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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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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 .

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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 .

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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 .

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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 .

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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

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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

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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 )

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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-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O 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-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR

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-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD 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-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O 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-GENEPROD O O O B-SUBCELLULAR

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 ) .

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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 .

O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O 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-SMALL_MOLECULE O 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-SUBCELLULAR I-SUBCELLULAR O O O O O O O O

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 .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O 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. 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 .

O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O

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 .

O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O

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 .

O O O B-ORGANISM B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(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 .

O O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE O O O O O O O O B-ORGANISM B-CELL_TYPE O B-CELL_TYPE O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O

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 .

O O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O

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 .

O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 .

B-EXP_ASSAY O B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

B-EXP_ASSAY O O B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O

(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 .

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O B-ORGANISM O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

(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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 .

O O O B-EXP_ASSAY O O B-TISSUE O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-SMALL_MOLECULE O B-TISSUE O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O B-GENEPROD O O O O O O

(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 .

O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O O O B-ORGANISM O O B-ORGANISM O O O O O O O O O O O O O O B-CELL_TYPE O O O O O 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) 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 .

O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O B-CELL_LINE O O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) The 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 .

O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-CELL_TYPE O B-CELL_TYPE O O O O O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O B-CELL_LINE O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-GENEPROD O

(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 .

O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O B-ORGANISM O B-EXP_ASSAY O

(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 .

O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_LINE O O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-GENEPROD O O

(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 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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.


Mif2Flag - wt or - ∆ sigAT directly from insect lysate was immobilized on M2 Flag Agarose , washed incubated with Mtw1 - Nnf1 ( MN ) . After washing , bound complexes were eluted by adding 3xFlag peptide . Western blotting against His - tag on Nnf1 confirmed increased binding of MN to Mif2 - ∆ sigAT , already visible in Coomassie stained SDS - PAGE gel .

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Mif2Flag-wt or -∆sigAT directly from insect cell lysate was immobilized on M2 Flag Agarose, washed and incubated with Mtw1-Nnf1 complex (MN). After washing, bound complexes were eluted by adding 3xFlag peptide. Western blotting against the His-tag on Nnf1 confirmed increased binding of MN to Mif2-∆sigAT, already visible in the Coomassie stained SDS-PAGE gel.


Analytical SEC chromatograms corresponding Coomassie stained SDS - PAGE gels of Mif2 - wt or Mif2 - ∆ sigAT MN alone in combination at 10 µM . combinations were incubated for 1 h at 4 ° C prior to run . MN ( blue ) , Mif2 ( red ) , combination ( green ) . Note that same MN elution profile SDS - PAGE ( upper panel ) is displayed in both sets to improve clarity . Dashed boxes highlighting corresponding fractions were included to improve comparability .

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Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mif2-wt or Mif2-∆sigAT and MN alone and in combination at 10 µM concentration. All combinations were incubated for 1 h at 4°C prior to the run. MN (blue), Mif2 (red), combination (green). Note that the same MN elution profile and SDS-PAGE (upper panel) is displayed in both sets to improve clarity. Dashed boxes highlighting corresponding fractions were included to improve comparability.


Analytical SEC chromatograms corresponding Coomassie stained SDS - PAGE gels of Cse4n ( blue ) , Mif2 - wt ( red ) , combination ( green ) . Note that same Cse4n ( upper panel ) Mif2 ( middle panel ) elution profiles SDS - PAGEs are also displayed as reference in B C ( Mif2 ) or C ( Cse4n ) to improve clarity .

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Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Cse4n (light blue), Mif2-wt (red), combination (green). Note that the same Cse4n (upper panel) and Mif2 (middle panel) elution profiles and SDS-PAGEs are also displayed as reference in B and C (Mif2) or C (Cse4n) to improve clarity.


Analytical SEC chromatograms corresponding Coomassie stained SDS - PAGE gels of Mtw1c ( blue ) , Mif2 - wt ( red ) , combination ( green ) . Note that same Mtw1c ( upper panel ) elution profile SDS - PAGE is also displayed as reference in C to improve clarity .

