figf3 ( a ) Immunoblot analysis of the WIPI4 protein ( apparent mobility at ∼ 35 kDa ) in LCLs ( top ) . Truncated forms were not detected ( middle ) . HSP90 was used as a loading control ( bottom ) . An asterisk indicates nonspecific immunoreactive bands . ( b ) Cells were treated with 250 nM Torin1 in the presence or absence of 20 μM chloroquine for 2 h . Cell lysates were analyzed by SDS - PAGE and immunoblotting using antibodies to LC3 , WIPI4 and HSP90 . The positions of LC3 - I ( cytosolic ) and LC3 - II ( membrane bound ) are indicated . ( c - e ) Cells were cultured in the presence of Torin1 for 2 h . ( c ) Cytospun cells were fixed and analyzed by immunofluorescence microscopy using antibodies to LC3 and ATG9A . Abnormal colocalization of LC3 with ATG9A was observed in the LCLs of affected subjects . Scale bars , 10 μm and 1 μm in the inset . ( d , e ) The numbers of LC3 + ( d ) and LC3 + ATG9A + ( e ) foci were quantified from more than 20 images from 3 independent samples ( Online Methods ) . Data are presented as mean ± s . e . m . * P 0 . 05 , ANOVA followed by Bonferroni - Dunn post - hoc test .

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figf3(a) Immunoblot analysis of the WIPI4 protein (apparent mobility at ∼35 kDa) in LCLs (top). Truncated forms were not detected (middle). HSP90 was used as a loading control (bottom). An asterisk indicates nonspecific immunoreactive bands.(b) Cells were treated with 250 nM Torin1 in the presence or absence of 20 μM chloroquine for 2 h. Cell lysates were analyzed by SDS-PAGE and immunoblotting using antibodies to LC3, WIPI4 and HSP90. The positions of LC3-I (cytosolic) and LC3-II (membrane bound) are indicated.(c-e) Cells were cultured in the presence of Torin1 for 2 h. (c) Cytospun cells were fixed and analyzed by immunofluorescence microscopy using antibodies to LC3 and ATG9A. Abnormal colocalization of LC3 with ATG9A was observed in the LCLs of affected subjects. Scale bars, 10 μm and 1 μm in the inset. (d,e) The numbers of LC3+ (d) and LC3+ATG9A+ (e) foci were quantified from more than 20 images from 3 independent samples (Online Methods). Data are presented as mean ± s.e.m. *P 0.05, ANOVA followed by Bonferroni-Dunn post-hoc test.


Figure 1 ( A ) Crystal structure of a dynamin monomer ( PDB ID : 3SNH ) and corresponding color - coded linear domain architecture of dynamin . The PHD is shown in both the closed ( 3SNH , solution structure ) and open ( 3ZYS , cryo - EM reconstruction of membrane - bound dynamin ) states . The approximate locations of terminal residues flanking different functional domains are identified with black arrowheads . The positions of the L1SP and L2SP loops are identified by red arrowheads . Changes in HDX behavior for dynamin analyzed ( B ) in solution the presence GMPPCP , ( C ) in solution in the presence of GDP • AlF4 - , ( D ) membrane - bound , or ( E ) membrane - bound in the presence of GMPPCP and mapped onto the structures of dynamin monomers . Color coding ( see scale below ) shows the extent of solvent exchange relative to apo dynamin in solution . All HDX MS data shown represents an average of 3 replicates and are color coded from red to blue with warm colors representing increased conformational dynamics ( red being the highest D2O uptake ) and cool colors representing decreased conformational dynamics ( blue being the lowest D2O uptake ) . Grey indicates no statistically significant change between the two states . Quantitative data on the individual peptides used to generate this map are provided in Appendix ( Figure S1 - S4 ) .

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Figure 1(A) Crystal structure of a dynamin monomer (PDB ID: 3SNH) and corresponding color-coded linear domain architecture of dynamin. The PHD is shown in both the closed (3SNH, solution structure) and open (3ZYS, cryo-EM reconstruction of membrane-bound dynamin) states. The approximate locations of terminal residues flanking different functional domains are identified with black arrowheads. The positions of the L1SP and L2SP loops are identified by red arrowheads.Changes in HDX behavior for dynamin analyzed (B) in solution the presence GMPPCP, (C) in solution in the presence of GDP•AlF4-, (D) membrane-bound, or (E) membrane-bound in the presence of GMPPCP and mapped onto the structures of dynamin monomers. Color coding (see scale below) shows the extent of solvent exchange relative to apo dynamin in solution. All HDX MS data shown represents an average of 3 replicates and are color coded from red to blue with warm colors representing increased conformational dynamics (red being the highest D2O uptake) and cool colors representing decreased conformational dynamics (blue being the lowest D2O uptake). Grey indicates no statistically significant change between the two states. Quantitative data on the individual peptides used to generate this map are provided in Appendix (Figure S1-S4).


Figure 2 ( A ) Changes in solvent exchange superimposed onto the structure ( PDB ID : 2SNH ) of the G domain or ( B ) the stalk / PHD of dynamin in the GMPPCP or lipid bound states , respectively . Key structural elements are identified and the color - coding shows the magnitude of solvent exchange relative to the apo state ( see scale below ) . ( C ) Differential kinetics of solvent exchange of selected peptides that were identified as altered in both GMPPCP and GDP • AlF4 - - bound states . The peptides are mapped to secondary structure elements on the polypeptide sequence . Differences in kinetics of solvent exchange of select peptides measured ( D ) for dynamin bound to GMPPCP in the presence or absence of lipid nanotubes ( E ) for dynamin bound to lipid nanotubes in the presence or absence of GMPPCP . HDX data are presented as an average of three independent triplicates and color coded as in Figure 1 .

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Figure 2(A) Changes in solvent exchange superimposed onto the structure (PDB ID: 2SNH) of the G domain or (B) the stalk/PHD of dynamin in the GMPPCP or lipid bound states, respectively. Key structural elements are identified and the color-coding shows the magnitude of solvent exchange relative to the apo state (see scale below).(C) Differential kinetics of solvent exchange of selected peptides that were identified as altered in both GMPPCP and GDP•AlF4--bound states. The peptides are mapped to secondary structure elements on the polypeptide sequence. Differences in kinetics of solvent exchange of select peptides measured (D) for dynamin bound to GMPPCP in the presence or absence of lipid nanotubes (E) for dynamin bound to lipid nanotubes in the presence or absence of GMPPCP. HDX data are presented as an average of three independent triplicates and color coded as in Figure 1.


Figure 3 ( A ) Design of a functional dynamin mutant to probe PHD stalk interactions . Minimal structure of the PHD and the PHD - Stalk interface showing the location of important residues discussed in this study . The unresolved loop L1NS in the stalk hosts the IAEDANS label and is sensitive to the two tryptophans ( W525 and W542 ) in the PHD . The distance between two introduced cysteines ( S357C and native C602 ) used to construct a double cysteine Dyn1 is also shown . ( B ) Comparison of emission spectra of various IAEDANS - labeled Dyn1 RCL variants ( D352C , Y354C , S357C , I365C , H367C ) , upon Trp excitation at 295 nm . ( C ) FRET transfer upon excitation is contributed by two neighboring Trps ( W525 and W542 ) ; Substitution by Phe resulted in significant loss of FRET . ( D ) IAEDANS emission kinetics upon addition of lipid nanotubes with or without PI4 , 5P2 ( ex = 295 ) . The arrow indicates addition of lipids .

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Figure 3(A) Design of a functional dynamin mutant to probe PHD stalk interactions. Minimal structure of the PHD and the PHD-Stalk interface showing the location of important residues discussed in this study. The unresolved loop L1NS in the stalk hosts the IAEDANS label and is sensitive to the two tryptophans (W525 and W542) in the PHD. The distance between two introduced cysteines (S357C and native C602) used to construct a double cysteine Dyn1 is also shown.(B) Comparison of emission spectra of various IAEDANS-labeled Dyn1 RCL variants (D352C, Y354C, S357C, I365C, H367C), upon Trp excitation at 295 nm.(C) FRET transfer upon excitation is contributed by two neighboring Trps (W525 and W542); Substitution by Phe resulted in significant loss of FRET.(D) IAEDANS emission kinetics upon addition of lipid nanotubes with or without PI4,5P2 (ex=295). The arrow indicates addition of lipids.


Figure 4 ( B ) Histogram shows basal and assembly - stimulated GTPase activities of non - crosslinked Dyn1CC vs crosslinked Dyn1Closed and Dyn1WT . Inset shows crosslinking of the PHD and stalk domains in Dyn1Y354C / S607C ( Dyn1CC ) with the bifunctional crosslinker , MTS - 2 - MTS , to yield Dyn1Closed , which migrates more slowly on SDS - PAGE , as well as liposome binding of Dyn1CC compared to Dyn1Closed ( Bound , B ; Unbound , UB ) . ( C ) Ability of Dyn1WT , Dyn1CC and Dyn1Closed to catalyze membrane fission and vesicle release from SUPER templates . ( D ) Size exclusion chromatographic profiles of Dyn1CC , Dyn1Closed at room temperature or after incubation for 10 min at 37 ° C . ( E ) Oligomeric state of Dyn1ΔΔ at room temperature . ( F ) Negative - stain electron micrographs of Dyn1WT , Dyn1ΔΔ and Dyn1Closed assembled onto lipid nanotubes . Scale bar = 200 nm ( G ) Curvature dependent liposome - stimulated GTPase activity of Dyn1WT and Dyn1ΔΔ . ( H ) Ability of Dyn1WT and Dyn1ΔΔ to catalyze membrane fission and vesicle release from SUPER templates .

