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 .

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


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 .

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

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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 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 .

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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 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 .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 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 .

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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 ) 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 .

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

(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 staining and PAS staining of lung sections from wild - type ( WT ) and 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 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 .

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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 ) 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 .

O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

(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 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 .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

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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 analysis 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 ) 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 .

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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 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 .

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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 analysis * P < 0 . 05 ( unpaired t test ) . 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

(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 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 .

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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 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 .

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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 the MN complex . Mif2 and Ame1 wild - type N - termini serve as a control , protein bands are highlighted in the 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.


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 .

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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 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 .

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

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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.


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 .

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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 levels 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 the indicated construct .

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


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 .

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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.


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 .

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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.


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 .

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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 levels 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 cells . Proteins 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 the 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 and corresponding Coomassie stained SDS - PAGE gels of Mtw1c ( blue ) , Cse4n ( light 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 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 .

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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 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 .

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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 and corresponding Coomassie stained SDS - PAGE gels of Mtw1c ( blue ) , Cse4n ( light 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 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 .

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

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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.


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 .

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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 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 .

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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 and Mtw1 - Myc from yeast cell extracts .

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


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 .

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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 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 ;

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

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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 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 .

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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 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 .

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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.


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 .

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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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

O 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

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 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 .

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 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

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

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.


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 .

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

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


Bar - plots show the distance of multiple - tethered ( O ) and docked ( P ) SVs from presynaptic density ( PD ) , 0 - 100 nm : MP - SV pool ( dark blue ) ; 100 - 500 nm : peripheral SV pool ( light blue ) . Upon stimulation , multiple - tethered and docked SVs are localized significantly closer to the PD in OtofPga / Pga . P - values for ( O ) and ( P ) are calculated by Wilcoxon Rank test , * p < 0 . 05 , * * * p < 0 . 001 , ns p > 0 . 05 and Ø inconclusive statistics because occurrence of docked SVs in wild - type resting condition is extremely rare . Multiple - tethered SVs , wild - type : inhib , n = 3 SVs ; rest , n = 13 SVs ; stim , n = 3 SVs ; OtofPga / Pga : inhib , n = 21 SVs ; rest , n = 34 SVs ; stim , n = 16 SVs ; docked SVs , wild - type : inhib , n = 2 SVs ; rest , n = 1 SVs ; stim , n = 7 SVs ; OtofPga / Pga : inhib , n = 20 SVs ; rest , n = 4 SVs ; stim , n = 18 SVs .

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

Bar-plots show the distance of multiple-tethered (O) and docked (P) SVs from presynaptic density (PD), 0-100 nm: MP-SV pool (dark blue); 100-500 nm: peripheral SV pool (light blue). Upon stimulation, multiple-tethered and docked SVs are localized significantly closer to the PD in OtofPga/Pga. P-values for (O) and (P) are calculated by Wilcoxon Rank test, * p < 0.05, *** p < 0.001, ns p > 0.05 and Ø inconclusive statistics because occurrence of docked SVs in wild-type resting condition is extremely rare. Multiple-tethered SVs, wild-type: inhib, n = 3 SVs; rest, n = 13 SVs; stim, n = 3 SVs; OtofPga/Pga: inhib, n = 21 SVs; rest, n = 34 SVs; stim, n = 16 SVs; docked SVs, wild-type: inhib, n = 2 SVs; rest, n = 1 SVs; stim, n = 7 SVs; OtofPga/Pga: inhib, n = 20 SVs; rest, n = 4 SVs; stim, n = 18 SVs.


Example virtual sections depict SVs with single membrane - ( orange arrowhead ) and presynaptic density ( pink arrowhead ) - attached tethers in resting wild - type . Magnification 12 , 000x ; scale bar , 40 nm . 3D reconstructions of same tethered SVs ( orange ) as shown in ( A ) and ( B ) ; membrane - attached ( A ' , yellow tether ) and presynaptic density ( PD ) - attached ( B ' , pink tether ) . Individual tether lengths are depicted . For clarity , ribbon ( R ) and AZ - membrane are shown in transparent . Scale bar , 20 nm .