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Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mtw1c (blue), Mif2-wt (red), combination (green). Note that the same Mtw1c (upper panel) elution profile and SDS-PAGE is also displayed as reference in C to improve clarity.


Analytical SEC chromatograms corresponding Coomassie stained SDS - PAGE gels of Mtw1c ( blue ) , Cse4n ( blue ) , Mif2 - wt ( red ) , combination ( green ) .

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Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mtw1c (blue), Cse4n (light blue), Mif2-wt (red), combination (green).


Analytical SEC chromatograms corresponding Coomassie stained SDS - PAGE gels of Cse4n ( blue ) , Mif2Ame - 1N swap ( red ) , combination ( green ) . Note that same Cse4n ( upper panel ) Mif2Ame - 1N swap ( middle panel ) elution profiles SDS - PAGEs are also displayed as reference in E F ( Mif2Ame - 1N swap ) or F ( Cse4n ) to improve clarity .

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Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Cse4n (light blue), Mif2Ame-1N swap (red), combination (green). Note that the same Cse4n (upper panel) and Mif2Ame-1N swap (middle panel) elution profiles and SDS-PAGEs are also displayed as reference in E and F (Mif2Ame-1N swap) or F (Cse4n) to improve clarity.


Analytical SEC chromatograms corresponding Coomassie stained SDS - PAGE gels of Mtw1c ( blue ) , Mif2Ame - 1N swap ( red ) , combination ( green ) . Note that same Mtw1c ( upper panel ) elution profile SDS - PAGE is also displayed as reference in F to improve clarity .

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Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mtw1c (blue), Mif2Ame-1N swap (red), combination (green). Note that the same Mtw1c (upper panel) elution profile and SDS-PAGE is also displayed as reference in F to improve clarity.


Analytical SEC chromatograms corresponding Coomassie stained SDS - PAGE gels of Mtw1c ( blue ) , Cse4n ( blue ) , Mif2Ame - 1N swap ( red ) , combination ( green ) .

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Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mtw1c (blue), Cse4n (light blue), Mif2Ame-1N swap (red), combination (green).


Phenotypic of Mif2 mutants using a galactose inducible overexpression system . Strains harboring indicated Mif2 expression constructs on pESC plasmids were plated in serial dilution on doHIS - Glucose or doHIS - Raffinose / Galactose plates incubated at 30 ° C .

O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O

Phenotypic analysis of Mif2 mutants using a galactose inducible overexpression system. Strains harboring the indicated Mif2 expression constructs on pESC plasmids were plated in serial dilution on doHIS-Glucose or doHIS-Raffinose/Galactose plates and incubated at 30 °C.


Serial dilution of chromosomally integrated Mif2 variants under control of pGAL promoter . Strains were plated on YEP - Glucose YEP - Raffinose / Galactose plates .

B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O

Serial dilution assay of chromosomally integrated Mif2 variants under the control of the pGAL promoter. Strains were plated on YEP-Glucose and YEP-Raffinose/Galactose plates.


of chromosome segregation in strains from ( B ) . Distribution of fluorescently labeled Chromosome III was analyzed in large - budded 16 hours after switching medium to Galactose . Scale bar represents 2 μm .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O

Analysis of chromosome segregation in strains from (B). Distribution of fluorescently labeled Chromosome III was analyzed in large-budded cells 16 hours after switching the medium to Galactose. Scale bar represents 2 μm.


Microscopic of kinetochores in Mif2 overexpressing visualizing Mtw1 - GFP Spc42 - mCherry . of representative large budded after 16h in Galactose are shown . One kinetochore cluster is enlarged in box for each . Scale bar represents 5 µm . Brightfield show of corresponding yeast . Only large budded were included in . of ( D ) in which large budded were classified according to their Mtw1 - GFP signal into four indicated groups . 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 2 independent experiments .