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Figure 4(B) Histogram shows basal and assembly-stimulated GTPase activities of non-crosslinked Dyn1CC vs crosslinked Dyn1Closed and Dyn1WT. Inset shows crosslinking of the PHD and stalk domains in Dyn1Y354C/S607C (Dyn1CC) with the bifunctional crosslinker, MTS-2-MTS, to yield Dyn1Closed, which migrates more slowly on SDS-PAGE, as well as liposome binding of Dyn1CC compared to Dyn1Closed (Bound, B; Unbound, UB).(C) Ability of Dyn1WT, Dyn1CC and Dyn1Closed to catalyze membrane fission and vesicle release from SUPER templates.(D) Size exclusion chromatographic profiles of Dyn1CC, Dyn1Closed at room temperature or after incubation for 10 min at 37°C.(E) Oligomeric state of Dyn1ΔΔ at room temperature.(F) Negative-stain electron micrographs of Dyn1WT, Dyn1ΔΔ and Dyn1Closed assembled onto lipid nanotubes. Scale bar = 200 nm(G) Curvature dependent liposome-stimulated GTPase activity of Dyn1WT and Dyn1ΔΔ.(H) Ability of Dyn1WT and Dyn1ΔΔ to catalyze membrane fission and vesicle release from SUPER templates.


Figure 5 ( A ) Temperature dependence of basal and lipid stimulated GTPase activity of Dyn1WT and Dyn2S619L . ( B ) Temperature sensitive membrane fission activity of Dyn1WT , Dyn2WT and Dyn2S619L measured using SUPER templates . ( C ) Detection of Dyn2 PHD - stalk interactions by FRET using IAEDANS - labeled Dyn2L354C . ( D ) Curvature dependent membrane binding and consequent opening of PHD in Dyn1Y354C - IAEDANS and Dyn2L354C - IAEDANS . ( E ) Differential temperature dependence of PHD - stalk FRET for WT and S619L mutant Dyn2IAEDANS . Data are presented as relative FRET distances assuming a single donor / acceptor pair . ( F ) Differential solvent exchange kinetics of Dyn1S619L at ambient temperature compared to Dyn1WT under identical conditions . Yellow coloring indicates a significant increase in HDX . Quantitative data on the individual peptides used to generate this map are provided in ( Appendix Fig . S5 ) .

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Figure 5(A) Temperature dependence of basal and lipid stimulated GTPase activity of Dyn1WT and Dyn2S619L.(B) Temperature sensitive membrane fission activity of Dyn1WT, Dyn2WT and Dyn2S619L measured using SUPER templates.(C) Detection of Dyn2 PHD-stalk interactions by FRET using IAEDANS-labeled Dyn2L354C.(D) Curvature dependent membrane binding and consequent opening of PHD in Dyn1Y354C-IAEDANS and Dyn2L354C-IAEDANS.(E) Differential temperature dependence of PHD-stalk FRET for WT and S619L mutant Dyn2IAEDANS. Data are presented as relative FRET distances assuming a single donor/acceptor pair.(F) Differential solvent exchange kinetics of Dyn1S619L at ambient temperature compared to Dyn1WT under identical conditions. Yellow coloring indicates a significant increase in HDX. Quantitative data on the individual peptides used to generate this map are provided in (Appendix Fig. S5).


Figure 6 ( A , B ) Transferrin receptor ( TfnR ) uptake , a measure of CME , shown as % uptake of total steady - state surface TfnR for control H1299 cells , and H1299 cells stabily expressing low amounts of siRNA - resistant Dyn2WT - EGFP ( A ) or Dyn2S619L - EGFP ( B ) with or without treatment of siRNA - directed towards endogenous Dyn2 . ( C ) Fixed cell TIRF - M images of Dyn2WT - EGFP or Dyn2S619L - EGFP expressing H1299 cells with or without Dyn2 - siRNA treatment showing co - localization with clathrin light chain immunostained rabbit anti - CLC and Alexa 547 - conjugated secondary antibodies . ( D ) Percentage of Dyn2 - EGFP positive clathrin coated pits detected by live cell TIRF - M and automated master / slave image analysis ( Aguet et al , 2013 ) . ( E ) Lifetime analysis of CCPs which are positive for EGFP - Dyn2WT or EGFP - Dyn2S619L with or without siRNA - mediated specific knockdown of endogenous Dyn2 .

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Figure 6(A,B) Transferrin receptor (TfnR) uptake, a measure of CME, shown as % uptake of total steady-state surface TfnR for control H1299 cells, and H1299 cells stabily expressing low amounts of siRNA-resistant Dyn2WT-EGFP (A) or Dyn2S619L-EGFP (B) with or without treatment of siRNA-directed towards endogenous Dyn2.(C) Fixed cell TIRF-M images of Dyn2WT-EGFP or Dyn2S619L-EGFP expressing H1299 cells with or without Dyn2-siRNA treatment showing co-localization with clathrin light chain immunostained rabbit anti-CLC and Alexa 547-conjugated secondary antibodies.(D) Percentage of Dyn2-EGFP positive clathrin coated pits detected by live cell TIRF-M and automated master/slave image analysis (Aguet et al, 2013).(E) Lifetime analysis of CCPs which are positive for EGFP-Dyn2WT or EGFP-Dyn2S619L with or without siRNA-mediated specific knockdown of endogenous Dyn2.


Figure 1B WT HeLa cells were incubated with HPG for 30 minutes in the presence or absence of Harr and / or Chloramphenicol ( Chl ) . After fixation , AF647 - azide ( magenta ) was used for click reaction ( see A ) , and cells were immunostained for TOM20 as mitochondrial marker ( green ) . Representative epifluorescent microscopy images are shown . Magenta images shown in the presence of only Chl and in the absence of both inhibitors ( none ) are presented at lower brightness level than the other panels , duo to their very strong intensities . An inset of Harr panel is zoomed in the panel below for better visualization . Met , methionine . Scale bars , 50 µm and 10 µm for the zoom panel .

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Figure 1B WT HeLa cells were incubated with HPG for 30 minutes in the presence or absence of Harr and/or Chloramphenicol (Chl). After fixation, AF647-azide (magenta) was used for click reaction (see A), and cells were immunostained for TOM20 as mitochondrial marker (green). Representative epifluorescent microscopy images are shown. Magenta images shown in the presence of only Chl and in the absence of both inhibitors (none) are presented at lower brightness level than the other panels, duo to their very strong intensities. An inset of Harr panel is zoomed in the panel below for better visualization. Met, methionine. Scale bars, 50 µm and 10 µm for the zoom panel.


Figure 2A Mitochondrial translation products were labeled in WT HeLa cells for indicated time lengths ( heat map ) and cells immunostained for TOM20 ( gray ) . Representative confocal images are shown for each time point . The same 5 - minute image was used in both rows with different brightness as a reference for intensity comparison of the other time points in the row . Scale bars , 10 µm . Mitochondrial protein synthesis was quantified over time . 30 cells from N = 3 independent experiments were analyzed for each time point ( Average of the HPG intensity in the mitochondrial area was calculated and the baseline was set to zero by subtracting the value of time point 0 from all the values . Error bars , + / - SEM ) . A hyperbolic graph was fitted to the data . R - squared , 0 . 97 . B As in A , Mitochondrial translation products were labeled for increasing time ( 10 and 20 minutes ) in HeLa cells . The number of analyzed cells was increased in the plots from 25 to 300 per time point . 150 or 300 random cells from N = 3 independent experiments were analyzed . Kruskal - Wallis followed by Fisher ' s test . * * * * p < 0 . 0001 . Error bars , + / - SEM . C Mitochondrial translation products were labeled for 30 minutes and clicked with AF647 ( magenta ) and cells immunostained for TOM20 ( green ) in fibroblasts , iPSCs - derived cardiomyocytes , and hippocampal neurons . Representative epifluorescent microscopy images are shown . The correlation of HPG and TOM20 channels is represented in fire lookup table , after calculating the Pearson Correlation values over each pixel of the images . Scale bars , 25 µm .

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Figure 2A Mitochondrial translation products were labeled in WT HeLa cells for indicated time lengths (heat map) and cells immunostained for TOM20 (gray). Representative confocal images are shown for each time point. The same 5-minute image was used in both rows with different brightness as a reference for intensity comparison of the other time points in the row. Scale bars, 10 µm. Mitochondrial protein synthesis was quantified over time. 30 cells from N = 3 independent experiments were analyzed for each time point (Average of the HPG intensity in the mitochondrial area was calculated and the baseline was set to zero by subtracting the value of time point 0 from all the values. Error bars, +/- SEM). A hyperbolic graph was fitted to the data. R-squared, 0.97.B As in A, Mitochondrial translation products were labeled for increasing time (10 and 20 minutes) in HeLa cells. The number of analyzed cells was increased in the plots from 25 to 300 per time point. 150 or 300 random cells from N = 3 independent experiments were analyzed. Kruskal-Wallis followed by Fisher's test. ****p < 0.0001. Error bars, +/- SEM.C Mitochondrial translation products were labeled for 30 minutes and clicked with AF647 (magenta) and cells immunostained for TOM20 (green) in fibroblasts, iPSCs-derived cardiomyocytes, and hippocampal neurons. Representative epifluorescent microscopy images are shown. The correlation of HPG and TOM20 channels is represented in fire lookup table, after calculating the Pearson Correlation values over each pixel of the images. Scale bars, 25 µm.


Figure 3A Mitochondrial translation products were labeled using a FUNCAT approach and [ 35S ] Met labeling in control and TSFM patient fibroblasts . Representative epifluorescent microscopy and the autoradiography images are shown . Scale bars , 50 µm . Changes in mitochondrial protein synthesis were quantified for both approaches . For microscopy image analysis , > 300 cells from N = 3 independent experiments were analyzed for each cell type . Mann - Whitney U test , * * * * p < 0 . 0001 . For radiolabeling analyses , N = 3 independent samples were quantified for each cell type . Student t test , * * p < 0 . 01 . Error bars , + / - SEM . B As in A using control and COA6 KO HEK cells . Scale bars , 25 µm . For microscopy image analysis , * * * * p < 0 . 0001 , and for radiolabeling experiments , * p < 0 . 05 were calculated . Error bars , + / - SEM .

