E , F ) Representative electron microscopy of cross sections of femoral quadriceps nerves . ( E ) Nerves from Ttll1 - / - Ccp1 - / - Ttll1 - / - mice show no abnormalities of myelinated axons ( green asterisks ) , indicating that loss of TTLL1 prevents axonal degeneration caused by absence of CCP1 . ( F ) Ccp1 - / - Ccp1 - / - Ttll7 - / - mice both show presence of foamy macrophages ( magenta arrowheads ) , which are signs for myelin phagocytosis during axon degeneration . This indicates that loss of TTLL7 cannot prevent CCP1 - induced degeneration of femoral quadriceps nerves .

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E,F) Representative electron microscopy images of cross sections of femoral quadriceps nerves. (E) Nerves from Ttll1-/- and Ccp1-/-Ttll1-/- mice show no abnormalities of myelinated axons (green asterisks), indicating that loss of TTLL1 prevents axonal degeneration caused by the absence of CCP1. (F) Ccp1-/- and Ccp1-/-Ttll7-/- mice both show the presence of foamy macrophages (magenta arrowheads), which are signs for myelin phagocytosis during axon degeneration. This indicates that loss of TTLL7 cannot prevent the CCP1-induced degeneration of femoral quadriceps nerves.


A ) Representative of DIV4 hippocampal neurons from wild type , Ttll1 - / - Ttll7 - / - mice were incubated with MitoTracker ® ( black dots ) mitochondria movements were recorded in longest neurite ( orange lines ) .

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A) Representative images of DIV4 primary hippocampal neurons from wild type, Ttll1-/- and Ttll7-/- mice Cells were incubated with MitoTracker® (black dots) and mitochondria movements were recorded in the longest neurite (orange lines).


B ) Kymographs of one representative experiment representing mitochondrial movements C - F ) of one single experiment shown in ( B ) . Scatter dot plots of ( C ) run ( D ) speed distributions , where each point represents a single run extracted from kymographs shown in ( B ) . black lines indicate median of distribution , with whiskers at interquartile range . Numerical values of medians are indicated below each plot . ( E ) A bar graph showing overall motility of mitochondria ( fraction of time mitochondria spent in movement relative to total number of mitochondria during recording time of 60 s ) . ( F ) A bar graph showing mitochondrial density in analysed axons . Values of overall motilities mitochondrial densities are given below bar plots .

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B) Kymographs of one representative experiment representing mitochondrial movements C - F) Analysis of one single experiment shown in (B). Scatter dot plots of (C) run length and (D) speed distributions, where each point represents a single run extracted from the kymographs shown in (B). The black lines indicate the median of the distribution, with whiskers at interquartile range. Numerical values of medians are indicated below each plot. (E) A bar graph showing the overall motility of mitochondria (the fraction of time mitochondria spent in movement relative to the total number of mitochondria during the recording time of 60 s). (F) A bar graph showing mitochondrial density in analysed axons. Values of the overall motilities and mitochondrial densities are given below the bar plots.


A ) Volcano plot of fold - change ( x - axis ) plotted against significance ( y - axis ) of lipids changing upon HF / HS feeding . Lipids are colored according to fold - change ( log2 , absolute ) > 1 ( orange ) , p - value < 0 . 05 ( red ) or both ( green ) . Pvalues calculated from differential expression using

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A) Volcano plot of the fold-change (x-axis) plotted against significance (y-axis) of lipids changing upon HF/HS feeding. Lipids are colored according to fold-change (log2, absolute) > 1 (orange), p-value <0.05 (red) or both (green). Pvalues calculated from differential expression using limma


B ) of fold - change ( log2 ) of each lipid in HF / HS compared to chow diet . Only lipid species detected in mice are shown .

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B) Heatmap of the fold-change (log2) of each lipid in HF/HS compared to chow diet. Only lipid species detected in all mice are shown.


C ) Examples of different hepatic lipids within one class that are regulated in different directions in HF / HS fed as compared chow fed mice .

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C) Examples of different hepatic lipids within one class that are regulated in different directions in HF/HS fed as compared chow fed mice.


A ) relative genetic variation of hepatic lipidome composition ; lipids were quantified in proportion to total lipidome . Each lipid class is shown in a different color where differences can be observed across strains .

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A) The relative genetic variation of hepatic lipidome composition; all lipids were quantified in proportion to the total lipidome. Each lipid class is shown in a different color where differences can be observed across the strains.


B ) showing correlations between different lipid species ( x - axis ) abundance of gut microbes ( y - axis ) . Microbes were summarized at of order ( o _ ) , genus ( g _ ) or family ( f _ ) . * = p < 0 . 05 , * * = p < 0 . 01 Pvalues were calculated on significance of regression ( students ) adjusted for multiple comparisons ( FDR = 0 . 05 ) .

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B) Heatmap showing correlations between different lipid species (x-axis) and the abundance of gut microbes (y-axis). Microbes were summarized at the levels of order (o_), genus (g_) or family (f_). *=p<0.05, **=p<0.01 Pvalues were calculated based on significance of regression (students test) and adjusted for multiple comparisons (FDR = 0.05).


A ) showing correlations among hepatic lipids .

A) Heatmap showing correlations among hepatic lipids.


B ) showing concordance between different lipid species ( class listed on y - axis ) certain phenotypic traits on x - axis .

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B) Heatmap showing concordance between different lipid species (class listed on y-axis) and certain phenotypic traits on the x-axis.


A ) Manhattan plot of genome - wide association for of hepatic Pex16 transcript , where only significant locus appears directly surrounding genomic location ( red arrow ) . Significant cut - offs are shown for FDR ( blue ) Bonferroni ( red ) . peak SNP ( rs27364570 ) is highlighted with a red box . Y - axis shows - log10 ( pvalue ) vs x - axis showing each SNP measured . P - values for GWAS associations were calculated using FaST - LMM .

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A) Manhattan plot of genome-wide association for the levels of hepatic Pex16 transcript, where the only significant locus appears directly surrounding the genomic location (red arrow). Significant cut-offs are shown for FDR (blue) and Bonferroni (red). The peak SNP (rs27364570) is highlighted with a dark red box. Y-axis shows the -log10(pvalue) vs x-axis showing each SNP measured. P-values for GWAS associations were calculated using FaST-LMM.


B ) Allelic distribution comparing GG vs TT ( x - axis ) for peak SNP of Pex16 association ( rs27364570 ) , where abundance of each LPC species ( y - axis ) showed significantly different depending on allele . P - values for GWAS associations were calculated using FaST - LMM . Boxplots show mean ( middle ) , 25 - 75 % quantiles ( colored box ) 5 - 95 % quantiles ( vertical lines )

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B) Allelic distribution comparing GG vs TT (x-axis) for the peak SNP of the Pex16 association (rs27364570), where the abundance of each LPC species (y-axis) showed significantly different levels depending on the allele. P-values for GWAS associations were calculated using FaST-LMM. Boxplots show mean (middle line), 25-75% quantiles (colored box) and 5-95% quantiles (vertical lines)


C ) Correlations between expression of hepatic Pex16 with LPC species phenotypic traits . Box color indicates bicor value , where relationships are positive number in each box shows p - value for each correlation . Pvalues were calculated on significance of regression ( students ) adjusted for multiple comparisons ( FDR = 0 . 05 )

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C) Correlations between expression of hepatic Pex16 with LPC species and phenotypic traits. Box color indicates bicor value, where all relationships are positive and number in each box shows p-value for each correlation. Pvalues were calculated based on significance of regression (students test) and adjusted for multiple comparisons (FDR = 0.05)


A ) Manhattan plot of genome - wide association for TAG ( 48 : 2 ) . Red shows Bonferroni - corrected threshold of significance calculated on FaST - LMM pvalues .

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A) Manhattan plot of genome-wide association for TAG(48:2). Red line shows Bonferroni-corrected threshold of significance calculated based on FaST-LMM pvalues.


B ) Locuszoom plots showing focused genomic region ( x - axis ) plotted against - log10 ( p - value ) of association for liver mRNA expression of Map2k6 . P - values for GWAS associations were calculated using FaST -

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B) Locuszoom plots showing the focused genomic region (x-axis) plotted against the -log10 (p-value) of association for liver mRNA expression of Map2k6. P-values for GWAS associations were calculated using FaST-LMM


( C ) Correlations between hepatic expression of Map2k6 TAGs identified in study . Blue represents negative correlations .

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(C) Correlations between hepatic expression of Map2k6 and all TAGs identified in the study. Blue represents negative correlations.


E ) Western blots of liver homogenate using anti - Map2k6 anti - β - actin .

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E) Western blots of liver homogenate using anti-Map2k6 and anti-β-actin.


F ) Comparison of total hepatic TAG between Map2k6 - overexpressing mice ( black bar ) control mice ( empty bar ) .

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F) Comparison of total hepatic TAG between Map2k6-overexpressing mice (black bar) and control mice (empty bar).


G ) Differences in total phospholipid ( PC ) , total cholesterol ( TC ) unesterified cholesterol ( UC ) between Map2k6 - overexpressing mice ( black bars ) control mice ( empty bars ) .

