( A , B ) Transferrin receptor ( TfnR ) uptake , a measure of CME , shown as % uptake of total steady - state surface TfnR for control H1299 , H1299 stabily expressing low amounts of siRNA - resistant Dyn2WT - EGFP ( A ) or Dyn2S619L - EGFP ( B ) with or without treatment of siRNA - directed towards endogenous Dyn2 .

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(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 TIRF - M of Dyn2WT - EGFP or Dyn2S619L - EGFP expressing H1299 with or without Dyn2 - siRNA treatment showing co - localization with clathrin chain immunostained rabbit anti - CLC Alexa 547 - conjugated secondary antibodies .

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(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 ) of Dyn2 - EGFP positive clathrin coated pits detected by live TIRF - M automated master / slave image ( Aguet et al , 2013 ) .

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(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 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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(E) Lifetime analysis of CCPs which are positive for EGFP-Dyn2WT or EGFP-Dyn2S619L with or without siRNA-mediated specific knockdown of endogenous Dyn2.


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

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B 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.


A Mitochondrial translation products were labeled in WT HeLa for indicated time lengths ( heat map ) immunostained for TOM20 ( gray ) . Representative confocal are shown for each time point . same 5 - minute image was used in both rows with different brightness as a reference for comparison of other time points in row . Scale bars , 10 µm . Mitochondrial synthesis was quantified over time . 30 from N = 3 independent experiments were analyzed for each time point ( Average of HPG in mitochondrial was calculated baseline was set to zero by subtracting value of time point 0 from values . Error bars , + / - ) . A hyperbolic graph was fitted to data . R - squared , 0 . 97 .

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A 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 20 minutes ) in HeLa . number of analyzed was increased in plots from 25 to 300 per time point . 150 or 300 random from N = 3 independent experiments were analyzed . Kruskal - Wallis followed by Fisher ' s . * * * * p < 0 . 0001 . Error bars , + / - .

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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 clicked with AF647 ( magenta ) immunostained for TOM20 ( green ) in fibroblasts , iPSCs - derived cardiomyocytes , hippocampal neurons . Representative epifluorescent microscopy are shown . correlation of HPG TOM20 channels is represented in fire lookup table , after calculating Pearson Correlation values over each pixel of . Scale bars , 25 µm .

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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.


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

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A 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.


A Mitochondrial translation products were labeled with HPG in hippocampal neurons , immunostained for ANK - G ( initial axon segment ) , plasma was stained with DiO . Representative epifluorescent zoomed insets of neural branches are shown . In both axon ( ANK - G positive , arrow ) dendrite ( star ) , HPG incorporation revealing mitochondrial synthesis was detected . Scale bars , 20 µm .

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A 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 of a dendrite ( top ) an axon ( bottom ) in which mitochondrial translation products were labeled with HPG ( magenta ) . HOMER1 VGLUT1 were used as post - presynapse markers , respectively ( green ) DiO stained randomly neurites ( gray ) . In each neurite , an exemplary region was selected DiO was outlined to mark outer edge of region . As a control , were treated with Chloramphenicol ( Chl ) to inhibit mitochondrial translation . Scale bars , 5 µm .

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

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

Figure 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.


( A - B ) NIH 3T3 were ( A ) co - transfected with constructs expressing full SPOP GFP - Gli31455 or ( B ) transfected with a construct expressing SC - 35 - GFP , GFP was monitored in live . ( A ) Snapshots were taken from timepoints as noted in Figure to show photobleaching ( at 0 s ) , recovery , fusion events . arrow in each panel points to that was photobleached subsequently fuses with another . These data were not used to calculate average recovery time as fusion event precludes accurate measurement of recovery . ( B ) Snapshots at indicated timepoints show a speckle fusion event . Additional are shown in Fig EV1 .

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(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 ) speckles were photobleached FRAP was monitored for 90 sec . Data from 45 FRAP events were corrected for background , normalized to minimum maximum , mean is shown with error bars representing standard error of mean . mean characteristic recovery time is indicated ± .

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(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 ) bodies are close to spherical . ( D ) Aspect ratios for speckles observed in NIH 3T3 transfected with constructs expressing HA - SPOP ( 151 ) ( D ) , or GFP - Gli31 - 455 HA - SPOP ( 155 ) ( E ) were calculated as described previously ( Brangwynne et al , 2011 ) . Representative of are shown as insets in each panel .

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(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.


( A ) SEC chromatograms of given loading concentrations of SPOP28 - 359 are shown . concentrations were normalized to monomer molecular , i . e . , identical concentrations of dimeric SPOP ΔBACK ( see Fig EV2 ) 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 . were injected at same loading ( 533 µM ) elution volume of globular molecular standards is noted above graph . ( C ) SEC chromatograms of SPOP28 - 359 ( 200 µM ) , SPOP mutBACK ( 718 µM ) , mixtures of two ( ratios given in Figure , SPOP at 200 µM ) are shown . elution volume of globular molecular standards is noted above graph .