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Figure 3A Mitochondrial translation products were labeled using a FUNCAT approach and [35S]Met labeling in control and TSFM patient fibroblasts. Representative epifluorescent microscopy and the autoradiography images are shown. Scale bars, 50 µm. Changes in mitochondrial protein synthesis were quantified for both approaches. For microscopy image analysis, >300 cells from N = 3 independent experiments were analyzed for each cell type. Mann-Whitney U test, ****p < 0.0001. For radiolabeling analyses, N =3 independent samples were quantified for each cell type. Student t test, **p < 0.01. Error bars, +/- SEM. B As in A using control and COA6 KO HEK cells. Scale bars, 25 µm. For microscopy image analysis, ****p < 0.0001, and for radiolabeling experiments, *p < 0.05 were calculated. Error bars, +/- SEM.


Figure 4A Mitochondrial translation products were labeled with HPG in hippocampal neurons , immunostained for ANK - G ( initial axon segment ) , and plasma membrane was stained with DiO . Representative epifluorescent images and zoomed insets of neural branches are shown . In both axon ( ANK - G positive , arrow ) and dendrite ( star ) , HPG incorporation revealing mitochondrial protein synthesis was detected . Scale bars , 20 µm . B Representative confocal images of a dendrite ( top ) and an axon ( bottom ) in which mitochondrial translation products were labeled with HPG ( magenta ) . HOMER1 and VGLUT1 were used as post - and presynapse markers , respectively ( green ) and DiO stained randomly neurites ( gray ) . In each neurite , an exemplary region was selected and the DiO was outlined to mark the outer edge of the region . As a control , cells were treated with Chloramphenicol ( Chl ) to inhibit mitochondrial translation . Scale bars , 5 µm .

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Figure 4A Mitochondrial translation products were labeled with HPG in hippocampal neurons, immunostained for ANK-G (initial axon segment), and plasma membrane was stained with DiO. Representative epifluorescent images and zoomed insets of neural branches are shown. In both axon (ANK-G positive, arrow) and dendrite (star), HPG incorporation revealing mitochondrial protein synthesis was detected. Scale bars, 20 µm.B Representative confocal images of a dendrite (top) and an axon (bottom) in which mitochondrial translation products were labeled with HPG (magenta). HOMER1 and VGLUT1 were used as post- and presynapse markers, respectively (green) and DiO stained randomly neurites (gray). In each neurite, an exemplary region was selected and the DiO was outlined to mark the outer edge of the region. As a control, cells were treated with Chloramphenicol (Chl) to inhibit mitochondrial translation. Scale bars, 5 µm.


Figure 2Figure 2 . SPOP localizes to nuclear speckles or other nuclear bodies . Confocal microscopy images of fixed NIH 3T3 cells transiently expressing ( A ) full length HA - SPOP , ( B ) GFP - Gli31455 , and ( C ) HA - SPOP + GFP - Gli31 - 455 are shown . DAPI was used to stain the nucleus , SPOP localization was identified using a anti - HA antibody , nuclear speckle localization was identified using a anti - SC - 35 antibody , and Gli31 - 455 localization was identified via GFP fluorescence . SPOP co - localizes with a marker for nuclear speckles or with the substrate Gli31455 . The areas with overlapping HA - and GFP signal contain 75 % of the punctate HA signal , and 100 % of the punctate GFP signal .

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Figure 2Figure 2. SPOP localizes to nuclear speckles or other nuclear bodies. Confocal microscopy images of fixed NIH 3T3 cells transiently expressing (A) full length HA-SPOP, (B) GFP-Gli31455, and (C) HA-SPOP+GFP-Gli31-455 are shown. DAPI was used to stain the nucleus, SPOP localization was identified using a anti-HA antibody, nuclear speckle localization was identified using a anti-SC-35 antibody, and Gli31-455 localization was identified via GFP fluorescence. SPOP co-localizes with a marker for nuclear speckles or with the substrate Gli31455. The areas with overlapping HA- and GFP signal contain 75% of the punctate HA signal, and 100% of the punctate GFP signal.


Figure 3 ( A - B ) NIH 3T3 cells were ( A ) co - transfected with constructs expressing full length SPOP and GFP - Gli31455 or ( B ) transfected with a construct expressing SC - 35 - GFP , and GFP fluorescence was monitored in live cells . ( A ) Snapshots were taken from individual timepoints as noted in the Figure to show photobleaching ( at 0 s ) , recovery , and nuclear body fusion events . The arrow in each panel points to the body that was photobleached and subsequently fuses with another body . These data were not used to calculate the average recovery time as the fusion event precludes accurate measurement of fluorescence recovery . ( B ) Snapshots at indicated timepoints show a nuclear speckle fusion event . Additional images are shown in Fig EV1 . ( C ) Individual nuclear speckles were photobleached and FRAP was monitored for 90 sec . Data from 45 individual cells and FRAP events were corrected for background , normalized to the minimum and maximum intensity , and the mean is shown with error bars representing the standard error of the mean . The mean characteristic recovery time is indicated ± SEM . ( D - E ) Nuclear bodies are close to spherical . ( D ) Aspect ratios for speckles observed in NIH 3T3 cells transfected with constructs expressing HA - SPOP ( 151 cells ) ( D ) , or GFP - Gli31 - 455 and HA - SPOP ( 155 cells ) ( E ) were calculated as described previously ( Brangwynne et al , 2011 ) . Representative individual images of cells are shown as insets in each panel .

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Figure 3(A-B) NIH 3T3 cells were (A) co-transfected with constructs expressing full length SPOP and GFP-Gli31455 or (B) transfected with a construct expressing SC-35-GFP, and GFP fluorescence was monitored in live cells. (A) Snapshots were taken from individual timepoints as noted in the Figure to show photobleaching (at 0 s), recovery, and nuclear body fusion events. The arrow in each panel points to the body that was photobleached and subsequently fuses with another body. These data were not used to calculate the average recovery time as the fusion event precludes accurate measurement of fluorescence recovery. (B) Snapshots at indicated timepoints show a nuclear speckle fusion event. Additional images are shown in Fig EV1.(C) Individual nuclear speckles were photobleached and FRAP was monitored for 90 sec. Data from 45 individual cells and FRAP events were corrected for background, normalized to the minimum and maximum intensity, and the mean is shown with error bars representing the standard error of the mean. The mean characteristic recovery time is indicated ± SEM.(D-E) Nuclear bodies are close to spherical. (D) Aspect ratios for speckles observed in NIH 3T3 cells transfected with constructs expressing HA-SPOP (151 cells) (D), or GFP-Gli31-455 and HA-SPOP (155 cells) (E) were calculated as described previously (Brangwynne et al, 2011). Representative individual images of cells are shown as insets in each panel.


Figure 4 ( A ) SEC chromatograms of given loading concentrations of SPOP28 - 359 are shown . The concentrations were normalized to the monomer molecular weight , i . e . , identical concentrations of dimeric SPOP ΔBACK ( see Fig EV2 ) and oligomeric SPOP28 - 359 contain identical numbers of protomers . ( B ) SEC chromatograms for SPOP constructs defective in self - association in one or both oligomerization domains are shown . Proteins were injected at the same loading concentration ( 533 µM ) and the elution volume of globular molecular weight standards is noted above the graph . ( C ) SEC chromatograms of SPOP28 - 359 ( 200 µM ) , SPOP mutBACK ( 718 µM ) , and mixtures of the two ( ratios given in the Figure , SPOP at 200 µM ) are shown . The elution volume of globular molecular weight standards is noted above the graph . ( D ) Constructs for expressing full - length HA - SPOP or HA - SPOP mutants capable of oligomerization through only one ( or neither ) of the self - association domains were transfected into NIH 3T3 cells . DAPI was used to stain the nucleus and SPOP localization was identified using an anti - HA antibody . Experiments were performed at least twice on four biological samples . Multiple cells were examined and representative cells are shown . For additional images see Appendix Fig S3 . Scale bar represents 5 µm .

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Figure 4(A) SEC chromatograms of given loading concentrations of SPOP28-359 are shown. The concentrations were normalized to the monomer molecular weight, i.e., identical concentrations of dimeric SPOP ΔBACK (see Fig EV2) and oligomeric SPOP28-359 contain identical numbers of protomers. (B) SEC chromatograms for SPOP constructs defective in self-association in one or both oligomerization domains are shown. Proteins were injected at the same loading concentration (533 µM) and the elution volume of globular molecular weight standards is noted above the graph. (C) SEC chromatograms of SPOP28-359 (200 µM), SPOP mutBACK (718 µM), and mixtures of the two (ratios given in the Figure, SPOP at 200 µM) are shown. The elution volume of globular molecular weight standards is noted above the graph.(D) Constructs for expressing full-length HA-SPOP or HA-SPOP mutants capable of oligomerization through only one (or neither) of the self-association domains were transfected into NIH 3T3 cells. DAPI was used to stain the nucleus and SPOP localization was identified using an anti-HA antibody. Experiments were performed at least twice on four biological samples. Multiple cells were examined and representative cells are shown. For additional images see Appendix Fig S3. Scale bar represents 5 µm.


Figure 5 ( A ) Substrate binding was assayed by anisotropy using a fluorescently labeled substrate peptide . All SPOP proteins bound substrate with an affinity similar to that of SPOP28 - 359 ( 4 - 8 µM , see Table 2 ) . Experimental data are shown as circles ; the solid lines are fits to eq . 2 ( Roehrl et al , 2004 ) . ( B ) In vitro crosslinking assays were performed for SPOP28 - 359 and each mutant at 30 μM protein with the amide - specific BS3 crosslinker . Crosslinking for SPOP ΔBACK and MATH domain are shown to demonstrate that crosslinking conditions do not lead to non - specific crosslinking of protein species . ( C ) Crosslinking reactions were performed on whole cell lysates from cells expressing wild type SPOP , SPOP mutBACK , SPOP mutBTB , or SPOP mutBTB - BACK . SPOP1 , SPOP2 and SPOPn identify SPOP monomers , dimers and larger species , respectively . For loading levels see Appendix Fig S4 .