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G) Differences in total phospholipid (PC), total cholesterol (TC) and unesterified cholesterol (UC) between Map2k6-overexpressing mice (black bars) and control mice (empty bars).


H ) fat % of both experimental cohorts prior to ( week 0 ) or 8 on a HF / HS diet .

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H) Body fat % of both experimental cohorts prior to (week 0) or 8 weeks on a HF/HS diet.


I , J ) Plasma of glucose ( I ) insulin ( J ) at end of 8 study .

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I,J) Plasma concentration of glucose (I) and insulin (J) at the end of the 8 weeks study.


A ) Manhattan plot of genome - wide association for expression of Ifi203 in liver . Red shows Bonferroni - corrected threshold blue shows an FDR = 0 . 01 pvalue of significance calculated on FaST - LMM pvalues .

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A) Manhattan plot of genome-wide association for expression of Ifi203 in liver. Red line shows Bonferroni-corrected threshold and blue shows an FDR =0.01 pvalue of significance calculated based on FaST-LMM pvalues.


B - D ) Allelic variation plots showing peak SNP for Ifi203 expression ( rs31614030 ) at CC or TT allele ( x - axis ) plotted against expression of Ifi203 ( B ) , of hepatic PC ( 38 : 3 ) ( C ) , plasma insulin ( D ) . Red shows Bonferroni - corrected threshold blue shows an FDR = 0 . 01 pvalue of significance calculated on FaST - LMM pvalues .

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B-D) Allelic variation plots showing the peak SNP for Ifi203 expression (rs31614030) at the CC or TT allele (x-axis) plotted against expression of Ifi203 (B), levels of hepatic PC(38:3) (C), and plasma insulin levels (D). Red line shows Bonferroni-corrected threshold and blue shows an FDR =0.01 pvalue of significance calculated based on FaST-LMM pvalues.


E - G ) Correlation between parameters listed above showing significant relationships between hepatic Ifi203 PC ( 38 : 3 ) ( E ) , hepatic Ifi203 plasma insulin ( F ) or PC ( 38 : 3 ) plasma insulin ( G ) . Pvalues were calculated on significance of regression ( students ) adjusted for multiple comparisons ( FDR = 0 . 05 )

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E-G) Correlation between the parameters listed above showing significant relationships between hepatic Ifi203 and PC(38:3) (E), hepatic Ifi203 and plasma insulin levels (F) or PC (38:3) and plasma insulin (G). Pvalues were calculated based on significance of regression (students test) and adjusted for multiple comparisons (FDR = 0.05)


Mice receiving control ( open bars ) or shIfi203 ( black bars ) were analyzed for liver expression of Ifi203 ( I ) , total PC in liver ( J ) P - values calculated using a students t - between groups . Data represent means ± ( n = 4 - 5 per group ) .

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Mice receiving the control virus (open bars) or shIfi203 (black bars) were analyzed for liver expression of Ifi203 (I), total PC levels in liver (J) P-values calculated using a students t-test between groups. Data represent means ± SEM (n =4-5 per group).


Mice receiving control ( open bars ) or shIfi203 ( black bars ) were analyzed for liver expression of Pemt ( K ) , total liver TAG content ( L ) , or plasma insulin ( M ) . P - values calculated using a students t - between groups . Data represent means ± ( n = 4 - 5 per group ) .

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Mice receiving the control virus (open bars) or shIfi203 (black bars) were analyzed for liver expression of Pemt (K), total liver TAG content (L), or plasma insulin levels (M). P-values calculated using a students t-test between groups. Data represent means ± SEM (n =4-5 per group).


B . Bimolecular complementation between subunits of Dsl COPI coat . BiFC pair β ' - COPVN • Dsl3pVC yielded foci that showed dramatically reduced in COPI binding - deficient dsl1 - 5xWA mutant . Schematic yeast representations depict typical BiFC foci localization patterns . Scale bar 10 μm .

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B. Bimolecular fluorescence complementation between subunits of the Dsl complex and the COPI coat. The BiFC pair β'-COPVN•Dsl3pVC yielded fluorescent foci that showed dramatically reduced fluorescence intensity in the COPI binding-deficient dsl1-5xWA mutant. Schematic yeast cell representations depict the typical BiFC foci localization patterns. Scale bar 10 μm.


C . of β ' - COPVN • Dsl3pVC BiFC signals in wild type in COPI binding - deficient dsl1 - 5xWA dsl1 - Δlasso mutants ( as shown exemplarily in ( B ) ) . Mean values + of at least three independent experiments ( n = 3 - 8 ) are displayed . Statistical was carried out by comparing number of with BiFC signals over integrated thresholds to those without signals ( * = p < 0 , 05 ) . dsl1 mutant strains show a significant decrease in brightness , spot number spot in comparison with their corresponding DSL1 wild type strains ( two - sample t - ) . dsl1 - 5xWA mutation also led to comparable signal reduction in carrying VNI152L variant in β ' - COPVN I152L • Dsl3pVC combination .

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C. Quantification of the β'-COPVN•Dsl3pVC BiFC signals in wild type cells and in COPI binding-deficient dsl1-5xWA and dsl1-Δlasso mutants (as shown exemplarily in (B)). Mean values + SEM of at least three independent experiments (n=3-8) are displayed. Statistical analysis was carried out by comparing the number of cells with BiFC signals over integrated thresholds to those without signals (*=p<0,05). dsl1 mutant strains show a significant decrease in brightness, spot number and spot size in comparison with their corresponding DSL1 wild type strains (two-sample t-test). The dsl1-5xWA mutation also led to comparable signal reduction in cells carrying the VNI152L variant in the β'-COPVN I152L•Dsl3pVC combination.


D . Subcellular localization of different BiFC - tagged pairs . Dsl1pVN • Dsl3pVC combination involving subunits of ER - resident Dsl1 exhibited typical ER localization . Sec16pVN • Sec16pVC signals in a diploid heterozygous displayed an pattern typical for ER exit sites , while diploid carrying α - COPVN • α - COPVC BiFC pair showed a Golgi - like pattern . Scale bar 10 μm .

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D. Subcellular localization of different BiFC-tagged protein pairs. The Dsl1pVN•Dsl3pVC combination involving subunits of the ER-resident Dsl1 complex exhibited typical ER localization. Sec16pVN•Sec16pVC signals in a diploid heterozygous strain displayed an pattern typical for ER exit sites, while diploid cells carrying the α-COPVN•α-COPVC BiFC pair showed a Golgi-like fluorescence pattern. Scale bar 10 μm.


E . localization of COPI • Dsl BiFC foci compared to autophagy marker Ape1p . Double of BiFC pair β ' - COPVN • Dsl3pVC autophagy marker mRFPApe1p are superimposed with DIC image . BiFC - YFP signals are pseudocolored green . signals appeared distinct from each other . Also no co - localization was observed when we analyzed COPI • COPII ( Sec16p ) BiFC spots in expressing mRFP tagged APE1 . Scale bar 10 µm .

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E. The localization of COPI•Dsl BiFC foci compared to the autophagy marker Ape1p. Double fluorescence micrographs of the BiFC pair β'-COPVN•Dsl3pVC and the autophagy marker mRFPApe1p are superimposed with the DIC image. BiFC-YFP signals are pseudocolored green. Fluorescent signals appeared distinct from each other. Also no co-localization was observed when we analyzed COPI•COPII(Sec16p) BiFC spots in cells expressing mRFP tagged APE1. Scale bar 10 µm.


F . Growth effects of Venus fragment tags in dsl1 mutant . Serial tenfold dilutions of liquid cultures were spotted on agar plates incubated at 30 ° C for two days . in second row show that β ' COPVN producing expressing dsl1 - 5xWA mutation could not grow . complementation of β ' COPVN by its cognate interaction partners Dsl1pVC Dsl3pVC , but not by non - cognate BiFC partners ( Sec24pVC , Sec16pVC or VCRer1p ) , suppressed these growth defects . Spot assays of exemplary BiFC partners are depicted . Asterisk : Dsl1pVC carrying dsl1 - 5xWA defect was expressed from a plasmid . See Fig EV1 , EV2 Appendix Fig S1 for full data display .

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F. Growth effects of Venus fragment tags in dsl1 mutant cells. Serial tenfold dilutions of liquid cell cultures were spotted on agar plates and incubated at 30 °C for two days. The images in the second row show that β'COPVN producing cells expressing the dsl1-5xWA mutation could not grow. The complementation of β'COPVN by its cognate interaction partners Dsl1pVC and Dsl3pVC, but not by non-cognate BiFC partners (Sec24pVC, Sec16pVC or VCRer1p), suppressed these growth defects. Spot assays of exemplary BiFC partners are depicted. Asterisk: Dsl1pVC carrying the dsl1-5xWA defect was expressed from a plasmid. See Fig EV1, EV2 and Appendix Fig S1 for full data display.


