid images of confocal live cell imaging of HeLa cells overexpressing GFP - Bax ( green ) and mito - dsRed ( magenta ) at 0 and 85 minutes after apoptosis induction with STS 1µM . Right panel shows zoomed overlaid images of HeLa cells following GFP - Bax translocation ( green ) and mitochondrial fragmentation ( magenta ) during apoptosis induction with STS 1µM . Scale bars , 10 µm ( left ) and 2 µm ( right ) ( B ) Reconstructed dual - color superresolution image of GFP - Bax overexpressing HeLa cells . GFP - Bax was imaged by immunosstaining with AF647 - anti - GFP - nanobodies and mitochondria were immunostained with CoxIV ( primary ) and cf680 ( secondary ) antibodies . Image was acquired 3h after STS treatment . Left panel shows the cell overview ( scale bar , 5 µm ) , right panel up shows the overview inset ( scale bar , 500nm ) and right panel down shows a non - random linear GFP - Bax assembly at mitochondria ( scale bar , 100nm ) . ( C ) Reconstructed dual - color superresolution image of HeLa cells co - overexpressing mMaple - mito and GFP - Bax and stained with AF647 - anti - GFP - nanobodies . Image was acquired 3h after STS treatment . Lower panel shows enlarged insets corresponding to the white rectangles in the upper panel . Scale bars , 1 µm ( overview ) and 100 nm ( insets )

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(A) Left panel shows the individual channels and overlaid images of confocal live cell imaging of HeLa cells overexpressing GFP-Bax (green) and mito-dsRed (magenta) at 0 and 85 minutes after apoptosis induction with STS 1µM. Right panel shows zoomed overlaid images of HeLa cells following GFP-Bax translocation (green) and mitochondrial fragmentation (magenta) during apoptosis induction with STS 1µM. Scale bars, 10 µm (left) and 2 µm (right)(B) Reconstructed dual-color superresolution image of GFP-Bax overexpressing HeLa cells. GFP-Bax was imaged by immunosstaining with AF647-anti-GFP-nanobodies and mitochondria were immunostained with CoxIV (primary) and cf680 (secondary) antibodies. Image was acquired 3h after STS treatment. Left panel shows the cell overview (scale bar, 5 µm), right panel up shows the overview inset (scale bar, 500nm) and right panel down shows a non-random linear GFP-Bax assembly at mitochondria (scale bar, 100nm).(C) Reconstructed dual-color superresolution image of HeLa cells co-overexpressing mMaple-mito and GFP-Bax and stained with AF647-anti-GFP-nanobodies. Image was acquired 3h after STS treatment. Lower panel shows enlarged insets corresponding to the white rectangles in the upper panel. Scale bars, 1 µm (overview) and 100 nm (insets)


( A ) AFM image of a control SLB prepared from LUVs with a mitochondrial - like lipid composition . The bilayer looks flat , without aggregates or defects . Scale bar , 1 µm . ( B ) AFM image of a SLB prepared from LUVs with a mitochondrial - like lipid composition pre - incubated with heat - activated Bax . The green arrows indicate the presence of membrane pores , which are heterogeneous in size and shape . The edges of the pores present protrusions corresponding to Bax clusters . Scale bar , 1 µm .

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(A) AFM image of a control SLB prepared from LUVs with a mitochondrial-like lipid composition. The bilayer looks flat, without aggregates or defects. Scale bar, 1 µm.(B) AFM image of a SLB prepared from LUVs with a mitochondrial-like lipid composition pre-incubated with heat-activated Bax. The green arrows indicate the presence of membrane pores, which are heterogeneous in size and shape. The edges of the pores present protrusions corresponding to Bax clusters. Scale bar, 1 µm.


( C ) 3D AFM topography of a Bax arc ( left ) and ring ( right ) . Both images reveal a circular dark hole that spans the lipid membrane ( green ) . Bax molecules around the pore rim ( magenta and white ) protrude 3 , 97 ± 1 , 02 nm above the membrane plane , as confirmed by the height profiles shown below each image ( corresponding to the white line in the 2D image insets ) . The topography of the arc structure reveals a pore only partially surrounded by Bax molecules , while lipids alone form the rest of the pore rim . Images are shown in a 42 ° tilted representation .

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(C) 3D AFM topography of a Bax arc (left) and ring (right). Both images reveal a circular dark hole that spans the lipid membrane (green). Bax molecules around the pore rim (magenta and white) protrude 3,97 ± 1,02 nm above the membrane plane, as confirmed by the height profiles shown below each image (corresponding to the white line in the 2D image insets). The topography of the arc structure reveals a pore only partially surrounded by Bax molecules, while lipids alone form the rest of the pore rim. Images are shown in a 42° tilted representation.


( D ) Quantitative analysis of the distinct structures found for activated Bax on SLBs . Data show the total number of structures in all the measurements .

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(D) Quantitative analysis of the distinct structures found for activated Bax on SLBs. Data show the total number of structures in all the measurements.


( C - D ) Snapshots from confocal live imaging movies of a representative embryo ( N = 7 ) during cellularisation co - expressing opto - Notch , Mito - Zdk , MCP : : GFP and sim - MS2 . Continuous photo - activation from the onset of cellularisation with a stack size of z = 25μm ( λ = 488 nm ) was alternated with mCherry excitation ( λ = 561 nm ) at 1 min intervals . Single confocal z - slices of the mCherry channel are shown to record opto - Notch localization before , and 10 minutes after sustained photo - activation . Magnified insets show opto - Notch bound to the mitochondrial anchor in the dark ( C ) and in the nucleus upon photo - activation ( D ) . Scale bar , 10 μm .

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(C-D) Snapshots from confocal live imaging movies of a representative embryo (N=7) during cellularisation co-expressing opto-Notch, Mito-Zdk, MCP::GFP and sim-MS2. Continuous photo-activation from the onset of cellularisation with a stack size of z= 25μm (λ=488 nm) was alternated with mCherry excitation (λ=561 nm) at 1 min intervals. Single confocal z-slices of the mCherry channel are shown to record opto-Notch localization before, and 10 minutes after sustained photo-activation. Magnified insets show opto-Notch bound to the mitochondrial anchor in the dark (C) and in the nucleus upon photo-activation (D). Scale bar, 10 μm.


( F - H ) Maximum intensity z - projections ( z = 25 μm ) of the same embryo simultaneously photoactivated and imaged in the GFP channel ( λ = 488 nm ) to record sim expression at a time resolution of 30 s . Shown are the first cycle of photo - activation , which approximates the dark state ( F ) , 15 min ( G ) , and 35 min ( H ) after continuous photo - activation . Magnified insets show that sim is not expressed in the dark at this stage ( F ) , but it is expressed in lateral ectodermal cells after 15 min of photo - activation ( G ) , and appeared to be shut off in most nuclei after 35 mins ( H ) . Scale bar , 10 μm .

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(F-H) Maximum intensity z-projections (z = 25 μm) of the same embryo simultaneously photoactivated and imaged in the GFP channel (λ=488 nm) to record sim expression at a time resolution of 30 s. Shown are the first cycle of photo-activation, which approximates the dark state (F), 15 min (G), and 35 min (H) after continuous photo-activation. Magnified insets show that sim is not expressed in the dark at this stage (F), but it is expressed in lateral ectodermal cells after 15 min of photo-activation (G), and appeared to be shut off in most nuclei after 35 mins (H). Scale bar, 10 μm.


The number of sim - expressing nuclei as counted by automated spot detection over time ( A ) Continuous opto - Notch activation initially induces sim expression across a large number of nuclei . Over time , the majority of nuclei adapt to the input and terminate expression .

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The number of sim-expressing nuclei as counted by automated spot detection over time (A) Continuous opto-Notch activation initially induces sim expression across a large number of nuclei. Over time, the majority of nuclei adapt to the input and terminate expression.


( D - E ) Quantification of nuclear localisation of mCherry : : NICD during opto - Notch photo - activation . Raw data was bleaching - corrected with an exponential fit and shown is the nuclear / total ratio , which further normalises bleaching and embryo - to - embryo variability . NICD displays persistent nuclear enrichment during continuous activation ( D ) and shuttles back and forth between nucleus and cytoplasm under the pulsatile protocol ( E ) .