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

Example virtual sections depict SVs with single membrane- (orange arrowhead) and presynaptic density (pink arrowhead) -attached tethers in resting wild-type. Magnification 12,000x; scale bar, 40 nm. 3D reconstructions of same tethered SVs (orange) as shown in (A) and (B); membrane-attached (A', yellow tether) and presynaptic density (PD) -attached (B', pink tether). Individual tether lengths are depicted. For clarity, ribbon (R) and AZ-membrane are shown in transparent. Scale bar, 20 nm.


The graph depicts the mean length of single membrane - , presynaptic density - attached and multiple - tethers . Data are presented as mean ± SEM . Stimulated OtofPga / Pga synapses exhibit a shorter average tether length compared to stimulated wild - 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 O O O O O O O O O O O O O O O O O

The graph depicts the mean length of single membrane-, presynaptic density-attached and multiple-tethers. Data are presented as mean ± SEM. Stimulated OtofPga/Pga synapses exhibit a shorter average tether length compared to stimulated wild-type.


The cumulative probability distribution of MP - SVs plotted against the tether length . The red dotted lines represent the mean MP tether length in resting wild - type for each sub - pool .

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

The cumulative probability distribution of MP-SVs plotted against the tether length. The red dotted lines represent the mean MP tether length in resting wild-type for each sub-pool.


Total mean tether length in the MP - SV pool . Data are presented as mean ± SEM . Longer tethers are present upon stimulation in wild - type . Resting OtofPga / Pga ribbon synapses harbor the longest tethers , and tether where shorter at stimulated OtofPga / Pga synapses compared to wild - type . The red dotted lines represent the mean MP tether length in resting wild - type for each sub - pool .

O O O O O O O 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 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

Total mean tether length in the MP-SV pool. Data are presented as mean ± SEM. Longer tethers are present upon stimulation in wild-type. Resting OtofPga/Pga ribbon synapses harbor the longest tethers, and tether where shorter at stimulated OtofPga/Pga synapses compared to wild-type. The red dotted lines represent the mean MP tether length in resting wild-type for each sub-pool.


( a ) Expression of H2B - mCherry under control of the CLV3 promoter in 14 - d - old seedlings . Whole - mount immunostaining using α - mCherry antibodies and laser scanning microscopy ( scale bar 10 μm ) .

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

(a) Expression of H2B-mCherry under control of the CLV3 promoter in 14-d-old seedlings. Whole-mount immunostaining using α-mCherry antibodies and laser scanning microscopy (scale bar 10 μm).


( c ) A representative example for enrichment of CLV3 transcript in mCherry - positive nuclei determined by qRT - PCR and normalized to wt ( N = 1 ) .

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

(c) A representative example for enrichment of CLV3 transcript in mCherry-positive nuclei determined by qRT-PCR and normalized to wt (N = 1).


( a ) Expression of CLV3 , mCherry , TEL2 , PAN , and the meristem marker genes STM and KNAT1 . Asterisks indicate timepoints of significantly different expression ( Wald test , Benjamini & Hochberg corrected , q < 0 . 05 ) between stem and non - stem cells for each time point ( N = 2 ) . + = stem cell nuclei ; - = non - stem cell nuclei , E = nuclei from embryos , D7 / 14 / 35 = nuclei from 7 / 14 / 35 day - old plants , S14 = nuclei from 14 d - old above - ground seedlings .

O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression of CLV3, mCherry, TEL2, PAN, and the meristem marker genes STM and KNAT1. Asterisks indicate timepoints of significantly different expression (Wald test, Benjamini & Hochberg corrected, q<0.05) between stem and non-stem cells for each time point (N = 2). + = stem cell nuclei; - = non-stem cell nuclei, E = nuclei from embryos, D7/14/35 = nuclei from 7/14/35 day-old plants, S14 = nuclei from 14 d-old above-ground seedlings.