B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Microscopic analysis of kinetochores in Mif2 overexpressing cells visualizing Mtw1-GFP and Spc42-mCherry. Images of representative large budded cells after 16h in Galactose are shown. One kinetochore cluster is enlarged in the white box for each strain. Scale bar represents 5 µm. Brightfield images show the morphology of the corresponding yeast cell. Only large budded cells were included in the analysis. Quantification of (D) in which large budded cells were classified according to their Mtw1-GFP signal into the four indicated groups. 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 2 independent experiments.


Co - IP experiment of indicated Mif2 - Flag variants Mtw1 - Myc from yeast extracts .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM O O

Co-IP experiment of indicated Mif2-Flag variants and Mtw1-Myc from yeast cell extracts.


Phenotypic of Mif2 - ∆ sigAT mutant using same galactose inducible overexpression system as in ( A ) . Strains harboring indicated Mif2 expression constructs on pESC plasmids were plated in serial dilution on doHIS - Glucose or doHIS - Raffinose / Galactose plates incubated at 30 ° C .

O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O

Phenotypic analysis of Mif2-∆sigAT mutant using the same galactose inducible overexpression system as in (A). Strains harboring the indicated Mif2 expression constructs on pESC plasmids were plated in serial dilution on doHIS-Glucose or doHIS-Raffinose/Galactose plates and incubated at 30 °C.


Comparable mean SV number per ribbon in wild - type at distal ( Ds . , H ) proximal ( Pr . , I ) part of ribbon . More SVs are observed at inhibitory OtofPga / Pga ribbons compared to inhibitory wild - type synapses at both ribbon parts , further at OtofPga / Pga synapses under in Pr . part . Data are presented as mean ± ; red dotted highlights wild - type mean SV number per ribbon under as a reference point ;

O O B-SUBCELLULAR B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-SUBCELLULAR O O O O O O

Comparable mean SV number per ribbon in wild-type at the distal (Ds., H) and the proximal (Pr., I) part of the ribbon. More SVs are observed at inhibitory OtofPga/Pga ribbons compared to inhibitory wild-type synapses at both ribbon parts, and further at OtofPga/Pga synapses under rest in the Pr. part. Data are presented as mean ± SEM; the red dotted line highlights the wild-type mean SV number per ribbon under rest as a reference point;


Representative virtual sections of wild - type ( J ) OtofPga / Pga ( K ) ribbon synapses under inhibition . Counted RA - SVs are marked with asterisks . Magnification , 12 , 000x ; scale bar , 100 nm .

O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O

Representative virtual sections of wild-type (J) and OtofPga/Pga (K) ribbon synapses under inhibition. Counted RA-SVs are marked with white asterisks. Magnification, 12,000x; scale bar, 100 nm.


Exemplary virtual sections of wild - type OtofPga / Pga under inhib ( L , M ) , ( L ' , M ' ) stim ( L ' ' , M ' ' ) . At OtofPga / Pga SVs are densely packed . Blue arrowheads indicate proteinaceous filaments between wild - type SVs . Magnification 12 , 000x ; scale bar , 100 nm .

O O O O O O O B-GENEPROD O O O O O 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-GENEPROD O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O

Exemplary virtual sections of wild-type and OtofPga/Pga under inhib (L,M), rest (L',M') and stim (L'',M''). At OtofPga/Pga SVs are densely packed. Blue arrowheads indicate proteinaceous filaments between wild-type SVs. Magnification 12,000x; scale bar, 100 nm.


Illustrations show two major SVs associated with filament / s in wild - type RA - SV pool at ; synaptic ribbon ( R , red ) , SVs ( green ) with filament / s ( blue ) . Not drawn to scale . ( A , B ) Ri - attached SVs ( Ri . ) ( C , D ) interconnected SVs ( In . ) harbor single ( A , C ) or multiple ( B , D ) filaments . Example virtual section from resting wild - type ; single Ri . ( A ' , A ' ' ) In . ( C ' , C ' ' ) , multiple Ri . ( B ' , B ' ' ) In . ( D ' , D ' ' ) . Magnification 12 , 000x ; scale bar , 40 nm . Structures are color - coded similarly to illustrations , number of ribbon - attached filaments per SV are indicated with red arrowhead / s interconnectors per SV with purple arrowhead / s in A ' ' , B ' ' , C ' ' D ' ' . Magnification 12 , 000x ; scale bar , 40 nm .