G . BiFC signal quantifications of strains presented in ( F ) . β ' - COPVN yielded BiFC signals with tested complementation partners . This rules out possibility that lack of suppression observed in F simply reflects inability of BiFC partners to form a , shows that successful complementation of split - YFP fragments does not per se suppress synthetic lethal effect of VN tag in dsl1 - 5xWA . Mean values + of at least three independent experiments ( n = 3 - 8 ) are displayed .

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G. BiFC signal quantifications of the strains presented in (F). β'-COPVN yielded BiFC fluorescence signals with all tested complementation partners. This rules out the possibility that the lack of suppression observed in F simply reflects the inability of the BiFC partners to form a complex, and shows that the successful complementation of the split-YFP fragments does not per se suppress the synthetic lethal effect of the VN tag in dsl1-5xWA cells. Mean values + SEM of at least three independent experiments (n=3-8) are displayed.


H . BiFC signal intensities greatly depend on which BiFC combination is analyzed . intensities of COPI • Dsl BiFC pairs ( left part of H ) were much higher than those of COPII • Dsl BiFC pairs ( right part of H ) .

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H. BiFC signal intensities greatly depend on which BiFC combination is analyzed. Fluorescence intensities of COPI•Dsl BiFC pairs (left part of H) were much higher than those of COPII•Dsl BiFC pairs (right part of H).


I . Localization of Dsl1pCFP in β ' - COPVN • Dsl3pVCcells . An overall ER localization of Dsl1pCFP was retained in β ' - COPVN • Dsl3pVCcells , while some of signal was found in foci . BiFC - YFP signals are pseudocolored red . Scale bar 10 µm .

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I. Localization of Dsl1pCFP in β'-COPVN•Dsl3pVCcells. An overall ER localization of Dsl1pCFP was retained in β'-COPVN•Dsl3pVCcells, while some of the signal was found in the foci. BiFC-YFP signals are pseudocolored red. Scale bar 10 µm.


A . Dsl1pVN • ε - COPVC BiFC signal localization pattern throughout budding cycle . Time - lapse of agarose - embedded were recorded at RT with continuous PM Glc + ura medium supply . Arrowheads mark positions of BiFC foci . signals exhibited a characteristic reproducible localization pattern , which favored sites of high material turnover during budding . Scale bar 10 µm .

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A. Dsl1pVN•ε-COPVC BiFC signal localization pattern throughout the budding cycle. Time-lapse images of agarose-embedded cells were recorded at RT with continuous PM Glc+ura medium supply. Arrowheads mark positions of BiFC foci. Fluorescence signals exhibited a characteristic and reproducible localization pattern, which favored sites of high material turnover during budding. Scale bar 10 µm.


B . Dsl1pVN • ε - COPVC BiFC foci showed different subcellular distribution depending on growth status of . were cultured in PM + ura medium with or without 2 % glucose at 30 ° C for 90 min , imaged . BiFC signals dispersed under glucose starvation , resulting in an ER exit site - like pattern . Scale bar 10 µm .

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 O O O B-CONTROLLED_VAR 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

B. Dsl1pVN•ε-COPVC BiFC foci showed different subcellular distribution depending on the growth status of cells. Cells were cultured in PM+ura medium with or without 2 % glucose at 30 °C for 90 min, and imaged. BiFC signals dispersed under glucose starvation, resulting in an ER exit site-like fluorescence pattern. Scale bar 10 µm.


C . effect of carbon source depletion on COPI • Dsl BiFC signal polarization . carrying Dsl1pVN • ε - COPVC were starved in glucose - free PM + ura medium for 90 min . Then medium was replaced by PM Glc + ura with 2 % glucose , while imaging continuously at 1 min intervals . Two exemplary are shown ( upper lower panel ) . Starvation - induced dispersed BiFC signals reversibly returned to polarized distribution within minutes after addition of glucose - containing medium . Arrowheads mark point of signal repolarization , arrows indicate resumed bud outgrowth . Scale bar 5 µm .

O O O 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 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 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 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

C. The effect of carbon source depletion on COPI•Dsl BiFC signal polarization. Cells carrying Dsl1pVN•ε-COPVC were starved in glucose-free PM+ura medium for 90 min. Then the medium was replaced by PM Glc+ura with 2 % glucose, while imaging the cells continuously at 1 min intervals. Two exemplary cells are shown (upper and lower panel). Starvation-induced dispersed BiFC signals reversibly returned to polarized distribution within minutes after addition of glucose-containing medium. Arrowheads mark the point of signal repolarization, arrows indicate resumed bud outgrowth. Scale bar 5 µm.


D . βʼ - COPVN • Dsl3pVC BiFC signals during mating . of BiFC pair β ' - COPVN • Dsl3pVC ( green ) are superimposed with DIC image ( grey ) . Two haploid strains carrying βʼ - COPVN • Dsl3pVC were allowed to mate in YEPD liquid culture for 2 h at 30 ° C . Then time - lapse recordings were made in agarose embedding at RT with continuous PM Glc + ura medium supply . Arrowheads mark positions of BiFC foci . signals exhibited a characteristic reproducible localization at sites of high material turnover during zygote formation . Scale bar 10 µm .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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 B-MEASURED_VAR I-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 O O O O O O O O O O O O O O O O O O O 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. βʼ-COPVN•Dsl3pVC BiFC signals during mating. Fluorescence micrographs of the BiFC pair β'-COPVN•Dsl3pVC (green) are superimposed with the DIC image (grey). Two haploid strains carrying βʼ-COPVN•Dsl3pVC were allowed to mate in YEPD liquid culture for 2 h at 30 °C. Then time-lapse recordings were made in agarose embedding at RT with continuous PM Glc+ura medium supply. Arrowheads mark positions of BiFC foci. Fluorescence signals exhibited a characteristic and reproducible localization at sites of high material turnover during zygote formation. Scale bar 10 µm.


E . Comparison of BiFC localization pattern in COPI • Dsl COPI • COPI strains . Time - lapse of agarose - embedded carrying COPI • Dsl BiFC pair βʼ - COPVN • Dsl3pVC or COPI • COPI BiFC pair βʼ - COPVN • α - COPVC were recorded at RT with continuous PM Glc + ura medium supply at 5 min intervals . Single pseudokymographs were generated by concatenating central section of ( sliced along pole axis , frames at double width of bud neck ) of a time - lapse dataset . COPI • Dsl showed one single dominant spot at bud tip later bud neck . In contrast , COPI • COPI additionally exhibited multiple spots with no apparent polarization tendency , which were localized mainly in mother . Schematic yeast representations depict typical BiFC foci localization pattern of strains . 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Comparison of the BiFC localization pattern in COPI•Dsl and COPI•COPI strains. Time-lapse images of agarose-embedded cells carrying the COPI•Dsl BiFC pair βʼ-COPVN•Dsl3pVC or the COPI•COPI BiFC pair βʼ-COPVN•α-COPVC were recorded at RT with continuous PM Glc+ura medium supply at 5 min intervals. Single cell pseudokymographs were generated by concatenating the central section of the cell (sliced along the pole axis, frames at double the width of the bud neck) of a time-lapse dataset. The COPI•Dsl strain showed one single dominant spot at the bud tip and later the bud neck. In contrast, the COPI•COPI strain additionally exhibited multiple spots with no apparent polarization tendency, which were localized mainly in the mother cell. Schematic yeast cell representations depict the typical BiFC foci localization pattern of the strains. Scale bar 10 µm.


F . recovery after photobleaching of BiFC signals . Time - lapse of agarose - embedded carrying β ' - COPVN • Dsl3pVC BiFC pair were recorded at RT with PM Glc + ura medium supply at 2 min intervals . of ROIs marked in dashed lines ( where full , full bud , or only bud foci were bleached ) were depleted by three repetitions of excitation at 177 µs / pixel 100 % . Arrowheads : reappearing signal ; scale bar 2 µm . G . Densitometric measurements of intensities in photobleached . Representative data sets from five independent experiments are displayed , featuring that had been bleached entirely ( black lines ) , at bud only ( blue lines ) , or at foci at bud tip only ( blue ) . Values are displayed in of initial , were corrected for photobleaching effects by normalization against of unbleached . recovery was typically not continued to full recovery stage because of photobleaching artefacts occurring during long - term imaging of samples .

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

F. Fluorescence recovery after photobleaching of BiFC signals. Time-lapse images of agarose-embedded cells carrying the β'-COPVN•Dsl3pVC BiFC pair were recorded at RT with PM Glc+ura medium supply at 2 min intervals. Fluorescence of ROIs marked in dashed lines (where the full cell, full bud, or only the bud foci were bleached) were depleted by three repetitions of fluorescence excitation at 177 µs/pixel and 100 % laser intensity. Arrowheads: reappearing fluorescence signal; scale bar 2 µm.G. Densitometric measurements of fluorescence intensities in photobleached cells. Representative data sets from five independent experiments are displayed, featuring cells that had been bleached entirely (black lines), at the bud only (dark blue lines), or at the fluorescent foci at the bud tip only (light blue line). Values are displayed in percentage of initial fluorescence, and were corrected for photobleaching effects by normalization against fluorescence of unbleached cells. Fluorescence recovery was typically not continued to full recovery stage because of the photobleaching artefacts occurring during long-term imaging of the samples.