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(D-E) Quantification of nuclear localisation of mCherry::NICD during opto-Notch photo-activation. Raw data was bleaching-corrected with an exponential fit and shown is the nuclear/total ratio, which further normalises bleaching and embryo-to-embryo variability. NICD displays persistent nuclear enrichment during continuous activation (D) and shuttles back and forth between nucleus and cytoplasm under the pulsatile protocol (E).


( D - E ) Results of continuous activation experiments in a Hairless heterozygous hypomorphic mutant ( Hairless H2 ) and under overexpression of Twist ( Twist OE ) The observed outcomes are most consistent with the predictions from the state - dependent inactivation model .

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(D-E) Results of continuous activation experiments in a Hairless heterozygous hypomorphic mutant (Hairless H2) and under overexpression of Twist (Twist OE) The observed outcomes are most consistent with the predictions from the state-dependent inactivation model.


( A ) In vitro pull - downs between strep ( II ) - tagged DDB1 - DCAF12 and untagged wild type ( WT ) CCT5 or CCT5 ( 1 - 529 ) ( Δ ( Ct ) ) seen on a Coomassie - stained SDS - PAGE gel .

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(A) In vitro pull-downs between strep(II)-tagged DDB1-DCAF12 and untagged wild type (WT) CCT5 or CCT5 (1-529) (Δ(Ct)) seen on a Coomassie-stained SDS-PAGE gel.


( B ) Titration curves between a fluorescent ATTO488CCT520 degron peptide and TbDDB1 - DCAF12 or terbium - coupled streptavidin ( Tb - SA ) ( n = 3 ) . Signal originating in the absence of TbDDB1 - DCAF12 is unspecific and was subtracted in subsequent experiments .

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(B) Titration curves between a fluorescent ATTO488CCT520 degron peptide and TbDDB1-DCAF12 or terbium-coupled streptavidin (Tb-SA) (n=3). Signal originating in the absence of TbDDB1-DCAF12 is unspecific and was subtracted in subsequent experiments.


( C ) TR - FRET counter - titration of unlabeled wild type ( WT ) CCT5 or an unlabeled CCT520 peptide into pre - assembled TbDDB1 - DCAF12488 ( n = 3 ) .

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(C) TR-FRET counter-titration of unlabeled wild type (WT) CCT5 or an unlabeled CCT520 peptide into pre-assembled TbDDB1-DCAF12488 (n=3).


( D ) TR - FRET counter - titrations of label - free CCT5 C - terminal peptides into TbDDB1 - DCAF12488 ( n = 3 ) . Sequences of the peptides and IC50 values for the titrations are listed on the table . Peptides are labeled for their degron position and corresponding CCT5 amino acid number . ( E ) Counter - titration of unlabeled CCT510 mutant peptides into TbDDB1 - DCAF12488 ( n = 3 ) . Sequences of the peptides and IC50 values for the titrations are listed on the table . Peptides are labeled for their degron position .

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(D) TR-FRET counter-titrations of label-free CCT5 C-terminal peptides into TbDDB1-DCAF12488 (n=3). Sequences of the peptides and IC50 values for the titrations are listed on the table. Peptides are labeled for their degron position and corresponding CCT5 amino acid number. (E) Counter-titration of unlabeled CCT510 mutant peptides into TbDDB1-DCAF12488 (n=3). Sequences of the peptides and IC50 values for the titrations are listed on the table. Peptides are labeled for their degron position. D


( D ) Titration curves between a fluorescent ATTO488CCT520 degron peptide and wild type ( WT ) or mutant TbDDB1 - DCAF12 complexes ( n = 3 ) . Unspecific signal arising from the Tb - SA label was subtracted .

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(D) Titration curves between a fluorescent ATTO488CCT520 degron peptide and wild type (WT) or mutant TbDDB1-DCAF12 complexes (n=3). Unspecific signal arising from the Tb-SA label was subtracted.


( E ) In vitro ubiquitination of CCT5 by wild type ( WT ) or mutant CRL4DCAF12 complexes in the presence of ubiquitin , ATP , E1 and E2 enzymes .

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(E) In vitro ubiquitination of CCT5 by wild type (WT) or mutant CRL4DCAF12 complexes in the presence of ubiquitin, ATP, E1 and E2 enzymes.


( A ) Counter - titration of unlabeled CCT5 or unlabeled TRiC into pre - assembled TbDDB1 - DCAF12488 ( n = 3 ) .

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(A) Counter-titration of unlabeled CCT5 or unlabeled TRiC into pre-assembled TbDDB1-DCAF12488 (n=3).


( B ) In vitro ubiquitination of monomeric wild type CCT5 or TRiC by CRL4DCAF12 in the presence of ubiquitin , ATP , E1 and E2 enzymes . CCT5 , but not TRiC , is modified with poly - ubiquitin ( Ubn ) chains by CRL4DCAF12 . Free CCT5 protein carries an N - terminal strep ( II ) tag and differs in electrophoretic mobility from untagged CCT5 purified in complex with TRiC .

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(B) In vitro ubiquitination of monomeric wild type CCT5 or TRiC by CRL4DCAF12 in the presence of ubiquitin, ATP, E1 and E2 enzymes. CCT5, but not TRiC, is modified with poly-ubiquitin (Ubn) chains by CRL4DCAF12. Free CCT5 protein carries an N-terminal strep(II) tag and differs in electrophoretic mobility from untagged CCT5 purified in complex with TRiC.


Representative false colour images of leaf surface temperature captured by a thermal camera from WT , stp1 - 1 , stp4 - 1 , stp1stp4 , stp13 , stp1stp13 and stp4stp13 plants .

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Representative false colour images of leaf surface temperature captured by a thermal camera from WT, stp1-1, stp4-1, stp1stp4, stp13, stp1stp13 and stp4stp13 plants.


Normalized leaf surface temperature over the phenotyping period . Data shown are means ± SEM ; n = 10 per genotype and time point .

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Normalized leaf surface temperature over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point.


Whole - plant recordings of changes in stomatal conductance ( gs ) from WT , stp1 - 1 , stp4 - 1 and stp1stp4 plants . Data shown are means ± SEM ; n ≥ 3 per genotype .

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Whole-plant recordings of changes in stomatal conductance (gs) from WT, stp1-1, stp4-1 and stp1stp4 plants. Data shown are means ± SEM; n ≥ 3 per genotype.


Whole - plant recordings of changes in stomatal conductance ( gs ) from self - grafted donor lines ( WT / WT , stp1stp4 / stp1stp4 ) and reciprocal grafting of shoot / root ( WT / stp1stp4 , stp1stp4 / WT ) plants . Data shown are means ± SEM ; n = 3 per genotype .

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Whole-plant recordings of changes in stomatal conductance (gs) from self-grafted donor lines (WT/WT, stp1stp4/stp1stp4) and reciprocal grafting of shoot/root (WT/stp1stp4, stp1stp4/WT) plants. Data shown are means ± SEM; n = 3 per genotype.


Content of soluble sugars in guard cell - enriched epidermal peels of WT and stp1stp4 plants at the end of the night ( EoN ) and after 40 min of illumination with white light at 150 µmol m - 2 s - 1 following the EoN . Data for two independent experiments are shown ; means ± SEM ; n ≥ 7 per genotype and time point .

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Content of soluble sugars in guard cell-enriched epidermal peels of WT and stp1stp4 plants at the end of the night (EoN) and after 40 min of illumination with white light at 150 µmol m-2 s-1 following the EoN. Data for two independent experiments are shown; means ± SEM; n ≥ 7 per genotype and time point.


Representative confocal laser microscopy images of propidium iodide - stained guard cell starch granules of intact leaves of WT , stp1 - 1 , stp4 - 1 , stp1stp4 , suc1 and suc3 plants . Scale bar , 10 µm .

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Representative confocal laser microscopy images of propidium iodide-stained guard cell starch granules of intact leaves of WT, stp1-1, stp4-1, stp1stp4, suc1 and suc3 plants. Scale bar, 10 µm.


Starch dynamics in guard cells of intact leaves of WT , stp1 - 1 , stp4 - 1 , stp1stp4 , suc1 and suc3 plants at the end of the night ( EoN ) and after one and three hours of illumination with 150 µmol m - 2 s - 1 of white light . Data for three independent experiments are shown ; means ± SEM ; n = 120 individual guard cells per genotype and time point .

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Starch dynamics in guard cells of intact leaves of WT, stp1-1, stp4-1, stp1stp4, suc1 and suc3 plants at the end of the night (EoN) and after one and three hours of illumination with 150 µmol m-2 s-1 of white light. Data for three independent experiments are shown; means ± SEM; n = 120 individual guard cells per genotype and time point.


