( h - i ) Quantification of cluster formation assays performed with mCherry - p62 and GST - 4xUb in presence of the indicated free ubiquitin chains . Ubiquitin chains were either pre - mixed with p62 before the addition of GST - 4xUb ( h ) , or added 3 minutes after the addition of GST - 4xUb to p62 ( i )

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(h-i) Quantification of cluster formation assays performed with mCherry-p62 and GST-4xUb in presence of the indicated free ubiquitin chains. Ubiquitin chains were either pre-mixed with p62 before the addition of GST-4xUb (h), or added 3 minutes after the addition of GST-4xUb to p62 (i)


( j ) Quantification of cluster formation assays conducted with mCherry - 2xFKBP - p62 in presence of AP20187 , GST - 4xUB and the indicated Ub

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(j) Quantification of cluster formation assays conducted with mCherry-2xFKBP-p62 in presence of AP20187, GST-4xUB and the indicated Ub chains


( k ) Representative micrographs of glutathione beads coated with the indicated GST - tagged proteins and incubated with the indicated mCherry - p62 variants . In all pictures the brightness was adjusted to highlight the profile of the beads . For all graphs averages and SDs from at least three independent replicates are

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(k) Representative micrographs of glutathione beads coated with the indicated GST-tagged proteins and incubated with the indicated mCherry-p62 variants. In all pictures the brightness was adjusted to highlight the profile of the beads. For all graphs averages and SDs from at least three independent replicates are shown


( a ) mCherry - p62 / GST - GFP - 4xUb clusters were photobleached and fluorescence recovery in the bleached area was monitored . The mCherry - tagged low complexity domain ( LCD ) of hnRNPA1 ( hnRNPA1LCD ) was included as a reference for a liquid droplet - forming protein . Left : representative micrographs of bleached particles . White arrows indicate the position and orientation of the kymographs shown in Figure EV4a . Right : quantification of fluorescence recovery . Individual traces of all particles analyzed from three independent replicates are displayed

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(a) mCherry-p62/GST-GFP-4xUb clusters were photobleached and fluorescence recovery in the bleached area was monitored. The mCherry-tagged low complexity domain (LCD) of hnRNPA1 (hnRNPA1LCD) was included as a reference for a liquid droplet-forming protein. Left: representative micrographs of bleached particles. White arrows indicate the position and orientation of the kymographs shown in Figure EV4a. Right: quantification of fluorescence recovery. Individual traces of all particles analyzed from three independent replicates are displayed


( b ) p62 puncta in STG - p62 cells were photobleached and the fluorescence recovery was monitored . Left : representative micrographs of bleached cells . White squares indicate the bleached puncta . Insert : magnification of the bleached puncta . Right : quantification of fluorescence recovery . Averages and SDs of 28 ( unbleached ) and 29 ( bleached ) puncta from three independent experiments are

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(b) p62 puncta in STG-p62 cells were photobleached and the fluorescence recovery was monitored. Left: representative micrographs of bleached cells. White squares indicate the bleached puncta. Insert: magnification of the bleached puncta. Right: quantification of fluorescence recovery. Averages and SDs of 28 (unbleached) and 29 (bleached) puncta from three independent experiments are shown


( c ) Representative electron micrographs of negatively stained p62 filaments incubated in presence or absence of GST - 4xUb for the indicated

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(c) Representative electron micrographs of negatively stained p62 filaments incubated in presence or absence of GST-4xUb for the indicated times


( a ) Top : schematic representation of the domain architecture of the GFP - NBR1 construct . PB1 : Phox and Bem1p domain , ZZ : Zinc finger , CC : coiled - coil , LIR : LC3 - interacting region , UBA : ubiquitin - associated domain . Bottom : GFP - NBR1 was recruited to glutathione beads coated with the indicated GST fusion proteins and imaged by spinning disc microscopy . Representative micrographs of at least 70 beads imaged per sample from two independent experiments

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(a) Top: schematic representation of the domain architecture of the GFP-NBR1 construct. PB1: Phox and Bem1p domain, ZZ: Zinc finger, CC: coiled-coil, LIR: LC3-interacting region, UBA: ubiquitin-associated domain. Bottom: GFP-NBR1 was recruited to glutathione beads coated with the indicated GST fusion proteins and imaged by spinning disc microscopy. Representative micrographs of at least 70 beads imaged per sample from two independent experiments


( b - c ) mCherry - p62 variants were recruited to GFP - or GFP - NBR1 - coated beads and imaged as in ( a ) ( b ) Representative micrographs of at least 70 beads per sample from three independent

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(b-c) mCherry-p62 variants were recruited to GFP- or GFP-NBR1-coated beads and imaged as in (a) (b) Representative micrographs of at least 70 beads per sample from three independent replicates


( d - e ) 2µM mCherry - p62 and / or GFP - NBR1 were incubated in presence of 5µM GST - 4xUb . Representative micrographs ( d ) and quantification of particle number for each channel ( e ) are shown . Averages and SDs from at least three independent experiments are

(d-e) 2µM mCherry-p62 and/or GFP-NBR1 were incubated in presence of 5µM GST-4xUb. Representative micrographs (d) and quantification of particle number for each channel (e) are shown. Averages and SDs from at least three independent experiments are shown


( a ) Cluster formation assays were conducted in presence or absence of 20µM mCherry - LC3B or mCherry , either pre - mixed with p62 or added 1 " after the addition of GST - 4xUb . Quantification of particle number ( left ) and size ( right ) are shown

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(a) Cluster formation assays were conducted in presence or absence of 20µM mCherry-LC3B or mCherry, either pre-mixed with p62 or added 1" after the addition of GST-4xUb. Quantification of particle number (left) and size (right) are shown


( b ) Cluster formation assays were conduced with mCherry - p62 wt or LIR mutant in presence of GST - 4xUb . Left : representative images of the samples two minutes after the addition of GST - 4xUb . Middle : quantification of particle count . The dashed line indicates the time point of images displayed on the left . Right : input gel of the samples after reaction . The relative position of marker bands is indicated . For all graphs , averages and SDs from at least three independent replicates are

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(b) Cluster formation assays were conduced with mCherry-p62 wt or LIR mutant in presence of GST-4xUb. Left: representative images of the samples two minutes after the addition of GST-4xUb. Middle: quantification of particle count. The dashed line indicates the time point of images displayed on the left. Right: input gel of the samples after reaction. The relative position of marker bands is indicated. For all graphs, averages and SDs from at least three independent replicates are shown


( c ) Top left : Western blots of lysates of CRISPR / Cas9 genome - edited STG - p62 wild type and LIR mutant cells . Clone Lir1B was used for the experiments shown in Figure 6c . Bottom left : Representative pictures of STG - p62 WT or LIR mutant Hap1 cells treated with Puromycin for 90 minutes and stained with anti - Ub conjugates antibody ( FK2 ) . Right : quantification of cytoplasmic p62 puncta per cell ( top ) , and of their co - localization upon puromycin treatment ( bottom ) . Averages and SDs from three independent experiments are shown . In total , at least 1440 number of cells were counted per

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

(c) Top left: Western blots of lysates of CRISPR/Cas9 genome-edited STG-p62 wild type and LIR mutant cells. Clone Lir1B was used for the experiments shown in Figure 6c. Bottom left: Representative pictures of STG-p62 WT or LIR mutant Hap1 cells treated with Puromycin for 90 minutes and stained with anti-Ub conjugates antibody (FK2). Right: quantification of cytoplasmic p62 puncta per cell (top), and of their co-localization upon puromycin treatment (bottom). Averages and SDs from three independent experiments are shown. In total, at least 1440 number of cells were counted per condition


A . SDS - PAGE analysis of purified T4SS3 - 10 + D4 complex . Left panel : Coomassie - stained SDS - PAGE gel of the T4SS3 - 10 + D4 complex . Right panel : Sypro Ruby - stained SDS - PAGE gel of the T4SS3 - 10m and T4SS3 - 10 + D4 complexes . Molecular weights of MW markers are shown on the left . Identification of bands is shown in the middle . " * " indicates TrwK / VirB4 degradation products . " * * " indicates minor contaminants OmpA and OmpC .

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A. SDS-PAGE analysis of purified T4SS3-10+D4 complex. Left panel: Coomassie-stained SDS-PAGE gel of the T4SS3-10+D4 complex. Right panel: Sypro Ruby-stained SDS-PAGE gel of the T4SS3-10m and T4SS3-10+D4 complexes. Molecular weights of MW markers are shown on the left. Identification of bands is shown in the middle. "*" indicates TrwK/VirB4 degradation products. "**" indicates minor contaminants OmpA and OmpC.


B . Stoichiometry measurement . Fluorescence scan of an SDS - PAGE gel analysis of the T4SS3 - 10 + D4 complex where cysteine residues were reacted with AlexaFluor 633 C5 maleimide . Signals corresponding to the proteins coupled to AlexaFluor 633 were detected at a wavelength of 633 nm . Fluorescent Trw protein bands are labelled and the derived stoichiometry for TrwB / VirD4 is indicated .

B. Stoichiometry measurement. Fluorescence scan of an SDS-PAGE gel analysis of the T4SS3-10+D4 complex where cysteine residues were reacted with AlexaFluor 633 C5 maleimide. Signals corresponding to the proteins coupled to AlexaFluor 633 were detected at a wavelength of 633 nm. Fluorescent Trw protein bands are labelled and the derived stoichiometry for TrwB/VirD4 is indicated.


C . SDS - PAGE analysis of complexes purified from cell expressing deletion mutants of the pBADM11 _ trwN / virB1 - trwE / virB10Strep _ trwD / virB11 _ HistrwB / virD4 constructs termed pBADM11 _ ΔtrwX / ΔvirBY ( see main text ) . The same molecular weights marker has been used on this gel as in the gel presented in Figure 1A . * indicates degradation products of TrwK / VirB4 .

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C. SDS-PAGE analysis of complexes purified from cell expressing deletion mutants of the pBADM11_trwN/virB1-trwE/virB10Strep_trwD/virB11_HistrwB/virD4 constructs termed pBADM11_ΔtrwX/ΔvirBY (see main text). The same molecular weights marker has been used on this gel as in the gel presented in Figure 1A. * indicates degradation products of TrwK/VirB4.


Figure 2 . Interaction between TrwB / VirD4 and TrwE / VirB10 . Left panel : Coomassie - stained SDS - PAGE gel of the T4SS3 - 10 + D4 complex ( lane 1 ) , T4SS3 - 10 + D4 : TrwEΔN42 complex ( lane 2 ) and T4SS3 - 10 + D4 : TrwEΔN64 complex ( lane 3 ) . Middle panel : Western Blot analysis using αStrep antibodies to detect the TrwE / VirB10 protein within the T4SS3 - 10 + D4 complex and its variants . Right panel : Western Blot analysis using αHis antibodies to detect the TrwB / VirD4 protein within the T4SS3 - 10 + D4 complex and its variants .