Time - lapse of agarose - embedded were taken at RT with fresh PM Glc + ura medium supply throughout one budding cycle . Representative of characteristically reoccurring localization stages are displayed . In combinations , foci localize to areas of outgrowth in bud . Often , another prominent spot is found in mother on side of bud neck . Scale bar 5 µm . Kymographs of time - lapse datasets shown in this Figure are presented in Fig EV3A . A - D . expressing complementary BiFC - tagged between subunits of Dsl COPI were analyzed . Panel ( D ) shows a BiFC combination that includes a mutated VC - tag . This mutation in VC part is equivalent to A206K mutation in GFP , which prevents formation of YPF dimers ( Zacharias et al . , 2002 ) .

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

Time-lapse micrographs of agarose-embedded cells were taken at RT with fresh PM Glc+ura medium supply throughout one budding cycle. Representative images of characteristically reoccurring fluorescence localization stages are displayed. In all combinations, fluorescence foci localize to areas of membrane outgrowth in the bud. Often, another prominent fluorescent spot is found in the mother cell on the side of the bud neck. Scale bar 5 µm. Kymographs of all time-lapse datasets shown in this Figure are presented in Fig EV3A.A-D. Cells expressing complementary BiFC-tagged proteins between subunits of the Dsl complex and COPI were analyzed. Panel (D) shows a BiFC combination that includes a mutated VC-tag. This mutation in the VC part is equivalent to the A206K mutation in GFP, which prevents the formation of YPF dimers (Zacharias et al., 2002).


Time - lapse of agarose - embedded were taken at RT with fresh PM Glc + ura medium supply throughout one budding cycle . Representative of characteristically reoccurring localization stages are displayed . In combinations , foci localize to areas of outgrowth in bud . Often , another prominent spot is found in mother on side of bud neck . Scale bar 5 µm . Kymographs of time - lapse datasets shown in this Figure are presented in Fig EV3A . E , F . VC - tagged COPI vesicle cargo Rer1p was examined in combination with VN - tagged COPI subunit β ' - COP or VN - tagged Dsl1 .

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

Time-lapse micrographs of agarose-embedded cells were taken at RT with fresh PM Glc+ura medium supply throughout one budding cycle. Representative images of characteristically reoccurring fluorescence localization stages are displayed. In all combinations, fluorescence foci localize to areas of membrane outgrowth in the bud. Often, another prominent fluorescent spot is found in the mother cell on the side of the bud neck. Scale bar 5 µm. Kymographs of all time-lapse datasets shown in this Figure are presented in Fig EV3A.E, F. The VC-tagged COPI vesicle cargo protein Rer1p was examined in combination with the VN-tagged COPI subunit β'-COP or the VN-tagged Dsl1 protein.


Time - lapse of agarose - embedded were taken at RT with fresh PM Glc + ura medium supply throughout one budding cycle . Representative of characteristically reoccurring localization stages are displayed . In combinations , foci localize to areas of outgrowth in bud . Often , another prominent spot is found in mother on side of bud neck . Scale bar 5 µm . Kymographs of time - lapse datasets shown in this Figure are presented in Fig EV3A . G . BiFC spots in COPI • COPII BiFC combination ( β ' - COPVN • Sec16pVC ) were also polarized .

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

Time-lapse micrographs of agarose-embedded cells were taken at RT with fresh PM Glc+ura medium supply throughout one budding cycle. Representative images of characteristically reoccurring fluorescence localization stages are displayed. In all combinations, fluorescence foci localize to areas of membrane outgrowth in the bud. Often, another prominent fluorescent spot is found in the mother cell on the side of the bud neck. Scale bar 5 µm.Kymographs of all time-lapse datasets shown in this Figure are presented in Fig EV3A.G. BiFC spots in COPI•COPII BiFC combination (β'-COPVN•Sec16pVC) were also polarized.


Time - lapse of agarose - embedded were taken at RT with fresh PM Glc + ura medium supply throughout one budding cycle . Representative of characteristically reoccurring localization stages are displayed . In combinations , foci localize to areas of outgrowth in bud . Often , another prominent spot is found in mother on side of bud neck . Scale bar 5 µm . Kymographs of time - lapse datasets shown in this Figure are presented in Fig EV3A . H . COPI • COPI ( α - COPVN • α - COPVC ) BiFC signals showed one spot that localized to bud tip , at later budding stages , to bud neck ( compare with A - G ) . Additionally , multiple spots with no apparent polarization tendency were observed , which are reminiscent of a Golgi pattern .

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

Time-lapse micrographs of agarose-embedded cells were taken at RT with fresh PM Glc+ura medium supply throughout one budding cycle. Representative images of characteristically reoccurring fluorescence localization stages are displayed. In all combinations, fluorescence foci localize to areas of membrane outgrowth in the bud. Often, another prominent fluorescent spot is found in the mother cell on the side of the bud neck. Scale bar 5 µm. Kymographs of all time-lapse datasets shown in this Figure are presented in Fig EV3A.H. COPI•COPI (α-COPVN•α-COPVC) BiFC signals showed one spot that localized to the bud tip and, at later budding stages, to the bud neck (compare with A-G). Additionally, multiple spots with no apparent polarization tendency were observed, which are reminiscent of a Golgi pattern.


Time - lapse of agarose - embedded were taken at RT with fresh PM Glc + ura medium supply throughout one budding cycle . Representative of characteristically reoccurring localization stages are displayed . In combinations , foci localize to areas of outgrowth in bud . Often , another prominent spot is found in mother on side of bud neck . Scale bar 5 µm . Kymographs of time - lapse datasets shown in this Figure are presented in Fig EV3A . I . β ' - COPmGFPfluorescence foci in Dsl1p - depleted GAL - DSL1 after incubation in glucose - containing medium ( YEPD ) for 4 h at 30 ° C .

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

Time-lapse micrographs of agarose-embedded cells were taken at RT with fresh PM Glc+ura medium supply throughout one budding cycle. Representative images of characteristically reoccurring fluorescence localization stages are displayed. In all combinations, fluorescence foci localize to areas of membrane outgrowth in the bud. Often, another prominent fluorescent spot is found in the mother cell on the side of the bud neck. Scale bar 5 µm. Kymographs of all time-lapse datasets shown in this Figure are presented in Fig EV3A.I. β'-COPmGFPfluorescence foci in Dsl1p-depleted GAL-DSL1 cells after incubation in glucose-containing medium (YEPD) for 4 h at 30 °C.


Time - lapse of agarose - embedded were taken at RT with supply of fresh PM Glc + ura medium throughout one budding cycle . Only ( F ) shows fixed immunostained for Sed5p . produced β ' - COPVN • Dsl3pVC BiFC pair . Most strains contained plasmids encoding different organelle markers as indicated below . BiFC signals are pseudocolored green , organelle markers are pseudocolored red . Scale bar 5 µm . A . RFPHDEL , ER

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

Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.A. RFPHDEL, ER marker


Time - lapse of agarose - embedded were taken at RT with supply of fresh PM Glc + ura medium throughout one budding cycle . Only ( F ) shows fixed immunostained for Sed5p . produced β ' - COPVN • Dsl3pVC BiFC pair . Most strains contained plasmids encoding different organelle markers as indicated below . BiFC signals are pseudocolored green , organelle markers are pseudocolored red . Scale bar 5 µm . B . Sec13pRFP , ER exit sites

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

Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.B. Sec13pRFP, ER exit sites marker


Time - lapse of agarose - embedded were taken at RT with supply of fresh PM Glc + ura medium throughout one budding cycle . Only ( F ) shows fixed immunostained for Sed5p . produced β ' - COPVN • Dsl3pVC BiFC pair . Most strains contained plasmids encoding different organelle markers as indicated below . BiFC signals are pseudocolored green , organelle markers are pseudocolored red . Scale bar 5 µm . C . RFPSed5p , cis - Golgi markers ( genomic )

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

Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.C. RFPSed5p, cis-Golgi markers (genomic)


Time - lapse of agarose - embedded were taken at RT with supply of fresh PM Glc + ura medium throughout one budding cycle . Only ( F ) shows fixed immunostained for Sed5p . produced β ' - COPVN • Dsl3pVC BiFC pair . Most strains contained plasmids encoding different organelle markers as indicated below . BiFC signals are pseudocolored green , organelle markers are pseudocolored red . Scale bar 5 µm . D . CFPGos1p , medial Golgi

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

Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.D. CFPGos1p, medial Golgi marker


Time - lapse of agarose - embedded were taken at RT with supply of fresh PM Glc + ura medium throughout one budding cycle . Only ( F ) shows fixed immunostained for Sed5p . produced β ' - COPVN • Dsl3pVC BiFC pair . Most strains contained plasmids encoding different organelle markers as indicated below . BiFC signals are pseudocolored green , organelle markers are pseudocolored red . Scale bar 5 µm . E . Sec7pCFP , trans - Golgi

Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.E. Sec7pCFP, trans-Golgi marker


Time - lapse of agarose - embedded were taken at RT with supply of fresh PM Glc + ura medium throughout one budding cycle . Only ( F ) shows fixed immunostained for Sed5p . produced β ' - COPVN • Dsl3pVC BiFC pair . Most strains contained plasmids encoding different organelle markers as indicated below . BiFC signals are pseudocolored green , organelle markers are pseudocolored red . Scale bar 5 µm . F . fixed carrying β ' - COPVN • Dsl3pVC BiFC pair along with trans - Golgi marker CFPSec7p were used for this panel . β ' - COPVN • Dsl3pVC BiFC signals are pseudocolored . Immunostaining was used to visualize cis - Golgi marker Sed5p ( red ) . trans - Golgi marker CFPSec7p is pseudocolored green .