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(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 - HA - SPOP or HA - SPOP mutants capable of oligomerization through only one ( or neither ) of self - association domains were transfected into NIH 3T3 . DAPI was used to stain nucleus SPOP localization was identified using an anti - HA antibody . Experiments were performed at least twice on four biological samples . Multiple were examined representative are shown . For additional see Appendix Fig S3 . Scale bar represents 5 µm .

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(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.


( A ) Substrate binding was assayed by anisotropy using a fluorescently labeled substrate peptide . SPOP bound substrate with an affinity similar to that of SPOP28 - 359 ( 4 - 8 µM , see Table 2 ) . Experimental data are shown as circles ; solid lines are fits to eq . 2 ( Roehrl et al , 2004 ) .

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(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 each mutant at 30 μM with amide - specific BS3 crosslinker . Crosslinking for SPOP ΔBACK MATH domain are shown to demonstrate that crosslinking conditions do not lead to non - specific crosslinking of species . ( C ) Crosslinking reactions were performed on whole lysates from expressing wild type SPOP , SPOP mutBACK , SPOP mutBTB , or SPOP mutBTB - BACK . SPOP1 , SPOP2 SPOPn identify SPOP monomers , dimers larger species , respectively . For loading see Appendix Fig S4 .

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(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.


( A ) SV - AUC data for 0 . 2 nM AF488 - SPOP ΔBACK . scans were collected for 12 h are plotted against from axis of rotation ( circles ) . data were subjected to standard c ( s ) in SEDFIT ( Schuck , 2000 ) results of this fit are shown ( solid lines , rainbow color scheme ) .

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(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 dilution series are shown .

(B) Sedimentation coefficient distributions for a selection of values from the dilution series are shown.


( C ) isotherm of weighted - average s - values vs . ( black circles ) , was fit to a dimer self - association model reveals a KD of 1 . 11 nM , [ 95 % confidence interval 0 . 88 , 1 . 40 nM ] ( solid ) .

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(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 ) BACK domain dimerizes with micromolar affinity . Experimental - 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 ) yielding a KD of 36 ± 3 µM ( average standard deviation from 3 independent experiments ) . Fits to monomer / dimer equilibrium model to alternative monomer / trimer , tetramer pentamer equilibrium models are depicted as black solid as dashed grey lines , respectively . Residuals for models are depicted in lower panel .

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(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.


( B ) Experimental - 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 self - associating unit ( orange ) . largest SPOP oligomer taken into account was an undecamer of SPOP dimers ( ( SPOP2 ) 11 ) . fits from three independent experiments yielded a KD2 of 2 . 4 ± 0 . 4 µM . Lines for fits of data to self - association models that assume formation of oligomeric species instead of isodesmic self - association are shown for reference ( gray lines ) .

O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 - average molar mass ( Mw ) from CG - MALS data ( circles ) fits ( solid lines ) are depicted for each SPOP construct assayed , showing that a progressive increase in SPOP oligomer is observed only when both self - association domains are interaction - competent . This Figure is comprised of data shown in Figures 6D , 7B , Appendix Fig S5A for direct comparison .

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(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.


( A ) Native MS spectrum of 30 µM SPOP28 - 359 confirms that SPOP self - associates through addition of dimers . increase in mass indicates that dimer is stable building block within assemblies .

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(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 ) - dependence of SPOP oligomer distribution ( mole fraction ) from isodesmic modeling of CG - MALS data .

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(C) Concentration-dependence of the SPOP oligomer size distribution (mole fraction) from isodesmic modeling of CG-MALS data.


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

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

Figure 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.


Representative H & E ( top ) immunofluorescent ( IF ) ( bottom ) of control Lats1 / 2 dKO airways at indicated time points post tamoxifen treatment . Tomato ( for Scgb1a1 lineage , red ) , CC10 ( for secretory , ) , Acetylated tubulin ( Act - Tub , for ciliated , green ) , DAPI ( blue ) . Scale bar , 100μm . Statistical of Scgb1a1 lineage - labeled tdTomato + CC10 + secretory in ( B ) .

O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-GENEPROD O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O B-CELL_TYPE O O O B-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O

Representative 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 of control Lats1 / 2 dKO airways at indicated time points post tamoxifen treatment . Tomato ( for Scgb1a1 lineage , red ) , CC10 ( green ) , AQP5 ( for AT1 , ) , DAPI ( blue ) . Scale bar , 100μm . of Scgb1a1 lineage - labeled tdTomato + AQP5 + AT1 in ( D ) . Data are presented as mean ± ( n = 5 mice for each group ) .