Representative false colour images of photosystem II ( PSII ) operating efficiency ( ΦPSII ) captured by chlorophyll fluorescence imaging from WT , stp1 - 1 , stp4 - 1 and stp1stp4 plants . ΦPSII was measured at a photosynthetically active radiation ( PAR ) of 440 µmol m - 2 s - 1 .

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Representative false colour images of photosystem II (PSII) operating efficiency (ΦPSII) captured by chlorophyll fluorescence imaging from WT, stp1-1, stp4-1 and stp1stp4 plants. ΦPSII was measured at a photosynthetically active radiation (PAR) of 440 µmol m-2 s-1.


ΦPSII quantified over the phenotyping period . Data shown are means ± SEM ; n = 10 per genotype and time point .

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ΦPSII quantified over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point.


Whole - plant recordings of changes in CO2 assimilation ( A ) from WT , stp1 - 1 , stp4 - 1 and stp1stp4 plants . Data shown are means ± SEM ; n ≥ 3 per genotype .

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Whole-plant recordings of changes in CO2 assimilation (A) from WT, stp1-1, stp4-1 and stp1stp4 plants. Data shown are means ± SEM; n ≥ 3 per genotype.


Whole - plant recordings of changes in CO2 assimilation ( A ) from self - grafted donor lines ( WT / WT , stp1stp4 / stp1stp4 ) and reciprocal grafting of shoot / root ( WT / stp1stp4 , stp1stp4 / WT ) plants . Data shown are means ± SEM ; n = 3 per genotype .

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Whole-plant recordings of changes in CO2 assimilation (A) from self-grafted donor lines (WT/WT, stp1stp4/stp1stp4) and reciprocal grafting of shoot/root (WT/stp1stp4, stp1stp4/WT) plants. Data shown are means ± SEM; n = 3 per genotype.


Quantification of leaf glucose ( Glc ) . Data shown are means ± SEM ; n = 8 per genotype and time point . Quantification of leaf fructose ( Fru ) . Data shown are means ± SEM ; n = 8 per genotype and time point . Quantification of leaf sucrose ( Suc ) . Data shown are means ± SEM ; n = 8 per genotype and time point .

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Quantification of leaf glucose (Glc). Data shown are means ± SEM; n = 8 per genotype and time point.   Quantification of leaf fructose (Fru). Data shown are means ± SEM; n = 8 per genotype and time point.   Quantification of leaf sucrose (Suc). Data shown are means ± SEM; n = 8 per genotype and time point.


Quantification of leaf starch . Data shown are means ± SEM ; n = 8 per genotype and time point .

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Quantification of leaf starch. Data shown are means ± SEM; n = 8 per genotype and time point.


Representative Red Green Blue ( RGB ) images of 3 - ( day 0 ) and 4 - week - old ( day 7 ) WT , stp1 - 1 , stp4 - 1 and stp1stp4 plants .

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Representative Red Green Blue (RGB) images of 3-(day 0) and 4-week-old (day 7) WT, stp1-1, stp4-1 and stp1stp4 plants.


Projected rosette area over the phenotyping period . Data shown are means ± SEM ; n = 10 per genotype and time point .

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Projected rosette area over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point.


Photosynthetic CO2 assimilation ( A ) in dark - adapted WT and stp1stp4 plants in response to a step increase in CO2 concentrations from 0 to 1000 ppm . Data shown are means ± SEM ; n = 3 per genotype .

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Photosynthetic CO2 assimilation (A) in dark-adapted WT and stp1stp4 plants in response to a step increase in CO2 concentrations from 0 to 1000 ppm. Data shown are means ± SEM; n=3 per genotype.


Quantification of leaf glucose ( Glc ) . Data for two independent experiments are shown ; means ± SEM ; n ≥ 17 per genotype and time point .

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Quantification of leaf glucose (Glc). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.


Quantification of leaf fructose ( Fru ) . Data for two independent experiments are shown ; means ± SEM ; n ≥ 17 per genotype and time point . Quantification of leaf sucrose ( Suc ) . Data for two independent experiments are shown ; means ± SEM ; n ≥ 17 per genotype and time point . Quantification of leaf starch . Data for two independent experiments are shown ; means ± SEM ; n ≥ 17 per genotype and time point .

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Quantification of leaf fructose (Fru). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.   Quantification of leaf sucrose (Suc). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.   Quantification of leaf starch. Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.


Representative Red Green Blue ( RGB ) images of 3 - ( day 0 ) and 4 - week - old ( day 6 ) WT and stp1stp4 plants grown under 600 ppm CO2 .

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Representative Red Green Blue (RGB) images of 3-(day 0) and 4-week-old (day 6) WT and stp1stp4 plants grown under 600 ppm CO2.


Projected rosette area over the phenotyping period . Data shown are from two independent experiments ; means ± SEM ; n ≥ 24 per genotype and time point .

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Projected rosette area over the phenotyping period. Data shown are from two independent experiments; means ± SEM; n ≥ 24 per genotype and time point.


Representative confocal laser microscopy images of propidium iodide - stained guard cell starch granules of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2 . Scale bar , 10 µm .

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Representative confocal laser microscopy images of propidium iodide-stained guard cell starch granules of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2. Scale bar, 10 µm.


Starch dynamics in guard cells of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2 at the end of the night ( EoN ) and after one and three hours of illumination with 150 µmol m - 2 s - 1 of white light . Data for two independent experiments are shown ; means ± SEM ; n = 80 individual guard cells per genotype and time point .

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Starch dynamics in guard cells of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2 at the end of the night (EoN) and after one and three hours of illumination with 150 µmol m-2 s-1 of white light. Data for two independent experiments are shown; means ± SEM; n = 80 individual guard cells per genotype and time point.


( A ) RNAscope fluorescence in - situ hybridization of Ch25h transcripts ( red ) in the spinal cord of non - immunized mice ( NI ) ( left panels ) and mice 17 days after EAE immunization ( peak disease ) ( 2 right panels ) shown in endothelial cells ( Isolectin B4 ( IsoB4 ) green top panels ) and activated macrophages / microglia ( Ionized calcium biding adaptor molecule 1 ( IBA1 ) green lower panels ) . Nuclei are shown in blue . ( B ) Quantitative analysis of Ch25h mRNA expression in the spinal cord of NI and EAE mice comparing expression in macrophages / microglia ( MG ) and endothelial cells ( EC ) , n = 6 biological replicates / group .

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(A) RNAscope fluorescence in-situ hybridization of Ch25h transcripts (red) in the spinal cord of non-immunized mice (NI) (left panels) and mice 17 days after EAE immunization (peak disease) (2 right panels) shown in endothelial cells (Isolectin B4 (IsoB4) green top panels) and activated macrophages/microglia (Ionized calcium biding adaptor molecule 1 (IBA1) green lower panels). Nuclei are shown in blue. (B) Quantitative analysis of Ch25h mRNA expression in the spinal cord of NI and EAE mice comparing expression in macrophages/microglia (MG) and endothelial cells (EC), n = 6 biological replicates/group.


( E ) Percentage of eGFP + CNS ECs in the same condition as in ( D ) . Symbols indicate individual mice and bars indicate mean ± SD . Ch25hfl / fl NI : n = 4 biological replicates , Ch25hfl / fl EAE : n = 5 biological replicates , Ch25hECKO NI : n = 4 biological replicates , Ch25hECKO EAE : n = 4 biological replicates . Representative results of 1 experiment .

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(E) Percentage of eGFP+ CNS ECs in the same condition as in (D). Symbols indicate individual mice and bars indicate mean ± SD. Ch25hfl/fl NI: n = 4 biological replicates, Ch25hfl/fl EAE: n = 5 biological replicates, Ch25hECKO NI: n = 4 biological replicates, Ch25hECKO EAE: n = 4 biological replicates. Representative results of 1 experiment.