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Figure 5(A) Substrate binding was assayed by anisotropy using a fluorescently labeled substrate peptide. All SPOP proteins bound substrate with an affinity similar to that of SPOP28-359 (4-8 µM, see Table 2). Experimental data are shown as circles; the solid lines are fits to eq. 2 (Roehrl et al, 2004).(B) In vitro crosslinking assays were performed for SPOP28-359 and each mutant at 30 μM protein with the amide-specific BS3 crosslinker. Crosslinking for SPOP ΔBACK and MATH domain are shown to demonstrate that crosslinking conditions do not lead to non-specific crosslinking of protein species. (C) Crosslinking reactions were performed on whole cell lysates from cells expressing wild type SPOP, SPOP mutBACK, SPOP mutBTB, or SPOP mutBTB-BACK. SPOP1, SPOP2 and SPOPn identify SPOP monomers, dimers and larger species, respectively. For loading levels see Appendix Fig S4.


Figure 6 ( A ) SV - AUC data for 0 . 2 nM AF488 - SPOP ΔBACK . Fluorescence scans were collected for 12 h and are plotted against distance from the axis of rotation ( circles ) . The data were subjected to standard c ( s ) analysis in SEDFIT ( Schuck , 2000 ) and the results of this fit are shown ( solid lines , rainbow color scheme ) . ( B ) Sedimentation coefficient distributions for a selection of values from the dilution series are shown . ( C ) The isotherm of weighted - average s - values vs . concentration ( black circles ) , was fit to a dimer self - association model and reveals a KD of 1 . 11 nM , [ 95 % confidence interval 0 . 88 , 1 . 40 nM ] ( solid line ) . ( D ) The BACK domain dimerizes with micromolar affinity . Experimental weight - average molar mass ( Mw ) from CG - MALS for SPOP mutBTB ( black circles ) was fitted to a self - association model ( see text for details ) for BACK - BACK dimerization ( black line ) yielding a KD of 36 ± 3 µM ( average and standard deviation from 3 independent experiments ) . Fits to the monomer / dimer equilibrium model and to alternative monomer / trimer , tetramer and pentamer equilibrium models are depicted as black solid line and as dashed and grey lines , respectively . Residuals for all models are depicted in the lower panel .

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Figure 6(A) SV-AUC data for 0.2 nM AF488-SPOP ΔBACK. Fluorescence scans were collected for 12 h and are plotted against distance from the axis of rotation (circles). The data were subjected to standard c(s) analysis in SEDFIT (Schuck, 2000) and the results of this fit are shown (solid lines, rainbow color scheme).(B) Sedimentation coefficient distributions for a selection of values from the dilution series are shown.(C) The isotherm of weighted-average s-values vs. concentration (black circles), was fit to a dimer self-association model and reveals a KD of 1.11 nM, [95% confidence interval 0.88, 1.40 nM] (solid line).(D) The BACK domain dimerizes with micromolar affinity. Experimental weight-average molar mass (Mw) from CG-MALS for SPOP mutBTB (black circles) was fitted to a self-association model (see text for details) for BACK-BACK dimerization (black line) yielding a KD of 36 ± 3 µM (average and standard deviation from 3 independent experiments). Fits to the monomer/dimer equilibrium model and to alternative monomer/trimer, tetramer and pentamer equilibrium models are depicted as black solid line and as dashed and grey lines, respectively. Residuals for all models are depicted in the lower panel.


Figure 7 ( B ) Experimental weight - average molar mass ( Mw ) from CG - MALS for SPOP28 - 359 ( orange circles ) was fitted to an isodesmic self - association model in which SPOP dimers are the self - associating unit ( orange line ) . The largest SPOP oligomer taken into account was an undecamer of SPOP dimers ( ( SPOP2 ) 11 ) . The fits from three independent experiments yielded a KD2 of 2 . 4 ± 0 . 4 µM . Lines for fits of the data to self - association models that assume formation of individual oligomeric species instead of isodesmic self - association are shown for reference ( gray lines ) . ( C ) Experimental weight - average molar mass ( Mw ) from CG - MALS data ( circles ) and fits ( solid lines ) are depicted for each SPOP construct assayed , showing that a progressive increase in SPOP oligomer size is observed only when both self - association domains are interaction - competent . This Figure is comprised of data shown in Figures 6D , 7B , and Appendix Fig S5A for direct comparison .

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Figure 7(B) Experimental weight-average molar mass (Mw) from CG-MALS for SPOP28-359 (orange circles) was fitted to an isodesmic self-association model in which SPOP dimers are the self-associating unit (orange line). The largest SPOP oligomer taken into account was an undecamer of SPOP dimers ((SPOP2)11). The fits from three independent experiments yielded a KD2 of 2.4 ± 0.4 µM. Lines for fits of the data to self-association models that assume formation of individual oligomeric species instead of isodesmic self-association are shown for reference (gray lines).(C) Experimental weight-average molar mass (Mw) from CG-MALS data (circles) and fits (solid lines) are depicted for each SPOP construct assayed, showing that a progressive increase in SPOP oligomer size is observed only when both self-association domains are interaction-competent. This Figure is comprised of data shown in Figures 6D, 7B, and Appendix Fig S5A for direct comparison.


Figure 8 ( A ) Native MS spectrum of 30 µM SPOP28 - 359 confirms that SPOP self - associates through addition of dimers . The increase in mass indicates that the dimer is the stable building block within the assemblies . ( C ) Concentration - dependence of the SPOP oligomer size distribution ( mole fraction ) from isodesmic modeling of CG - MALS data .

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Figure 8(A) Native MS spectrum of 30 µM SPOP28-359 confirms that SPOP self-associates through addition of dimers. The increase in mass indicates that the dimer is the stable building block within the assemblies.(C) Concentration-dependence of the SPOP oligomer size distribution (mole fraction) from isodesmic modeling of CG-MALS data.


Figure 9Figure 9 . The self - association deficient SPOP mutant mutBTB has a substrate degradation defect in vivo . UAS transgenes were expressed under the control of an epithelial driver C765 - Gal4 in Drosophila melanogaster . ( A ) UAS vector served as a control and yielded a normal wing . Longitudinal veins are denoted by numbers 1 - 5 . Expression of ( B ) SPOP WT resulted in a modest Hh loss - of function phenotype with fusion of LV3 and LV4 ( arrow ) . ( C ) mutBTB acts in a dominant - negative manner , as evidenced by a modest Hh gain - of function phenotype with expansion of the LV3 - LV4 intervein space . Expression of ( D ) mutBACK and ( E ) mutBTB - BACK does not induce a wing patterning defect . ~ 50 animals each were analyzed from two independent crosses .

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Figure 9Figure 9. The self-association deficient SPOP mutant mutBTB has a substrate degradation defect in vivo. UAS transgenes were expressed under the control of an epithelial driver C765-Gal4 in Drosophila melanogaster. (A) UAS vector served as a control and yielded a normal wing. Longitudinal veins are denoted by numbers 1-5. Expression of (B) SPOP WT resulted in a modest Hh loss-of function phenotype with fusion of LV3 and LV4 (arrow). (C) mutBTB acts in a dominant-negative manner, as evidenced by a modest Hh gain-of function phenotype with expansion of the LV3-LV4 intervein space. Expression of (D) mutBACK and (E) mutBTB-BACK does not induce a wing patterning defect. ~50 animals each were analyzed from two independent crosses.


Figure 1Representative H & E ( top ) and immunofluorescent ( IF ) images ( bottom ) of control and Lats1 / 2 dKO airways at indicated time points post tamoxifen treatment . Tomato ( for Scgb1a1 lineage , red ) , CC10 ( for secretory cell , white ) , Acetylated tubulin ( Act - Tub , for ciliated cell , green ) , and DAPI ( blue ) . Scale bar , 100μm . Statistical quantification of Scgb1a1 lineage - labeled tdTomato + CC10 + secretory cells in ( B ) . Representative IF images of control and Lats1 / 2 dKO airways at indicated time points post tamoxifen treatment . Tomato ( for Scgb1a1 lineage , red ) , CC10 ( green ) , AQP5 ( for AT1 cell , white ) , and DAPI ( blue ) . Scale bar , 100μm . Quantification of Scgb1a1 lineage - labeled tdTomato + AQP5 + AT1 cells in ( D ) . Data are presented as mean ± SEM ( n = 5 mice for each group ) . Representative H & E ( top ) and IF images ( bottom ) of control , dKO , and qKO airways at day 21 post tamoxifen treatment . Tomato ( for Scgb1a1 lineage , red ) , CC10 ( green ) , AQP5 ( white ) , and DAPI ( blue ) . Quantification of Scgb1a1 lineage - labeled tdTomato + CC10 + secretory cells in ( G ) . Data are presented as mean ± SEM ( n = 3 mice for each genotype ) .

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Figure 1Representative H&E (top) and immunofluorescent (IF) images (bottom) of control and Lats1/2 dKO airways at indicated time points post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (for secretory cell, white), Acetylated tubulin (Act-Tub, for ciliated cell, green), and DAPI (blue). Scale bar, 100μm. Statistical quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells in (B).Representative IF images of control and Lats1/2 dKO airways at indicated time points post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (green), AQP5 (for AT1 cell, white), and DAPI (blue). Scale bar, 100μm. Quantification of Scgb1a1 lineage-labeled tdTomato+AQP5+ AT1 cells in (D). Data are presented as mean ± SEM (n=5 mice for each group).Representative H&E (top) and IF images (bottom) of control, dKO, and qKO airways at day 21 post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (green), AQP5 (white), and DAPI (blue). Quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells in (G). Data are presented as mean ± SEM (n=3 mice for each genotype).


Figure 2Representative IF images of control and Lats1 / 2 dKO airways at indicated time points post tamoxifen treatment . Tomato ( for Scgb1a1 lineage , red ) , CLDN4 ( for DATPs , white ) , CC10 ( G , green ) , AGER ( H , for AT1 cell , green ) , and DAPI ( blue ) . Quantification of Scgb1a1 lineage - labeled tdTomato + CLDN4 + DATPs in ( G and H ) . Data are presented as mean ± SEM ( n = 5 mice for each genotype ) .