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

Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.F. Individual fixed cells carrying the β'-COPVN•Dsl3pVC BiFC pair along with trans-Golgi marker CFPSec7p were used for this panel. The β'-COPVN•Dsl3pVC BiFC signals are pseudocolored white. Immunostaining was used to visualize the cis-Golgi marker Sed5p (red). The trans-Golgi marker CFPSec7p is pseudocolored green.


Time - lapse of agarose - embedded were taken at RT with supply of fresh PM Glc + ura medium throughout one budding cycle . Only ( F ) shows fixed immunostained for Sed5p . produced β ' - COPVN • Dsl3pVC BiFC pair . Most strains contained plasmids encoding different organelle markers as indicated below . BiFC signals are pseudocolored green , organelle markers are pseudocolored red . Scale bar 5 µm . G . CFPRer1 , COPI

Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.G. CFPRer1, COPI cargo


Time - lapse of agarose - embedded were taken at RT with supply of fresh PM Glc + ura medium throughout one budding cycle . Only ( F ) shows fixed immunostained for Sed5p . produced β ' - COPVN • Dsl3pVC BiFC pair . Most strains contained plasmids encoding different organelle markers as indicated below . BiFC signals are pseudocolored green , organelle markers are pseudocolored red . Scale bar 5 µm . H . CFPSec4p , marker for secretory

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

Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.H. CFPSec4p, marker for secretory vesicles


A . of two different COPI • COPII BiFC pairs are shown : Sec16pVN • α - COPVC or Sec16pVN • ε - COPVC . DSL1 show dispersed punctate patterns resembling ER exit site patterns . In dsl1 - 5xWA mutant , exhibit additional bright spots . Scale bar 10 µm . B . of differences of strains shown in ( A ) . COPI • COPII BiFC is significantly increased in carrying dsl1 - 5xWA mutant as compared to DSL1 wild type . Mean values + of at least nine independent experiments ( n = 918 ) are displayed ( two - sample unpaired twotailed ttest ) .

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 B-MEASURED_VAR 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 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 B-CONTROLLED_VAR 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

A. Fluorescence micrographs of two different COPI•COPII BiFC pairs are shown: Sec16pVN•α-COPVC or Sec16pVN•ε-COPVC. DSL1 cells show dispersed punctate patterns resembling ER exit site patterns. In the dsl1-5xWA mutant, cells exhibit additional bright spots. Scale bar 10 µm.B. Quantification of fluorescence intensity differences of the strains shown in (A). COPI•COPII BiFC fluorescence is significantly increased in cells carrying the dsl1-5xWA mutant as compared to DSL1 wild type. Mean values + SEM of at least nine independent experiments (n=918) are displayed (two-sample unpaired twotailed ttest).


C . Time - lapse of expressing β ' - COPVN • Dsl3pVC BiFC pair in a dsl1 - 5xWA background . were imaged embedded in agarose at RT with fresh PM Glc + ura medium supply throughout one budding cycle . signals retain characteristic polarization pattern as seen DSL1 wild type ( see Fig 3 ) . Scale bar 10 μm .

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C. Time-lapse micrographs of cells expressing the β'-COPVN•Dsl3pVC BiFC pair in a dsl1-5xWA background. Cells were imaged embedded in agarose at RT with fresh PM Glc+ura medium supply throughout one budding cycle. Fluorescence signals retain the characteristic polarization pattern as seen DSL1 wild type cells (see Fig 3). Scale bar 10 μm.


A - B . Involvement of filamentous cytoskeletal structures in BiFC foci polarization . Time - lapse of agarose - embedded expressing Dsl1pVN • ε - COPVC BiFC combination at RT with fresh PM Glc + ura medium supply . ( A ) Upon disruption of actin filaments with 1 , 25 μM latrunculin A , COPI • Dsl BiFC signals dispersed . Arrowheads : polarized signal , asterisks : unpolarized localization . are displayed with inverted brightness values . Scale bar 10 μm .

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A-B. Involvement of filamentous cytoskeletal structures in BiFC foci polarization. Time-lapse micrographs of agarose-embedded cells expressing the Dsl1pVN•ε-COPVC BiFC combination at RT with fresh PM Glc+ura medium supply. (A) Upon disruption of actin filaments with 1,25 μM latrunculin A, COPI•Dsl BiFC signals dispersed. Arrowheads: polarized signal, asterisks: unpolarized localization. Fluorescent images are displayed with inverted brightness values. Scale bar 10 μm.


A - B . Involvement of filamentous cytoskeletal structures in BiFC foci polarization . Time - lapse of agarose - embedded expressing Dsl1pVN • ε - COPVC BiFC combination at RT with fresh PM Glc + ura medium supply . ( B ) Microtubuli disruption through 24 μg / ml nocodazole did not affect polarization of COPI • Dsl BiFC signals . Arrowheads : polarized signal , asterisks : unpolarized localization . are displayed with inverted brightness values . Scale bar 10 μm .

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A-B. Involvement of filamentous cytoskeletal structures in BiFC foci polarization. Time-lapse micrographs of agarose-embedded cells expressing the Dsl1pVN•ε-COPVC BiFC combination at RT with fresh PM Glc+ura medium supply. (B) Microtubuli disruption through 24 μg/ml nocodazole did not affect polarization of the COPI•Dsl BiFC signals. Arrowheads: polarized signal, asterisks: unpolarized localization. Fluorescent images are displayed with inverted brightness values. Scale bar 10 μm.


C - E . Involvement of myosin motors in BiFC foci polarization . Time - lapse of agarose - embedded carrying COPI • Dsl BiFC pairs Dsl1pVN • ε - COPVC or β ' COPVN • Dsl3pVC were taken . Either wild type ( E ) or mutants carrying myosin V mutations myo2 - 66 ( C ) or myo4 ∆ ( D ) were analyzed . were grown at RT with fresh PM Glc + ura medium supply . In myo2 - 66 , BiFC signal polarization was hardly detectable even at permissive , while it was unaffected in myo4 ∆ compared to control .

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C-E. Involvement of myosin motors in BiFC foci polarization. Time-lapse micrographs of agarose-embedded cells carrying the COPI•Dsl BiFC pairs Dsl1pVN•ε-COPVC or β'COPVN•Dsl3pVC were taken. Either wild type cells (E) or mutants carrying the myosin V mutations myo2-66 (C) or myo4∆ (D) were analyzed. Cells were grown at RT with fresh PM Glc+ura medium supply. In the myo2-66 cells, the BiFC signal polarization was hardly detectable even at permissive temperature, while it was unaffected in myo4∆ compared to the control cells.


F . of β ' - COPVN • Dsl3pVCcells carrying myo2 - 66 defect . COPI • Dsl BiFC , as well as ER or Golgi markers were analyzed . While BiFC spots were dispersed in these mutants , they retained their association with ER Golgi . Scale bar 5 μm .

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F. Fluorescence micrographs of β'-COPVN•Dsl3pVCcells carrying the myo2-66 defect. COPI•Dsl BiFC, as well as ER or Golgi markers were analyzed. While the BiFC spots were dispersed in these mutants, they retained their association with ER and Golgi. Scale bar 5 μm.


A - B . Immuno - electron showing buds neck region of two β ' - COPVN • Dsl3pVCcells . Both parts contain an overview image ( left ) two zoomed details ( right ) . Slides were decorated with antibody against COPI visualized with A - conjugated 10 nm gold particles . Gold particles mark electron - dense areas in apposition to membranes / or vesicular structures , which are localized at side of bud bud neck of mother . Scale bar 100 nm .

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A-B. Immuno-electron micrographs showing the buds and the neck region of two β'-COPVN•Dsl3pVCcells. Both parts contain an overview image (left) and two zoomed details (right). Slides were decorated with antibody against COPI and visualized with protein A-conjugated 10 nm gold particles. Gold particles mark electron-dense areas in apposition to membranes and/or vesicular structures, which are localized at the side of the bud and the bud neck of the mother cell. Scale bar 100 nm.


C . micrograph of a budding carrying β ' - COPVN • Dsl3pVC ( green ) RFPHDEL ( red ) . Arrowheads mark BiFC signals which may correspond to localizations found in ( A ) ( B ) . Scale bar 2 µm .

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C. Fluorescence micrograph of a budding cell carrying β'-COPVN•Dsl3pVC (green) and RFPHDEL (red). Arrowheads mark BiFC signals which may correspond to the localizations found in (A) and (B). Scale bar 2 µm.