( A ) EAE disease course in Ch25hECKO and Cre negative littermates ( Ch25hfl / fl ) . Top panel : EAE clinical score . Bars indicate mean ± SEM . Bottom panel : Survival analysis of EAE , depicting disease incidence . Representative results of 2 experiments with a minimum of n = 5 biological replicates / group as indicated on the graph . ( B ) As in ( A ) except that Ch25h fl / fl - Pdgfb - iCreERT2 mice that express Cre recombinase in blood endothelial cells ( Ch25hBECKO ) are shown . Representative results of 2 experiments with a minimum of n = 5 biological replicates / group as indicated on the graph . ( C ) As in ( A ) except that Ch25hfl / fl - Prox1 - CreERT2 mice that express Cre recombinase in lymphatic endothelial cells ( Ch25hLECKO ) are shown . Representative results of 2 experiments with a minimum of n = 5 biological replicates / group as indicated on the graph .

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(A) EAE disease course in Ch25hECKO and Cre negative littermates (Ch25hfl/fl). Top panel: EAE clinical score. Bars indicate mean ± SEM. Bottom panel: Survival analysis of EAE, depicting disease incidence. Representative results of 2 experiments with a minimum of n = 5 biological replicates/group as indicated on the graph. (B) As in (A) except that Ch25h fl/fl- Pdgfb-iCreERT2 mice that express Cre recombinase in blood endothelial cells (Ch25hBECKO) are shown. Representative results of 2 experiments with a minimum of n = 5 biological replicates/group as indicated on the graph. (C) As in (A) except that Ch25hfl/fl- Prox1-CreERT2 mice that express Cre recombinase in lymphatic endothelial cells (Ch25hLECKO) are shown. Representative results of 2 experiments with a minimum of n = 5 biological replicates/group as indicated on the graph.


( D , E ) Histopathological quantifications ( D ) and representatives staining ( E ) of spinal cord sections of immunized Ch25hfl / fl mice ( n = 7 biological replicates ) and Ch25hECKO mice ( n = 10 biological replicates ) at day 21 post - immunization for cellular infiltration ( HE ) . Five sections per mouse were quantified . Scale bars 200 µm ( top panels ) , 100 µm ( bottom panels ) .

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(D, E) Histopathological quantifications (D) and representatives staining (E) of spinal cord sections of immunized Ch25hfl/fl mice (n = 7 biological replicates) and Ch25hECKO mice (n = 10 biological replicates) at day 21 post-immunization for cellular infiltration (HE). Five sections per mouse were quantified. Scale bars 200 µm (top panels), 100 µm (bottom panels).


( A ) RT - qPCR analysis of Ch25h expression in primary mouse brain microvascular endothelial cells ( pMBMECs ) isolated from Ch25hfl / fl and Ch25hECKO mice . Cells were left unstimulated ( NS ) or stimulated with IL - 1β . n = 5 biological replicates / group . The experiment was performed 3 times .

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(A) RT-qPCR analysis of Ch25h expression in primary mouse brain microvascular endothelial cells (pMBMECs) isolated from Ch25hfl/fl and Ch25hECKO mice. Cells were left unstimulated (NS) or stimulated with IL-1β. n = 5 biological replicates/group. The experiment was performed 3 times.


( F ) RT - qPCR of pMBMECs isolated from Ch25hECKO and Ch25hfl / fl mice and stimulated with IL - 1β . n = 4 biological replicates / group . The experiment was realized once .

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(F) RT-qPCR of pMBMECs isolated from Ch25hECKO and Ch25hfl/fl mice and stimulated with IL-1β. n = 4 biological replicates/group. The experiment was realized once.


( G ) RT - qPCR of CNS endothelial cells sorted from Ch25hECKO and Ch25hfl / fl mice during EAE ( day 10 after immunization ) . Ch25hECKO n = 3 biological replicates , Ch25hfl / fl n = 4 biological replicates . The experiment was performed once .

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(G) RT-qPCR of CNS endothelial cells sorted from Ch25hECKO and Ch25hfl/fl mice during EAE (day 10 after immunization). Ch25hECKO n = 3 biological replicates, Ch25hfl/fl n = 4 biological replicates. The experiment was performed once.


( C ) 25 - OHC , 7 - keto - 25 - OHC , and 24 ( S ) - OHC concentration in the same conditions as in ( B ) . Bars represent mean ± SD . n = 6 biological replicates / group except for Ch25fl / fl IL - 1β were n = 5 biological replicates . The experiment was performed once . ( D ) 25 - OHC , 7 - keto - 25 - OHC , and 24 ( S ) - OHC levels in spinal cord tissue extracted from Ch25hfl / fl ( n = 8 ) , Ch25hECKO ( n = 3 ) and Ch25hBBBKO ( n = 6 ) at the peak of EAE .

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(C) 25-OHC, 7-keto-25-OHC, and 24(S)-OHC concentration in the same conditions as in (B). Bars represent mean ± SD. n = 6 biological replicates/group except for Ch25fl/fl IL-1β were n = 5 biological replicates. The experiment was performed once. (D) 25-OHC, 7-keto-25-OHC, and 24(S)-OHC levels in spinal cord tissue extracted from Ch25hfl/fl (n = 8), Ch25hECKO (n = 3) and Ch25hBBBKO (n = 6) at the peak of EAE.


( G ) Prostaglandin concentration ( nM ) in the supernatant of pMBMECs comparing conditions mentioned in ( A ) . n = 5 biological replicates / group and bars represent mean ± SD .

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(G) Prostaglandin concentration (nM) in the supernatant of pMBMECs comparing conditions mentioned in (A). n = 5 biological replicates/group and bars represent mean ± SD.


( C ) Impact of CNS PMN - MDSC and BM - PMN on CD4 + T cell proliferation assessed by CFSE dilution using flow cytometry . PMN - MDSC were FACS - sorted from the CNS ( right panel ) and BM ( left panel ) of WT mice at the peak of EAE . NS = non stimulated . n = 4 biological replicates / group . Bars indicate mean ± SD . CNS PMN - MDSC / CD4 + T cells : representative results of 2 independent experiments ; BM - PMN : 1 experiment .

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(C) Impact of CNS PMN-MDSC and BM-PMN on CD4+ T cell proliferation assessed by CFSE dilution using flow cytometry. PMN-MDSC were FACS-sorted from the CNS (right panel) and BM (left panel) of WT mice at the peak of EAE. NS = non stimulated. n = 4 biological replicates/group. Bars indicate mean ± SD. CNS PMN-MDSC/CD4+ T cells: representative results of 2 independent experiments; BM-PMN: 1 experiment.


( H ) Percentage ( upper left panel ) and absolute number ( lower left panel ) of CNS live cells CD45 + CD11b + Ly6CintLy6G + PMN - MDSC ; Percentage of live CD4 + CD44 + Ki67 + cells ( upper right panel ) and ratio ( lower right panel ) of absolute number of PMN - MDSC and proliferating CD4 + T cells in the CNS at the peak of the disease of Ch25hfl / fl ( n = 9 ) and Ch25hECKO mice ( n = 8 ) . Symbols depict individual mice and bars indicate mean ± SD . Combined results of 2 independent experiments .

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(H) Percentage (upper left panel) and absolute number (lower left panel) of CNS live cells CD45+CD11b+Ly6CintLy6G+ PMN-MDSC; Percentage of live CD4+CD44+Ki67+ cells (upper right panel) and ratio (lower right panel) of absolute number of PMN-MDSC and proliferating CD4+ T cells in the CNS at the peak of the disease of Ch25hfl/fl (n = 9) and Ch25hECKO mice (n = 8). Symbols depict individual mice and bars indicate mean ± SD. Combined results of 2 independent experiments.


( I ) EAE disease course in Ch25hBBBKO mice ( n = 11 ) where the Cre - recombinase is expressed in endothelial cells of the CNS and Cre negative littermates ( Ch25hfl / fl : n = 7 and Ch25hfl / wt : n = 2 ) . Bars indicate mean ± SEM . Representative results of 2 independent experiments .

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(I) EAE disease course in Ch25hBBBKO mice (n = 11) where the Cre-recombinase is expressed in endothelial cells of the CNS and Cre negative littermates (Ch25hfl/fl: n = 7 and Ch25hfl/wt : n = 2). Bars indicate mean ± SEM. Representative results of 2 independent experiments.


( B ) Survival analysis in the same conditions as in ( A ) . Ch25hfl / fl isotype : n = 7 , Ch25hECKO isotype : n = 8 , Ch25hfl / fl Combo : n = 8 , Ch25hECKO Combo : n = 9 .

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(B) Survival analysis in the same conditions as in (A). Ch25hfl/fl isotype: n = 7, Ch25hECKO isotype: n = 8, Ch25hfl/fl Combo: n = 8, Ch25hECKO Combo: n = 9.