( c ) Number of DEGs between stem and non - stem cells at each timepoint . The banded portion of the bars indicates the number of transcription factor genes ( also in parenthesis ) .

O O O O O O O O O O O O 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) Number of DEGs between stem and non-stem cells at each timepoint. The banded portion of the bars indicates the number of transcription factor genes (also in parenthesis).


( b ) Expression of individual significantly up - regulated genes marked with # in ( a ) . Asterisks indicate timepoints of significantly different expression ( Wald test , Benjamini & Hochberg corrected , q < 0 . 05 ) between stem and non - stem cells for each time point ( N = 2 ) . + = stem cells ; - = non - stem cells , E = nuclei from embryos , D7 / 14 / 35 = nuclei from 7 / 14 / 35 day - old plants , S14 = nuclei from 14 d - old above - ground seedlings .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression of individual significantly up-regulated genes marked with # in (a). Asterisks indicate timepoints of significantly different expression (Wald test, Benjamini & Hochberg corrected, q<0.05) between stem and non-stem cells for each time point (N = 2).+ = stem cells; - = non-stem cells, E = nuclei from embryos, D7/14/35 = nuclei from 7/14/35 day-old plants, S14 = nuclei from 14 d-old above-ground seedlings.


( a ) Box plot summarizing differential expression of unambiguously mapped individual TEs , compared with all protein - coding genes , or housekeeping genes . Box plots outline the interquartile range ( IQR ) with the median and whiskers + / - 1 . 5 IQR . For all p - values , see also Appendix Table EV6 . * * indicate a p - value smaller than 1 . 01e - 11 ( two sided non paired Wilcoxon rank sum test ) .

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

(a) Box plot summarizing differential expression of unambiguously mapped individual TEs, compared with all protein-coding genes, or housekeeping genes. Box plots outline the interquartile range (IQR) with the median and whiskers +/- 1.5 IQR . For all p-values, see also Appendix Table EV6. ** indicate a p-value smaller than 1.01e-11 (two sided non paired Wilcoxon rank sum test).


( a ) Increased expression of TEs in stem cells of D7 seedlings according to qRT - PCR . Box plots outline the interquartile range ( IQR ) with the median and whiskers + / - 1 . 5 IQR . N = 2 ( ATLANTYS3 , ATCOPIA39 , ATCOPIA49 , ATCOPIA29 , ATGP1 - 1 ) , N = 3 ( VANDAL6 , ATGP2 , ATGP1 - 2 , ATLINE1 , ATGP1 - 3 ) , N = 4 ( ATHILA6A , ATCOPIA83 ) , N = 5 ( VANDAL12 , ATHILA3 , ATCOPIA22 ) , and N = 6 for CLAVATA . * p < 0 . 01 , * * p < 0 . 001 ( t - test ) .

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

(a) Increased expression of TEs in stem cells of D7 seedlings according to qRT-PCR. Box plots outline the interquartile range (IQR) with the median and whiskers +/- 1.5 IQR. N = 2 (ATLANTYS3, ATCOPIA39, ATCOPIA49, ATCOPIA29, ATGP1-1), N = 3 (VANDAL6, ATGP2, ATGP1-2, ATLINE1, ATGP1-3), N = 4 (ATHILA6A, ATCOPIA83), N = 5 (VANDAL12, ATHILA3, ATCOPIA22), and N = 6 for CLAVATA. *p<0.01, **p<0.001 (t-test).


( b ) Heatmap of TE expression in several silencing mutants determined by qRT - PCR relative to a housekeeping gene . To be able to display data in log - scale also for samples with zero expression , a value of 1 . 0e - 6 ( factor 10 smaller than the smallest value ) was added to all values . Data for nuclei and CLV3 : : H2BmCherry are the same as in ( a ) . For seedling samples , N = 2 ( ATCOPIA49 , ATHILA3 ) , N = 3 ( ATCOPIA39 , VANDAL6 , ATGP2 , ATLINE1 , ATCOPIA29 , ATGP1 - 1 ) , and N = 4 ( ATLANTYS3 , VANDAL12 , ATGP1 - 2 , ATHILA6A ) .