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Figure 2. Interaction between TrwB/VirD4 and TrwE/VirB10. Left panel: Coomassie-stained SDS-PAGE gel of the T4SS3-10+D4 complex (lane 1), T4SS3-10+D4:TrwEΔN42 complex (lane 2) and T4SS3-10+D4:TrwEΔN64 complex (lane 3). Middle panel: Western Blot analysis using αStrep antibodies to detect the TrwE/VirB10 protein within the T4SS3-10+D4 complex and its variants. Right panel: Western Blot analysis using αHis antibodies to detect the TrwB/VirD4 protein within the T4SS3-10+D4 complex and its variants.


A . NS - EM micrograph of the T4SS3 - 10 + D4 complex . A few representative particles of the T4SS3 - 10 + D4 complex are circled in yellow . Scale bar 50nm . B . Representative class averages of T4SS3 - 10 + D4 complex obtained after initial alignment and classification .

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A. NS-EM micrograph of the T4SS3-10+D4 complex. A few representative particles of the T4SS3-10+D4 complex are circled in yellow. Scale bar 50nm. B. Representative class averages of T4SS3-10+D4 complex obtained after initial alignment and classification.


C . The upper row represents typical class averages of the T4SS3 - 10 + D4 IMC complex ( i . e . with the outer membrane core complex ( OMC ) masked out ) . The second row shows projections of the T4SS3 - 10 + D4 IMC complex in the same directions determined for the classes above . The third row displays projections of the T4SS3 - 10 IMC complex ( EMD - 2567 ) in the same directions . The bottom row shows the differences between the projections above ( plus or minus TrwB / VirD4 ) corresponding to positions of the TrwB / VirD4 protein .

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C. The upper row represents typical class averages of the T4SS3-10+D4 IMC complex (i.e. with the outer membrane core complex (OMC) masked out). The second row shows projections of the T4SS3-10+D4 IMC complex in the same directions determined for the classes above. The third row displays projections of the T4SS3-10 IMC complex (EMD-2567) in the same directions. The bottom row shows the differences between the projections above (plus or minus TrwB/VirD4) corresponding to positions of the TrwB/VirD4 protein.


D . FSC of the electron density map of the T4SS3 - 10 + D4 complex . The 0 . 5 line crosses the FSC at a resolution of 28 Å .

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D. FSC of the electron density map of the T4SS3-10+D4 complex. The 0.5 line crosses the FSC at a resolution of 28 Å.


A . Side views of the NS - EM structure of the T4SS3 - 10 ( left ) and T4SS3 - 10 + D4 ( right ) complexes . The arches , the TrwK / VirB4 barrels , and the TrwB / VirD4 density are shown in green , yellow , and blue , respectively . The boundaries of the 3 - tiers structure of the TrwK / VirB4 barrel are indicated in the middle of the panels and each tier is labelled as lower , middle , and upper , respectively .

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A. Side views of the NS-EM structure of the T4SS3-10 (left) and T4SS3-10+D4 (right) complexes. The arches, the TrwK/VirB4 barrels, and the TrwB/VirD4 density are shown in green, yellow, and blue, respectively. The boundaries of the 3-tiers structure of the TrwK/VirB4 barrel are indicated in the middle of the panels and each tier is labelled as lower, middle, and upper, respectively.


B . Side view of the T4SS3 - 10 + D4 structure ( upper panel ) and derived schematic representation ( lower panel ) . Overall dimensions are indicated C . Same as in B except that the side - view shown is rotated 90 degrees along a vertical axis compared to the view shown in panel B . Overall dimension is indicated . D . Bottom view of the NS - EM structure of the T4SS3 - 10 ( upper panel ) and the corresponding schematic representation ( lower panel ) . Angle between the axes connecting the TrwK / VirB4 hexamer pair and the TrwB / VirD4 dimer pair is shown .

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B. Side view of the T4SS3-10+D4 structure (upper panel) and derived schematic representation (lower panel). Overall dimensions are indicated C. Same as in B except that the side-view shown is rotated 90 degrees along a vertical axis compared to the view shown in panel B. Overall dimension is indicated. D. Bottom view of the NS-EM structure of the T4SS3-10 (upper panel) and the corresponding schematic representation (lower panel). Angle between the axes connecting the TrwK/VirB4 hexamer pair and the TrwB/VirD4 dimer pair is shown.


B . Class averages of antibody - bound T4SS3 - 10 + D4FLAG . Class averages of the aligned NS - EM images of the T4SS3 - 10 + D4FLAG with 3 - 4 particles per class . Red circles indicate the position of the antibody .

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B. Class averages of antibody-bound T4SS3-10+D4FLAG. Class averages of the aligned NS-EM images of the T4SS3-10+D4FLAG with 3-4 particles per class. Red circles indicate the position of the antibody.


C Representative D - type motor neurons in wild - type and mxl - 1 mutant animals 24 h after laser surgery . In wild - type animals , a severed axon has regenerated a growth cone ( arrow ) . In mutants , proximal ends of axons failed to regenerate ( arrowhead ) . Scale bar = 10 μm .

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C Representative D-type motor neurons in wild-type and mxl-1 mutant animals 24 h after laser surgery. In wild-type animals, a severed axon has regenerated a growth cone (arrow). In mutants, proximal ends of axons failed to regenerate (arrowhead). Scale bar = 10 μm.


D Percentages of axons that initiated regeneration 24 h after laser surgery . The numbers ( n ) of animals examined are shown . Error bars indicate 95 % confidence intervals ( CI ) . Data information statistical significance was determined by Fisher ' s exact test ; * P < 0 . 05 ; * * * P < 0 . 001 ; NS ; not significant .

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D Percentages of axons that initiated regeneration 24 h after laser surgery. The numbers (n) of animals examined are shown. Error bars indicate 95% confidence intervals (CI). Data information statistical significance was determined by Fisher's exact test; *P < 0.05; ***P <0.001; NS; not significant.


Percentages of axons that initiated regeneration 24 h after laser surgery . The numbers ( n ) of animals examined are shown . Error bars indicate 95 % CI . Data information : In ( A ) , statistical significance was determined by Fisher ' s exact test ; * * * P < 0 . 001 ; NS ; not significant .

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Percentages of axons that initiated regeneration 24 h after laser surgery. The numbers (n) of animals examined are shown. Error bars indicate 95% CI. Data information: In (A), statistical significance was determined by Fisher's exact test; ***P <0.001; NS; not significant.


B Induction of Psvh - 2 : : nls : : venus expression in D - type motor neurons by laser surgery . Expression of fluorescent proteins in D - type motor neurons of wild - type , mxl - 1 , tdpt - 1 and mxl - 1 ; tdpt - 1 animals 3 h after laser surgery are shown . Yellow arrowheads and white arrows indicate axon and cell bodies , respectively , of D - type neurons . White arrows indicate the sites of laser surgery . D neurons are visualized by CFP under control of the unc - 25 promoter . Cell bodies of D - type neurons are magnified and shown within the red boxes . Most of the intestinal fluorescence in these photos is from endogenous and variable background autofluorescence . A schematic representation of D - type motor neurons is shown on the bottom . Scale bars = 10 μm .

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B Induction of Psvh-2::nls::venus expression in D-type motor neurons by laser surgery. Expression of fluorescent proteins in D-type motor neurons of wild-type, mxl-1, tdpt-1 and mxl-1; tdpt-1 animals 3 h after laser surgery are shown. Yellow arrowheads and white arrows indicate axon and cell bodies, respectively, of D-type neurons. White arrows indicate the sites of laser surgery. D neurons are visualized by CFP under control of the unc-25 promoter. Cell bodies of D-type neurons are magnified and shown within the red boxes. Most of the intestinal fluorescence in these photos is from endogenous and variable background autofluorescence. A schematic representation of D-type motor neurons is shown on the bottom. Scale bars = 10 μm.


Percentages of axons that initiated regeneration 24 h after laser surgery . The numbers ( n ) of animals examined are shown . Error bars indicate 95 % CI . Data information : statistical significance was determined by Fisher ' s exact test ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; NS ; not significant .

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Percentages of axons that initiated regeneration 24 h after laser surgery. The numbers (n) of animals examined are shown. Error bars indicate 95% CI. Data information: statistical significance was determined by Fisher's exact test; *P < 0.05; **P < 0.01; ***P < 0.001; NS; not significant.


B Interaction of MXL - 1 with TDPT - 1 by yeast two - hybrid assays . The reporter yeast strain L40u was co - transformed with expression vectors encoding LexA DBD - MXL - 1 and GAL4 AD - TDPT - 1 as indicated . Yeasts carrying the indicated plasmids were grown on a selective plate lacking histidine and containing 10 mM 5 - aminotriazole for 4 days .

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B Interaction of MXL-1 with TDPT-1 by yeast two-hybrid assays. The reporter yeast strain L40u was co-transformed with expression vectors encoding LexA DBD-MXL-1 and GAL4 AD-TDPT-1 as indicated. Yeasts carrying the indicated plasmids were grown on a selective plate lacking histidine and containing 10 mM 5-aminotriazole for 4 days.


B Interaction between ETS - 4 and TDPT - 1 . COS - 7 cells were transfected with plasmids encoding T7 - TDPT - 1 , HA - ETS - 4 ( WT ) , HA - ETS - 4 ( S73E ) ( SE ) and HA - ETS - 4 ( S73A ) ( SA ) , as indicated . Whole - cell extracts ( WCE ) and immunoprecipitated complexes obtained with anti - T7 antibody ( IP : T7 ) were analyzed by immunoblotting ( IB ) .

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B Interaction between ETS-4 and TDPT-1. COS-7 cells were transfected with plasmids encoding T7-TDPT-1, HA-ETS-4 (WT), HA-ETS-4(S73E) (SE) and HA-ETS-4(S73A) (SA), as indicated. Whole-cell extracts (WCE) and immunoprecipitated complexes obtained with anti-T7 antibody (IP: T7) were analyzed by immunoblotting (IB).


C In vitro SUMOylation of ETS - 4 . COS - 7 cells were transfected with plasmids encoding HA - ETS - 4 ( WT ) and HA - ETS - 4 ( K32A ; K83A ) ( 2KA ) , as indicated . Cell lysates were immunoprecipitated with anti - HA antibody . The immunoprecipitates were subjected to in vitro SUMOylation assays . Reaction mixtures were analyzed by immunoblotting ( IB ) with anti - SUMO - 2 / 3 and anti - HA antibodies . Arrows mark the SUMOylated - ETS - 4 bands .