( C ) Representative contour plots of PMN - MDSC in the CNS at day 22 of EAE assessed by flow cytometry in the same conditions as in ( A ) . ( D ) As in ( C ) except that statistical analysis and percentage of PMN - MDSC in live cells are shown . Symbols depict individual mice and bars mean ± SD . Ch25hfl / fl Isotype n = 4 , Ch25hECKO isotype n = 4 , Ch25hfl / fl Combo , n = 3 , Ch25hECKO Combo n = 4 . (

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(C) Representative contour plots of PMN-MDSC in the CNS at day 22 of EAE assessed by flow cytometry in the same conditions as in (A). (D) As in (C) except that statistical analysis and percentage of PMN-MDSC in live cells are shown. Symbols depict individual mice and bars mean ± SD. Ch25hfl/fl Isotype n = 4, Ch25hECKO isotype n = 4, Ch25hfl/fl Combo, n = 3, Ch25hECKO Combo n = 4. (


( E ) Representative contour plots of CD101 and representative histograms of Ly6G expression in blood neutrophils at day 16 of EAE assessed by flow cytometry . Ch25hECKO and Ch25hfl / fl mice were treated with isotype or Combo treatment which was initiated at the first symptoms of EAE ( day 14 after immunization ) . ( F ) As in ( E ) except that statistical analysis is shown . Symbols depict individual mice , bars mean ± SD . Ch25hfl / lfl isotype n = 7 , Ch25hECKO isotype n = 5 , Ch25hfl / lfl Combo n = 6 , Ch25hECKO Combo n = 5 . The experiment was performed once . (

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(E) Representative contour plots of CD101 and representative histograms of Ly6G expression in blood neutrophils at day 16 of EAE assessed by flow cytometry. Ch25hECKO and Ch25hfl/fl mice were treated with isotype or Combo treatment which was initiated at the first symptoms of EAE (day 14 after immunization). (F) As in (E) except that statistical analysis is shown. Symbols depict individual mice, bars mean ± SD. Ch25hfl/lfl isotype n = 7, Ch25hECKO isotype n = 5, Ch25hfl/lfl Combo n = 6, Ch25hECKO Combo n = 5. The experiment was performed once. (


Reverse Transcriptase PCR showing that bd1971 , and control gene dnaK , are expressed throughout the predatory cycle of Bdellovibrio bacteriovorus . RNA was isolated at the time points indicated across the top of the gel during one round of synchronous Bdellovibrio infection of E . coli cells . Primers were designed to anneal specifically to the gene of interest . L = 100bp DNA ladder , AP = Attack Phase cells , 15 - 45 = 15 - 45 minutes respectively since infection , 1h - 4h = hours respectively since infection . Ec = E . coli strain S17 - 1 RNA ( negative control : no Bdellovibrio RNA ) ; NT = control with no template RNA ; gen = B . bacteriovorus HD100 genomic DNA ( positive control ) . The cartoon above represents each stage in the predatory cycle .

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Reverse Transcriptase PCR showing that bd1971, and control gene dnaK, are expressed throughout the predatory cycle of Bdellovibrio bacteriovorus. RNA was isolated at the time points indicated across the top of the gel during one round of synchronous Bdellovibrio infection of E. coli cells. Primers were designed to anneal specifically to the gene of interest. L = 100bp DNA ladder, AP = Attack Phase cells, 15-45 = 15-45 minutes respectively since infection, 1h-4h = hours respectively since infection. Ec = E. coli strain S17-1 RNA (negative control: no Bdellovibrio RNA); NT = control with no template RNA; gen = B. bacteriovorus HD100 genomic DNA (positive control). The cartoon above represents each stage in the predatory cycle.


Epifluorescent images of E . coli invaded by B . bacteriovorus HD100 with mCherry tagged Bd1971 at the times indicated throughout the invasive process during one round of synchronous infection . Fluorescence was acquired with a two second exposure and maximum sensitivity gain but different balance and contrast levels in the fluorescence channel were used to produce these images ( see methods ) as the fluorescence was fainter at later timepoints but we wished to show whether or not the cellular localization within the Bdellovibrio altered during predation . Fluorescence images and merges with phase contrast images are shown . Scale bars are 2 µm . Images are representative of three independent experiments .

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Epifluorescent images of E. coli invaded by B. bacteriovorus HD100 with mCherry tagged Bd1971 at the times indicated throughout the invasive process during one round of synchronous infection. Fluorescence was acquired with a two second exposure and maximum sensitivity gain but different balance and contrast levels in the fluorescence channel were used to produce these images (see methods) as the fluorescence was fainter at later timepoints but we wished to show whether or not the cellular localization within the Bdellovibrio altered during predation. Fluorescence images and merges with phase contrast images are shown. Scale bars are 2 µm. Images are representative of three independent experiments.


Relative levels of c - di - GMP in matched cell biomass host - independent ( HI ) cultures of bd1971 : : kan compared with fliC1 merodiploid KnR HI control strain ( Wild - Type for bd1971 ) . The bd1971 mutant strain contains significantly more c - di - GMP than the control . Data are from 3 independent experiments .

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

Relative levels of c-di-GMP in matched cell biomass host-independent (HI) cultures of bd1971::kan compared with fliC1 merodiploid KnR HI control strain (Wild-Type for bd1971). The bd1971 mutant strain contains significantly more c-di-GMP than the control. Data are from 3 independent experiments.


Relative levels of c - di - GMP in matched cell biomass wild - type predatory cultures of HD100 strain , silent Bd1971 deletion strain ( Δ1971 ) , Bd1971D306307A strain ( bd1971DD ) and Bd1971R67D strain ( bd1971R ) . All mutant strains had higher levels of c - di - GMP compared to the wild type strain . Error bars show SEM . Data are from six independent experiments for HD100 and ΔBd1971 and from four independent experiments for Bd1971DD and Bd1971R mutants .

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

Relative levels of c-di-GMP in matched cell biomass wild-type predatory cultures of HD100 strain, silent Bd1971 deletion strain (Δ1971), Bd1971D306307A strain (bd1971DD) and Bd1971R67D strain (bd1971R). All mutant strains had higher levels of c-di-GMP compared to the wild type strain. Error bars show SEM. Data are from six independent experiments for HD100 and ΔBd1971 and from four independent experiments for Bd1971DD and Bd1971R mutants.


Apo - Bd1971 was supplemented with 10 mM MgCl2 or CaCl2 in conjunction with saturating concentrations ( 2 . 5 mM ) of cAMP , cGMP or no nucleotide and phosphodiesterase activity with respect to the generic substrate PNPP ( 5 mM ) was measured spectrophotometrically . Values are the average of three repeats , error bars represent standard deviation .

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

Apo-Bd1971 was supplemented with 10 mM MgCl2 or CaCl2 in conjunction with saturating concentrations (2.5 mM) of cAMP, cGMP or no nucleotide and phosphodiesterase activity with respect to the generic substrate PNPP (5 mM) was measured spectrophotometrically. Values are the average of three repeats, error bars represent standard deviation.


HPLC traces of reaction products generated from 100 µM c - di - GMP incubated with apo - Bd1971 ( Control ) , or protein supplemented with either 100 µM cAMP or cGMP . Peaks were identified by reference to standards also resolved by HPLC and are labeled in the figure .

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

HPLC traces of reaction products generated from 100 µM c-di-GMP incubated with apo-Bd1971 (Control), or protein supplemented with either 100 µM cAMP or cGMP. Peaks were identified by reference to standards also resolved by HPLC and are labeled in the figure.


ITC titration curves and relative heats of binding for ( A ) cAMP binding to Bd1971ΔEAL . Successive injections of 6 µl of cNMP ( 0 . 5 - 1 . 6 mM ) were made into 1 . 4 ml of apo - Bd1971ΔEAL ( 50 - 150 µM ) and heat changes measured . The top panels show the ITC titrations and the bottom panels show the binding isotherm associated with each ITC experiment .

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

ITC titration curves and relative heats of binding for (A) cAMP binding to Bd1971ΔEAL. Successive injections of 6 µl of cNMP (0.5 - 1.6 mM) were made into 1.4 ml of apo-Bd1971ΔEAL (50 - 150 µM) and heat changes measured. The top panels show the ITC titrations and the bottom panels show the binding isotherm associated with each ITC experiment.