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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 ) IF ( bottom ) of control , dKO , qKO airways at day 21 post tamoxifen treatment . Tomato ( for Scgb1a1 lineage , red ) , CC10 ( green ) , AQP5 ( ) , DAPI ( blue ) . of Scgb1a1 lineage - labeled tdTomato + CC10 + secretory in ( G ) . Data are presented as mean ± ( n = 3 mice for each genotype ) .

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

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).


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

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Representative 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).


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

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

Representative 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.


Representative H & E IF of control , Lats1 / 2 dKO , Raptor ; Lats1 / 2 tKO airways at day 21 post tamoxifen treatment . Tomato ( for Scgb1a1 lineage , red ) , CC10 ( green , upper ) , AGER ( green , lower ) , DAPI ( blue ) . Scale bar , 100μm .

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Representative 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.


of Scgb1a1 lineage - labeled tdTomato + CC10 + secretory ( left ) tdTomato + AGER + AT1 ( right ) in ( E ) . Data are presented as mean ± ( n = 2 - 3 mice for each genotype ) . * * * p < 0 . 001 ( Student ' s t - ) .

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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 - acid or essential ( 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 ± ( n = 3 technical replicates for each genotype ) . * p < 0 . 05 , * * p < 0 . 001 ( Student ' s t - ) .

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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).


Western blot for expression of YAP , ATF4 , LAT1 / Slc7a5 in control or YAP5SA overexpressed C22 airway epithelial . Knockdown of ATF4 was induced by using lentiviral - mediated inducible scramble shRNA ( shScramble , control ) or shATF4 .

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Western 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.


of L - ( L - AA , left ) or EAA ( right ; leucine , isoleucine , valine ) in lines in ( A ) . Data are presented as mean ± ( n = 3 biological replicates for each group ) . * p < 0 . 05 , NS = not significant ( Student ' s t - ) .

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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 IF showing expression of secretory marker CC10 , DATP marker CLDN4 , AT1 marker CAV1 in organoids derived from Lats1 / 2 dKO secretory with shscramble , shAtf4 , or shSlc7a5 . CC10 ( green ) , CLDN4 ( ) , CAV1 ( ) DAPI ( blue ) . of Scgb1a1 + lineage - labeled tdTomato + CC10 + secretory ( red ) , tdTomato + CLDN4 + DATP ( green ) , tdTomato + CAV1 + AT1 ( blue ) in ( E ) . Representative H & E IF showing expression of secretory marker CC10 , DATP marker CLDN4 , or AT1 marker CAV1 in organoids derived from Scgb1a1 lineage - labeled secretory with or without hydroxytamoxifen - induced ATF4 overexpression . CC10 ( green ) , CLDN4 ( ) , CAV1 ( ) , DAPI ( blue ) . of Scgb1a1 lineage - labeled tdTomato + CC10 + secretory ( red ) , tdTomato + CLDN4 + DATP ( green ) , tdTomato + CAV1 + AT1 ( blue ) in ( G ) .

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

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).


Representative H & E of human lung tissues from normal background bronchiolitis obliterans ( BO ) with progressive fibrosis .

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Representative H&E images of human lung tissues from normal background and bronchiolitis obliterans (BO) with progressive fibrosis.


Representative IF showing expressions of YAP , DATP marker CLDN4 , AT1 marker AQP5 in airways of normal background ( top ) BO ( bottom ) human lungs . CLDN4 ( ) , AQP5 ( red ) , YAP ( green ) , DAPI ( blue ) . Of note , a flattened airway layer in BO lungs . Scale bars , 100μm .

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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 showing expressions of secretory marker CC10 mTORC1 activation marker p - S6 in airways of normal background ( top ) BO ( bottom ) human lungs . CC10 ( green ) , p - S6 ( ) , DAPI ( blue ) . Scale bars , 100μm .

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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 showing expression of YAP , ATF4 , AQP5 in airways of normal background ( top ) BO ( bottom ) human lungs . YAP ( green ) , ATF4 ( ) , AQP5 ( red ) , DAPI ( blue ) . Of note , co - expressions of YAP , ATF4 , AQP5 in airways of BO lungs . Scale bars , 100μm .

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

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 showing expression of p - S6 ( ) in airway losing CC10 ( green ) expression of BO human lungs . Scale bars , 100μm .

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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.


A - C , Immunofluorescence ( inverted ) of P14 - P15 IHCs from indicated genotypes , revealing differences in otoferlin distribution . Maximum projection of confocal stacks , scale bar 10µm .