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C In vitro SUMOylation of ETS-4. COS-7 cells were transfected with plasmids encoding HA-ETS-4 (WT) and HA-ETS-4(K32A; K83A) (2KA), as indicated. Cell lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates were subjected to in vitro SUMOylation assays. Reaction mixtures were analyzed by immunoblotting (IB) with anti-SUMO-2/3 and anti-HA antibodies. Arrows mark the SUMOylated-ETS-4 bands.


E SUMOylation of ETS - 4 in mammalian cells . HEK293 cells were transfected with plasmids encoding HA - ETS - 4 ( WT ) , HA - ETS - 4 ( K32A ; K83A ) ( 2KA ) , T7 - TDPT - 1 , His - SUMO - 1 , His - SUMO - 2 and Myc - MXL - 1 , as indicated . SUMO - conjugated proteins were isolated by cobalt affinity chromatography . ETS - 4 was detected by immunoblotting with anti - HA antibody . Whole - cell extracts ( WCE ) were analyzed by immunoblotting ( IB ) .

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E SUMOylation of ETS-4 in mammalian cells. HEK293 cells were transfected with plasmids encoding HA-ETS-4 (WT), HA-ETS-4(K32A; K83A) (2KA), T7-TDPT-1, His-SUMO-1, His-SUMO-2 and Myc-MXL-1, as indicated. SUMO-conjugated proteins were isolated by cobalt affinity chromatography. ETS-4 was detected by immunoblotting with anti-HA antibody. Whole-cell extracts (WCE) were analyzed by immunoblotting (IB).


A Effect of phosphorylation of ETS - 4 on SUMOylation of ETS - 4 in mammalian cells . HEK293 cells were transfected with plasmids encoding HA - ETS - 4 ( WT ) , HA - ETS - 4 ( K32A ; K83A ) ( 2KA ) , HA - ETS - 4 ( S73A ) ( SA ) , HA - ETS - 4 ( S73E ) ( SE ) , T7 - TDPT - 1 and His - SUMO - 2 , as indicated . SUMO - conjugated proteins were isolated by cobalt affinity chromatography . ETS - 4 was detected by immunoblotting with anti - HA antibody . Whole - cell extracts ( WCE ) were analyzed by immunoblotting ( IB ) .

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A Effect of phosphorylation of ETS-4 on SUMOylation of ETS-4 in mammalian cells. HEK293 cells were transfected with plasmids encoding HA-ETS-4 (WT), HA-ETS-4(K32A; K83A) (2KA), HA-ETS-4(S73A) (SA), HA-ETS-4(S73E) (SE), T7-TDPT-1 and His-SUMO-2, as indicated. SUMO-conjugated proteins were isolated by cobalt affinity chromatography. ETS-4 was detected by immunoblotting with anti-HA antibody. Whole-cell extracts (WCE) were analyzed by immunoblotting (IB).


B Effect of SUMOylation of ETS - 4 on PKA phosphorylation of ETS - 4 in vitro . COS - 7 cells were transfected with plasmids encoding HA - ETS - 4 ( WT ) , HA - ETS - 4 ( S73A ) ( SA ) and HA - ETS - 4 ( K32A ; K83A ) ( 2KA ) , as indicated . Cell lysates were immunoprecipitated with anti - HA antibody . The immunoprecipitates were subjected to in vitro PKA kinase assays or in vitro SUMOylation and then in vitro PKA kinase assays , using recombinant GST - fused active PKA , as indicated . Reaction mixtures were analyzed by immunoblotting ( IB ) with anti - phospho - PKA substrate , anti - SUMO - 2 / 3 and anti - HA antibodies . Arrows mark the SUMOylated - ETS - 4 bands . Asterisk indicates the non - SUMOylated - and phosphorylated - ETS - 4 bands .

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B Effect of SUMOylation of ETS-4 on PKA phosphorylation of ETS-4 in vitro. COS-7 cells were transfected with plasmids encoding HA-ETS-4 (WT), HA-ETS-4(S73A) (SA) and HA-ETS-4(K32A; K83A) (2KA), as indicated. Cell lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates were subjected to in vitro PKA kinase assays or in vitro SUMOylation and then in vitro PKA kinase assays, using recombinant GST-fused active PKA, as indicated. Reaction mixtures were analyzed by immunoblotting (IB) with anti-phospho-PKA substrate, anti-SUMO-2/3 and anti-HA antibodies. Arrows mark the SUMOylated-ETS-4 bands. Asterisk indicates the non-SUMOylated- and phosphorylated-ETS-4 bands.


Percentages of axons that initiated regeneration 24 h after laser surgery are shown . The numbers ( n ) of animals examined are shown . Error bars indicate 95 % CI . Data information statistical significance was determined by Fisher ' s exact test ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; NS ; not significant .

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Percentages of axons that initiated regeneration 24 h after laser surgery are shown. The numbers (n) of animals examined are shown. Error bars indicate 95% CI. Data information statistical significance was determined by Fisher's exact test; *P < 0.05; **P < 0.01; ***P < 0.001; NS; not significant.


Percentages of axons that initiated regeneration 24 h after laser surgery are shown . The numbers ( n ) of animals examined are shown . Error bars indicate 95 % CI . Data information : statistical significance was determined by Fisher ' s exact test ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; NS ; not significant .

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Percentages of axons that initiated regeneration 24 h after laser surgery are shown. The numbers (n) of animals examined are shown. Error bars indicate 95% CI. Data information: statistical significance was determined by Fisher's exact test; *P < 0.05; **P < 0.01; ***P < 0.001; NS; not significant.


( C ) Cell morphology and SA - β - Gal activity of mouse or NMR fibroblasts 12 or 21 days after DXR treatment . Arrowheads indicate dying cells . The number in the upper left corner indicates Hoechst - positive nuclei . Scale bar , 100 μm .

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(C) Cell morphology and SA-β-Gal activity of mouse or NMR fibroblasts 12 or 21 days after DXR treatment. Arrowheads indicate dying cells. The number in the upper left corner indicates Hoechst-positive nuclei. Scale bar, 100 μm.


Time - course analysis of mouse or NMR fibroblasts after DXR treatment quantification of BrdU - positive cells ( % ) ( E )

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Time-course analysis of mouse or NMR fibroblasts after DXR treatment quantification of BrdU-positive cells (%) (E)


Time - course analysis of mouse or NMR fibroblasts after DXR treatment qRT - PCR analysis of the expression of INK4a ( F ) normalized to ACTB mRNA

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Time-course analysis of mouse or NMR fibroblasts after DXR treatment qRT-PCR analysis of the expression of INK4a (F) normalized to ACTB mRNA levels


qRT - PCR analysis of the expression of INK4a ( J ) in shINK4a - transduced NMR fibroblasts at 21 days after DXR treatment .

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qRT-PCR analysis of the expression of INK4a (J) in shINK4a-transduced NMR fibroblasts at 21 days after DXR treatment.


quantification of Annexin V - positive cells ( % ) ( K ) in shINK4a - transduced NMR fibroblasts at 21 days after DXR treatment .

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quantification of Annexin V-positive cells (%) (K) in shINK4a-transduced NMR fibroblasts at 21 days after DXR treatment.


( B ) qRT - PCR analysis of the expression of INK4a at 12 days after INK4a or mock transduction , normalized to ACTB mRNA levels .

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(B) qRT-PCR analysis of the expression of INK4a at 12 days after INK4a or mock transduction, normalized to ACTB mRNA levels.


( C ) Cell morphology and SA - β - Gal activity of mouse or NMR fibroblasts 12 days after INK4a transduction . Arrowheads indicate dying cells . The number in the upper left corner indicates Hoechst - positive nuclei . Scale bar , 100 μm .

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(C) Cell morphology and SA-β-Gal activity of mouse or NMR fibroblasts 12 days after INK4a transduction. Arrowheads indicate dying cells. The number in the upper left corner indicates Hoechst-positive nuclei. Scale bar, 100 μm.


( F ) Western blot analysis of RB in mouse or NMR fibroblasts at 12 days after INK4a transduction ( p , phospho - specific antibody ) . ACTIN was used as a loading control . The arrowhead indicates a non - specific band .

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(F) Western blot analysis of RB in mouse or NMR fibroblasts at 12 days after INK4a transduction (p, phospho-specific antibody). ACTIN was used as a loading control. The arrowhead indicates a non-specific band.


( I ) SA - β - Gal activity in the adherent living cell population and floating dead cell population in mouse or NMR fibroblast cultures at 12 days after INK4a transduction . Scale bar , 100 μm . ( J ) Quantification of SA - β - Gal - positive cells in the adherent living cell population and floating dead cell population after the same treatment as in I .

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(I) SA-β-Gal activity in the adherent living cell population and floating dead cell population in mouse or NMR fibroblast cultures at 12 days after INK4a transduction. Scale bar, 100 μm. (J) Quantification of SA-β-Gal-positive cells in the adherent living cell population and floating dead cell population after the same treatment as in I.


( C and D ) Quantification of SA - β - Gal - positive cells ( % ) ( C ) and Annexin V - positive cells ( % ) ( Annexin V + / PI - as early apoptotic and Annexin V + / PI + double - positive as late apoptotic ) ( D ) in NMR - fibroblasts transduced with different forms of SV40 Large T antigen ( LT , LT ∆ , and LTK1 ) and INK4a . OE ; overexpression .

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(C and D) Quantification of SA-β-Gal-positive cells (%) (C) and Annexin V-positive cells (%) (Annexin V+/PI- as early apoptotic and Annexin V+/PI+ double-positive as late apoptotic) (D) in NMR-fibroblasts transduced with different forms of SV40 Large T antigen (LT, LT∆, and LTK1) and INK4a. OE; overexpression.


( F ) Quantification of SA - β - Gal - positive cells ( % ) at 21 days after DXR treatment in NMR - fibroblasts transduced with different forms of SV40 Large T antigen ( LT , LT ∆ , and LTK1 ) . ( G ) Quantification of Annexin V - positive cells after the same treatment as in F . Annexin V + / PI - cells were counted as early apoptotic cells and Annexin V + / PI - cells as late apoptotic cells .

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(F) Quantification of SA-β-Gal-positive cells (%) at 21 days after DXR treatment in NMR-fibroblasts transduced with different forms of SV40 Large T antigen (LT, LT∆, and LTK1). (G) Quantification of Annexin V-positive cells after the same treatment as in F. Annexin V+/PI- cells were counted as early apoptotic cells and Annexin V+/PI- cells as late apoptotic cells.