ITC titration curves and relative heats of binding for cGMP binding to Bd1971ΔEAL . Successive injections of 6 µl of cNMP ( 0 . 5 - 1 . 6 mM ) were made into 1 . 4 ml of apo - Bd1971ΔEAL ( 50 - 150 µM ) and heat changes measured . The top panels show the ITC titrations and the bottom panels show the binding isotherm associated with each ITC experiment .

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

ITC titration curves and relative heats of binding for cGMP binding to Bd1971ΔEAL. Successive injections of 6 µl of cNMP (0.5 - 1.6 mM) were made into 1.4 ml of apo-Bd1971ΔEAL (50 - 150 µM) and heat changes measured. The top panels show the ITC titrations and the bottom panels show the binding isotherm associated with each ITC experiment.


Data were fitted with an allosteric sigmoidal model in GraphPad Prism software and used to determine the kinetic parameters associated with cNMP dependent phosphodiesterase activity . Data points for both sets of results are the average of three experimental replicates with the standard deviation shown by intersecting bars . Results are representative of at least three independent experiments .

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

Data were fitted with an allosteric sigmoidal model in GraphPad Prism software and used to determine the kinetic parameters associated with cNMP dependent phosphodiesterase activity.Data points for both sets of results are the average of three experimental replicates with the standard deviation shown by intersecting bars. Results are representative of at least three independent experiments.


Spot tests of co - transformants of either Bd0367 ( DgcA ; GGDEF protein ) response regulator domain or Bd3125 ( CdgA ; a degenerate GGDEF protein likely involved in c - di - GMP binding and signalling ) with full length Bd1971 , or the N - terminal ( 1 - 145 ) , or C - terminal ( 145 - 400 ) portions of Bd1971 in either pUT18c or pKT25 as indicated . Positive control was co - transformants with pUT18c - zip and pKT25 - zip , negative control was the empty vectors . The co - transformants were plated on LB - X - Gal medium with positive interaction resulting in blue colouration , which can be seen for all of the interactions in both plasmid combinations , but not for the negative control . Images are representative of at least 4 independent experiments .

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

Spot tests of co-transformants of either Bd0367 (DgcA; GGDEF protein) response regulator domain or Bd3125 (CdgA; a degenerate GGDEF protein likely involved in c-di-GMP binding and signalling) with full length Bd1971, or the N-terminal (1-145), or C-terminal (145-400) portions of Bd1971 in either pUT18c or pKT25 as indicated. Positive control was co-transformants with pUT18c-zip and pKT25-zip, negative control was the empty vectors. The co-transformants were plated on LB-X-Gal medium with positive interaction resulting in blue colouration, which can be seen for all of the interactions in both plasmid combinations, but not for the negative control. Images are representative of at least 4 independent experiments.


Confirmation of BTH interactions by β - Galactosidase activity assays . The interaction is significantly stronger between Bd3125 and the N - terminal domain of Bd1971 than the interaction with the C - terminus of Bd1971 . The response regulator domain of Bd0367 showed significant interaction with Bd1971 , but the full length Bd0367 did not . * p > 0 . 05 by Student ' s t - test N / S - not significant . Data are from at least three independent experiments , error bars are the standard error of the

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

Confirmation of BTH interactions by β-Galactosidase activity assays. The interaction is significantly stronger between Bd3125 and the N-terminal domain of Bd1971 than the interaction with the C-terminus of Bd1971. The response regulator domain of Bd0367 showed significant interaction with Bd1971, but the full length Bd0367 did not. *p>0.05 by Student's t-test N/S- not significant. Data are from at least three independent experiments, error bars are the standard error of the mean


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


( a ) HeLa cell lysates were immunoprecipitated with Atg16L1 antibody ( Atg16L1 ) or no antibody ( No Ab ) in control , and subjected to SDS - PAGE ; proteins were stained with SimplyBlue safe stain ( Invitrogen ) in accordance with the manufacturer ' s protocol . Bands indicated in the box were cut out and digested with trypsin , and identified by MALDI - TOF MS . HC , heavy chain .

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

(a) HeLa cell lysates were immunoprecipitated with Atg16L1 antibody (Atg16L1) or no antibody (No Ab) in control, and subjected to SDS-PAGE; proteins were stained with SimplyBlue safe stain (Invitrogen) in accordance with the manufacturer's protocol. Bands indicated in the box were cut out and digested with trypsin, and identified by MALDI-TOF MS. HC, heavy chain.


( b ) HeLa cells transfected for 24 h with control vector , or wild - type or different deletion mutants ( residues 2 - 77 , 2 - 275 and 232 - 607 ) of Flag - Atg16L1 were immunoprecipitated with anti - Flag antibody ( Atg16L1 ) and immunoblotted with anti - clathrin , anti - Flag and anti - Atg5 antibodies . Clathrin interacts with the N terminus of Atg16L1 , in a similar manner to Atg12 - Atg5 . Total lysates were run alongside as controls for protein input . FL , full - length .

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

(b) HeLa cells transfected for 24 h with control vector, or wild-type or different deletion mutants (residues 2-77, 2-275 and 232-607) of Flag-Atg16L1 were immunoprecipitated with anti-Flag antibody (Atg16L1) and immunoblotted with anti-clathrin, anti-Flag and anti-Atg5 antibodies. Clathrin interacts with the N terminus of Atg16L1, in a similar manner to Atg12-Atg5. Total lysates were run alongside as controls for protein input. FL, full-length.


( c ) HeLa cell lysates were immunoprecipitated ( IP ) with Atg16L1 antibody ( + Ab ) or no antibody ( no Ab ) and immunoblotted with anti - clathrin , anti - AP2 and anti - Atg16L1 antibodies . Total cell lysates ( TL ) were also blotted for input controls . Endogenous Atg16L1 interacts with clathrin heavy chain and AP2 .

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(c) HeLa cell lysates were immunoprecipitated (IP) with Atg16L1 antibody (+Ab) or no antibody (no Ab) and immunoblotted with anti-clathrin, anti-AP2 and anti-Atg16L1 antibodies. Total cell lysates (TL) were also blotted for input controls. Endogenous Atg16L1 interacts with clathrin heavy chain and AP2.


( d ) HeLa cells transfected for 24 h with Flag - Atg16L1 and increasing concentrations of Atg5 were immunoprecipitated ( IP ) with anti - Flag antibody ( Atg16L1 ) and immunoblotted with anti - clathrin , anti - Flag and anti - Atg5 antibodies . Atg5 overexpression neither interferes with nor increases clathrin - Atg16L1 binding . Uncropped images of blots are shown in Supplementary Information , Fig . S9 .

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

(d) HeLa cells transfected for 24 h with Flag-Atg16L1 and increasing concentrations of Atg5 were immunoprecipitated (IP) with anti-Flag antibody (Atg16L1) and immunoblotted with anti-clathrin, anti-Flag and anti-Atg5 antibodies. Atg5 overexpression neither interferes with nor increases clathrin-Atg16L1 binding. Uncropped images of blots are shown in Supplementary Information, Fig. S9.


( a ) HeLa cells transfected for 5 days with two rounds of control , epsin 1 ( eps . ) , clathrin heavy chain ( cla . ) , AP2 or AP1 siRNA were either left untreated or treated for 4 h with bafilomycin A1 ( + / - BafA1 ) , after which they were lysed for western blot analysis with anti - LC3 and anti - tubulin antibodies . Note that LC3 - I is often very faint compared with LC3 - II in HeLa cells under the protein extraction conditions we use . However , this is not a problem because it is advisable to relate LC3 - II to tubulin / actin .

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

(a) HeLa cells transfected for 5 days with two rounds of control, epsin 1 (eps.), clathrin heavy chain (cla.), AP2 or AP1 siRNA were either left untreated or treated for 4 h with bafilomycin A1 (+/- BafA1), after which they were lysed for western blot analysis with anti-LC3 and anti-tubulin antibodies. Note that LC3-I is often very faint compared with LC3-II in HeLa cells under the protein extraction conditions we use. However, this is not a problem because it is advisable to relate LC3-II to tubulin/actin.


( b ) Ratio of LC3 - II to tubulin on knockdown ( KD ) of control , epsin , clathrin heavy chain , AP2 or AP1 , with or without bafilomycin A1 , quantified from three independent experiments ( two for epsin ) and represented in the graph . Asterisk , P 0 . 01 ; two asterisks , P 0 . 001 ; three asterisks , P 0 . 0001 .