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Figure 2Representative IF images of control and Lats1/2 dKO airways at indicated time points post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CLDN4 (for DATPs, white), CC10 (G, green), AGER (H, for AT1 cell, green), and DAPI (blue). Quantification of Scgb1a1 lineage-labeled tdTomato+CLDN4+ DATPs in (G and H). Data are presented as mean ± SEM (n=5 mice for each genotype).


Figure 3Representative brightfield and H & E images of 3D organoids . Arrow indicates transitioned 3D organoids retaining DATP and AT1 cells . Scale bars , 1mm for brightfield images , 200μm for H & E images . Representative IF images of secretory , DATP , or AT1 cells in secretory organoids in indicated culture conditions . Tomato ( for Scgb1a1 lineage , red ) , CC10 ( E , green ) , CLDN4 ( E , white ) , AGER ( F , green ) , and DAPI ( blue ) . Quantification of transitioned 3D organoids in 3D normal media ( 3D ) , amino acid - limited Basal MEM media ( BME ) , or affluent repletion of EAA in BME media ( BME + EAA ) with 4 - OHT or EtOH treatment . Representative IF images of YAP and TAZ expression of secretory organoids in indicated culture conditions . Tomato ( for Scgb1a1 lineage , red ) , YAP ( green ) , TAZ ( white ) , and DAPI ( blue ) . Scale bars , 100μm .

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Figure 3Representative brightfield and H&E images of 3D organoids. Arrow indicates transitioned 3D organoids retaining DATP and AT1 cells. Scale bars, 1mm for brightfield images, 200μm for H&E images. Representative IF images of secretory, DATP, or AT1 cells in secretory organoids in indicated culture conditions. Tomato (for Scgb1a1 lineage, red), CC10 (E, green), CLDN4 (E, white), AGER (F, green), and DAPI (blue). Quantification of transitioned 3D organoids in 3D normal media (3D), amino acid-limited Basal MEM media (BME), or affluent repletion of EAA in BME media (BME+EAA) with 4-OHT or EtOH treatment. Representative IF images of YAP and TAZ expression of secretory organoids in indicated culture conditions. Tomato (for Scgb1a1 lineage, red), YAP (green), TAZ (white), and DAPI (blue). Scale bars, 100μm.


Figure 4Representative H & E and IF images of control , Lats1 / 2 dKO , and Raptor ; Lats1 / 2 tKO airways at day 21 post tamoxifen treatment . Tomato ( for Scgb1a1 lineage , red ) , CC10 ( green , upper ) , AGER ( green , lower ) , and DAPI ( blue ) . Scale bar , 100μm . Quantification of Scgb1a1 lineage - labeled tdTomato + CC10 + secretory cells ( left ) and tdTomato + AGER + AT1 cells ( right ) in ( E ) . Data are presented as mean ± SEM ( n = 2 - 3 mice for each genotype ) . * * * p < 0 . 001 ( Student ' s t - test ) . Measurement of L - amino acid or essential amino acids ( leucine , isoleucine , valine ) contents in Scgb1a1 lineage - labeled tdTomato + organoids derived from control , Lats1 / 2 dKO or Raptor ; Lats1 / 2 tKO mice . Data are presented as mean ± SEM ( n = 3 technical replicates for each genotype ) . * p < 0 . 05 , * * p < 0 . 001 ( Student ' s t - test ) .

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Figure 4Representative H&E and IF images of control, Lats1/2 dKO, and Raptor;Lats1/2 tKO airways at day 21 post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (green, upper), AGER (green, lower), and DAPI (blue). Scale bar, 100μm.Quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells (left) and tdTomato+AGER+ AT1 cells (right) in (E). Data are presented as mean ± SEM (n=2-3 mice for each genotype). ***p<0.001 (Student's t-test).Measurement of L-amino acid or essential amino acids (leucine, isoleucine, valine) contents in Scgb1a1 lineage-labeled tdTomato+ organoids derived from control, Lats1/2 dKO or Raptor;Lats1/2 tKO mice. Data are presented as mean ± SEM (n=3 technical replicates for each genotype). *p<0.05, **p<0.001 (Student's t-test).


Figure 5Western blot images for the expression of YAP , ATF4 , and LAT1 / Slc7a5 in control or YAP5SA overexpressed C22 airway epithelial cell line . Knockdown of ATF4 was induced by using lentiviral - mediated inducible scramble shRNA ( shScramble , control ) or shATF4 . Concentration of L - amino acids ( L - AA , left ) or EAA ( right ; leucine , isoleucine , valine ) in cell lines in ( A ) . Data are presented as mean ± SEM ( n = 3 biological replicates for each group ) . * p < 0 . 05 , NS = not significant ( Student ' s t - test ) . Representative H & E images and IF images showing the expression of secretory cell marker CC10 , DATP cell marker CLDN4 , and AT1 cell marker CAV1 in organoids derived from Lats1 / 2 dKO secretory cells with shscramble , shAtf4 , or shSlc7a5 . CC10 ( green ) , CLDN4 ( white ) , CAV1 ( white ) and DAPI ( blue ) . Quantification of Scgb1a1 + lineage - labeled tdTomato + CC10 + secretory cells ( red ) , tdTomato + CLDN4 + DATP cells ( green ) , and tdTomato + CAV1 + AT1 cells ( blue ) in ( E ) . Representative H & E images and IF images showing the expression of secretory cell marker CC10 , DATP cell marker CLDN4 , or AT1 cell marker CAV1 in organoids derived from Scgb1a1 lineage - labeled secretory cells with or without hydroxytamoxifen - induced ATF4 overexpression . CC10 ( green ) , CLDN4 ( white ) , CAV1 ( white ) , and DAPI ( blue ) . Quantification of Scgb1a1 lineage - labeled tdTomato + CC10 + secretory cells ( red ) , tdTomato + CLDN4 + DATP cells ( green ) , and tdTomato + CAV1 + AT1 cells ( blue ) in ( G ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Western blot images for the expression of YAP, ATF4, and LAT1/Slc7a5 in control or YAP5SA overexpressed C22 airway epithelial cell line. Knockdown of ATF4 was induced by using lentiviral-mediated inducible scramble shRNA (shScramble, control) or shATF4.Concentration of L-amino acids (L-AA, left) or EAA (right; leucine, isoleucine, valine) in cell lines in (A). Data are presented as mean ± SEM (n=3 biological replicates for each group). *p<0.05, NS=not significant (Student's t-test).Representative H&E images and IF images showing the expression of secretory cell marker CC10, DATP cell marker CLDN4, and AT1 cell marker CAV1 in organoids derived from Lats1/2 dKO secretory cells with shscramble, shAtf4, or shSlc7a5. CC10 (green), CLDN4 (white), CAV1 (white) and DAPI (blue). Quantification of Scgb1a1+ lineage-labeled tdTomato+CC10+ secretory cells (red), tdTomato+CLDN4+ DATP cells (green), and tdTomato+CAV1+ AT1 cells (blue) in (E). Representative H&E images and IF images showing the expression of secretory cell marker CC10, DATP cell marker CLDN4, or AT1 cell marker CAV1 in organoids derived from Scgb1a1 lineage-labeled secretory cells with or without hydroxytamoxifen-induced ATF4 overexpression. CC10 (green), CLDN4 (white), CAV1 (white), and DAPI (blue). Quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells (red), tdTomato+CLDN4+ DATP cells (green), and tdTomato+CAV1+ AT1 cells (blue) in (G).


Figure 6Representative H & E images of human lung tissues from normal background and bronchiolitis obliterans ( BO ) with progressive fibrosis . Representative IF images showing the expressions of nuclear YAP , DATP cell marker CLDN4 , and AT1 cell marker AQP5 in the airways of normal background ( top ) and BO ( bottom ) human lungs . CLDN4 ( white ) , AQP5 ( red ) , YAP ( green ) , and DAPI ( blue ) . Of note , a flattened airway layer in BO lungs . Scale bars , 100μm . Representative IF images showing the expressions of secretory cell marker CC10 and mTORC1 activation marker p - S6 in the airways of normal background ( top ) and BO ( bottom ) human lungs . CC10 ( green ) , p - S6 ( white ) , and DAPI ( blue ) . Scale bars , 100μm . Representative IF images showing the expression of nuclear YAP , ATF4 , and AQP5 in the airways of normal background ( top ) and BO ( bottom ) human lungs . YAP ( green ) , ATF4 ( white ) , AQP5 ( red ) , and DAPI ( blue ) . Of note , co - expressions of nuclear YAP , nuclear ATF4 , and AQP5 in the airways of BO lungs . Scale bars , 100μm . Representative IF images showing the expression of p - S6 ( white ) in the airway cells losing CC10 ( green ) expression of BO human lungs . Scale bars , 100μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Representative H&E images of human lung tissues from normal background and bronchiolitis obliterans (BO) with progressive fibrosis.Representative IF images showing the expressions of nuclear YAP, DATP cell marker CLDN4, and AT1 cell marker AQP5 in the airways of normal background (top) and BO (bottom) human lungs. CLDN4 (white), AQP5 (red), YAP (green), and DAPI (blue). Of note, a flattened airway layer in BO lungs. Scale bars, 100μm.Representative IF images showing the expressions of secretory cell marker CC10 and mTORC1 activation marker p-S6 in the airways of normal background (top) and BO (bottom) human lungs. CC10 (green), p-S6 (white), and DAPI (blue). Scale bars, 100μm.Representative IF images showing the expression of nuclear YAP, ATF4, and AQP5 in the airways of normal background (top) and BO (bottom) human lungs. YAP (green), ATF4 (white), AQP5 (red), and DAPI (blue). Of note, co-expressions of nuclear YAP, nuclear ATF4, and AQP5 in the airways of BO lungs. Scale bars, 100μm.Representative IF images showing the expression of p-S6 (white) in the airway cells losing CC10 (green) expression of BO human lungs. Scale bars, 100μm.