( C ) Quantification of PI - positive cells in NMR and mouse fibroblasts 18 h after 6 h of treatment with the indicated doses of hydrogen peroxide ( H2O2 ) . Data are expressed as the mean ± SD from n = 3 biological replicates .

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(C) Quantification of PI-positive cells in NMR and mouse fibroblasts 18 h after 6 h of treatment with the indicated doses of hydrogen peroxide (H2O2). Data are expressed as the mean ± SD from n = 3 biological replicates.


( B ) Left , representative images of lipid peroxidation ( green ) staining in NMR fibroblasts at 12 days after INK4a - transduction . Scale bar , 50 μm . Right , quantification of signal intensity of lipid peroxidation staining . Data are expressed as the mean ± SD from n = 3 biological replicates .

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(B) Left, representative images of lipid peroxidation (green) staining in NMR fibroblasts at 12 days after INK4a-transduction. Scale bar, 50 μm. Right, quantification of signal intensity of lipid peroxidation staining. Data are expressed as the mean ± SD from n = 3 biological replicates.


( C ) Quantification of PI - positive cells in NMR fibroblasts treated for 24 h with N - acetyl L - cysteine ( NAC ) , Trolox , or Tempol at 20 days after INK4a transduction ( % ) . Data are expressed as the mean ± SD from n = 6 biological replicates except for Trolox and Tempol ( n = 4 ) .

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(C) Quantification of PI-positive cells in NMR fibroblasts treated for 24 h with N-acetyl L-cysteine (NAC), Trolox, or Tempol at 20 days after INK4a transduction (%). Data are expressed as the mean ± SD from n = 6 biological replicates except for Trolox and Tempol (n = 4).


( D ) Western blot of monoamine oxidase ( MAO ) - A and MAO - B in mouse or NMR fibroblasts at 20 days after INK4a transduction . ACTIN was used as a loading control . Numbers below the gel images indicate quantification of MAO - A or - B / ACTIN intensity ( n = 3 average ) .

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(D) Western blot of monoamine oxidase (MAO)-A and MAO-B in mouse or NMR fibroblasts at 20 days after INK4a transduction. ACTIN was used as a loading control. Numbers below the gel images indicate quantification of MAO-A or -B/ACTIN intensity (n = 3 average).


( G ) Western blot of MAO - A and MAO - B in mouse or NMR fibroblasts at 21 days after doxorubicin ( DXR ) treatment . ACTIN was used as a loading control . Numbers below the gel images indicate quantification of MAO - A or - B / ACTIN intensity ( n = 3 average ) .

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(G) Western blot of MAO-A and MAO-B in mouse or NMR fibroblasts at 21 days after doxorubicin (DXR) treatment. ACTIN was used as a loading control. Numbers below the gel images indicate quantification of MAO-A or -B/ACTIN intensity (n = 3 average).


( B ) qRT - PCR analysis of INK4a expression in the skin , muscle , and white adipose tissue ( WAT ) of 6 - week - old mice ( young ; 6 w ) , 1 - year - old mice ( middle - aged ; 1 y ) , 1 - year - old NMRs ( young ; 1 y ) , and 15 - year - old NMRs ( middle - aged ; 15 y ) .

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(B) qRT-PCR analysis of INK4a expression in the skin, muscle, and white adipose tissue (WAT) of 6-week-old mice (young; 6 w), 1-year-old mice (middle-aged; 1 y), 1-year-old NMRs (young; 1 y), and 15-year-old NMRs (middle-aged; 15 y).


( D ) SA - β - Gal activity ( SA - β - Gal , blue ; nuclei , green ) and TUNEL staining ( TUNEL , red ; nuclei , blue ) in lungs of mice or NMRs at 2 , 7 , 14 , and 21 days after Bleo administration .

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(D) SA-β-Gal activity (SA-β-Gal, blue; nuclei, green) and TUNEL staining (TUNEL, red; nuclei, blue) in lungs of mice or NMRs at 2, 7, 14, and 21 days after Bleo administration.


Time - course analysis of mice or NMR lungs after Bleo administration : qRT - PCR analysis of the expression of INK4a normalized to ACTB mRNA levels ( F )

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Time-course analysis of mice or NMR lungs after Bleo administration: qRT-PCR analysis of the expression of INK4a normalized to ACTB mRNA levels (F)


Time - course analysis of mice or NMR lungs after Bleo administration ; quantification of TUNEL - positive cells ( % ) ( G ) .

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Time-course analysis of mice or NMR lungs after Bleo administration ; quantification of TUNEL-positive cells (%) (G).


( B ) SA - β - Gal activity ( SA - β - Gal , blue ; nuclei , green ) and TUNEL staining ( TUNEL , red ; nuclei , blue ) in lungs of mice or NMRs at 21 days after Bleo administration , with or without Phe are shown .

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(B) SA-β-Gal activity (SA-β-Gal, blue; nuclei, green) and TUNEL staining (TUNEL, red; nuclei, blue) in lungs of mice or NMRs at 21 days after Bleo administration, with or without Phe are shown.


qRT - PCR analysis of INK4a expression normalized to ACTB mRNA levels ( D ) in mouse or NMR lungs at 21 days after Bleo administration , with or without Phe .

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qRT-PCR analysis of INK4a expression normalized to ACTB mRNA levels (D) in mouse or NMR lungs at 21 days after Bleo administration, with or without Phe.


quantification of TUNEL - positive cells ( % ) ( E ) in mouse or NMR lungs at 21 days after Bleo administration , with or without Phe .

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quantification of TUNEL-positive cells (%) (E) in mouse or NMR lungs at 21 days after Bleo administration, with or without Phe.


Identification of the ENU - induced hearing loss pedigree MPC169 , subsequently named clarinet . ABR phenotyping of pedigree Muta - Ped - C3PDE - 169 at 3 - months of age identified 8 mice with elevated hearing thresholds ( red triangles ) compared to their normal hearing colony mates ( n = 61 , black triangles ) . Indeed , all eight affected mice were found to not respond to the highest intensity stimulus ( 90 dB SPL ) at the three frequencies tested , or the click stimulus , and so their thresholds are shown as 95 dB SPL .

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Identification of the ENU-induced hearing loss pedigree MPC169, subsequently named clarinet. ABR phenotyping of pedigree Muta-Ped-C3PDE-169 at 3-months of age identified 8 mice with elevated hearing thresholds (red triangles) compared to their normal hearing colony mates (n=61, black triangles). Indeed, all eight affected mice were found to not respond to the highest intensity stimulus (90 dB SPL) at the three frequencies tested, or the click stimulus, and so their thresholds are shown as 95 dB SPL.


Averaged ABR thresholds for Clrn2clarinet / del629 compound heterozygotes at P21 , showing significantly elevated thresholds compared to Clrn2 + / + , Clrn2clarinet / + and Clrn2del629 / + control colony mates . All five Clrn2clarinet / del629 mice were found to not respond at the highest intensity stimulus ( 90 dB SPL ) for at least one frequency / click stimulus . Data shown are mean ± s . d . * * * p < 0 . 001 , one - way ANOVA ( Please see Appendix supplemental table 1 for exact P - values ) .

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Averaged ABR thresholds for Clrn2clarinet/del629 compound heterozygotes at P21, showing significantly elevated thresholds compared to Clrn2+/+, Clrn2clarinet/+ and Clrn2del629/+ control colony mates. All five Clrn2clarinet/del629 mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least one frequency/click stimulus. Data shown are mean ± s.d. ***p<0.001, one-way ANOVA (Please see Appendix supplemental table 1 for exact P-values).


Regional plot of p values for SNP association with hearing difficulty around the CLRN2 gene locus . The genes within the region are annotated , and the direction of the transcripts is shown by arrows . Colouring is based on linkage disequilibrium ( LD ) across the region with the most associated SNP , rs35414371 , shown in purple .

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Regional plot of p values for SNP association with hearing difficulty around the CLRN2 gene locus. The genes within the region are annotated, and the direction of the transcripts is shown by arrows. Colouring is based on linkage disequilibrium (LD) across the region with the most associated SNP, rs35414371, shown in purple.


RT - PCR analysis in P30 mice showing the presence of Clrn2 transcripts in the inner ear , eye , but not in brain or muscle . β - actin was used as a positive control .

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RT-PCR analysis in P30 mice showing the presence of Clrn2 transcripts in the inner ear, eye, but not in brain or muscle. β-actin was used as a positive control.


Clrn2 transcripts could be detected in both inner ( IHCs ) and outer ( OHCs ) hair cells of P15 wild - type mice . Otoferlin ( Otof ) and oncomodulin ( Ocm ) transcripts were used as positive controls for IHCs and OHCs , respectively . Ocm transcripts were only present in the OHC lysate , demonstrating that the IHC sample had not been contaminated with OHCs .

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Clrn2 transcripts could be detected in both inner (IHCs) and outer (OHCs) hair cells of P15 wild-type mice. Otoferlin (Otof) and oncomodulin (Ocm) transcripts were used as positive controls for IHCs and OHCs, respectively. Ocm transcripts were only present in the OHC lysate, demonstrating that the IHC sample had not been contaminated with OHCs.


Auditory phenotyping of clarinet mice at P16 ( C ) , P21 ( D ) ABR threshold measurements show that Clrn2clarinet / clarinet mice ( red ) exhibit a severe - to - profound hearing loss affecting all frequencies tested . At 16 kHz in Clrn2clarinet / clarinet mice , the mean ABR hearing thresholds vary from 55 - 65 dB SPL at P16 , 60 - 90 dB SPL at P21 , and to 80 - 100 dB SPL at P28 and P42 . Age - matched Clrn2 + / + ( black ) and Clrn2clarinet / + ( grey ) controls display thresholds within the expected range ( 15 - 45 dB SPL ) at all frequencies and time - points tested . At P16 , all eight Clrn2clarinet / clarinet mice exhibited recordable ABR responses for each frequency tested and the click stimulus . For the longitudinal ABR study , at P21 and P28 three of the seven Clrn2clarinet / clarinet mice were found to not respond at the highest intensity stimulus ( 90 dB SPL ) for at least one frequency - specific / click stimulus . By P42 , five of the Clrn2clarinet / clarinet mice were found to not respond at the highest intensity stimulus ( 90 dB SPL ) for at least two frequency - specific / click stimuli . ABR data shown are mean ± s . d . * * * p < 0 . 001 , one - way ANOVA .