D . COPI - positive , electron - dense structures in close apposition to perinuclear ER . 10 nm gold particles mark COPI . Scale bar 100 nm .

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D. COPI-positive, electron-dense structures in close apposition to perinuclear ER. 10 nm gold particles mark COPI. Scale bar 100 nm.


E . Accumulation of COPI - positive vesicles . 10 nm gold particles mark COPI . PM : plasma , nuc : nucleus . Scale bar 100 nm .

E. Accumulation of COPI-positive vesicles. 10 nm gold particles mark COPI. PM: plasma membrane, nuc: nucleus. Scale bar 100 nm.


( A ) Human myeloid lymphoid were purified from PBMCs using a sorter . Total was isolated from these AECs to perform chip hybridization microarray .

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(A) Human myeloid cells and lymphoid cells were purified from PBMCs using a cell sorter. Total RNA was isolated from these primary cells and primary AECs to perform chip hybridization and microarray analysis.


profile of EphA2 expression in different is indicated . ( B ) Murine B T were purified from spleens using a sorter , total was isolated from murine B , T , BMDCs , BMDMs , AMs AECs . Shown is mean ± SD . Data represent three independent biological replicates .

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The profile of EphA2 expression in different cells is indicated. (B) Murine B cells and T cells were purified from spleens using a cell sorter, total RNA was isolated from murine B cells, T cells, BMDCs, BMDMs, AMs and AECs. Shown is mean ± SD. Data represent three independent biological replicates.


profile of EphA2 expression in different is indicated . ( C ) Immunoblot of EphA2 β - actin in BMDCs , BMDMs , AMs , AECs .

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The profile of EphA2 expression in different cells is indicated. (C) Immunoblot analysis of EphA2 and β-actin in BMDCs, BMDMs, AMs, and AECs.


( A - D ) ELISA of IL - 1β , IL - 18 , IFN - β IL - 6 in supernatant of AEC , which were stimulated for 16 h with reovirus ( MOI = 5 ) or HSV - 1 ( MOI = 5 ) . n = 3 samples . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate average of triplicates . Mock indicated unstimulated . * * P < 0 . 01 * * * P < 0 . 001 ( unpaired t ) . NS means no statistical difference . Data represent three independent biological replicates .

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(A-D) ELISA of IL-1β, IL-18, IFN-β and IL-6 in supernatant of AEC cells, which were stimulated for 16 h with RNA virus reovirus (MOI=5) or DNA virus HSV-1 (MOI=5). n = 3 primary samples. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. Mock indicated unstimulated cells. **P <0.01 and ***P<0.001 (unpaired t test). NS means no statistical difference. Data represent three independent biological replicates.


( E ) Immunoblot of IL - 1β caspase - 1 in AECs lysate or supernatant , β - actin served as a loading control throughout . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate average of triplicates . Mock indicated unstimulated . * * P < 0 . 01 * * * P < 0 . 001 ( unpaired t ) . NS means no statistical difference . Data represent three independent biological replicates .

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(E) Immunoblot analysis of IL-1β and caspase-1 in AECs cell lysate or cell supernatant, β-actin served as a loading control throughout. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. Mock indicated unstimulated cells. **P <0.01 and ***P<0.001 (unpaired t test). NS means no statistical difference. Data represent three independent biological replicates.


( A - B ) ELISA of IL - 1β IL - 18 in BALF samples were from wild - type ( WT ) EphA2 - kockout ( KO ) mice ( n = 3 per ) on day 1 day 3 after intranasal infection with reovirus ( 4 × 104 PFU per mouse ) . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate average of triplicates . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 ( unpaired t ) . Data represent three independent biological replicates .

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(A-B) ELISA of IL-1β and IL-18 in BALF samples were from wild-type (WT) and EphA2-kockout (KO) mice (n=3 per strain) on day 1 and day 3 after intranasal infection with reovirus (4×104 PFU per mouse). Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.


number of BALF samples were from wild - type ( WT ) EphA2 - kockout ( KO ) mice ( n = 3 per ) on day 1 day 3 after intranasal infection with reovirus ( 4 × 104 PFU per mouse ) . Shown is mean ± SD . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate average of triplicates . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 ( unpaired t ) . Data represent three independent biological replicates .

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Cell number of BALF samples were from wild-type (WT) and EphA2-kockout (KO) mice (n=3 per strain) on day 1 and day 3 after intranasal infection with reovirus (4×104 PFU per mouse). Shown is mean ± SD. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.


number category of BALF samples were from wild - type ( WT ) EphA2 - kockout ( KO ) mice ( n = 3 per ) on day 1 day 3 after intranasal infection with reovirus ( 4 × 104 PFU per mouse ) . Shown is mean ± SD . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate average of triplicates . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 ( unpaired t ) . Data represent three independent biological replicates .

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Cell number and category of BALF samples were from wild-type (WT) and EphA2-kockout (KO) mice (n=3 per strain) on day 1 and day 3 after intranasal infection with reovirus (4×104 PFU per mouse). Shown is mean ± SD. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.


( F ) Hematoxylin eosin ( H & E ) - of lung sections were from EphA2 + / + EphA2 − / − mice intranasally infected with reovirus ( 4 × 104 PFU per mouse ) on day 0 , day 1 day 3 . Scale bars represent 200μm .

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(F) Hematoxylin and eosin (H&E)-staining of lung sections were from EphA2+/+ and EphA2−/− mice intranasally infected with reovirus (4×104 PFU per mouse) on day 0, day 1 and day 3. Scale bars represent 200μm.


( G ) Immunoblot of EphA2 IL - 1β in lung homogenates were from wild - type ( WT ) EphA2 - kockout ( KO ) mice on day 1 day 3 of intranasal infection with reovirus ( 4 × 104 PFU per mouse ) , β - actin served as a loading control throughout .

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(G) Immunoblot analysis of EphA2 and IL-1β in lung homogenates were from wild-type (WT) and EphA2-kockout (KO) mice on day 1 and day 3 of intranasal infection with reovirus (4×104 PFU per mouse), β-actin served as a loading control throughout.


Quantitative RT - PCR of mRNA encoding various cytokines ( IL - 4 , IL - 5 , IL - 13 in lung samples from wild - type ( WT ) EphA2 - knockout ( KO ) mice ( n = 4 per ) sensitized with ova ; results presented relative to β - actin . Shown is mean ± SD . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate average of triplicates . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 ( unpaired t ) . Data represent three or four independent biological replicates .

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Quantitative RT-PCR analysis of mRNA encoding various cytokines (IL-4, IL-5, IL-13 in lung samples from wild-type (WT) and EphA2-knockout (KO) mice (n=4 per strain) sensitized with ova; results presented relative to β-actin. Shown is mean ± SD. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates.


Quantitative RT - PCR of mRNA encoding various cytokines IL - 33 ) in lung samples from wild - type ( WT ) EphA2 - knockout ( KO ) mice ( n = 4 per ) sensitized with ova ; results presented relative to β - actin . Shown is mean ± SD . ( C ) ELISA of IL - 33 in lung tissue homogenate samples from wild - type ( WT ) EphA2 - knockout ( KO ) mice ( n = 3 per ) sensitized with ova . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate average of triplicates . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 ( unpaired t ) . Data represent three or four independent biological replicates .

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Quantitative RT-PCR analysis of mRNA encoding various cytokines IL-33) in lung samples from wild-type (WT) and EphA2-knockout (KO) mice (n=4 per strain) sensitized with ova; results presented relative to β-actin. Shown is mean ± SD. (C) ELISA analysis of IL-33 in lung tissue homogenate samples from wild-type (WT) and EphA2-knockout (KO) mice (n=3 per strain) sensitized with ova. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates.


( D - G ) ELISA of IL - 4 , IL - 13 , IL - 1β IL - 18 in lung tissue homogenate samples from wild - type ( WT ) EphA2 - knockout ( KO ) mice ( n = 3 per ) sensitized with ova . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate average of triplicates . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 ( unpaired t ) . Data represent three or four independent biological replicates .

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(D-G) ELISA analysis of IL-4, IL-13, IL-1β and IL-18 in lung tissue homogenate samples from wild-type (WT) and EphA2-knockout (KO) mice (n=3 per strain) sensitized with ova. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates.


( H ) number category of BALF samples from wild - type ( WT ) EphA2 - knockout ( KO ) mice ( n = 3 per ) sensitized with ova . Shown is mean ± SD . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate average of triplicates . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 ( unpaired t ) . Data represent three or four independent biological replicates .

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(H) Cell number and category of BALF samples from wild-type (WT) and EphA2-knockout (KO) mice (n=3 per strain) sensitized with ova. Shown is mean ± SD. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates.


( I ) H & E PAS of lung sections from wild - type ( WT ) EphA2 - knockout ( KO ) mice sensitized with ova . Scale bars represent 200μm .

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(I) H&E staining and PAS staining of lung sections from wild-type (WT) and EphA2-knockout (KO) mice sensitized with ova. Scale bars represent 200μm.