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

(b) Heatmap of TE expression in several silencing mutants determined by qRT-PCR relative to a housekeeping gene. To be able to display data in log-scale also for samples with zero expression, a value of 1.0e-6 (factor 10 smaller than the smallest value) was added to all values. Data for nuclei and CLV3::H2BmCherry are the same as in (a). For seedling samples, N = 2 (ATCOPIA49, ATHILA3), N = 3 (ATCOPIA39, VANDAL6, ATGP2, ATLINE1, ATCOPIA29, ATGP1-1), and N = 4 (ATLANTYS3, VANDAL12, ATGP1-2, ATHILA6A).


( a ) CG , CHG , and CHH methylation at chromosome 3 in stem and non - stem cells . The inset for CHG methylation shows a magnification of the pericentromeric region .

O O O O O O O O O O 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) CG, CHG, and CHH methylation at chromosome 3 in stem and non-stem cells. The inset for CHG methylation shows a magnification of the pericentromeric region.


( a ) DNA methylation in % at CHG ( left ) and CHH ( right ) sites in TE with increased expression at D7 , compared between developmental stages ( D7 / 14 / 35 = 7 / 14 / 35 day - old plants ) and nuclei of stem ( + ) and non - stem ( - ) cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) DNA methylation in % at CHG (left) and CHH (right) sites in TE with increased expression at D7, compared between developmental stages (D7/14/35 = 7/14/35 day-old plants) and nuclei of stem (+) and non-stem (-) cells.


( b ) Genomic location of the TEs ( blue ) from ( a ) on the five chromosomes . Dark grey bars indicate the location of centromeres and blue lines indicate global TE density .

(b) Genomic location of the TEs (blue) from (a) on the five chromosomes. Dark grey bars indicate the location of centromeres and blue lines indicate global TE density.


( a ) DNA methylation differences between stem and non - stem cells and their enrichment within DMRs of different epigenetic mutants using permutation tests ( data from Stroud , Greenberg , Feng , Bernatavichute and Jacobsen 2013 ) . Mutants are in alphabetical order according to the gene acronym . Red indicates an enrichment of methylated DNA in the respective context and blue a depletion .

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(a) DNA methylation differences between stem and non-stem cells and their enrichment within DMRs of different epigenetic mutants using permutation tests (data from Stroud, Greenberg, Feng, Bernatavichute and Jacobsen 2013). Mutants are in alphabetical order according to the gene acronym. Red indicates an enrichment of methylated DNA in the respective context and blue a depletion.


( K TUNEL staining of testis sections from WT and KO mice at four months of age ( K ) White dotted line indicates the outline of testis tubules .

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(K TUNEL staining of testis sections from WT and KO mice at four months of age (K) White dotted line indicates the outline of testis tubules.


L ) TUNEL staining of testis sections from WT and KO mice at four months of age with apoptotic cell number per tubule ( L ) . n = 3 in ( L ) .

L) TUNEL staining of testis sections from WT and KO mice at four months of age with apoptotic cell number per tubule (L). n=3 in (L).


( A ) Immunostaining of SYCP3 and γH2AX in sections from adult WT mice . Arrow and arrowhead indicate diffuse and dot - like γH2AX staining , respectively . White dotted line indicates the outline of testis tubules . ( B ) Proportion of tubules of γH2AX expression patterns . At least 100 tubules of each mouse were counted . n = 4 for WT and n = 5 for KO . ( C ) Average number of spermatocytes with γH2AX expression patterns per tubule . At least 50 tubules of each mouse were counted . n = 3 .