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Auditory phenotyping of clarinet mice at P16 (C), P21 (D) ABR threshold measurements show that Clrn2clarinet/clarinet mice (red) exhibit a severe-to-profound hearing loss affecting all frequencies tested. At 16 kHz in Clrn2clarinet/clarinet mice, the mean ABR hearing thresholds vary from 55-65 dB SPL at P16, 60-90 dB SPL at P21, and to 80-100 dB SPL at P28 and P42. Age-matched Clrn2+/+ (black) and Clrn2clarinet/+ (grey) controls display thresholds within the expected range (15-45 dB SPL) at all frequencies and time-points tested. At P16, all eight Clrn2clarinet/clarinet mice exhibited recordable ABR responses for each frequency tested and the click stimulus. For the longitudinal ABR study, at P21 and P28 three of the seven Clrn2clarinet/clarinet mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least one frequency-specific/click stimulus. By P42, five of the Clrn2clarinet/clarinet mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least two frequency-specific/click stimuli. ABR data shown are mean ± s.d. ***p<0.001, one-way ANOVA.


Auditory phenotyping of clarinet mice at P28 ( E ) and P42 ( F ) . ABR threshold measurements show that Clrn2clarinet / clarinet mice ( red ) exhibit a severe - to - profound hearing loss affecting all frequencies tested . At 16 kHz in Clrn2clarinet / clarinet mice , the mean ABR hearing thresholds vary from 55 - 65 dB SPL at P16 , 60 - 90 dB SPL at P21 , and to 80 - 100 dB SPL at P28 and P42 . Age - matched Clrn2 + / + ( black ) and Clrn2clarinet / + ( grey ) controls display thresholds within the expected range ( 15 - 45 dB SPL ) at all frequencies and time - points tested . At P16 , all eight Clrn2clarinet / clarinet mice exhibited recordable ABR responses for each frequency tested and the click stimulus . For the longitudinal ABR study , at P21 and P28 three of the seven Clrn2clarinet / clarinet mice were found to not respond at the highest intensity stimulus ( 90 dB SPL ) for at least one frequency - specific / click stimulus . By P42 , five of the Clrn2clarinet / clarinet mice were found to not respond at the highest intensity stimulus ( 90 dB SPL ) for at least two frequency - specific / click stimuli . ABR data shown are mean ± s . d . * * * p < 0 . 001 , one - way ANOVA .

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

Auditory phenotyping of clarinet mice at P28 (E) and P42 (F). ABR threshold measurements show that Clrn2clarinet/clarinet mice (red) exhibit a severe-to-profound hearing loss affecting all frequencies tested. At 16 kHz in Clrn2clarinet/clarinet mice, the mean ABR hearing thresholds vary from 55-65 dB SPL at P16, 60-90 dB SPL at P21, and to 80-100 dB SPL at P28 and P42. Age-matched Clrn2+/+ (black) and Clrn2clarinet/+ (grey) controls display thresholds within the expected range (15-45 dB SPL) at all frequencies and time-points tested. At P16, all eight Clrn2clarinet/clarinet mice exhibited recordable ABR responses for each frequency tested and the click stimulus. For the longitudinal ABR study, at P21 and P28 three of the seven Clrn2clarinet/clarinet mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least one frequency-specific/click stimulus. By P42, five of the Clrn2clarinet/clarinet mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least two frequency-specific/click stimuli. ABR data shown are mean ± s.d. ***p<0.001, one-way ANOVA.


Averaged DPOAE responses for clarinet mice at P28 , showing significantly reduced responses in Clrn2clarinet / clarinet mutants at all frequencies tested . DPOAE data shown are mean ± s . d . * p < 0 . 02 , * * p < 0 . 01 , one - way ANOVA . Please see Appendix supplemental table 1 for exact P - values .

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

Averaged DPOAE responses for clarinet mice at P28, showing significantly reduced responses in Clrn2clarinet/clarinet mutants at all frequencies tested. DPOAE data shown are mean ± s.d. *p<0.02, **p<0.01, one-way ANOVA. Please see Appendix supplemental table 1 for exact P-values.


Vestibular behavioural tests ( swim tests , contact - righting , trunk - curl , and platform ) . The Clrn2clarinet / clarinet mice ( red , P28 , n = 5 , and P60 , n = 7 ) have no vestibular dysfunction , displaying similar performances to age - matched control Clrn2clarinet / + mice ( grey , P28 , n = 3 , and P60 , n = 7 ) . Being similar , the values at P28 and P60 were combined . Electroretinogram ( ERG ) measurements from control Clrn2clarinet / + ( grey ) and mutant Clrn2clarinet / clarinet ( red ) mice . Each trace in B and C is representative of an ERG response from the eye of age - matched Clrn2clarinet / + and Clrn2clarinet / clarinet mice , showing no significant difference in a - or b - wave amplitudes . ( D - F ) The lack of change in ERG amplitude responses in Clrn2clarinet / clarinet mice ( aged 3 or 6 - 7 months ) , regardless of the test conditions : scotopic ( D , E ) , or photopic ( F ) , indicate normal photoreceptor kinetics and no change in the sensitivity of photoreceptor 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Vestibular behavioural tests (swim tests, contact-righting, trunk-curl, and platform). The Clrn2clarinet/clarinet mice (red, P28, n=5, and P60, n=7) have no vestibular dysfunction, displaying similar performances to age-matched control Clrn2clarinet/+ mice (grey, P28, n=3, and P60, n=7). Being similar, the values at P28 and P60 were combined. Electroretinogram (ERG) measurements from control Clrn2clarinet/+ (grey) and mutant Clrn2clarinet/clarinet (red) mice. Each trace in B and C is representative of an ERG response from the eye of age-matched Clrn2clarinet/+ and Clrn2clarinet/clarinet mice, showing no significant difference in a- or b-wave amplitudes. (D-F) The lack of change in ERG amplitude responses in Clrn2clarinet/clarinet mice (aged 3 or 6-7 months), regardless of the test conditions: scotopic (D,E), or photopic (F), indicate normal photoreceptor kinetics and no change in the sensitivity of photoreceptor cells.


Electroretinogram ( ERG ) measurements from control Clrn2clarinet / + ( grey ) and mutant Clrn2clarinet / clarinet ( red ) mice . Each trace in B and C is representative of an ERG response from the eye of age - matched Clrn2clarinet / + and Clrn2clarinet / clarinet mice , showing no significant difference in a - or b - wave amplitudes . ( D - F ) The lack of change in ERG amplitude responses in Clrn2clarinet / clarinet mice ( aged 3 or 6 - 7 months ) , regardless of the test conditions : scotopic ( D , E ) , or photopic ( F ) , indicate normal photoreceptor kinetics and no change in the sensitivity of photoreceptor 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Electroretinogram (ERG) measurements from control Clrn2clarinet/+ (grey) and mutant Clrn2clarinet/clarinet (red) mice. Each trace in B and C is representative of an ERG response from the eye of age-matched Clrn2clarinet/+ and Clrn2clarinet/clarinet mice, showing no significant difference in a- or b-wave amplitudes. (D-F) The lack of change in ERG amplitude responses in Clrn2clarinet/clarinet mice (aged 3 or 6-7 months), regardless of the test conditions: scotopic (D,E), or photopic (F), indicate normal photoreceptor kinetics and no change in the sensitivity of photoreceptor cells.


Confocal microscopy images of whole - mount preparations of mid - basal cochlear sensory epithelia from Clrn2clarinet / + ( A ) and Clrn2clarinet / clarinet ( B ) P6 mice immunostained for actin . Despite lack of clarin - 2 , the developing sensory epithelium of mutants is similar to that of heterozygous controls .

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

Confocal microscopy images of whole-mount preparations of mid-basal cochlear sensory epithelia from Clrn2clarinet/+ (A) and Clrn2clarinet/clarinet (B) P6 mice immunostained for actin. Despite lack of clarin-2, the developing sensory epithelium of mutants is similar to that of heterozygous controls.


Confocal microscopy images of whole - mount preparations of cochlear sensory epithelia from Clrn2clarinet / + ( C ) , Clrn1 - / - ( D ) , and Clrn2clarinet / clarinet ( E ) P6 mice immunostained for stereocilin ( green ) and actin ( red ) . Unlike the fragmented immunostaining in Clrn1 - / - mice ( mirroring the fragmentation of the hair bundle ) , stereocilin immunostaining in Clrn2clarinet / clarinet mice reflects the normally V - shaped bundles of OHCs , similar to Clrn2clarinet / + OHCs .

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

Confocal microscopy images of whole-mount preparations of cochlear sensory epithelia from Clrn2clarinet/+ (C), Clrn1-/- (D), and Clrn2clarinet/clarinet (E) P6 mice immunostained for stereocilin (green) and actin (red). Unlike the fragmented immunostaining in Clrn1-/- mice (mirroring the fragmentation of the hair bundle), stereocilin immunostaining in Clrn2clarinet/clarinet mice reflects the normally V-shaped bundles of OHCs, similar to Clrn2clarinet/+ OHCs.


Representative scanning electron micrographs of the sensory epithelium of Clrn2clarinet / + ( F ) and Clrn2clainet / clarinet ( G ) P8 mice , showing no apparent differences in the gross patterning of IHCs and OHCs . Arrowheads in a , b ( white ) , f and g ( black ) illustrate the convex shape of the apical circumference of OHCs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 scanning electron micrographs of the sensory epithelium of Clrn2clarinet/+ (F) and Clrn2clainet/clarinet (G) P8 mice, showing no apparent differences in the gross patterning of IHCs and OHCs. Arrowheads in a,b (white),f and g (black) illustrate the convex shape of the apical circumference of OHCs.


Pseudo - coloured scanning electron micrographs of individual outer and inner hair cell bundles from clarinet mice at P8 and P16 , showing that gross morphology of OHC and IHC bundles is similar between Clrn2 + / + and Clrn2clarinet / clarinet mice . Representative images from the mid region of the cochlear spiral are shown and close - up views illustrate the three full rows , tallest ( red ) , middle ( blue ) and short ( yellow ) , of stereocilia in IHC and OHC hair bundles . At P8 and P16 ( upper panels in A and B ) , the three stereocilia rows are observed in Clrn2clarinet / clarinet OHCs ( A ) and IHCs ( B ) , although the shortest and middle row of IHC stereocilia appear less prolate compared to controls . ( C , D ) Distribution of individual stereocilia measures across genotypes at P8 and P16 .

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

Pseudo-coloured scanning electron micrographs of individual outer and inner hair cell bundles from clarinet mice at P8 and P16, showing that gross morphology of OHC and IHC bundles is similar between Clrn2+/+ and Clrn2clarinet/clarinet mice. Representative images from the mid region of the cochlear spiral are shown and close-up views illustrate the three full rows, tallest (red), middle (blue) and short (yellow), of stereocilia in IHC and OHC hair bundles. At P8 and P16 (upper panels in A and B), the three stereocilia rows are observed in Clrn2clarinet/clarinet OHCs (A) and IHCs (B), although the shortest and middle row of IHC stereocilia appear less prolate compared to controls. (C,D) Distribution of individual stereocilia measures across genotypes at P8 and P16.