( A ) Immunoblot of precipitated with anti - EphA2 or IgG ( control ) were from whole - lysates of AECs from wild - type mice stimulated with reovirus ( MOI = 5 ) for 3 h . Input , 10 % of AECs lysate . Data information : position of markers ( shown in kDa ) is indicated on right - side . Data are representative of three independent biological replicates .

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(A) Immunoblot analysis of proteins precipitated with anti-EphA2 or IgG (control) were from whole-cell lysates of AECs from wild-type mice stimulated with reovirus (MOI=5) for 3 h. Input, 10% of the AECs lysate. Data information: The position of protein markers (shown in kDa) is indicated on the right-hand side. Data are representative of three independent biological replicates.


( C ) indicated plasmids of HA - tagged full - EphA2 EphA2 truncation mutants were respectively transfected into HEK293T along with Myc - tagged NLRP3 , then lysates were immunoprecipitated with anti - Myc antibody , followed by western blot with anti - HA antibody anti - Myc Antibody . Data information : position of markers ( shown in kDa ) is indicated on right - side . Data are representative of three independent biological replicates .

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(C) The indicated plasmids of HA-tagged full-length EphA2 and EphA2 truncation mutants were respectively transfected into HEK293T cells along with Myc-tagged NLRP3, and then cell lysates were immunoprecipitated with anti-Myc antibody, followed by western blot analysis with anti-HA antibody and anti-Myc Antibody. Data information: The position of protein markers (shown in kDa) is indicated on the right-hand side. Data are representative of three independent biological replicates.


( D ) indicated plasmids of HA - tagged full - NLRP3 NLRP3 truncation mutants were respectively transfected into HEK293T along with Myc - tagged EphA2 , then lysates were immunoprecipitated with anti - Myc antibody , followed by western blot with anti - HA antibody anti - Myc antibody . Data information : position of markers ( shown in kDa ) is indicated on right - side . Data are representative of three independent biological replicates .

(D) The indicated plasmids of HA-tagged full-length NLRP3 and NLRP3 truncation mutants were respectively transfected into HEK293T cells along with Myc-tagged EphA2, and then cell lysates were immunoprecipitated with anti-Myc antibody, followed by western blot analysis with anti-HA antibody and anti-Myc antibody. Data information: The position of protein markers (shown in kDa) is indicated on the right-hand side. Data are representative of three independent biological replicates.


( E ) Colocalization of endogenous EphA2 NLRP3 in AEC . AEC were unstimulated or stimulated with reovirus for 2h before confocal microscopy . EphA2 was stained with rabbit anti - EphA2 ( 1 : 500 ) , followed by Alexa Fluor 594 goat anti - rabbit secondary antibody ( red ) , while NLRP3 was stained with mouse anti - NLRP3 ( 1 : 500 ) , followed by Alexa Fluor 488 goat anti - mouse secondary antibody ( green ) . DAPI ( 4 ′ , 6 - diamidino - 2 - phenylindole ) served as nuclei marker ( blue ) . Scale bars represent 20μm . Pearson ' s correlation coefficient Mander ' s overlap coefficient in upper red box are 0 . 5 , 0 . 95 respectively , lower red box 0 . 78 , 0 . 94 respectively .

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(E) Colocalization of endogenous EphA2 and NLRP3 in AEC cells. AEC cells were unstimulated or stimulated with reovirus for 2h before confocal microscopy analysis. EphA2 was stained with rabbit anti-EphA2 (1:500), followed by Alexa Fluor 594 goat anti-rabbit secondary antibody (red), while NLRP3 was stained with mouse anti-NLRP3 (1:500), followed by Alexa Fluor 488 goat anti-mouse secondary antibody (green). DAPI (4′,6-diamidino-2-phenylindole) served as the nuclei marker (blue). Scale bars represent 20μm. The Pearson's correlation coefficient and the Mander's overlap coefficient in the upper red box are 0.5, and 0.95 respectively, and the lower red box 0.78, and 0.94 respectively.


( A ) indicated plasmids of Myc - tagged NLRP3 GFP - tagged EphA2 were transfected into HEK293T then lysates were immunoprecipitated with anti - Myc antibody , followed by western blot with anti - p - Tyr antibody , anti - GFP anti - Myc .

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(A) The indicated plasmids of Myc-tagged NLRP3 and GFP-tagged EphA2 were transfected into HEK293T cells and then cell lysates were immunoprecipitated with the anti-Myc antibody, followed by western blot analysis with the anti-p-Tyr antibody, anti-GFP and anti-Myc.


( B ) Immunoblot of p - Tyr of NLRP3 precipitated with anti - NLRP3 antibody in AECs without ( Ctrl ) or with LPS stimulation or reovirus ( Reo ) infection .

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(B) Immunoblot analysis of p-Tyr of NLRP3 precipitated with anti-NLRP3 antibody in AECs without (Ctrl) or with LPS stimulation or reovirus (Reo) infection.


( C ) indicated plasmids of Myc - tagged wild - type NLRP3 or its mutants Y132F , Y164F , Y251F , Y570F Y589F were respectively transfected into HEK293T along with GFP - tagged EphA2 , then lysates were immunoprecipitated with anti - Myc antibody , followed by western blot with anti - p - Tyr antibody , anti - HA antibody anti - Myc antibody .

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(C) The indicated plasmids of Myc-tagged wild-type NLRP3 or its mutants Y132F, Y164F, Y251F, Y570F and Y589F were respectively transfected into HEK293T cells along with GFP-tagged EphA2, and then cell lysates were immunoprecipitated with the anti-Myc antibody, followed by western blot analysis with the anti-p-Tyr antibody, anti-HA antibody and anti-Myc antibody.


( D ) Immunoblot of EphA2 , ASC , NLRP3 , caspase - 1 IL - 1β in HEK293T were co - transfected with 200ng EphA2 , 40ng ASC , 40ng caspase - 1 200ng IL - 1β , vector or wild NLPR3 or Y132F as indicated . position of markers ( shown in kDa ) is indicated on right - side .

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(D) Immunoblot analysis of EphA2, ASC, NLRP3, caspase-1 and IL-1β in HEK293T cells were co-transfected with 200ng EphA2, 40ng ASC, 40ng caspase-1 and 200ng IL-1β, vector or wild NLPR3 or Y132F as indicated. The position of protein markers (shown in kDa) is indicated on the right-hand side.


( E ) Densitometry * P < 0 . 05 ( unpaired t ) . NS means no statistical difference . Shown is mean ± SD . Data represent three independent biological replicates .

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(E) Densitometry analysis *P<0.05 (unpaired t test). NS means no statistical difference. Shown is mean ± SD. Data represent three independent biological replicates.


( A - B ) Confocal microscopy of AEC uninfected or infected with reovirus at multiplicity of infection ( MOI ) of 20 for 3h . AECs primed with LPS stimulated with ATP for 30 min severed as a positive control . ASC was stained with mouse anti - ASC ( 1 : 200 ) , followed by Alexa Fluor 488 goat anti - mouse secondary antibody ( green ) . DAPI served as nuclei marker ( blue ) . Data information : Scale bars represent 50μm . Each symbol represents an independent experiment ; small horizontal lines indicate average of triplicates . * * P < 0 . 01 * * * P < 0 . 001 ( unpaired t ) . Data represent three independent biological replicates .

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(A-B) Confocal microscopy analysis of AEC cells uninfected or infected with reovirus at multiplicity of infection (MOI) of 20 for 3h. AECs primed with LPS and stimulated with ATP for 30 min severed as a positive control. ASC was stained with mouse anti-ASC (1:200), followed by Alexa Fluor 488 goat anti-mouse secondary antibody (green). DAPI served as the nuclei marker (blue). Data information: Scale bars represent 50μm. Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.


( C - D ) Confocal microscopy of HEK293T transfected with flag - ASC Myc - tagged vector or Myc - tagged NLRP3 or Myc - tagged NLRP3 Y132F . were stained with anti - flag ( 1 : 200 ) , anti - Myc ( 1 : 200 ) , followed by Alexa Fluor 488 goat anti - rabbit secondary antibody ( green ) or Alexa Fluor 594 goat anti - mouse secondary antibody ( red ) . DAPI served as nuclei marker ( blue ) . Data information : Scale bars represent 50μm . Each symbol represents an independent experiment ; small horizontal lines indicate average of triplicates . * * P < 0 . 01 * * * P < 0 . 001 ( unpaired t ) . Data represent three independent biological replicates .

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(C-D) Confocal microscopy analysis of HEK293T cells transfected with flag-ASC and Myc-tagged vector or Myc-tagged NLRP3 or Myc-tagged NLRP3 Y132F. Cells were stained with anti-flag (1:200), anti-Myc (1:200), followed by Alexa Fluor 488 goat anti-rabbit secondary antibody (green) or Alexa Fluor 594 goat anti-mouse secondary antibody (red). DAPI served as the nuclei marker (blue). Data information: Scale bars represent 50μm. Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.