O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O 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-EXP_ASSAY O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Illustrations show the two major SVs associated with filament/s in the wild-type RA-SV pool at rest; synaptic ribbon (R, red), SVs (dark green) with filament/s (blue). Not drawn to scale. (A,B) Ri-attached SVs (Ri.) and (C,D) interconnected SVs (In.) harbor single (A,C) or multiple (B,D) filaments. Example virtual section from resting wild-type; single Ri. (A',A'') and In. (C',C''), multiple Ri. (B',B'') and In. (D',D''). Magnification 12,000x; scale bar, 40 nm. Structures are color-coded similarly to the illustrations, the number of ribbon-attached filaments per SV are indicated with red arrowhead/s and interconnectors per SV with purple arrowhead/s in A'', B'', C'' and D''. Magnification 12,000x; scale bar, 40 nm.


graph is representing of RA - SVs around whole ribbon at wild - type under inhib , stim . Upon stimulation , of SVs without filaments ribbon - attached SVs are significantly reduced , while interconnected SVs increased significantly .

O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O

The graph is representing the percentage of RA-SVs around the whole ribbon at wild-type under inhib, rest and stim. Upon stimulation, the percentage of SVs without filaments and ribbon-attached SVs are significantly reduced, while interconnected SVs increased significantly.


Depicted are fractions of SVs without filaments , ribbon - attached SVs ( Ri . ) , interconnected SVs ( In . ) SVs with Ri . + In . per ribbon , at ( A ) distal ( Ds . ) ( B ) proximal ( Pr . ) part of ribbon . Data are represented as mean ± of wild - type OtofPga / Pga IHCs under inhib , stim . ( A ) Upon stimulation , in Ds . part at wild - type , fraction of SVs without filaments reduces , but of SVs with Ri . + In . increases significantly . At OtofPga / Pga IHCs fraction of SVs without filaments is reduced when comparing stim vs . inhib . fraction of interconnected SVs increases with stimulation compared to inhibition . In mutant ( inhib ) , fraction of interconnected SVs decreases compared to wild - type ( inhib ) , Ri . + In . SVs reduce vs . wild - type ( stim ) . ( B ) fraction of SVs without filaments increased at inhibited OtofPga / Pga synapses . red dotted lines in ( A ) ( B ) represent mean fraction of RA - SVs / ribbon part of resting wild - type for each defined sub - pool .

O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O

Depicted are the fractions of SVs without filaments, ribbon-attached SVs (Ri.), interconnected SVs (In.) and SVs with Ri. + In. per ribbon, at (A) the distal (Ds.) and (B) the proximal (Pr.) part of the ribbon. Data are represented as mean ± SEM of wild-type and OtofPga/Pga IHCs under inhib, rest and stim. (A) Upon stimulation, in the Ds. part at wild-type, the fraction of SVs without filaments reduces, but of SVs with Ri. + In. increases significantly. At OtofPga/Pga IHCs the fraction of SVs without filaments is reduced when comparing stim vs. inhib. The fraction of interconnected SVs increases with stimulation compared to inhibition and rest. In the mutant (inhib), the fraction of interconnected SVs decreases compared to wild-type (inhib), and Ri. + In. SVs reduce vs. wild-type (stim). (B) The fraction of SVs without filaments increased at inhibited OtofPga/Pga synapses. The red dotted lines in (A) and (B) represent the mean fraction of RA-SVs/ribbon part of resting wild-type for each defined sub-pool.