The OHC stereocilia measurements at both P8 and P16 ( C ) show a significant difference in height of Clrn2 + / + stereocilia compared to Clrn2clarinet / clarinet in : e . g . for the shortest row at P8 : Clrn2 + / + : 0 . 6188 ± 0 . 0944 p . µm ( n = 27 ) ; Clrn2clarinet / clarinet : 0 . 2948 ± 0 . 1029 p . µm ( n = 27 ) ( p < 0 . 0001 , Mann - Whitney ranks comparison ) , and at P16 : Clrn2 + / + : 0 . 579 ± 0 . 06179 p . µm ( n = 27 ) ; Clrn2clarinet / clarinet : 0 . 07282 ± 0 . 1626 p . µm ( n = 27 ) ( p < 0 . 0001 , Mann - Whitney ranks comparison ) . Note the amount of value points equal to zero at P16 . ( D ) Conversely , IHC stereocilia measurements at P8 and P16 did not show a difference between Clrn2 + t / + and Clrn2clarinet / clarinet in : for the shortest row at P8 : Clrn2 + / + : 0 . 8808 ± 0 . 1753 p . µm ( n = 27 ) ; Clrn2clarinet / clarinet : 0 . 9313 ± 0 . 1217 p . µm ( n = 27 ) ( p = 0 . 2244 , unpaired t - test ) ; and at P16 : Clrn2 + / + : 0 . 6784 ± 0 . 1171 p . µm ( n = 23 ) ; Clrn2clarinet / clarinet : 0 . 6063 ± 0 . 1855 p . µm ( n = 26 ) ( p = 0 . 2436 , Mann - Whitney ranks comparison ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 OHC stereocilia measurements at both P8 and P16 (C) show a significant difference in height of Clrn2+/+ stereocilia compared to Clrn2clarinet/clarinet in: e.g. for the shortest row at P8: Clrn2+/+: 0.6188 ± 0.0944 p.µm (n=27); Clrn2clarinet/clarinet: 0.2948 ± 0.1029 p.µm (n=27) (p<0.0001, Mann-Whitney ranks comparison), and at P16: Clrn2+/+: 0.579 ± 0.06179 p.µm (n=27); Clrn2clarinet/clarinet: 0.07282 ± 0.1626 p.µm (n=27) (p<0.0001, Mann-Whitney ranks comparison). Note the amount of value points equal to zero at P16. (D) Conversely, IHC stereocilia measurements at P8 and P16 did not show a difference between Clrn2+t/+ and Clrn2clarinet/clarinet in: for the shortest row at P8: Clrn2+/+: 0.8808 ± 0.1753 p.µm (n=27); Clrn2clarinet/clarinet: 0.9313 ± 0.1217 p.µm (n=27) (p=0.2244, unpaired t-test); and at P16: Clrn2+/+: 0.6784 ± 0.1171 p.µm (n=23); Clrn2clarinet/clarinet: 0.6063 ± 0.1855 p.µm (n=26) (p=0.2436, Mann-Whitney ranks comparison).


Sensory hair cell bundle patterning and measurements in clarinet mice Pseudo - coloured scanning electron micrographs of individual OHC ( left panels ) and IHC ( right panels ) hair bundles from clarinet mice at P28 . Clrn2clarinet / clarinet mutants have only two rows of OHC stereocilia , and the middle row of stereocilia is less uniform in height compared to controls . The middle and short rows of stereocilia in Clrn2clarinet / clarinet IHC bundles appear fewer in number , and heterogeneous in height . Scale - 2 µm .

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

Sensory hair cell bundle patterning and measurements in clarinet mice Pseudo-coloured scanning electron micrographs of individual OHC (left panels) and IHC (right panels) hair bundles from clarinet mice at P28. Clrn2clarinet/clarinet mutants have only two rows of OHC stereocilia, and the middle row of stereocilia is less uniform in height compared to controls. The middle and short rows of stereocilia in Clrn2clarinet/clarinet IHC bundles appear fewer in number, and heterogeneous in height. Scale - 2 µm.


Schematic representation of the auditory sensory organ , illustrating the positioning of the electrode used for injectoporation of GFP - tagged clarin - 2 construct into cochlear supporting and hair cells . ( B - F ) Top and side views of representative images of supporting cells ( asterisk ) and hair cells expressing clarin - 2 . In supporting cells , GFP - tagged clarin - 2 ( green ) was distributed diffusely throughout the cytoplasm ( A , B ) . By contrast , in all injectoporated hair cells , the majority of clarin - 2 was observed in the apical stereocilia ( C - E ) . Scale - 2 µm .

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

Schematic representation of the auditory sensory organ, illustrating the positioning of the electrode used for injectoporation of GFP-tagged clarin-2 construct into cochlear supporting and hair cells. (B-F) Top and side views of representative images of supporting cells (asterisk) and hair cells expressing clarin-2. In supporting cells, GFP-tagged clarin-2 (green) was distributed diffusely throughout the cytoplasm (A,B). By contrast, in all injectoporated hair cells, the majority of clarin-2 was observed in the apical stereocilia (C-E). Scale - 2 µm.


Confocal images of IHC and OHC hair bundles of Clrn2clarinet / clarinet mice and heterozygous littermates at P6 immunostained for PDZD7 ( A , green ) and whirlin ( B , green ) and actin ( red in both figures ) . The PDZD7 immunostaining is normally restricted to the base of stereocilia in both Clrn2clarinet / + and Clrn2clarinet / clarinet P6 mice ( A ) . Whirlin immunostaining is properly located at the stereocilia tips of IHC and OHC hair bundles ( B ) .

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

Confocal images of IHC and OHC hair bundles of Clrn2clarinet/clarinet mice and heterozygous littermates at P6 immunostained for PDZD7 (A, green) and whirlin (B, green) and actin (red in both figures). The PDZD7 immunostaining is normally restricted to the base of stereocilia in both Clrn2clarinet/+ and Clrn2clarinet/clarinet P6 mice (A). Whirlin immunostaining is properly located at the stereocilia tips of IHC and OHC hair bundles (B).


Harmonin - b immunostaining in IHC hair bundles . In Clrn2clarinet / clarinet mice ( D ) , the harmonin - b immunoreactive puncta ( green ) were still observable on the stereocilia , but unlike in age - matched heterozygote littermates ( C ) , were located much closer to the tip of stereocilia . The two diagrams in ( C ) and ( D ) illustrate the position of harmonin - b immunostaining ( green ) corresponding to the site of the upper attachment point of the tip link ( UAPTL ) , facing the tip link . The bright green signal outside stereociliary bundles are non - specific . The change of harmonin - b localization along the stereocilium in Clrn2clarinet / clarinet mice is illustrated further by line scan ( E ) and quantification ( F ) analyses . The insets in ( E ) show images of individual stereocilia used for the line scan signal analysis . The harmonin - b immunoreactive puncta were located within 850 ± 28 nm distance from the tip of the tallest stereocilia in Clrn2clarine / + mice ( n = 31 hair bundles from 5 mice ) ( F ) , and within 575 ± 23 nm in Clrn2clarinet / clarinet mice ( n = 35 hair bundles from 5 mice ) .

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

Harmonin-b immunostaining in IHC hair bundles. In Clrn2clarinet/clarinet mice (D), the harmonin-b immunoreactive puncta (green) were still observable on the stereocilia, but unlike in age-matched heterozygote littermates (C), were located much closer to the tip of stereocilia. The two diagrams in (C) and (D) illustrate the position of harmonin-b immunostaining (green) corresponding to the site of the upper attachment point of the tip link (UAPTL), facing the tip link. The bright green signal outside stereociliary bundles are non-specific. The change of harmonin-b localization along the stereocilium in Clrn2clarinet/clarinet mice is illustrated further by line scan (E) and quantification (F) analyses. The insets in (E) show images of individual stereocilia used for the line scan signal analysis. The harmonin-b immunoreactive puncta were located within 850 ± 28 nm distance from the tip of the tallest stereocilia in Clrn2clarine/+ mice (n=31 hair bundles from 5 mice) (F), and within 575 ± 23 nm in Clrn2clarinet/clarinet mice (n=35 hair bundles from 5 mice).


Cochlear whole - mounts from P21 clarinet mice , labelled with the IHC pre - synaptic ribbon marker Ribeye ( red ) and the post - synaptic density marker GluR2 ( green ) , showing a similar number of total and matched Ribeye - positive and GluR2 - postive puncta in Clrn2clarinet / clarinet mutant cochleae compared to Clrn2clarinet / + littermates , which is reflected in puncta counted per five hair cells ( H ) n = 4 per genotype .

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

Cochlear whole-mounts from P21 clarinet mice, labelled with the IHC pre-synaptic ribbon marker Ribeye (red) and the post-synaptic density marker GluR2 (green), showing a similar number of total and matched Ribeye-positive and GluR2-postive puncta in Clrn2clarinet/clarinet mutant cochleae compared to Clrn2clarinet/+ littermates, which is reflected in puncta counted per five hair cells (H) n = 4 per genotype.


Saturating MET currents recorded from P6 Clrn2clarinet / + ( A ) and Clrn2clarinet / clarinet ( B ) apical - coil OHCs by applying sinusoidal force stimuli of 50 Hz to the hair bundles at − 121 mV and + 99 mV . The driver voltage ( DV ) signal of ± 40 V to the fluid jet is shown above the traces ( positive deflections of the DV are excitatory ) . The holding potential was − 81 mV . Extracellular Ca2 + concentration was 1 . 3 mM . Arrows and arrowheads indicate the closure of the MET currents ( i . e . resting MET current ) elicited during inhibitory bundle displacements at hyperpolarized and depolarized membrane potentials , respectively . Dashed lines indicate the holding current , which is the current at the holding membrane potential .

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

Saturating MET currents recorded from P6 Clrn2clarinet/+ (A) and Clrn2clarinet/clarinet (B) apical-coil OHCs by applying sinusoidal force stimuli of 50 Hz to the hair bundles at −121 mV and +99 mV. The driver voltage (DV) signal of ± 40 V to the fluid jet is shown above the traces (positive deflections of the DV are excitatory). The holding potential was −81 mV. Extracellular Ca2+ concentration was 1.3 mM. Arrows and arrowheads indicate the closure of the MET currents (i.e. resting MET current) elicited during inhibitory bundle displacements at hyperpolarized and depolarized membrane potentials, respectively. Dashed lines indicate the holding current, which is the current at the holding membrane potential.