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(A) Immunostaining of SYCP3 and γH2AX in sections from adult WT mice. Arrow and arrowhead indicate diffuse and dot-like γH2AX staining, respectively. White dotted line indicates the outline of testis tubules. (B) Proportion of tubules of γH2AX expression patterns. At least 100 tubules of each mouse were counted. n=4 for WT and n=5 for KO. (C) Average number of spermatocytes with γH2AX expression patterns per tubule. At least 50 tubules of each mouse were counted. n=3.


( D - H ) Representative nuclear spreads of WT and KO spermatocytes at leptonema ( D ) , zygonema ( E ) , early pachynema ( F ) , late pachynema ( G ) and diplonema ( H ) . Spermatocytes were immunostained for SYCP3 , γH2AX , and H1t . Magnified views are indicated by dashed areas . Schematic representation shows homolog characteristic revealed by SYCP3 signals at different backgrounds and stages . Arrowheads indicate bivalent stretches lacking SYCP3 signals in KO . ( I ) Proportion statistics of spermatocytes with discontinuous SYCP3 signals . At least 200 cells of each mouse were counted . n = 3 for WT and n = 4 for KO . ( J ) Meiotic stage frequencies in ( D - H ) . At least 200 cells of each mouse were counted . n = 3 .

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(D-H) Representative nuclear spreads of WT and KO spermatocytes at leptonema (D), zygonema (E), early pachynema (F), late pachynema (G) and diplonema (H). Spermatocytes were immunostained for SYCP3, γH2AX, and H1t. Magnified views are indicated by dashed areas. Schematic representation shows homolog characteristic revealed by SYCP3 signals at different backgrounds and stages. Arrowheads indicate bivalent stretches lacking SYCP3 signals in KO. (I) Proportion statistics of spermatocytes with discontinuous SYCP3 signals. At least 200 cells of each mouse were counted. n=3 for WT and n=4 for KO. (J) Meiotic stage frequencies in (D-H). At least 200 cells of each mouse were counted. n=3.


( C ) Nuclear - spread spermatocytes from WT and KO mice were immunostained for SYCP3 and MLH1 . Representative images of pachynema are shown . Magnified views are indicated by dashed areas . ( D ) Number of bivalents without an MLH1 focus per pachytene spermatocyte in ( C ) . n = 3 mice . Nor , normal pachynema ; L 1 , pachynema - like 1 ; L 2 , pachynema - like 2 .

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(C) Nuclear-spread spermatocytes from WT and KO mice were immunostained for SYCP3 and MLH1. Representative images of pachynema are shown. Magnified views are indicated by dashed areas. (D) Number of bivalents without an MLH1 focus per pachytene spermatocyte in (C). n=3 mice. Nor, normal pachynema; L 1, pachynema-like 1; L 2, pachynema-like 2.


( E - H ) Nuclear - spread spermatocytes from WT and KO mice were immunostained for SYCP3 together with RAD51 ( E ) or DMC1 ( G ) and quantification of RAD51 ( F ) or DMC1 ( H ) focus numbers at early zygonema , late zygonema , and pachynema . Representative images of early zygonema are shown . n = 3 mice .

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(E-H) Nuclear-spread spermatocytes from WT and KO mice were immunostained for SYCP3 together with RAD51 (E) or DMC1 (G) and quantification of RAD51 (F) or DMC1 (H) focus numbers at early zygonema, late zygonema, and pachynema. Representative images of early zygonema are shown. n=3 mice.


( A - D ) Representative nuclear spreads of WT and KO spermatocytes at leptonema ( A ) , zygonema ( B ) , pachynema ( C ) and diplonema ( D ) . Spermatocytes were immunostained for REC8 and SYCP3 . REC8 foci distribute along SYCP3 - labeled structures . Magnified views are indicated by dashed areas .

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(A-D) Representative nuclear spreads of WT and KO spermatocytes at leptonema (A), zygonema (B), pachynema (C) and diplonema (D). Spermatocytes were immunostained for REC8 and SYCP3. REC8 foci distribute along SYCP3-labeled structures. Magnified views are indicated by dashed areas.