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A-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 ) Vglut3 ( green ) position of for scans ; maximum projection of few optical sections , scale bars 5µm . Lower panels , for of , was normalized to for each fluorophore , then average of five parallel scans through middle of for sum of both values ( black ) was used to determine position of basal . At most basal point along this which exceeds threshold value of 2 ( yellow diamond ) , otoferlin - Vglut3 difference ( blue ) gave value for relative otoferlinplasma ( orange diamond ) . Insets : enlargements of basal regions . G , Otoferlin were reduced in OtofI515T / I515T IHCs ( indicates numbers represent numbers of ) OtofPga / Pga IHCs compared to wild - type ( Otof + / + ) controls ( mean ± ) . H , of apical / basal ( above / below midline depicted as green in A ) indicates an apical shift of otoferlin in OtofPga / Pga IHCs . I , Relative of - bound otoferlin at basal pole of IHCs . K , Absolute amount of otoferlin at basal IHCplasma , gained by multiplication of relative plasma ( I ) x total otoferlin ( G ) . Kruskal - Wallis ; * * * indicates p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 .

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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.


A , ABR thresholds in OtofI515T / I515T ( red , n = 5 ) Otof - / I515T ( blue , n = 7 ) mice were elevated compared to Otof + / + mice ( black , n = 5 ) at an of 3 - 4 . grey dotted indicates maximum loudspeaker output of 90dB ; thresholds exceeding this value were set to 100dB for calculation of mean ± . At 12kHz , only threshold increase for Otof - / I515T vs Otof + / + is significant ( Kruskal - Wallis with Dunn ' s multiple comparisons between 3 groups ) . B , Grand averages of ABR waveforms ± in response to 80dB click stimulation of mice analyzed in A : small wave preceding ABR wave I probably represents summating potential ( SP , hair 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 ± for different stimulus intensities ( differences between genotypes are significant ; 2 - way ANOVA with Tukey ' s multiple comparison ) . D , At 8 ( circles ) 25 ( 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 - ) . Grey dotted as in A . E , Grand averages of ABR waveforms ± in response to 80dB Click stimulation in mice aged 8 . 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 ± for different stimulus intensities for 8 week 25 week old OtofI515T / I515T Otof + / + mice ( p < 0 . 001 , 2 - way ANOVA ) .

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A, 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).


A , B , No difference in voltage dependent Ca2 + - currents ( A ) fractional activation of ICa - channels ( B ) between OtofI515T / I515T IHCs ( n = 16 ) IHCs of Otof + / + ( n = 13 ) .

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A,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 capacitance ( ΔCm , lower panel ) in response to depolarization ( left , 20ms ; right , 100ms ) to voltage where maximum Ca2 + - currents were elicited ( upper panel ) , typically - 14mV .

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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 ) Otof + / + ( n = 11 ) IHCs was similar , sustained exocytosis , representing most likely release of replenished vesicles , was significantly reduced in OtofI515T / I515T IHCs ( t - ) . Lower panel , Ca2 + - current integrals were of similar .

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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 .

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E, Flash photolysis of caged Ca2+ elicited a smaller exocytic response in OtofI515T/I515T IHCs.


F , kinetics of fast component from E was comparable between OtofI515T / I515T IHCs Otof + / + ( b ) . Open circles represent previously published data on IHCs of hearing wild - type mice ( a ; Beutner et al , 2001 ; Pangrsic et al , 2010 ) .

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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).


A , Spontaneous rates of SGNs from OtofI515T / I515T ( red , n = 35 ) Otof + / + ( black , n = 39 ) were not significantly different ( p = 0 . 83 , Kolmogorov - Smirnov ) .

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A, 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 ± from OtofI515T / I515T mice ( n = 25 - 32 ) Otof + / + littermate SGNs ( n = 13 - 27 ) to stimulation with 50ms tone bursts at characteristic frequency ( CF ) of each fiber , 30dB above threshold at indicated stimulus rates .

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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 , of onset ( largest 0 . 5ms bin , Mann - Whitney U ) adapted responses ( averaged 35 - 45ms from response onset , t - ) from data in B - D .

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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 , jitter of first sound - evoked spike was significantly increased ( p < 0 . 001 , Mann - Whitney U ) but retained its inverse correlation with spike rates .

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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 were significantly shallower in OtofI515T / I515T SGNs , both for repetitive stimulation with 50ms tone bursts ( left , p = 0 . 014 , t - ) for continuous stimulation with amplitude modulated tones ( right , p < 0 . 001 , Mann - Whitney U ) .

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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 maximal synchronization index ) was typically less precise in OtofI515T / I515T SGNs than in Otof + / + SGNs ( p = 0 . 09 , Mann - Whitney U ) .

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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 15ms probe tones ( both at CF , 30dB above threshold ) were separated by silent intervals of variable duration . Inter - masker intervals were 500ms for Otof + / + , 1000ms for OtofI515T / I515T . of probe masker onset responses revealed enhanced RRP depletion after stimulation in OtofI515T / I515T ( pink ; mean ± red ) compared to Otof + / + ( grey ; mean ± black ; for 4ms interval : p = 0 . 001 , t - ) a slowed time course of recovery ( x : half time of recovery , taken from normalized recovery functions ; p < 0 . 001 , Mann - Whitney U ) .