Figure 1A - C , Immunofluorescence images ( inverted intensity ) of P14 - P15 IHCs from indicated genotypes , revealing differences in otoferlin fluorescence intensity and distribution . Maximum projection of confocal stacks , scale bar 10µm . D - F , Upper panels , examples for IHCs , co - labelled for otoferlin ( magenta ) and Vglut3 ( green ) and position of the line for line scans ; maximum projection of few optical sections , scale bars 5µm . Lower panels , for quantification of membrane staining , the fluorescence was normalized to the cellular fluorescence for each fluorophore , then the average of five parallel line scans through the middle of the cells for the sum of both fluorescence values ( black line ) was used to determine the position of the basal membrane . At the most basal cellular point along this line which exceeds the threshold value of 2 ( yellow diamond ) , the otoferlin - Vglut3 fluorescence difference ( blue line ) gave the value for relative otoferlinplasma membrane levels ( orange diamond ) . Insets : enlargements of basal regions . G , Otoferlin protein levels were reduced in OtofI515T / I515T IHCs ( indicates numbers represent numbers of cells ) and OtofPga / Pga IHCs compared to wild - type ( Otof + / + ) controls ( mean ± SEM ) . H , ratio of apical / basal protein levels ( above / below nuclear midline depicted as green line in A ) indicates an apical shift of otoferlin in OtofPga / Pga IHCs . I , Relative levels of membrane - bound otoferlin at the basal pole of IHCs . K , Absolute amount of otoferlin at the basal IHCplasma membrane , gained by multiplication of relative plasma membrane levels ( I ) x total cellular otoferlin protein levels ( G ) . Kruskal - Wallis test ; * * * indicates p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 .

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Figure 1A-C, Immunofluorescence images (inverted intensity) of P14-P15 IHCs from indicated genotypes, revealing differences in otoferlin fluorescence intensity and distribution. Maximum projection of confocal stacks, scale bar 10µm.D-F, Upper panels, examples for IHCs, co-labelled for otoferlin (magenta) and Vglut3 (green) and position of the line for line scans; maximum projection of few optical sections, scale bars 5µm. Lower panels, for quantification of membrane staining, the fluorescence was normalized to the cellular fluorescence for each fluorophore, then the average of five parallel line scans through the middle of the cells for the sum of both fluorescence values (black line) was used to determine the position of the basal membrane. At the most basal cellular point along this line which exceeds the threshold value of 2 (yellow diamond), the otoferlin-Vglut3 fluorescence difference (blue line) gave the value for relative otoferlinplasma membrane levels (orange diamond). Insets: enlargements of basal regions.G, Otoferlin protein levels were reduced in OtofI515T/I515T IHCs (indicates numbers represent numbers of cells) and OtofPga/Pga IHCs compared to wild-type (Otof+/+) controls (mean±SEM).H, ratio of apical/basal protein levels (above/below nuclear midline depicted as green line in A) indicates an apical shift of otoferlin in OtofPga/Pga IHCs.I, Relative levels of membrane-bound otoferlin at the basal pole of IHCs.K, Absolute amount of otoferlin at the basal IHCplasma membrane, gained by multiplication of relative plasma membrane levels (I) x total cellular otoferlin protein levels (G). Kruskal-Wallis test; *** indicates p<0.001; **, p<0.01; *, p<0.05.


Figure 2A , ABR thresholds in OtofI515T / I515T ( red , n = 5 ) and Otof - / I515T ( blue , n = 7 ) mice were elevated compared to Otof + / + mice ( black , n = 5 ) at an age of 3 - 4 weeks . The grey dotted line indicates the maximum loudspeaker output of 90dB ; thresholds exceeding this value were set to 100dB for calculation of the mean ± SEM . At 12kHz , only the threshold increase for Otof - / I515T vs Otof + / + is significant ( Kruskal - Wallis test with Dunn ' s multiple comparisons test between all 3 groups ) . B , Grand averages of ABR waveforms ± SEM in response to 80dB click stimulation of the mice analyzed in A : The small wave preceding ABR wave I probably represents the summating potential ( SP , hair cell receptor potential ) , which is intact in Otof mutants . ABR wave I is reduced in amplitude while subsequent peaks are better preserved in OtofI515T / I515T mice ( Fig EV1 ) . C , Mean ABR wave I amplitude ± SEM for different stimulus intensities ( all differences between genotypes are significant ; 2 - way ANOVA with Tukey ' s multiple comparison test ) . D , At 8 weeks ( circles ) and 25 weeks ( open squares ) , OtofI515T / I515T mice showed highly elevated ABR thresholds compared to Otof + / + mice ( n = 7 - 8 each ; p < 0 . 001 at 12kHz , Mann - Whitney - U - test ) . Grey dotted line as in A . E , Grand averages of ABR waveforms ± SEM in response to 80dB Click stimulation in mice aged 8 weeks . OtofI515T / I515T ( n = 8 ) have drastically reduced ABR amplitudes compared to Otof + / + mice ( n = 7 ) . Otof - / - mice have no ABR ( green , n = 9 ) . F , Mean ABR wave I amplitude ± SEM for different stimulus intensities for 8 week and 25 week old OtofI515T / I515T and Otof + / + mice ( p < 0 . 001 , 2 - way ANOVA ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A, ABR thresholds in OtofI515T/I515T (red, n=5) and Otof-/I515T (blue, n=7) mice were elevated compared to Otof+/+ mice (black, n=5) at an age of 3-4 weeks. The grey dotted line indicates the maximum loudspeaker output of 90dB; thresholds exceeding this value were set to 100dB for calculation of the mean ± SEM. At 12kHz, only the threshold increase for Otof-/I515T vs Otof+/+ is significant (Kruskal-Wallis test with Dunn's multiple comparisons test between all 3 groups).B, Grand averages of ABR waveforms ± SEM in response to 80dB click stimulation of the mice analyzed in A: The small wave preceding ABR wave I probably represents the summating potential (SP, hair cell receptor potential), which is intact in Otof mutants. ABR wave I is reduced in amplitude while subsequent peaks are better preserved in OtofI515T/I515T mice (Fig EV1).C, Mean ABR wave I amplitude ± SEM for different stimulus intensities (all differences between genotypes are significant; 2-way ANOVA with Tukey's multiple comparison test).D, At 8 weeks (circles) and 25 weeks (open squares), OtofI515T/I515T mice showed highly elevated ABR thresholds compared to Otof+/+ mice (n=7-8 each; p<0.001 at 12kHz, Mann-Whitney-U-test). Grey dotted line as in A.E, Grand averages of ABR waveforms ± SEM in response to 80dB Click stimulation in mice aged 8 weeks. OtofI515T/I515T (n=8) have drastically reduced ABR amplitudes compared to Otof+/+ mice (n=7). Otof-/- mice have no ABR (green, n=9).F, Mean ABR wave I amplitude ± SEM for different stimulus intensities for 8 week and 25 week old OtofI515T/I515T and Otof+/+ mice (p<0.001, 2-way ANOVA).


Figure 3A , B , No difference in voltage dependent Ca2 + - currents ( A ) and fractional activation of ICa - channels ( B ) between OtofI515T / I515T IHCs ( n = 16 ) and IHCs of Otof + / + littermates ( n = 13 ) . C , Exocytosis was recorded by measures of changes in membrane capacitance ( ΔCm , lower panel ) in response to depolarization ( left , 20ms ; right , 100ms ) to the voltage where maximum Ca2 + - currents were elicited ( upper panel ) , typically - 14mV . D , Upper panel , while for stimuli up to 20ms exocytosis from OtofI515T / I515T ( n = 13 ) and Otof + / + ( n = 11 ) IHCs was similar , sustained exocytosis , representing most likely the release of replenished vesicles , was significantly reduced in OtofI515T / I515T IHCs ( t - test ) . Lower panel , Ca2 + - current integrals were of similar size . E , Flash photolysis of caged Ca2 + elicited a smaller exocytic response in OtofI515T / I515T IHCs . F , The kinetics of the fast component from E was comparable between OtofI515T / I515T IHCs and Otof + / + littermates ( b ) . Open circles represent previously published data on IHCs of hearing wild - type mice ( a ; Beutner et al , 2001 ; Pangrsic et al , 2010 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A,B, No difference in voltage dependent Ca2+-currents (A) and fractional activation of ICa-channels (B) between OtofI515T/I515T IHCs (n=16) and IHCs of Otof+/+ littermates (n=13).C, Exocytosis was recorded by measures of changes in membrane capacitance (ΔCm, lower panel) in response to depolarization (left, 20ms; right, 100ms) to the voltage where maximum Ca2+-currents were elicited (upper panel), typically -14mV.D, Upper panel, while for stimuli up to 20ms exocytosis from OtofI515T/I515T (n=13) and Otof+/+ (n=11) IHCs was similar, sustained exocytosis, representing most likely the release of replenished vesicles, was significantly reduced in OtofI515T/I515T IHCs (t-test). Lower panel, Ca2+-current integrals were of similar size.E, Flash photolysis of caged Ca2+ elicited a smaller exocytic response in OtofI515T/I515T IHCs.F, The kinetics of the fast component from E was comparable between OtofI515T/I515T IHCs and Otof+/+ littermates (b). Open circles represent previously published data on IHCs of hearing wild-type mice (a; Beutner et al, 2001; Pangrsic et al, 2010).