Average peak - to - peak current - voltage curves recorded from Clrn2clarinet / + ( grey , P6 , n = 7 ) and Clrn2clarinet / clarinet ( red , P6 - 7 , n = 9 ) apical coil OHCs .

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

Average peak-to-peak current-voltage curves recorded from Clrn2clarinet/+ (grey, P6, n=7) and Clrn2clarinet/clarinet (red, P6-7, n=9) apical coil OHCs.


Resting open probability ( Po ) of the MET current at the membrane potential of − 121 mV and + 99 mV from apical - ( D ) and basal - coil ( E ) OHCs . Number of OHCs investigated is shown above the columns .

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

Resting open probability (Po) of the MET current at the membrane potential of −121 mV and + 99 mV from apical- (D) and basal-coil (E) OHCs. Number of OHCs investigated is shown above the columns.


Saturating MET currents recorded from a P7 Clrn2clarinet / + ( F ) and a P8 Clrn2clarinet / clarinet ( G ) apical - coil IHC using the same experimental protocol described above .

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

Saturating MET currents recorded from a P7 Clrn2clarinet/+ (F) and a P8 Clrn2clarinet/clarinet (G) apical-coil IHC using the same experimental protocol described above.


Average Po of the MET current measured in apical coil IHCs at the membrane potential of − 121 mV and + 99 mV from Clrn2clarinet / + ( P7 , n = 3 ) and Clrn2clarinet / clarinet ( P7 - 8 , n = 6 ) apical - coil IHCs .

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

Average Po of the MET current measured in apical coil IHCs at the membrane potential of −121 mV and + 99 mV from Clrn2clarinet/+ (P7, n=3) and Clrn2clarinet/clarinet (P7-8, n=6) apical-coil IHCs.


Potassium currents recorded from mature Clrn2clarinet / + ( A , P15 ) and Clrn2clarinet / clarinet ( B , P16 ) apical - coil OHCs . Currents were elicited by depolarizing voltage steps ( 10 mV nominal increments ) from - 144 mV to more depolarized values from the holding potential of - 84 mV . Note that the current characteristic of mature OHCs , The size of IK , n , measured in isolation as the deactivating tail currents ( difference between instantaneous and steady - state inward currents ) for voltage steps from the holding potential to − 124 mV , was 545 ± 115 pA , ( n = 5 ) in Clrn2clarinet / + and 595 ± 67 pA , ( n = 7 ) in Clrn2clarinet / clarinet OHCs .

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

Potassium currents recorded from mature Clrn2clarinet/+ (A, P15) and Clrn2clarinet/clarinet (B, P16) apical-coil OHCs. Currents were elicited by depolarizing voltage steps (10 mV nominal increments) from -144 mV to more depolarized values from the holding potential of -84 mV. Note that the current characteristic of mature OHCs, The size of IK,n, measured in isolation as the deactivating tail currents (difference between instantaneous and steady-state inward currents) for voltage steps from the holding potential to −124 mV, was 545 ± 115 pA, (n=5) in Clrn2clarinet/+ and 595 ± 67 pA, (n=7) in Clrn2clarinet/clarinet OHCs.


Average peak current - voltage relation for the total K + current recorded from Clrn2clarinet / + ( P15 - 16 , n = 7 ) and Clrn2clarinet / clarinet ( P15 - 16 , n = 5 ) OHCs .

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

Average peak current-voltage relation for the total K+ current recorded from Clrn2clarinet/+ (P15-16, n=7) and Clrn2clarinet/clarinet (P15-16, n=5) OHCs.


Potassium currents recorded from P22 mature Clrn2clarinet / + and Clrn2clarinet / clarinet apical - coil IHCs , respectively , using the same voltage protocol described above . Holding potential of - 64 mV .

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

Potassium currents recorded from P22 mature Clrn2clarinet/+ and Clrn2clarinet/clarinet apical-coil IHCs, respectively, using the same voltage protocol described above. Holding potential of -64 mV.


Peak current - voltage relation for the total K + current recorded from the

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

Peak current-voltage relation for the total K+ current recorded from the IHCs


Voltage responses from P22 Clrn2clarinet / + ( G ) and Clrn2clarinet / clarinet ( H , I ) IHCs . Note that Ca2 + - dependent action potentials could be induced in mature IHCs ( I ) .

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

Voltage responses from P22 Clrn2clarinet/+ (G) and Clrn2clarinet/clarinet (H,I) IHCs. Note that Ca2+-dependent action potentials could be induced in mature IHCs (I).


( A - D ) A . castellanii was infected ( MOI 5 ) with L . pneumophila JR32 producing ( A ) GFP under the control of PflaA ( Ptac - mCherry - PflaA - gfp ) and mCherry constitutively , ( B ) GFP under the control of PralF ( PralF - gfp ) and stained with DAPI , ( C ) GFP under the control of PsidC ( PsidC - gfp ) and stained with DAPI , or ( D ) Timer constitutively ( pNP107 ) . The infected amoebae were fixed at different time points post - infection , analyzed by confocal microscopy and 3D reconstructed ( scale bars : 5 µm ) . Motile and virulent bacteria ( GFP - positive ) emerge at the colony cluster periphery only after bacterial replication has ceased ( red fluorescent Timer ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A. castellanii was infected (MOI 5) with L. pneumophila JR32 producing (A) GFP under the control of PflaA (Ptac-mCherry-PflaA-gfp) and mCherry constitutively, (B) GFP under the control of PralF (PralF-gfp) and stained with DAPI, (C) GFP under the control of PsidC (PsidC-gfp) and stained with DAPI, or (D) Timer constitutively (pNP107). The infected amoebae were fixed at different time points post-infection, analyzed by confocal microscopy and 3D reconstructed (scale bars: 5 µm). Motile and virulent bacteria (GFP-positive) emerge at the colony cluster periphery only after bacterial replication has ceased (red fluorescent Timer).


( G ) Transmission electron micrographs of A . castellanii infected with L . pneumophila . A . castellanii was infected ( MOI 5 , 42 h ) with L . pneumophila wild - type strain JR32 and subjected to high - pressure freezing and electron microscopy . Many LCVs were still intact at 42 h post infection ( left panel ; arrow : intact LCV membrane , arrowhead plasma membrane ) , and occasionally , the bacteria were flagellated ( middle panel , arrow ) . Some LCVs were already ruptured , and the bacteria were released into the host cell cytosol ( right panel ; arrows : ruptured LCV membrane ) . Scale bars : 2 µm , 0 . 5 µm ( insets ) .

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

(G) Transmission electron micrographs of A. castellanii infected with L. pneumophila. A. castellanii was infected (MOI 5, 42 h) with L. pneumophila wild-type strain JR32 and subjected to high-pressure freezing and electron microscopy. Many LCVs were still intact at 42 h post infection (left panel; arrow: intact LCV membrane, arrowhead plasma membrane), and occasionally, the bacteria were flagellated (middle panel, arrow). Some LCVs were already ruptured, and the bacteria were released into the host cell cytosol (right panel; arrows: ruptured LCV membrane). Scale bars: 2 µm, 0.5 µm (insets).


( A ) L . pneumophila subpopulation proteomics . A . castellanii was infected ( MOI 5 , 42 h ) with L . pneumophila JR32 ( Ptac - mCherry - PflaA - gfp ) . After cell lysis , released intracellular bacteria were sorted by flow cytometry in mCherry - positive / GFP - negative and mCherry - positive / GFP - positive L . pneumophila subpopulations , and their proteome was determined and compared . Protein abundance in each subset is depicted as a volcano plot . Proteins enriched in the GFP - producing subpopulation have a positive log2 ratio ( q - value ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) L. pneumophila subpopulation proteomics. A. castellanii was infected (MOI 5, 42 h) with L. pneumophila JR32 (Ptac-mCherry-PflaA-gfp). After cell lysis, released intracellular bacteria were sorted by flow cytometry in mCherry-positive/GFP-negative and mCherry-positive/GFP-positive L. pneumophila subpopulations, and their proteome was determined and compared. Protein abundance in each subset is depicted as a volcano plot. Proteins enriched in the GFP-producing subpopulation have a positive log2 ratio (q-value).


( D , E ) Expression of select L . pneumophila genes in amoebae . A . castellanii was infected ( MOI 5 ; 6 h and 48 h ) with L . pneumophila JR32 harboring reporter constructs for ( D ) PfleQ - mCherry - PflaA - gfp or ( E ) PlegC8 - mCherry - PflaA - gfp , respectively . The infected amoebae were fixed and imaged by confocal microscopy ( scale bars : 5 µm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E) Expression of select L. pneumophila genes in amoebae. A. castellanii was infected (MOI 5; 6 h and 48 h) with L. pneumophila JR32 harboring reporter constructs for (D) PfleQ-mCherry-PflaA-gfp or (E) PlegC8-mCherry-PflaA-gfp, respectively. The infected amoebae were fixed and imaged by confocal microscopy (scale bars: 5 µm).


( A - E ) Representative live cell microscopy images of D . discoideum infected with PflaA - GFP - producing L . pneumophila . D . discoideum Ax3 producing cytosolic mCherry ( pDM1042 ) was infected ( MOI 5 ) with ( A , B ) wild - type L . pneumophila JR32 ( Ptac - mCherry - PflaA - gfp ) , ( C , D ) JR32 ( Ptac - mCerulean - PflaA - gfp ) or ( E ) ∆ flaA ( Ptac - mCerulean - PflaA - gfp ) and embedded in agarose for 48 h . PflaA - GFP - positive L . pneumophila ( A ) escapes the LCV ( 0 sec ) , followed by other bacteria , which are contained in the cytosol and localize to the plasma membrane , where a PflaA - GFP - positive L . pneumophila first leaves the ruptured host cell ( 185 sec ) , or ( B ) triggers host cell death ( loss of cytosolic mCherry ) upon exit of the amoeba . PflaA - GFP - positive L . pneumophila ( C ) escape the LCV after 75 sec and spread within the cytosol for several minutes , and ( D ) are the first to exit the host cell , leading to the loss of cytosolic mCherry and host cell death . ( E ) L . pneumophila ∆ flaA lyses the LCV and host cell less vigorously . Scale bars : 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Representative live cell microscopy images of D. discoideum infected with PflaA-GFP-producing L. pneumophila. D. discoideum Ax3 producing cytosolic mCherry (pDM1042) was infected (MOI 5) with (A, B) wild-type L. pneumophila JR32 (Ptac-mCherry-PflaA-gfp), (C, D) JR32 (Ptac-mCerulean-PflaA-gfp) or (E) ∆flaA (Ptac-mCerulean-PflaA-gfp) and embedded in agarose for 48 h. PflaA-GFP-positive L. pneumophila (A) escapes the LCV (0 sec), followed by other bacteria, which are contained in the cytosol and localize to the plasma membrane, where a PflaA-GFP-positive L. pneumophila first leaves the ruptured host cell (185 sec), or (B) triggers host cell death (loss of cytosolic mCherry) upon exit of the amoeba. PflaA-GFP-positive L. pneumophila (C) escape the LCV after 75 sec and spread within the cytosol for several minutes, and (D) are the first to exit the host cell, leading to the loss of cytosolic mCherry and host cell death. (E) L. pneumophila ∆flaA lyses the LCV and host cell less vigorously. Scale bars: 5 µm.