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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 noise was interrupted by silent gaps . 5 OtofI515T / I515T mice ( pink , mean ± red ) avoided drinking less efficiently than 2 Otof + / + mice ( grey , mean ± black ) for shorter gap durations . See also Figs S2 / S3 .

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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.


A , 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 .

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A, 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 ) Otof + / + mice ( B , Click 80dB ) decreased with increasing in bulla . Each color represents a different experiment , dashed lines are fits . Open symbols indicate beginning of experiments ; subsequent recordings are connected by lines . four indicated data points in B correspond to ABRs in A .

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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 ) Vglut3 ( green ) . Note intense immunostaining of otoferlin at plasma . Scale bar 10 µm . E - J , Explanted organs of Corti from Otof - / - mice at P4 were transfected by GeneGun with otoferlin cDNA eGFP immunostained at DIV2 . E , Example transfected with wild - type mouseotoferlin cDNA . F , G , Representative transfected with mouseotoferlin including 20 of RXR motif ( see Fig EV4 ) . in G were incubated at 38 . 5 ° C for 30 min prior to fixation . RXR - otoferlin transfected show a localization of otoferlin surrounding Vglut3 immunofluorescence / eGFP at basal pole of ( arrows ) , similar as in controls ( A , B ) . H , J , Both human RXR motif Ile515Thr mutation were introduced in mouse cDNA , were incubated at 37 ° C ( H ) or for 30min at 38 . 5 ° C ( J ) before fixation . Here , green surrounds otoferlin immunofluorescence ( arrows ) , suggesting loss of otoferlin from plasma . K , of relative plasma of otoferlin ( as in Fig 1 ) revealed normal plasma abundance when human RXR motif was present , but a strong reduction for otoferlin with RXR Ile515Thr ( ; mean ± ; t - ) .

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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).


A - E , Capacitance increments recorded from Otof + / + ( black ) OtofI515T / I515T ( red ) IHCs . recorded at indicated temperatures ( circles ) , mean ± for perforated patch clamp experiments ( t - ) .

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A-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 capacitance changes Ca2 + - current integrals for Otof + / + ( F ) OtofI515T / I515T IHCs ( G ) at different temperatures illustrates drastic increase in exocytosis for physiological . * Significant differences compared to RT measurements are indicated with colors of respective , between PT high in violet ( t - ) .

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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 ) OtofI515T / I515T IHCs ( middle lower panels ) of explanted organs of Corti at P7 - P8 after incubation at indicated temperatures for 24h ; maximum projections of z - stacks , inverted , scale bar 10µm . same imaging settings have been applied in experiments , same lookup table was applied for upper middle panels . of lower panels are enhanced compared to middle panels ( lookup table covering full data range of only this genotype ) to visualize otoferlin distribution .

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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 ) OtofI515T / I515T IHCs ( middle lower panels ) of explanted organs of Corti at P7 - P8 after incubation at indicated temperatures for 24h ; maximum projections of z - stacks , inverted , scale bar 10µm . same imaging settings have been applied in experiments , same lookup table was applied for upper middle panels . of lower panels are enhanced compared to middle panels ( lookup table covering full data range of only this genotype ) to visualize otoferlin distribution . K , of otoferlin immunofluorescence in Otof + / + IHCs ( black bars , n = 98 - 137 , 4 - 5 experiments ) OtofI515T / I515T IHCs ( red bars , n = 90 - 97 ; Kruskal - Wallis as in L - N ) , indicate reduced with increasing . L , Apical / basal otoferlin distribution , revealing a significant apical shift of otoferlin Vglut3 for OtofI515T / I515T IHCs ( red symbols ) at 38 . 5 ° C compared to Otof + / + ( grey / black symbols ) . M , Relative of bound otoferlin were lowered with increasing . N , Absolute otoferlin immunostaining strongly decreased with .

O O O O B-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O

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.


A - C , Pre - embedding immunogold labelling for EM visualizes otoferlin localization in random ultrathin sections through basal part of IHCs in Otof + / + ( A ) OtofI515T / I515T ( B ) but not in Otof - / - ( C ) mice . IHCs are highlighted in beige . Scale bar 100nm .

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A-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 ) postsynaptic afferent boutons of SGNs ( Aff ) , displaying otoferlin immunogold labelling at active zone membranes ( pink arrowheads ) but not around synaptic ribbon , using saponin permeabilization ( D - F ) , or Triton - X - 100 treatment ( G ) .

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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 ribbon as expected for synaptic vesicle . Scale bar 200nm .