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

(b) Ratio of LC3-II to tubulin on knockdown (KD) of control, epsin, clathrin heavy chain, AP2 or AP1, with or without bafilomycin A1, quantified from three independent experiments (two for epsin) and represented in the graph. Asterisk, P 0.01; two asterisks, P 0.001; three asterisks, P 0.0001.


( c ) HeLa cells stably expressing GFP - mRFP - LC3 ( ref . 17 ) were transfected for 5 days with two rounds of control or clathrin heavy chain siRNA , during which they were either left untreated ( UT ) or treated with bafilomycin A1 ( BafA1 ) for the last 15 h . Cells were then fixed and analysed on a Cellomics ArrayScan system . Quantification of autophagic vacuoles ( AV ) or autolysosomes ( AL ) per cell in the different conditions is shown in the graph . Asterisk , P 0 . 01 ; n . s . , not significant . n = 2 , 000 cells .

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

(c) HeLa cells stably expressing GFP-mRFP-LC3 (ref. 17) were transfected for 5 days with two rounds of control or clathrin heavy chain siRNA, during which they were either left untreated (UT) or treated with bafilomycin A1 (BafA1) for the last 15 h. Cells were then fixed and analysed on a Cellomics ArrayScan system. Quantification of autophagic vacuoles (AV) or autolysosomes (AL) per cell in the different conditions is shown in the graph. Asterisk, P 0.01; n.s., not significant. n = 2,000 cells.


( d ) HeLa cells transfected for 4 days with control , clathrin heavy chain , AP1 or AP2 siRNA were grown in Hanks balanced salt solution ( HBSS ) for 6 h , after which the cells were fixed and immunostained for endogenous LC3 . The percentages of HeLa cells with more than 50 LC3 vesicles ( marked with arrows ) are quantified in the graph . End . , endogenous . Three asterisks , P 0 . 0001 . n = 500 cells . All error bars represent s . e . m . Scale bars , 10 μm . Uncropped images of blots are shown in Supplementary Information , Fig . S9 .

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

(d) HeLa cells transfected for 4 days with control, clathrin heavy chain, AP1 or AP2 siRNA were grown in Hanks balanced salt solution (HBSS) for 6 h, after which the cells were fixed and immunostained for endogenous LC3. The percentages of HeLa cells with more than 50 LC3 vesicles (marked with arrows) are quantified in the graph. End., endogenous. Three asterisks, P 0.0001. n = 500 cells. All error bars represent s.e.m. Scale bars, 10 μm. Uncropped images of blots are shown in Supplementary Information, Fig. S9.


( a - d ) HeLa cells transfected for 72 h with control , epsin 1 , clathrin heavy chain , AP2 or AP1 siRNA were treated with HBSS ( to induce autophagy ) for 6 h , after which they were fixed and immunostained for endogenous Atg16L1 . Representative cells for the control and epsin 1 siRNA are in a , and percentages of HeLa cells with Atg16L1vesicles are quantified in b ( for epsin 1 ) , c ( for clathrin ) and d ( for AP1 and AP2 ) . Three asterisks , P 0 . 0001 . Scale bars , 10 μm . ( Note that we used single siRNAs for all experiments and the effects were confirmed with two independent sequences for clathrin heavy chain ) . n = 600 cells .

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

(a-d) HeLa cells transfected for 72 h with control, epsin 1, clathrin heavy chain, AP2 or AP1 siRNA were treated with HBSS (to induce autophagy) for 6 h, after which they were fixed and immunostained for endogenous Atg16L1. Representative cells for the control and epsin 1 siRNA are in a, and percentages of HeLa cells with Atg16L1vesicles are quantified in b (for epsin 1), c (for clathrin) and d (for AP1 and AP2). Three asterisks, P 0.0001. Scale bars, 10 μm. (Note that we used single siRNAs for all experiments and the effects were confirmed with two independent sequences for clathrin heavy chain). n = 600 cells.


( e , f ) HeLa cells transfected for 24 h with Flag - tagged wild - type Atg16L1 or the Atg16L1 deletion mutants 2 - 275 Atg16L1 or 232 - 607 Atg16L1 were fixed and immunostained with anti - Flag antibody ( e ) ; the proportions of cells with Flag - Atg16L1 vesicles are represented in f . Representative images of Atg16L1 vesicles with the different mutant constructs are shown in e . The large boxes in each panel are higher magnifications of regions in the smaller boxes . Three asterisks , P 0 . 0001 ; n . s . , not significant . n = 100 cells .

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

(e, f) HeLa cells transfected for 24 h with Flag-tagged wild-type Atg16L1 or the Atg16L1 deletion mutants 2-275 Atg16L1 or 232-607 Atg16L1 were fixed and immunostained with anti-Flag antibody (e); the proportions of cells with Flag-Atg16L1 vesicles are represented in f. Representative images of Atg16L1 vesicles with the different mutant constructs are shown in e. The large boxes in each panel are higher magnifications of regions in the smaller boxes. Three asterisks, P 0.0001; n.s., not significant. n = 100 cells.


( g - i ) HeLa cells transfected with control , epsin 1 , clathrin heavy chain , AP2 or AP1 siRNA , as above , were transfected with GFP - Atg16L1 and tomato LC3 for a further 24 h , after which the cells were fixed . g shows representative cells for control , epsin 1 and clathrin siRNA . The percentage of GFP - Atg16L1 ( green ) that co - localized ( yellow , marked by arrows ) with tomato LC3 vesicles ( red ) is quantified as shown in the graph in h ( control , epsin 1 and clathrin siRNA ) and i ( control , AP2 and AP1 siRNA ) . Three asterisks , P 0 . 0001 . n = 21 cells . Scale bars , 10 μm . All error bars represent s . e . m .

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

(g-i) HeLa cells transfected with control, epsin 1, clathrin heavy chain, AP2 or AP1 siRNA, as above, were transfected with GFP-Atg16L1 and tomato LC3 for a further 24 h, after which the cells were fixed. g shows representative cells for control, epsin 1 and clathrin siRNA. The percentage of GFP-Atg16L1 (green) that co-localized (yellow, marked by arrows) with tomato LC3 vesicles (red) is quantified as shown in the graph in h (control, epsin 1 and clathrin siRNA) and i (control, AP2 and AP1 siRNA). Three asterisks, P 0.0001. n = 21 cells. Scale bars, 10 μm. All error bars represent s.e.m.


( a ) HeLa cells transfected for 24 h with GFP - Atg16L1 were fixed to reveal the Atg16L1 vesicles ( green ) . Scale bar , 10 μm .

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(a) HeLa cells transfected for 24 h with GFP-Atg16L1 were fixed to reveal the Atg16L1 vesicles (green). Scale bar, 10 μm.


( b ) HeLa cells transiently transfected for 20 h with GFP - Atg16L1 and mRFP - GPi were cultured in starvation medium for a further 2 h . TIRF image with GFP - Atg16L1 ( green ) and mRFP - GPi ( red ) is shown . Arrows indicate co - localization between Atg16L1 and GPi . The percentage co - localization of GFP - Atg16L1 with mRFP - GPi is shown in the graph . n = 2 cells . Scale bar , 5 μm .

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

(b) HeLa cells transiently transfected for 20 h with GFP-Atg16L1 and mRFP-GPi were cultured in starvation medium for a further 2 h. TIRF image with GFP-Atg16L1 (green) and mRFP-GPi (red) is shown. Arrows indicate co-localization between Atg16L1 and GPi. The percentage co-localization of GFP-Atg16L1 with mRFP-GPi is shown in the graph. n = 2 cells. Scale bar, 5 μm.


( c ) HeLa cells transfected for 24 h with GFP - Atg16L1 were either left untreated or treated with 50 μM dynasore ( Sigma ) for 4 h and processed for immunogold electron microscopy with anti - GFP antibodies ( 15 - nm gold particles ) and anti - clathrin antibodies ( 10 - nm gold particles ) . Co - localization can be seen in the boxed areas . Quantification of clathrin - coated structures ( CCS ) that were associated with Atg16L1 per 1 , 000 μm2 is shown in the graph . Three asterisks , P 0 . 0001 . Scale bar , 100 nm . All error bars represent s . e . m .