Representative wild - type virtual section under depicts SVs with r - attached fil ( Ra . fil . , red arrowheads ) one interconnector ( Intercon . , purple arrowhead ) , around ribbon ( R ) . Magnification 12 , 000x ; scale bar , 40 nm . Example 3D reconstruction of RA - SVs with filaments ( green ) in resting wild - type . Larger average is found for ribbon - attached filaments ( Ra . fil . , red ) vs . SV interconnectors ( Intercon . , purple ) . For clear visualization , ribbon ( R ) is transparent same SV pair is shown in a front ( A ' ) , side ( A ' ' ) top view ( A ' ' ' ) . Scale bar , 50 nm .

O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative wild-type virtual section under rest depicts SVs with r-attached fil (Ra.fil., red arrowheads) and one interconnector (Intercon., purple arrowhead), around the ribbon (R). Magnification 12,000x; scale bar, 40 nm. Example 3D reconstruction of the RA-SVs with filaments (green) in resting wild-type. Larger average length is found for ribbon-attached filaments (Ra.fil., red) vs. SV interconnectors (Intercon., purple). For clear visualization, the ribbon (R) is transparent and the same SV pair is shown in a front (A'), side (A'') and top view (A'''). Scale bar, 50 nm.


Increased mean of ribbon - attached filaments vs . SV interconnectors in conditions for both genotypes . red dotted lines represent mean RA - filament in resting wild - type for each sub - pool .

O O O B-SUBCELLULAR O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O

Increased mean length of ribbon-attached filaments vs. SV interconnectors in all conditions for both genotypes. The red dotted lines represent the mean RA-filament length in resting wild-type for each sub-pool.


cumulative probability distribution of RA - SVs plotted against filament . graph illustrates shorter filaments in wild - type OtofPga / Pga ribbon synapses upon stimulation .

O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O

The cumulative probability distribution of RA-SVs plotted against the individual filament length. The graph illustrates shorter filaments in wild-type and OtofPga/Pga ribbon synapses upon stimulation.


Decreased mean filament upon stimulation in wild - type OtofPga / Pga ribbon synapses . Resting OtofPga / Pga exhibit shortest mean filament . red dotted lines represent mean RA - filament in resting wild - type for each sub - pool .

O O B-SUBCELLULAR O O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O

Decreased mean filament length upon stimulation in wild-type and OtofPga/Pga ribbon synapses. Resting OtofPga/Pga exhibit the shortest mean filament length. The red dotted lines represent the mean RA-filament length in resting wild-type for each sub-pool.


Representative tomographic virtual sections from resting wild - type . Five different types of MP - SVs are observed that we classify as sub - pools : non - tethered SVs ( A ' , A ' ' ; yellow ) , single presynaptic density - attached SVs ( B ' , B ' ' ; Pd . a , orange , pink arrowhead ) or single m - attached SVs ( C ' , C ' ' ; Ma . , orange , orange arrowhead ) , or multiple - tethered SVs ( D ' , D ' ' ; orange , brown arrowheads ) docked SVs ( E ' , E ' ' ; orange ) . Ribbon ( R ) , presynaptic density ( PD , pink ) AZ - ( blue ) . For clarity , a simplified illustration is provided for each sub - pool . Magnification : 12 , 000x ; scale bar , 40 nm . graph is showing of MP sub - pools shown in A - E of MP - SVs at inhibitory , resting stimulatory wild - type synapses .

O B-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O

Representative tomographic virtual sections from resting wild-type. Five different types of MP-SVs are observed that we classify as sub-pools: non-tethered SVs (A',A''; yellow), single presynaptic density-attached SVs (B',B''; Pd.a, orange, pink arrowhead) or single m-attached SVs (C',C''; Ma., orange, orange arrowhead), or multiple-tethered SVs (D',D''; orange, brown arrowheads) and docked SVs (E',E''; light orange). Ribbon (R), presynaptic density (PD, pink) and AZ-membrane (blue). For clarity, a simplified illustration is provided for each sub-pool. Magnification: 12,000x; scale bar, 40 nm. The graph is showing the percentage of the MP sub-pools shown in A-E of all MP-SVs at inhibitory, resting and stimulatory wild-type synapses.