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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 ; Otof + / I + , n = 82 vesicles , 6 ; Wilcoxon - rank - sum - , as in L - M ) . L , of immunogold clusters at plasma revealed a strong trend towards reduced of otoferlin in OtofI515T / I515T ( 6 each ) . M , Distal from plasma , immunogold labels were reduced in OtofI515T / I515T IHCs .

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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 of IHCribbon synapses in OtofI515T / I515Tand Otof + / ( conventional embedding without immunogold ) after pre - incubation for 10min at indicated followed by high K + stimulation or 0 Ca2 + inhibition for 1min 45sec . Scale bars 100nm .

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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 - proximal ( P ; Kruskal - Wallis ) ribbon - associated ( Q ; one way ANOVA followed by Tukey ' s ) synaptic vesicles ( see also Appendix Table S1 ) .

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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).


A - D , Exemplary virtual sections through EM tomograms for stimulatory condition at 36 ° C 39oC . Scale bar 100nm .

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A-D, Exemplary virtual sections through EM tomograms for stimulatory condition at 36°C and 39oC. Scale bar 100nm.


F - I , 3D reconstruction of ribbon - associated pool of vesicles ( front view ) . Active zone is depicted in blue , presynaptic density in pink , ribbons in red , scale bars 100nm .

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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 - proximal pool ( top view ) J - M , ribbon - associated pool of vesicles ( front view ) . Active zone is depicted in blue , presynaptic density in pink , ribbons in red , scale bars 100nm . N - Q , Cumulative probability distribution of diameter of round vesicles as well as longest axis of flattened vesicles in - proximal pool ( N - O ) ribbon - associated pool ( P - Q ) . N - P , one way ANOVA followed by Tukey ' s ; Q , Kruskal - Wallis . Round vesicles tested in both pools appeared larger in OtofI515T / I515T synapses but long axis of flattened vesicles were comparable for both genotypes ( see also Table EV1 ) .

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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).


( A ) Rescue of growth phenotype . Wild - type ( WT ) or mdm10Δ were transformed with empty plasmid pYX142 ( ) . In addition , mdm10Δ were transformed with pYX142 encoding MDM10 or MCP3 . were grown to an OD600 of 1 . 0 spotted on YPD or YPG plates in a 1 : 5 dilution series . Plates were incubated for growth at indicated temperatures .

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(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 mitochondrial defect in mdm10Δ . described in ( A ) expressing mitochondrially targeted GFP ( mtGFP ) were analysed by microscopy . Typical ( brightfield , BF ) of four different strains are shown ( scale bar = 5 µm ) . shows average with standard deviation bars of three independent experiments ( n = 3 ; SD ; * , p < 0 . 05 as indicated ) with at least 100 per experiment .

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(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.


( A ) Respiratory chain super - are slightly restored by over - expression of Mcp3 . Mitochondria isolated from indicated strains were lysed in 1 % digitonin subjected to a 4 - 8 % BN - PAGE . were analysed by immunodecoration with antibodies against either Cor1 of III or Cox2 of IV .

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(A) Respiratory chain super-complex levels are slightly restored by over-expression of Mcp3. Mitochondria isolated from the indicated strains were lysed in 1% digitonin and subjected to a 4-8% BN-PAGE. Proteins were analysed by immunodecoration with antibodies against either Cor1 of complex III or Cox2 of complex IV.


( B ) Assembly defects in TOM TOB complexes in mdm10 are partially restored by Mcp3 over - expression . Mitochondria of indicated were lysed in 1 % digitonin subjected to a 6 - 13 % BN - PAGE immunoblotting with antibodies against Tom40 ( left ) or Tob55 ( right ) . assembled TOM an unassembled Tom40 species at ca . 100 kDa ( arrowhead ) are indicated as well as TOB an additional higher molecular species of TOB ( asterisk ) .

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(B) Assembly defects in TOM and TOB complexes in mdm10 cells are partially restored by Mcp3 over-expression. Mitochondria of the indicated cells were lysed in 1% digitonin and subjected to a 6-13% BN-PAGE and immunoblotting with antibodies against Tom40 (left) or Tob55 (right). The assembled TOM complex and an unassembled Tom40 species at ca. 100 kDa (arrowhead) are indicated as well as the TOB complex and an additional higher molecular weight species of the TOB complex (asterisk).


( C ) Over - expression of Mcp3 partially rescues alterations in lipid composition of lacking Mdm10 . Lipids were extracted from highly pure mitochondria isolated from indicated then analysed by mass spectrometry . level of each phospholipid species in wild - type mitochondria was set to 100 % ERG / PL to 1 . Relative changes in mitochondria from other are presented . mean with standard deviations of three biological repeats with two technical repeats for each ( n = 6 ; SD ; * , p < 0 . 05 ) is given .