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

(c) HeLa cells transfected for 24 h with GFP-Atg16L1 were either left untreated or treated with 50 μM dynasore (Sigma) for 4 h and processed for immunogold electron microscopy with anti-GFP antibodies (15-nm gold particles) and anti-clathrin antibodies (10-nm gold particles). Co-localization can be seen in the boxed areas. Quantification of clathrin-coated structures (CCS) that were associated with Atg16L1 per 1,000 μm2 is shown in the graph. Three asterisks, P 0.0001. Scale bar, 100 nm. All error bars represent s.e.m.


( a ) HeLa cells transfected for 24 h with GFP - Atg16L1 ( green ) and CFP - LC3 ( blue ) were incubated with Alexa Fluor - 555 - conjugated cholera toxin subunit B ( red ) for 15 min at 4 ° C ( allowing toxin to bind to the plasma membrane ) . Then cells were incubated at 37 ° C ( allowing internalization of cholera toxin ) for 10 min and fixed for confocal analysis . Vesicles positive for both Atg16L1 and cholera toxin are yellow ( also see high - magnification images at the right ) . Note that the small Atg16L1 vesicles co - localizing with cholera toxin are negative for LC3 ( as marked with yellow arrows ) , and the Atg16L1 vesicles co - localizing with both cholera toxin and LC3 ( marked with blue arrows ) are shown in the magnified panels at the right . Scale bar , 10 μm .

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

(a) HeLa cells transfected for 24 h with GFP-Atg16L1 (green) and CFP-LC3 (blue) were incubated with Alexa Fluor-555-conjugated cholera toxin subunit B (red) for 15 min at 4 °C (allowing toxin to bind to the plasma membrane). Then cells were incubated at 37 °C (allowing internalization of cholera toxin) for 10 min and fixed for confocal analysis. Vesicles positive for both Atg16L1 and cholera toxin are yellow (also see high-magnification images at the right). Note that the small Atg16L1 vesicles co-localizing with cholera toxin are negative for LC3 (as marked with yellow arrows), and the Atg16L1 vesicles co-localizing with both cholera toxin and LC3 (marked with blue arrows) are shown in the magnified panels at the right. Scale bar, 10 μm.


( b ) HeLa cells transfected for 24 h with GFP - Atg16L1 ( green ) were incubated with Alexa Fluor - 555 - conjugated cholera toxin subunit B ( red ) as in a , after which they were fixed , immunostained for endogenous ( end . ) EEA1 ( blue ) and analysed by confocal microscopy . Vesicles positive for Atg16L1 and cholera toxin B are yellow ( white arrows ) and vesicles positive for both EEA1 and cholera toxin are purple ( blue arrows ) and are shown in the high - magnification images in the middle . The graph shows percentage co - localization . Cho . tox . , cholera toxin . n = 20 cells . Scale bar , 10 μm .

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

(b) HeLa cells transfected for 24 h with GFP-Atg16L1 (green) were incubated with Alexa Fluor-555-conjugated cholera toxin subunit B (red) as in a, after which they were fixed, immunostained for endogenous (end.) EEA1 (blue) and analysed by confocal microscopy. Vesicles positive for Atg16L1 and cholera toxin B are yellow (white arrows) and vesicles positive for both EEA1 and cholera toxin are purple (blue arrows) and are shown in the high-magnification images in the middle. The graph shows percentage co-localization. Cho. tox., cholera toxin. n = 20 cells. Scale bar, 10 μm.


( c ) HeLa cells treated with HBSS for 6 h were incubated with Alexa Fluor - 555 - labelled cholera toxin subunit B ( red ) as in a . Cells were then fixed , and immunostained for endogenous Atg16L1 ( green ) and endogenous EEA1 ( blue ) . Cells were analysed as in b ; the graph shows quantification of the results . Scale bar , 10 μm . Arrows indicate Atg16L1 - cholera toxin co - localization ; arrowheads indicate EEA1 - cholera toxin co - localization . n = 20 cells .

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(c) HeLa cells treated with HBSS for 6 h were incubated with Alexa Fluor-555-labelled cholera toxin subunit B (red) as in a. Cells were then fixed, and immunostained for endogenous Atg16L1 (green) and endogenous EEA1 (blue). Cells were analysed as in b; the graph shows quantification of the results. Scale bar, 10 μm. Arrows indicate Atg16L1-cholera toxin co-localization; arrowheads indicate EEA1-cholera toxin co-localization. n = 20 cells.


( d ) HeLa cells treated with HBSS for 6 h were incubated with Alexa Fluor - 555 - labelled cholera toxin subunit B ( red ) as in a . Cells were then fixed and immunostained for endogenous Atg5 ( green ) or endogenous Atg12 ( green ) . Co - localization of the Atg5 ( n = 18 cells ) or Atg12 vesicles ( n = 39 cells ) with cholera toxin is shown in yellow ( arrows ) and is quantified in the graph . Scale bar , 10 μm . All error bars represent s . e . m .

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

(d) HeLa cells treated with HBSS for 6 h were incubated with Alexa Fluor-555-labelled cholera toxin subunit B (red) as in a. Cells were then fixed and immunostained for endogenous Atg5 (green) or endogenous Atg12 (green). Co-localization of the Atg5 (n = 18 cells) or Atg12 vesicles (n = 39 cells) with cholera toxin is shown in yellow (arrows) and is quantified in the graph. Scale bar, 10 μm. All error bars represent s.e.m.


( a ) HeLa cells transfected for 24 h with Flag - tagged wild - type Atg16L1 or the Atg16L1 deletion mutants 2 - 275 or 232 - 607 Atg16L1 , together with GFP - Atg5 and CFP - LC3 , were fixed and immunostained with anti - Flag antibody before confocal analysis . Co - localization of wild - type or 2 - 275 Atg16L1 ( red ) with GFP - Atg5 ( green ) is marked by arrows in the insets . Scale bars , 10 μm .

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

(a) HeLa cells transfected for 24 h with Flag-tagged wild-type Atg16L1 or the Atg16L1 deletion mutants 2-275 or 232-607 Atg16L1, together with GFP-Atg5 and CFP-LC3, were fixed and immunostained with anti-Flag antibody before confocal analysis. Co-localization of wild-type or 2-275 Atg16L1 (red) with GFP-Atg5 (green) is marked by arrows in the insets. Scale bars, 10 μm.


( b ) HeLa cells transfected for 24 h with GFP - Atg16L1 were incubated for 15 min with horseradish peroxidase ( HRP ) - conjugated cholera toxin subunit B at 4 ° C . The cells were then incubated for 10 min at 37 ° C , after which they were processed for double immunogold labelling with anti - HRP antibodies ( 15 - nm gold particles ) and anti - GFP antibodies ( 10 - nm gold particles ) for electron microscopy . PM , plasma membrane . Scale bar , 100 nm . Co - localization can be seen in boxed areas . n = 49 fields . All error bars represent s . e . m .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells transfected for 24 h with GFP-Atg16L1 were incubated for 15 min with horseradish peroxidase (HRP)-conjugated cholera toxin subunit B at 4 °C. The cells were then incubated for 10 min at 37 °C, after which they were processed for double immunogold labelling with anti-HRP antibodies (15-nm gold particles) and anti-GFP antibodies (10-nm gold particles) for electron microscopy. PM, plasma membrane. Scale bar, 100 nm. Co-localization can be seen in boxed areas. n = 49 fields. All error bars represent s.e.m.


( a ) HeLa cells transfected for 24 h with GFP - Atg16L1 ( green ) were incubated with CellMask Orange plasma membrane stain for 5 min at 37 ° C , after which they were imaged immediately ( in an incubated chamber at 37 ° C ) . A time series after fusion of a CellMask Orange vesicle ( red ) with a GFP - Atg16L1 vesicle ( green ) is shown . Scale bar , 5 μm . A higher - magnification image showing co - localization ( yellow ) of GFP - Atg16L1 vesicle ( green ) with CellMask Orange - positive vesicle ( red ) , indicated by arrows , is also shown in the top and bottom panels .

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

(a) HeLa cells transfected for 24 h with GFP-Atg16L1 (green) were incubated with CellMask Orange plasma membrane stain for 5 min at 37 °C, after which they were imaged immediately (in an incubated chamber at 37 °C). A time series after fusion of a CellMask Orange vesicle (red) with a GFP-Atg16L1 vesicle (green) is shown. Scale bar, 5 μm. A higher-magnification image showing co-localization (yellow) of GFP-Atg16L1 vesicle (green) with CellMask Orange-positive vesicle (red), indicated by arrows, is also shown in the top and bottom panels.