GST - Mif2 _ 1 - 41 was immobilized on GSH Sepharose beads incubated with Mtw1 - Nnf1 ( MN ) in presence of increasing concentrations of Ame1 _ 1 - 20 peptide ( lanes 4 - 9 ) . GST - Ame1 _ 1 - 30 served as control ( lane 2 3 ) . After washing , bound complexes were eluted by adding SDS sample buffer . bands in Coomassie stained gel are highlighted in indicated color . Integrated density of Mtw1 bands was quantified using ImageJ correlated to GST - Mif2 amounts in same lane .

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GST-Mif2_1-41 was immobilized on GSH Sepharose beads and incubated with Mtw1-Nnf1 complex (MN) in the presence of increasing concentrations of Ame1_1-20 peptide (lanes 4-9). GST-Ame1_1-30 served as control (lane 2 and 3). After washing, bound complexes were eluted by adding SDS sample buffer. Protein bands in Coomassie stained gel are highlighted in the indicated color. Integrated density of Mtw1 bands was quantified using ImageJ and correlated to GST-Mif2 amounts in the same lane.


Binding of FITC - Mif21 - 41 to MN was directly determined in a polarization ( FP ) . This interaction at EC50 was than competed with increasing of Ame11 - 20 unlabeled peptide . Both experiments were performed in triplicates of which mean standard deviation is shown .

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Binding of FITC-Mif21-41 to MN was directly determined in a fluorescence polarization (FP) assay. This interaction at EC50 was than competed with increasing concentration of Ame11-20 unlabeled peptide. Both experiments were performed in triplicates of which the mean and standard deviation is shown.


Equivalent point mutations replacing Arg11 in Mif2 or Arg10 in Ame1 with Alanine prevent binding to MN . Mif2 Ame1 wild - type N - termini serve as a control , bands are highlighted in indicated color .

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Equivalent point mutations replacing Arg11 in Mif2 or Arg10 in Ame1 with Alanine prevent binding to the MN complex. Mif2 and Ame1 wild-type N-termini serve as a control, protein bands are highlighted in the indicated color.


of these Mtw1 mutants in vivo using anchor - away technique . Mtw1 - FRB strains containing RPL13 - FKBP12 for anchoring additionally harboring indicated rescue alleles were plated in serial dilution on YEPD or YEPD + Rapamycin plates incubated at 30 ° C .

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Analysis of these Mtw1 mutants in vivo using the anchor-away technique. Mtw1-FRB strains containing RPL13-FKBP12 for cytoplasmic anchoring and additionally harboring the indicated rescue alleles were plated in serial dilution on YEPD or YEPD + Rapamycin plates incubated at 30 °C.


Tetrad of Mtw1 - wt or mutants . Surviving spores in which Mtw1 - wt or mutant rescue construct was only source of Mtw1 are indicated in pink circles .

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Tetrad analysis of Mtw1-wt or mutants. Surviving spores in which the Mtw1-wt or mutant rescue construct was the only source of Mtw1 are indicated in pink circles.


Western blotting confirming expression of Mtw1 - 6x - Flag wt or mutant in Mtw1 - FRB background .

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Western blotting confirming the expression of Mtw1-6x-Flag wt or mutant proteins in the Mtw1-FRB strain background.


Analytical SEC runs accompanying SDS - PAGE of Mtw1c - wt or - 5A mutant ( blue ) , AO ( grey ) or a stoichiometric combination of both complexes ( green ) at 10 µM each . Boxes indicate corresponding fractions in left right panel . Note that same Ame1 - Okp1 elution profile SDS - PAGE ( middle panel ) is displayed in both sets to improve clarity .

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Analytical SEC runs and accompanying SDS-PAGE of Mtw1c-wt or -5A mutant (blue), AO (grey) or a stoichiometric combination of both complexes (green) at 10 µM each. Boxes indicate corresponding fractions in left and right panel. Note that the same Ame1-Okp1 elution profile and SDS-PAGE (middle panel) is displayed in both sets to improve clarity.


of Ame1 N - terminal deletion swap mutant using anchor - away technique . Ame1 - FRB strains containing RPL13 - FKBP12 for ribosome anchoring additionally harboring indicated rescue alleles were plated in serial dilution on YEPD or YEPD + Rapamycin plates incubated at 30 ° C .

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Analysis of Ame1 N-terminal deletion and swap mutant using the anchor-away technique. Ame1-FRB strains containing RPL13-FKBP12 for ribosome anchoring and additionally harboring the indicated rescue alleles were plated in serial dilution on YEPD or YEPD + Rapamycin plates incubated at 30 °C.


Expression of 3xHA tagged Ame1 rescue constructs from ( B ) analyzed by western blotting .

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Expression levels of 3xHA tagged Ame1 rescue constructs from (B) analyzed by western blotting.


Tetrad dissection of strains where one endogenous copy of Ame1 was replaced with indicated construct .

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Tetrad dissection of strains where one endogenous copy of Ame1 was replaced with the indicated construct.


Phenotypic of different Ame1 rescue constructs using microscopy . of representative large budded immediately 4 h after Rapamycin addition are shown , indicated as t = 0 h t = 4 h . Brightfield show of corresponding yeast . Only large budded were included in . One kinetochore cluster is enlarged in box . Scale bar represents 5 µm .

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Phenotypic analysis of different Ame1 rescue constructs using fluorescence microscopy. Images of representative large budded cells immediately and 4 h after Rapamycin addition are shown, indicated as t = 0 h and t = 4 h. Brightfield images show the morphology of the corresponding yeast cell. Only large budded cells were included in the analysis. One kinetochore cluster is enlarged in the white box. Scale bar represents 5 µm.


of ( E ) in which large budded were classified according to their Mtw1 - GFP signal into three indicated groups . Scale bar represents 5 µm . A total of 100 was quantified for every . Mean values of three indicated groups were calculated plotted in a bar chart . Error bars indicate of 3 independent experiments .

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Quantification of (E) in which large budded cells were classified according to their Mtw1-GFP signal into the three indicated groups. Scale bar represents 5 µm. A total of 100 cells was quantified for every strain. Mean values of the three indicated groups were calculated and plotted in a bar chart. Error bars indicate the SEM of 3 independent experiments.


of Mif2 N - terminal swap mutant using anchor - away technique as for ( B ) . of indicated Mif2 constructs were observed in an otherwise wild - type or ctf19 ∆ background .

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Analysis of Mif2 N-terminal and swap mutant using the anchor-away technique as for (B). Phenotypes of the indicated Mif2 constructs were observed in an otherwise wild-type or ctf19∆ background.


Western blotting showing 6xFlag tagged Mif2 expression of strains from ( G ) .

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Western blotting showing 6xFlag tagged Mif2 expression levels of strains from (G).


Co - purification of Strep - Mtw1c through Flag - Mif2 after co - expression in insect . were detected by western blotting using their respective tags .

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Co-purification of Strep-Mtw1c through Flag-Mif2 after co-expression in insect cells. Proteins were detected by western blotting using their respective tags.


Western blotting of elution fractions from an SEC experiment of Mif2 - wt or Mif2Ame1 - N swap in absence ( upper panel ) or presence ( lower panel ) of Mtw1c .

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Western blotting of elution fractions from an SEC experiment of Mif2-wt or Mif2Ame1-N swap in the absence (upper panel) or presence (lower panel) of Mtw1c.


ITC of Mif21 - 41 or Ame11 - 20 binding to MN . Experiments were performed 3 or 4 times , respectively . One representative binding curve is shown , whereas KD value represents average of experiments .

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ITC analysis of Mif21-41 or Ame11-20 binding to MN. Experiments were performed 3 or 4 times, respectively. One representative binding curve is shown, whereas the KD value represents the average of all experiments.


Cross - link ( XL ) MS of Mif2wt in isolation or bound to Cse4n using cross - linker Bis [ sulfosuccinimidyl ] glutarate . Mif2wt alone or in combination with Cse4n were subjected to analytical exclusion chromatography at 10 µM . corresponding Coomassie - stained SDS - PAGE gels of Mif2 - wt alone or in combination with Cse4n are shown . Fractions used for cross - linking are highlighted . Topological map of Mif2 on identified intra - molecular cross - links . Common cross links that are found in both samples are shown in red , whereas unique crosslinks that can be only found in one of samples are highlighted in blue . Conserved regions their described functions are indicated . A list of cross - links that link N - terminal residues to C - terminal regions of Mif2 is shown for Mif2 alone bound to Cse4n leading to a proposed conformation of Mif2 .

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Cross-link (XL) MS analysis of the Mif2wt protein in isolation or bound to Cse4n using the cross-linker Bis[sulfosuccinimidyl] glutarate. Mif2wt alone or in combination with Cse4n were subjected to analytical size exclusion chromatography at 10 µM concentration. The corresponding Coomassie-stained SDS-PAGE gels of Mif2-wt alone or in combination with Cse4n are shown. Fractions used for cross-linking are highlighted. Topological map of Mif2 based on the identified intra-molecular cross-links. Common cross links that are found in both samples are shown in red, whereas unique crosslinks that can be only found in one of the samples are highlighted in blue. Conserved regions and their described functions are indicated. A list of cross-links that link N-terminal residues to C-terminal regions of Mif2 is shown for Mif2 alone and bound to Cse4n leading to a proposed conformation of Mif2.