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(C) Over-expression of Mcp3 partially rescues the alterations in lipid composition of cells lacking Mdm10. Lipids were extracted from highly pure mitochondria isolated from the indicated cells and then analysed by mass spectrometry analysis. The level of each phospholipid species in wild-type mitochondria was set to 100% and the ERG/PL ratio to 1. Relative changes in mitochondria from other cells are presented. The mean with standard deviations of three biological repeats with two technical repeats for each (n=6; SD; *, p < 0.05) is given.


( D ) Mcp3 rescues loss of Mdm12 . Wild - type ( WT ) or mdm12Δ transformed with empty plasmid pYX142 ( ) or mdm12Δ over - expressing either Mcp3 or Mdm12 ( as a control ) were analysed at 30 ° C or 37 ° C by drop dilution on rich medium containing glycerol ( YPG ) .

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

(D) Mcp3 rescues loss of Mdm12. Wild-type (WT) or mdm12Δ cells transformed with the empty plasmid pYX142 () or mdm12Δ cells over-expressing either Mcp3 or Mdm12 (as a control) were analysed at 30°C or 37°C by drop dilution assay on rich medium containing glycerol (YPG).


( E ) Mcp3 rescues loss of Mmm1 . WT mmm1Δ were transformed analysed as described in ( D ) .

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(E) Mcp3 rescues loss of Mmm1. WT and mmm1Δ cells were transformed and analysed as described in (D).


( F ) Mcp3 rescues loss of Mdm34 . WT mdm34Δ were transformed analysed as described in ( D ) .

(F) Mcp3 rescues loss of Mdm34. WT and mdm34Δ cells were transformed and analysed as described in (D).


( A ) Growth phenotype of mcp3 . WT or mcp3 ∆ were grown to an OD600 of 1 . 0 in YPD medium spotted on YPD or YPG plates in a 1 : 5 dilution series . Plates were incubated for growth at indicated temperatures .

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

(A) Growth phenotype of mcp3 cells. WT or mcp3∆ cells were grown to an OD600 of 1.0 in YPD medium and spotted on YPD or YPG plates in a 1:5 dilution series. Plates were incubated for growth at the indicated temperatures.


( B ) mcp3 have normal mitochondrial . WT mcp3 ∆ expressing mitochondrially targeted GFP ( mtGFP ) were grown to mid - logarithmic phase then analysed by microscopy . Typical of two different strains are shown ( scale bar = 5 µm ) . of depicted strains shows average with standard deviation bars of three independent experiments with at least 100 per experiment ( n = 3 ; SD ; p as indicated ) .

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(B) mcp3 cells have normal mitochondrial morphology. WT and mcp3∆ cells expressing mitochondrially targeted GFP (mtGFP) were grown to mid-logarithmic phase then analysed by fluorescence microscopy. Typical images of the two different strains are shown (scale bar = 5 µm). The quantification of the depicted strains shows the average percentage with standard deviation bars of three independent experiments with at least 100 cells per experiment (n=3; SD; p as indicated).


( C , D ) Loss of Mcp3 has no effect on phospholipid composition of mitochondria . Highly - pure mitochondria were isolated from indicated yeast . Lipids were extracted then analysed by mass spectrometry . level of each phospholipid species in wild - type mitochondria was set to 100 % ERG / PL to 1 . Relative changes in mitochondria from mcp3 are presented . mean with standard deviations of three biological repeats with two technical repeats for each ( n = 6 ; SD ) is given .

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

(C, D) Loss of Mcp3 has no effect on the phospholipid composition of mitochondria. Highly-pure mitochondria were isolated from the indicated yeast cells. Lipids were extracted and then analysed by mass spectrometry analysis. The level of each phospholipid species in wild-type mitochondria was set to 100% and the ERG/PL ratio to 1. Relative changes in mitochondria from mcp3 cells are presented. The mean with standard deviations of three biological repeats with two technical repeats for each (n=6; SD) is given.


( E ) Over - expression of Mcp3 leads to alterations of tubular mitochondrial network . Wild - type expressing mitochondrially targeted GFP ( mtGFP ) were transformed with a plasmid over - expressing Mcp3 ( MCP3 ) or an empty plasmid as control ( ) . were grown to mid - logarithmic phase then analysed by microscopy . Typical of two different strains are shown ( scale bar = 5 µm ) .

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

(E) Over-expression of Mcp3 leads to alterations of the tubular mitochondrial network. Wild-type cells expressing mitochondrially targeted GFP (mtGFP) were transformed with a plasmid over-expressing Mcp3 (MCP3) or an empty plasmid as control (). Cells were grown to mid-logarithmic phase and then analysed by fluorescence microscopy. Typical images of the two different strains are shown (scale bar = 5 µm).