Figure 4A , Spontaneous rates of SGNs from OtofI515T / I515T ( red , n = 35 ) and Otof + / + littermates ( black , n = 39 ) were not significantly different ( p = 0 . 83 , Kolmogorov - Smirnov test ) . B - D , Averaged poststimulus time histograms ± SEM from OtofI515T / I515T mice ( n = 25 - 32 ) and Otof + / + littermate SGNs ( n = 13 - 27 ) to stimulation with 50ms tone bursts at the characteristic frequency ( CF ) of each fiber , 30dB above threshold at indicated stimulus rates . E , Quantification of onset ( largest 0 . 5ms bin , Mann - Whitney U test ) and adapted responses ( averaged 35 - 45ms from response onset , t - test ) from data in B - D . F , The jitter of the first sound - evoked spike was significantly increased ( p < 0 . 001 , Mann - Whitney U test ) but retained its inverse correlation with spike rates . G , Spike rate increases with rising stimulus intensity were significantly shallower in OtofI515T / I515T SGNs , both for repetitive stimulation with 50ms tone bursts ( left , p = 0 . 014 , t - test ) and for continuous stimulation with amplitude modulated tones ( right , p < 0 . 001 , Mann - Whitney U test ) . H , Phase locking to amplitude modulated tones ( assessed as the maximal synchronization index ) was typically less precise in OtofI515T / I515T SGNs than in Otof + / + SGNs ( p = 0 . 09 , Mann - Whitney U test ) . I , 100ms masker tone and 15ms probe tones ( both at CF , 30dB above threshold ) were separated by silent intervals of variable duration . Inter - masker intervals were 500ms for Otof + / + , and 1000ms for OtofI515T / I515T . The ratio of probe and masker onset responses revealed enhanced RRP depletion after stimulation in OtofI515T / I515T ( pink ; mean ± SEM red ) compared to Otof + / + ( grey ; mean ± SEM black ; for 4ms interval : p = 0 . 001 , t - test ) and a slowed time course of recovery ( x : half time of recovery , taken from normalized recovery functions ; p < 0 . 001 , Mann - Whitney U test ) . K , Mice learned to drink water when continuous noise was present but avoided drinking when the noise was interrupted by silent gaps . 5 OtofI515T / I515T mice ( pink , mean ± SEM red ) avoided drinking less efficiently than 2 Otof + / + mice ( grey , mean ± SEM black ) for shorter gap durations . See also Figs S2 / S3 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A, Spontaneous rates of SGNs from OtofI515T/I515T (red, n=35) and Otof+/+ littermates (black, n=39) were not significantly different (p=0.83, Kolmogorov-Smirnov test).B-D, Averaged poststimulus time histograms ±SEM from OtofI515T/I515T mice (n=25-32) and Otof+/+ littermate SGNs (n=13-27) to stimulation with 50ms tone bursts at the characteristic frequency (CF) of each fiber, 30dB above threshold at indicated stimulus rates.E, Quantification of onset (largest 0.5ms bin, Mann-Whitney U test) and adapted responses (averaged 35-45ms from response onset, t-test) from data in B-D.F, The jitter of the first sound-evoked spike was significantly increased (p<0.001, Mann-Whitney U test) but retained its inverse correlation with spike rates.G, Spike rate increases with rising stimulus intensity were significantly shallower in OtofI515T/I515T SGNs, both for repetitive stimulation with 50ms tone bursts (left, p=0.014, t-test) and for continuous stimulation with amplitude modulated tones (right, p<0.001, Mann-Whitney U test).H, Phase locking to amplitude modulated tones (assessed as the maximal synchronization index) was typically less precise in OtofI515T/I515T SGNs than in Otof+/+SGNs (p=0.09, Mann-Whitney U test).I, 100ms masker tone and 15ms probe tones (both at CF, 30dB above threshold) were separated by silent intervals of variable duration. Inter-masker intervals were 500ms for Otof+/+, and 1000ms for OtofI515T/I515T. The ratio of probe and masker onset responses revealed enhanced RRP depletion after stimulation in OtofI515T/I515T (pink; mean ±SEM red) compared to Otof+/+ (grey; mean ±SEM black; for 4ms interval: p=0.001, t-test) and a slowed time course of recovery (x: half time of recovery, taken from normalized recovery functions; p<0.001, Mann-Whitney U test).K, Mice learned to drink water when continuous noise was present but avoided drinking when the noise was interrupted by silent gaps. 5 OtofI515T/I515T mice (pink, mean ±SEM red) avoided drinking less efficiently than 2 Otof+/+ mice (grey, mean ±SEM black) for shorter gap durations. See also Figs S2/S3.


Figure 5A , Exemplary ABR traces ( Click 100dB ) from one OtofI515T / I515T mouse recorded at indicated bullatemperatures during local heating . Note that ABRs never disappeared completely , but wave I amplitude changed reversibly with temperature . B , C , ABR wave I amplitudes in OtofI515T / I515T ( A , Click 100dB ) and Otof + / + mice ( B , Click 80dB ) decreased with increasing temperature in the bulla . Each color represents a different experiment , dashed lines are line fits . Open symbols indicate the beginning of the experiments ; subsequent recordings are connected by lines . The four indicated data points in B correspond to ABRs in A . D , Explanted organ of Corti from an Otof + / + mouse at P4 after two days in vitro ( DIV2 ) at 37 ° C immunostained against otoferlin ( magenta ) and Vglut3 ( green ) . Note the intense immunostaining of otoferlin at the plasma membrane . Scale bar 10 µm . E - J , Explanted organs of Corti from Otof - / - mice at P4 were transfected by GeneGun with otoferlin cDNA and eGFP and immunostained at DIV2 . E , Example cell transfected with wild - type mouseotoferlin cDNA . F , G , Representative cells transfected with mouseotoferlin including the 20 amino acids of the RXR motif ( see Fig EV4 ) . Cells in G were incubated at 38 . 5 ° C for 30 min prior to fixation . The RXR - otoferlin transfected cells show a membrane localization of otoferlin surrounding the Vglut3 immunofluorescence / eGFP fluorescence at the basal pole of the cells ( arrows ) , similar as in controls ( A , B ) . H , J , Both the human RXR motif and the Ile515Thr mutation were introduced in mouse cDNA , and cells were incubated at 37 ° C ( H ) or for 30min at 38 . 5 ° C ( J ) before fixation . Here , green fluorescence surrounds the otoferlin immunofluorescence ( arrows ) , suggesting loss of otoferlin from the plasma membrane . K , Quantification of the relative plasma membrane levels of otoferlin ( as in Fig 1 ) revealed normal plasma membrane abundance when the human RXR motif was present , but a strong reduction for otoferlin with RXR and Ile515Thr ( individual cells ; mean ± SEM ; t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A, Exemplary ABR traces (Click 100dB) from one OtofI515T/I515T mouse recorded at indicated bullatemperatures during local heating. Note that ABRs never disappeared completely, but wave I amplitude changed reversibly with temperature.B,C, ABR wave I amplitudes in OtofI515T/I515T (A, Click 100dB) and Otof+/+ mice (B, Click 80dB) decreased with increasing temperature in the bulla. Each color represents a different experiment, dashed lines are line fits. Open symbols indicate the beginning of the experiments; subsequent recordings are connected by lines. The four indicated data points in B correspond to ABRs in A.D, Explanted organ of Corti from an Otof+/+ mouse at P4 after two days in vitro (DIV2) at 37°C immunostained against otoferlin (magenta) and Vglut3 (green). Note the intense immunostaining of otoferlin at the plasma membrane. Scale bar 10 µm.E-J, Explanted organs of Corti from Otof-/- mice at P4 were transfected by GeneGun with otoferlin cDNA and eGFP and immunostained at DIV2.E, Example cell transfected with wild-type mouseotoferlin cDNA.F,G, Representative cells transfected with mouseotoferlin including the 20 amino acids of the RXR motif (see Fig EV4). Cells in G were incubated at 38.5°C for 30 min prior to fixation. The RXR-otoferlin transfected cells show a membrane localization of otoferlin surrounding the Vglut3 immunofluorescence/eGFP fluorescence at the basal pole of the cells (arrows), similar as in controls (A,B).H,J, Both the human RXR motif and the Ile515Thr mutation were introduced in mouse cDNA, and cells were incubated at 37°C (H) or for 30min at 38.5°C (J) before fixation. Here, green fluorescence surrounds the otoferlin immunofluorescence (arrows), suggesting loss of otoferlin from the plasma membrane.K, Quantification of the relative plasma membrane levels of otoferlin (as in Fig 1) revealed normal plasma membrane abundance when the human RXR motif was present, but a strong reduction for otoferlin with RXR and Ile515Thr (individual cells; mean ±SEM; t-test).