Depicts fraction of non - tethered single - tethered MP - SVs per ribbon . Data are presented in mean ± . fraction of non - tethered SVs reduces in wild - type OtofPga / Pga after stimulation fraction of single - attached tethers increases in wild - type upon stimulation . In resting wild - type , most single - tethered SVs are attached to presynaptic density .

O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O 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-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O

Depicts the fraction of non-tethered and single-tethered MP-SVs per ribbon. Data are presented in mean ± SEM. The fraction of non-tethered SVs reduces in wild-type and OtofPga/Pga after stimulation and the fraction of single membrane-attached tethers increases in wild-type upon stimulation. In resting wild-type, most single-tethered SVs are attached to the presynaptic density.


Example tomographic virtual sections from inhibitory : wild - type ( A ) OtofPga / Pga ( B ) ; stimulatory : wild - type ( C ) OtofPga / Pga ( D ) . Tethered SVs are highlighted in orange with orange tether ( s ) ; single - tethers ( with orange arrowhead ) multiple - tethers ( with brown arrowhead ) . number of tethers per SV is indicated . Magnification : 12 , 000x ; scale bar , 40 nm . Virtual sections from inhibitory : wild - type ( E ) OtofPga / Pga ( F ) ; stimulatory : wild - type ( G ) OtofPga / Pga ( H ) . Docked SVs at AZ - ( blue ) are highlighted ( orange asterisk ) . Some docked SVs are localized close to presynaptic density ( PD , pink outline ) , anchoring ribbon ( R ) . Magnification : 12 , 000x ; scale bar , 100 nm .

O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O 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-EXP_ASSAY O O O B-SUBCELLULAR O O 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-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O

Example tomographic virtual sections from inhibitory: wild-type (A) and OtofPga/Pga (B); stimulatory: wild-type (C) and OtofPga/Pga (D). Tethered SVs are highlighted in orange with orange tether(s); single-tethers (with orange arrowhead) and multiple-tethers (with brown arrowhead). The number of tethers per SV is indicated. Magnification: 12,000x; scale bar, 40 nm. Virtual sections from inhibitory: wild-type (E) and OtofPga/Pga (F); stimulatory: wild-type (G) and OtofPga/Pga (H). Docked SVs at the AZ-membrane (blue line) are highlighted (orange asterisk). Some docked SVs are localized close to the presynaptic density (PD, pink outline), anchoring the ribbon (R). Magnification: 12,000x; scale bar, 100 nm.


of multiple - tethered MP - SVs per ribbon of total MP - SV pool show more multiple - tethered SVs in OtofPga / Pga IHCs . of docked MP - SVs per ribbon of total MP - pool . number of docked SVs is significantly increased at OtofPga / Pga inhibited specifically at stimulated AZs . P - values for ( I ) ( J ) are calculated by Fisher ' s exact Chi - square , wild - type : inhib , n = 102 SVs ; , n = 82 SVs ; stim , n = 99 SVs ; OtofPga / Pga : inhib , n = 123 SVs ; , n = 96 SVs ; stim , n = 111 SVs ; ns p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O B-GENEPROD O O B-CELL_TYPE O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O 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-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Percentage of multiple-tethered MP-SVs per ribbon of the total MP-SV pool show more multiple-tethered SVs in OtofPga/Pga IHCs. Percentage of docked MP-SVs per ribbon of the total MP-pool. The number of docked SVs is significantly increased at OtofPga/Pga inhibited and specifically at stimulated AZs. P-values for (I) and (J) are calculated by Fisher's exact Chi-square test, wild-type: inhib, n = 102 SVs; rest, n = 82 SVs; stim, n = 99 SVs; OtofPga/Pga: inhib, n = 123 SVs; rest, n = 96 SVs; stim, n = 111 SVs; ns p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001