( E ) Inter - REC8 distances along axial elements . Five stretches or homologs were measured and the average distance was calculated for each nucleus . Each point represents one nucleus . The upper panel is the schematic representation for distance measurement . n = 3 mice .

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(E) Inter-REC8 distances along axial elements. Five stretches or homologs were measured and the average distance was calculated for each nucleus. Each point represents one nucleus. The upper panel is the schematic representation for distance measurement. n=3 mice.


( F ) Western blot analyses for REC8 and SEPARASE in spermatocytes isolated by FACS after Hoechst staining . n = 3 mice . ( G ) Normalized density of REC8 in ( F ) . n = 3 mice .

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(F) Western blot analyses for REC8 and SEPARASE in spermatocytes isolated by FACS after Hoechst staining. n=3 mice. (G) Normalized density of REC8 in (F). n=3 mice.


( E ) Western blot analyses for SECURIN and CYCLIN B1 in spermatocytes isolated by FACS after Hoechst staining . n = 3 mice . ( F ) Normalized density of SECURIN and CYCLIN B1 in ( E ) . n = 3 mice .

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(E) Western blot analyses for SECURIN and CYCLIN B1 in spermatocytes isolated by FACS after Hoechst staining. n=3 mice. (F) Normalized density of SECURIN and CYCLIN B1 in (E). n=3 mice.


( F ) Western blot analyses for REC8 and SEPARASE for cells in ( E ) . n = 3 biological replicates . ( G ) Normalized density of REC8 in ( F ) . n = 3 biological replicates .

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(F) Western blot analyses for REC8 and SEPARASE for cells in (E). n=3 biological replicates. (G) Normalized density of REC8 in (F). n=3 biological replicates.


( H ) Western blot analyses for SECURIN and CYCLIN B1 for cells in ( E ) . n = 3 biological replicates . ( I ) Normalized density of SECURIN and CYCLIN B1 in ( H ) . n = 3 biological replicates .

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(H) Western blot analyses for SECURIN and CYCLIN B1 for cells in (E). n=3 biological replicates. (I) Normalized density of SECURIN and CYCLIN B1 in (H). n=3 biological replicates.


Expression of Tardbp , total Sort1 and exon 17b Sort1 mRNAs quantified by Q - PCR in primary hippocampal neurons infected with either scrambled or Tardbp shRNA lentiviruses . Values were first normalized to Gapdh mRNA levels , then plotted as average ± SEM relative to the value of scrambled shRNA ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . * * * , p < 0 . 001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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Expression of Tardbp, total Sort1 and exon 17b Sort1 mRNAs quantified by Q-PCR in primary hippocampal neurons infected with either scrambled or Tardbp shRNA lentiviruses. Values were first normalized to Gapdh mRNA levels, then plotted as average ± SEM relative to the value of scrambled shRNA (N=3 independent experiments; n=3 wells per condition in each experiment). ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Western blots of cell lysates and culture supernatants showing TDP - 43 and Sortilin expression in primary hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses . Uninfected cultures were used as an additional control . β - actin immunoblotting and Coomasise Blue staining were used to control equal loading of lysates and supernatants , respectively . Molecular weights are indicated in kDa . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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Western blots of cell lysates and culture supernatants showing TDP-43 and Sortilin expression in primary hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses. Uninfected cultures were used as an additional control. β-actin immunoblotting and Coomasise Blue staining were used to control equal loading of lysates and supernatants, respectively. Molecular weights are indicated in kDa. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Representative photomicrographs of cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses , immunolabled for BDNF ( green ) , SCG2 ( red ) and counterstained with DAPI ( blue ) in orthogonal views ( dashed lines ) showing colocalization of BDNF and SCG2 in the soma ( white arrows ) .