Figure 6A - E , Capacitance increments recorded from Otof + / + ( black ) and OtofI515T / I515T ( red ) IHCs . Individual cells recorded at indicated temperatures ( light circles ) , mean ± SEM for perforated patch clamp experiments ( t - test ) . F , G , Summary of the capacitance changes and Ca2 + - current integrals for Otof + / + ( F ) and OtofI515T / I515T IHCs ( G ) at the different temperatures illustrates the drastic increase in exocytosis for physiological temperature . * Significant differences compared to RT measurements are indicated with colors of the respective temperature , and between PT and high temperature in violet ( t - test ) . H - J , Otoferlin immunofluorescence in Otof + / + IHCs ( upper panel ) and OtofI515T / I515T IHCs ( middle and lower panels ) of explanted organs of Corti at P7 - P8 after incubation at indicated temperatures for 24h ; maximum projections of z - stacks , inverted images , scale bar 10µm . The same imaging settings have been applied in all experiments , and the same lookup table was applied for the upper and middle panels . Images of lower panels are enhanced compared to middle panels ( lookup table covering full data range of only this genotype ) to visualize otoferlin distribution . H - J , Otoferlin immunofluorescence in Otof + / + IHCs ( upper panel ) and OtofI515T / I515T IHCs ( middle and lower panels ) of explanted organs of Corti at P7 - P8 after incubation at indicated temperatures for 24h ; maximum projections of z - stacks , inverted images , scale bar 10µm . The same imaging settings have been applied in all experiments , and the same lookup table was applied for the upper and middle panels . Images of lower panels are enhanced compared to middle panels ( lookup table covering full data range of only this genotype ) to visualize otoferlin distribution . K , Quantification of otoferlin immunofluorescence in Otof + / + IHCs ( black bars , n = 98 - 137 cells , 4 - 5 experiments ) and OtofI515T / I515T IHCs ( red bars , n = 90 - 97 cells ; Kruskal - Wallis test as in L - N ) , indicate reduced protein levels with increasing temperature . L , Apical / basal otoferlin protein distribution , revealing a significant apical shift of otoferlin and Vglut3 for OtofI515T / I515T IHCs ( red symbols ) at 38 . 5 ° C compared to Otof + / + ( grey / black symbols ) . M , Relative levels of membrane bound otoferlin were lowered with increasing temperature . N , Absolute otoferlin membrane immunostaining strongly decreased with temperature .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A-E, Capacitance increments recorded from Otof+/+ (black) and OtofI515T/I515T (red) IHCs. Individual cells recorded at indicated temperatures (light circles), mean ±SEM for perforated patch clamp experiments (t-test).F,G, Summary of the capacitance changes and Ca2+-current integrals for Otof+/+ (F) and OtofI515T/I515T IHCs (G) at the different temperatures illustrates the drastic increase in exocytosis for physiological temperature. *Significant differences compared to RT measurements are indicated with colors of the respective temperature, and between PT and high temperature in violet (t-test).H-J, Otoferlin immunofluorescence in Otof+/+ IHCs (upper panel) and OtofI515T/I515T IHCs (middle and lower panels) of explanted organs of Corti at P7-P8 after incubation at indicated temperatures for 24h; maximum projections of z-stacks, inverted images, scale bar 10µm. The same imaging settings have been applied in all experiments, and the same lookup table was applied for the upper and middle panels. Images of lower panels are enhanced compared to middle panels (lookup table covering full data range of only this genotype) to visualize otoferlin distribution.H-J, Otoferlin immunofluorescence in Otof+/+ IHCs (upper panel) and OtofI515T/I515T IHCs (middle and lower panels) of explanted organs of Corti at P7-P8 after incubation at indicated temperatures for 24h; maximum projections of z-stacks, inverted images, scale bar 10µm. The same imaging settings have been applied in all experiments, and the same lookup table was applied for the upper and middle panels. Images of lower panels are enhanced compared to middle panels (lookup table covering full data range of only this genotype) to visualize otoferlin distribution.K, Quantification of otoferlin immunofluorescence in Otof+/+ IHCs (black bars, n=98-137 cells, 4-5 experiments) and OtofI515T/I515T IHCs (red bars, n=90-97 cells; Kruskal-Wallis test as in L-N), indicate reduced protein levels with increasing temperature.L, Apical/basal otoferlin protein distribution, revealing a significant apical shift of otoferlin and Vglut3 for OtofI515T/I515T IHCs (red symbols) at 38.5°C compared to Otof+/+ (grey/black symbols).M, Relative levels of membrane bound otoferlin were lowered with increasing temperature.N, Absolute otoferlin membrane immunostaining strongly decreased with temperature.


Figure 7A - C , Pre - embedding immunogold labelling for EM visualizes otoferlin localization in random ultrathin sections through the basal part of IHCs in Otof + / + ( A ) and OtofI515T / I515T ( B ) but not in Otof - / - ( C ) mice . IHCs are highlighted in beige . Scale bar 100nm . D - G , Magnified synaptic ribbons ( R ) and postsynaptic afferent boutons of SGNs ( Aff ) , displaying otoferlin immunogold labelling at active zone membranes ( pink arrowheads ) but not around the synaptic ribbon , using saponin permeabilization ( D - F ) , or Triton - X - 100 treatment ( G ) . H , Vglut3 immunogold labelling adjacent to the ribbon as expected for synaptic vesicle . Scale bar 200nm . I , J , Some otoferlin labelled structures bear clathrin coats ( red arrowheads ) , suggesting that they originate from bulk endocytosis . Scale bar 100nm . K , Otoferlin labelled vesicles were on average larger in OtofI515T / I515T IHCs ( OtofI515T / I515T , n = 86 vesicles , 18 images ; Otof + / I + , n = 82 vesicles , 6 images ; Wilcoxon - rank - sum - test , as in L - M ) . L , Quantification of immunogold clusters at the plasma membrane revealed a strong trend towards reduced levels of otoferlin in OtofI515T / I515T ( 6 images each ) . M , Distal from the plasma membrane , immunogold labels were reduced in OtofI515T / I515T IHCs . N , Representative electron micrographs of IHCribbon synapses in OtofI515T / I515Tand Otof + / ( conventional embedding without immunogold ) after pre - incubation for 10min at the indicated temperature followed by high K + stimulation or 0 Ca2 + inhibition for 1min 45sec . Scale bars 100nm . P - Q , Synaptic vesicles with > 45 nm diameter appeared at 39 ° C in random sections ; fraction of large membrane - proximal ( P ; Kruskal - Wallis test ) and ribbon - associated ( Q ; one way ANOVA followed by Tukey ' s test ) synaptic vesicles ( see also Appendix Table S1 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A-C, Pre-embedding immunogold labelling for EM visualizes otoferlin localization in random ultrathin sections through the basal part of IHCs in Otof+/+ (A) and OtofI515T/I515T (B) but not in Otof-/- (C) mice. IHCs are highlighted in beige. Scale bar 100nm.D-G, Magnified synaptic ribbons (R) and postsynaptic afferent boutons of SGNs (Aff), displaying otoferlin immunogold labelling at active zone membranes (pink arrowheads) but not around the synaptic ribbon, using saponin permeabilization (D-F), or Triton-X-100 treatment (G).H, Vglut3 immunogold labelling adjacent to the ribbon as expected for synaptic vesicle. Scale bar 200nm.I,J, Some otoferlin labelled structures bear clathrin coats (red arrowheads), suggesting that they originate from bulk endocytosis. Scale bar 100nm.K, Otoferlin labelled vesicles were on average larger in OtofI515T/I515T IHCs (OtofI515T/I515T, n=86 vesicles, 18 images; Otof+/I+, n=82 vesicles, 6 images; Wilcoxon-rank-sum-test, as in L-M).L, Quantification of immunogold clusters at the plasma membrane revealed a strong trend towards reduced levels of otoferlin in OtofI515T/I515T (6 images each).M, Distal from the plasma membrane, immunogold labels were reduced in OtofI515T/I515T IHCs.N, Representative electron micrographs of IHCribbon synapses in OtofI515T/I515Tand Otof+/ (conventional embedding without immunogold) after pre-incubation for 10min at the indicated temperature followed by high K+ stimulation or 0 Ca2+ inhibition for 1min 45sec. Scale bars 100nm.P-Q, Synaptic vesicles with >45 nm diameter appeared at 39°C in random sections; fraction of large membrane-proximal (P; Kruskal-Wallis test) and ribbon-associated (Q; one way ANOVA followed by Tukey's test) synaptic vesicles (see also Appendix Table S1).


Figure 8A - D , Exemplary virtual sections through EM tomograms for stimulatory condition at 36 ° C and 39oC . Scale bar 100nm . F - I , 3D reconstruction of the ribbon - associated pool of vesicles ( front view ) . Active zone membrane is depicted in blue , presynaptic density in pink , and ribbons in red , scale bars 100nm . F - I , 3D reconstruction of the membrane - proximal pool ( top view ) and J - M , the ribbon - associated pool of vesicles ( front view ) . Active zone membrane is depicted in blue , presynaptic density in pink , and ribbons in red , scale bars 100nm . N - Q , Cumulative probability distribution of the diameter of round vesicles as well as the longest axis of flattened vesicles in the membrane - proximal pool ( N - O ) and ribbon - associated pool ( P - Q ) . N - P , one way ANOVA followed by Tukey ' s test ; Q , Kruskal - Wallis test . Round vesicles tested in both the pools appeared larger in OtofI515T / I515T IHC synapses but the long axis of flattened vesicles were comparable for both the genotypes ( see also Table EV1 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A-D, Exemplary virtual sections through EM tomograms for stimulatory condition at 36°C and 39oC. Scale bar 100nm.F-I, 3D reconstruction of the ribbon-associated pool of vesicles (front view). Active zone membrane is depicted in blue, presynaptic density in pink, and ribbons in red, scale bars 100nm.F-I, 3D reconstruction of the membrane-proximal pool (top view) and J-M, the ribbon-associated pool of vesicles (front view). Active zone membrane is depicted in blue, presynaptic density in pink, and ribbons in red, scale bars 100nm.N-Q, Cumulative probability distribution of the diameter of round vesicles as well as the longest axis of flattened vesicles in the membrane-proximal pool (N-O) and ribbon-associated pool (P-Q). N-P, one way ANOVA followed by Tukey's test; Q, Kruskal-Wallis test. Round vesicles tested in both the pools appeared larger in OtofI515T/I515T IHC synapses but the long axis of flattened vesicles were comparable for both the genotypes (see also Table EV1).


Figure 1 ( A ) Rescue of growth phenotype . Wild - type ( WT ) or mdm10Δ cells were transformed with the empty plasmid pYX142 ( ) . In addition , mdm10Δ cells were transformed with pYX142 encoding MDM10 or MCP3 . Cells were grown to an OD600 of 1 . 0 and spotted on YPD or YPG plates in a 1 : 5 dilution series . Plates were incubated for growth at the indicated temperatures . ( B ) Over - expression of Mcp3 partially rescues the mitochondrial morphology defect in mdm10Δ cells . Cells described in ( A ) expressing mitochondrially targeted GFP ( mtGFP ) were analysed by fluorescence microscopy . Typical images ( fluorescence and brightfield , BF ) of the four different strains are shown ( scale bar = 5 µm ) . The quantification shows the average percentage with standard deviation bars of three independent experiments ( n = 3 ; SD ; * , p < 0 . 05 and as indicated ) with at least 100 cells per experiment .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Rescue of growth phenotype. Wild-type (WT) or mdm10Δ cells were transformed with the empty plasmid pYX142 (). In addition, mdm10Δ cells were transformed with pYX142 encoding MDM10 or MCP3. Cells were grown to an OD600 of 1.0 and spotted on YPD or YPG plates in a 1:5 dilution series. Plates were incubated for growth at the indicated temperatures.(B) Over-expression of Mcp3 partially rescues the mitochondrial morphology defect in mdm10Δ cells. Cells described in (A) expressing mitochondrially targeted GFP (mtGFP) were analysed by fluorescence microscopy. Typical images (fluorescence and brightfield, BF) of the four different strains are shown (scale bar = 5 µm). The quantification shows the average percentage with standard deviation bars of three independent experiments (n=3; SD; *, p < 0.05 and as indicated) with at least 100 cells per experiment.