( B ) L . pneumophila bacteria lacking flaA or lqsA are impaired for spreading from amoebae . A . castellanii amoebae were infected ( MOI 5 , 52 h ) with L . pneumophila JR32 , ∆ flaA or ∆ lqsA harboring Ptac - mCherry - PflaA - gfp and embedded in 0 . 5 % agarose / PYG medium . The fluorescence images are representative for the L . pneumophila strains indicated : JR32 ( n = 25 ) , ∆ flaA ( n = 13 ) or ∆ lqsA ( n = 19 ) . Scale bars : 20 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) L. pneumophila bacteria lacking flaA or lqsA are impaired for spreading from amoebae. A. castellanii amoebae were infected (MOI 5, 52 h) with L. pneumophila JR32, ∆flaA or ∆lqsA harboring Ptac-mCherry-PflaA-gfp and embedded in 0.5% agarose/PYG medium. The fluorescence images are representative for the L. pneumophila strains indicated: JR32 (n = 25), ∆flaA (n = 13) or ∆lqsA (n = 19). Scale bars: 20 µm.


( A , B ) Role of the Lqs system for PflaA expression in intracellular L . pneumophila . A . castellanii was infected ( MOI 5 , 48 h ) with L . pneumophila JR32 , ∆ lqsA , ∆ lqsR , ∆ lqsS , ∆ lqsT , or ∆ lqsS - ∆ lqsT harboring Ptac - mCherry - PflaA - gfp , and ( A ) fixed and analyzed by confocal microscopy ( representative 3D reconstructions , scale bars : 10 µm ) , or ( B ) lysed , fixed and analyzed by flow cytometry .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Role of the Lqs system for PflaA expression in intracellular L. pneumophila. A. castellanii was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆lqsA, ∆lqsR, ∆lqsS, ∆lqsT, or ∆lqsS-∆lqsT harboring Ptac-mCherry-PflaA-gfp, and (A) fixed and analyzed by confocal microscopy (representative 3D reconstructions, scale bars: 10 µm), or (B) lysed, fixed and analyzed by flow cytometry.


( C - F ) Role of the Lqs system for LCV escape and cytosolic localization of L . pneumophila . ( C , D ) A . castellanii or ( E , F ) D . discoideum was infected ( MOI 5 , 48 h ) with L . pneumophila JR32 , ∆ lqsA , ∆ lqsR , ∆ lqsS , ∆ lqsT , or ∆ lqsS - ∆ lqsT harboring Ptac - mCherry - PflaA - gfp , fixed and treated with an anti ‐ ubiquitin antibody . Amoebae containing bacteria decorated with ubiquitin were quantified by confocal microscopy . Representative images of ( C ) A . castellanii ( scale bars : 10 µm ) or ( E ) D . discoideum ( scale bars : 5 μm ) infected with L . pneumophila JR32 escaping or not from the LCV are shown . The percentage of amoebae containing ubiquitinated bacteria was quantified by counting the total number of infected cells and the number of ubiquitinated cells : ( D ) JR32 : n = 482 ; ΔlqsA : n = 834 ; ΔlqsR : n = 572 ; ΔlqsS : n = 729 ; ΔlqsT : n = 1469 ; ΔlqsS - ΔlqsT : n = 693 ; ( F ) : JR32 : n = 136 ; ΔlqsA : n = 201 ; ΔlqsR : n = 303 ; ΔlqsS : n = 183 ; ΔlqsT : n = 534 ; ΔlqsS - ΔlqsT : n = 180 .

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

(C-F) Role of the Lqs system for LCV escape and cytosolic localization of L. pneumophila. (C, D) A. castellanii or (E, F) D. discoideum was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆lqsA, ∆lqsR, ∆lqsS, ∆lqsT, or ∆lqsS-∆lqsT harboring Ptac-mCherry-PflaA-gfp, fixed and treated with an anti‐ubiquitin antibody. Amoebae containing bacteria decorated with ubiquitin were quantified by confocal microscopy. Representative images of (C) A. castellanii (scale bars: 10 µm) or (E) D. discoideum (scale bars: 5 μm) infected with L. pneumophila JR32 escaping or not from the LCV are shown. The percentage of amoebae containing ubiquitinated bacteria was quantified by counting the total number of infected cells and the number of ubiquitinated cells: (D) JR32: n = 482; ΔlqsA: n = 834; ΔlqsR: n = 572; ΔlqsS: n = 729; ΔlqsT: n = 1469; ΔlqsS-ΔlqsT: n = 693; (F): JR32: n = 136; ΔlqsA: n = 201; ΔlqsR: n = 303; ΔlqsS: n = 183; ΔlqsT: n = 534; ΔlqsS-ΔlqsT: n = 180.


( G , H ) D . discoideum producing the LCV / PtdIns ( 4 ) P probe P4C - GFP and cytosolic - mCherry was infected ( MOI 5 , 48 h ) with L . pneumophila JR32 , ∆ lqsA , ∆ lqsR , ∆ lqsS , ∆ lqsT , ∆ lqsS - ∆ lqsT , or ∆ flaA harboring Ptac - mCerulean . Representative images of ( G ) D . discoideum infected with L . - pneumophila JR32 in intact cells with intact LCV ( 18 . 5 % ) , lysed cells and lysed LCVs ( 60 . 2 % ) , intact cells with lysed LCVs ( 15 . 5 % ) and lysed cells with intact LCVS ( 5 . 8 % ) are shown ( scale bars : 5 µm ) . ( H ) The percentage of amoebae with intact LCVs was quantified by counting the total number of infected cells and the number of cells with intact LCV membrane : JR32 : n = 426 ; ΔlqsA : n = 468 ; ΔlqsR : n = 344 ; ΔlqsS : n = 325 ; ΔlqsT : n = 287 ; ΔlqsS - ΔlqsT : n = 289 ; ΔflaA : n = 248 .

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

(G, H) D. discoideum producing the LCV/PtdIns(4)P probe P4C-GFP and cytosolic-mCherry was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆lqsA, ∆lqsR, ∆lqsS, ∆lqsT, ∆lqsS-∆lqsT, or ∆flaA harboring Ptac-mCerulean. Representative images of (G) D. discoideum infected with L.-pneumophila JR32 in intact cells with intact LCV (18.5%), lysed cells and lysed LCVs (60.2%), intact cells with lysed LCVs (15.5%) and lysed cells with intact LCVS (5.8%) are shown (scale bars: 5 µm). (H) The percentage of amoebae with intact LCVs was quantified by counting the total number of infected cells and the number of cells with intact LCV membrane: JR32: n = 426; ΔlqsA: n = 468; ΔlqsR: n = 344; ΔlqsS: n = 325; ΔlqsT: n = 287; ΔlqsS-ΔlqsT: n = 289; ΔflaA: n = 248.


D . discoideum producing the LCV / PtdIns ( 4 ) P probe P4C - mCherry was infected ( MOI 5 , 48 h ) with L . pneumophila wild - type strains ( JR32 , Corby ) , or phospholipase triple mutants ( ∆ plcABC , ∆ plaACD ) harboring the Ptac - mCerulean - PflaA - gfp reporter construct , and fixed and treated with an anti ‐ ubiquitin antibody , LCV escape and cytosolic localization of the bacteria was assessed by confocal microscopy through quantification of amoeba containing intact , P4C - mCherry - positive LCVs or ubiquitin - decorated , cytosolic bacteria . ( A ) Representative images of amoebae harboring L . pneumophila confined within LCVs or released from the LCV to the cytosol are shown ( scale bars : 5 μm ) .

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

D. discoideum producing the LCV/PtdIns(4)P probe P4C-mCherry was infected (MOI 5, 48 h) with L. pneumophila wild-type strains (JR32, Corby), or phospholipase triple mutants (∆plcABC, ∆plaACD) harboring the Ptac-mCerulean-PflaA-gfp reporter construct, and fixed and treated with an anti‐ubiquitin antibody, LCV escape and cytosolic localization of the bacteria was assessed by confocal microscopy through quantification of amoeba containing intact, P4C-mCherry-positive LCVs or ubiquitin-decorated, cytosolic bacteria. (A) Representative images of amoebae harboring L. pneumophila confined within LCVs or released from the LCV to the cytosol are shown (scale bars: 5 μm).


( a , b ) Histograms of TDP43 ( WT ) - EGFP ( a ) and TDP43A315T - EGFP ( b ) levels in individual neurons . Increasing amounts of DNA significantly shifted the distribution of log - transformed expression levels toward higher values ( P 0 . 01 for all comparisons , two - sided Kolmogorov - Smirnov test ) . TDP43 ( WT ) - EGFP , n = 1 , 287 neurons , with 290 - 342 cells per group ; TDP43A315T - EGFP , n = 1 , 290 , with 315 - 332 neurons per group . Values were pooled from 12 wells per condition , with experiments performed in duplicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Histograms of TDP43(WT)-EGFP (a) and TDP43A315T-EGFP (b) levels in individual neurons. Increasing amounts of DNA significantly shifted the distribution of log-transformed expression levels toward higher values (P 0.01 for all comparisons, two-sided Kolmogorov-Smirnov test). TDP43(WT)-EGFP, n = 1,287 neurons, with 290-342 cells per group; TDP43A315T-EGFP, n = 1,290, with 315-332 neurons per group. Values were pooled from 12 wells per condition, with experiments performed in duplicate.


( c ) Automated fluorescence microscopy . Primary rodent cortical neurons were transfected with mApple and TDP43 - EGFP , and survival was determined by repeated imaging at regular intervals . The last time at which the cell was noted to be alive ( red arrows ) was used as the time of death . Cells that survive the entire length of the experiment ( cyan arrow ) were censored . Scale bar , 25 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Automated fluorescence microscopy. Primary rodent cortical neurons were transfected with mApple and TDP43-EGFP, and survival was determined by repeated imaging at regular intervals. The last time at which the cell was noted to be alive (red arrows) was used as the time of death. Cells that survive the entire length of the experiment (cyan arrow) were censored. Scale bar, 25 μm.