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Representative photomicrographs of cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses, immunolabled for BDNF (green), SCG2 (red) and counterstained with DAPI (blue) in orthogonal views (dashed lines) showing colocalization of BDNF and SCG2 in the soma (white arrows).


Colocalization between BDNF and SCG2 - positive vesicles expressed as percentage of total BDNF immunoreactivity ± SEM in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses ( N = 3 independent experiments , n > 50 individual cells per condition in each experiment ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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Colocalization between BDNF and SCG2-positive vesicles expressed as percentage of total BDNF immunoreactivity ±SEM in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses (N=3 independent experiments, n>50 individual cells per condition in each experiment). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Colocalization between BDNF and SCG2 - positive vesicles in hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses together with either EGFP or Sortilin rescue viruses . Histogram bars show average ± SEM BDNF / SCG2 colocalization expressed as percentage of total BDNF immunoreactivity ( N = 3 independent experiments , n > 50 individual cells per condition in each experiment ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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Colocalization between BDNF and SCG2-positive vesicles in hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses together with either EGFP or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF/SCG2 colocalization expressed as percentage of total BDNF immunoreactivity (N=3 independent experiments, n>50 individual cells per condition in each experiment). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


BDNF secretion induced by KCl treatment assessed by in - situ BDNF ELISA in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses . Histogram bars show average ± SEM BDNF levels normalized to a standard curve ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . * , p < 0 . 05 ; * * * , p < 0 . 001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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BDNF secretion induced by KCl treatment assessed by in-situ BDNF ELISA in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=3 independent experiments; n=3 wells per condition in each experiment). *, p<0.05; ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


BDNF secretion in cultured hippocampal neurons infected with scrambled ( left ) or Tardbp ( right ) shRNA lentiviruses together with either EGFP , Sort1 shRNA or Sortilin rescue viruses . Histogram bars show average ± SEM BDNF levels normalized to a standard curve ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . * * * , p < 0 . 001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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BDNF secretion in cultured hippocampal neurons infected with scrambled (left) or Tardbp (right) shRNA lentiviruses together with either EGFP, Sort1 shRNA or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=3 independent experiments; n=3 wells per condition in each experiment). ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Representative photomicrographs of TDP - 43 ( green ) and NeuN ( red ) immunohistochemistry in hippocampal CA1 of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Arrows denote areas of depleted TDP - 43 immunoreactivity in the mutant .

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Representative photomicrographs of TDP-43 (green) and NeuN (red) immunohistochemistry in hippocampal CA1 of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Arrows denote areas of depleted TDP-43 immunoreactivity in the mutant.


Percentage of TDP - 43 positive cells relative to NeuN cells in CA1 and the remaining hippocampus ( HC [ - CA1 ] ) of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± SEM ( N = 3 mice per condition ; 5 sections per mouse ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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Percentage of TDP-43 positive cells relative to NeuN cells in CA1 and the remaining hippocampus (HC [-CA1]) of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SEM (N=3 mice per condition; 5 sections per mouse). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


TDP - 43 intensity in TDP - 43 / NeuN double - positive cells in CA1 and the remaining hippocampus of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± SEM ( N = 3 mice per condition ; 5 sections per mouse ) . * , p < 0 . 05 ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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TDP-43 intensity in TDP-43/NeuN double-positive cells in CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SEM (N=3 mice per condition; 5 sections per mouse). *, p<0.05; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Expression of Tardbp mRNA in micro - dissected CA1 and the remaining hippocampus of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice quantified by Q - PCR . Results are presented as average ± SEM relative to levels in Tardbpfx / fx mice , after normalization to Gapdh mRNA . ( N = 3 mice per condition ; * * , p < 0 . 01 ) . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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Expression of Tardbp mRNA in micro-dissected CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice quantified by Q-PCR. Results are presented as average ± SEM relative to levels in Tardbpfx/fx mice, after normalization to Gapdh mRNA. (N=3 mice per condition; **, p<0.01). Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.