( B ) Internally tagged HA - Mcp3 is functional . mdm10 ∆ were transformed with empty plasmid pYX142 ( ) or pYX142 encoding Mdm10 , Mcp3 , or an internally HA - tagged Mcp3 ( MDM10 , MCP3 , HA - MCP3 ) . were grown to an OD600 of 1 . 0 spotted on YPG plates in a 1 : 5 dilution series . Plates were incubated for growth at 37 ° C for 4 days .

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(B) Internally tagged HA-Mcp3 is functional. mdm10∆ cells were transformed with the empty plasmid pYX142 () or pYX142 encoding Mdm10, Mcp3, or an internally HA-tagged Mcp3 (MDM10, MCP3, HA-MCP3). Cells were grown to an OD600 of 1.0 and spotted on YPG plates in a 1:5 dilution series. Plates were incubated for growth at 37°C for 4 days.


( C ) HA - Mcp3 is processed in vivo . Mitochondria isolated from mdm10 ∆ ( ∆ ) containing empty plasmid or expressing HA - Mcp3 were analysed by SDS - PAGE immunodecoration with antibodies against indicated . Two different bands are detectable in case of HA - Mcp3 ( p , precursor ; m , mature ) . MOM Tom40 serves as loading control .

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(C) HA-Mcp3 is processed in vivo. Mitochondria isolated from mdm10∆ (∆) cells containing the empty plasmid or expressing HA-Mcp3 were analysed by SDS-PAGE and immunodecoration with antibodies against the indicated proteins. Two different bands are detectable in case of HA-Mcp3 (p, precursor; m, mature protein). The MOM protein Tom40 serves as loading control.


( D ) Mcp3 is a mitochondrial . Whole lysate ( WCL ) fractions corresponding to cytosol ( Cyt . ) , microsomal fraction ( ER ) mitochondria ( Mito . ) of mcp3 ∆ either containing empty plasmid ( ) or expressing HA - Mcp3 were analysed by SDS - PAGE immunodecoration with antibodies against HA - tag , mitochondrial Tom70 , a marker for cytosol ( Bmh1 ) an ER marker ( Erv2 ) . m , mature .

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(D) Mcp3 is a mitochondrial protein. Whole cell lysate (WCL) and fractions corresponding to cytosol (Cyt.), light microsomal fraction (ER) and mitochondria (Mito.) of mcp3∆ cells either containing the empty plasmid () or expressing HA-Mcp3 were analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag, the mitochondrial protein Tom70, a marker protein for the cytosol (Bmh1) and an ER marker protein (Erv2). m, mature protein.


( E ) Mcp3 co - localizes with mitochondrial marker AAC . over - expressing HA - Mcp3 were fixed analysed by microscopy using antibodies against HA - epitope ( αHA ) mitochondrial ADP / ATP carrier ( αAAC ) . ( scale bar = 5 µm ) .

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(E) Mcp3 co-localizes with the mitochondrial marker protein AAC. Cells over-expressing HA-Mcp3 were fixed and analysed by fluorescence microscopy using antibodies against the HA-epitope (αHA) and the mitochondrial ADP/ATP carrier (αAAC). (scale bar = 5 µm).


( F ) C - terminally tagged Mcp3 - HA is located to mitochondria . A mitochondria enriched fraction ( Mito . ) post mitochondrial supernatant ( PMS ) of wild - type carrying empty plasmid ( ) or over - expressing C - terminally HA - tagged Mcp3 ( Mcp3 - HA ) were analysed by SDS - PAGE immunodecortion with antibodies against HA - Tag , mitochondrial Tom40 a marker for cytosol ( Hxk1 ) . m , mature .

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

(F) C-terminally tagged Mcp3-HA is located to mitochondria. A mitochondria enriched fraction (Mito.) and the post mitochondrial supernatant (PMS) of wild-type cells carrying the empty plasmid () or over-expressing the C-terminally HA-tagged Mcp3 (Mcp3-HA) were analysed by SDS-PAGE and immunodecortion with antibodies against the HA-Tag, the mitochondrial protein Tom40 and a marker protein for the cytosol (Hxk1). m, mature protein.


( G ) Mcp3 is - embedded . Mitochondria isolated from mcp3Δ expressing HA - Mcp3 were left untreated ( I , input ) or subjected to alkaline extraction . supernatant ( SN ) pellet ( P ) fractions were analysed by SDS - PAGE immunodecoration with antibodies against indicated . Tom20 an integral MOM ; Aco1 , soluble matrix . m , mature .

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

(G) Mcp3 is membrane-embedded. Mitochondria isolated from mcp3Δ cells expressing HA-Mcp3 were left untreated (I, input) or subjected to alkaline extraction. The supernatant (SN) and pellet (P) fractions were analysed by SDS-PAGE and immunodecoration with antibodies against the indicated proteins. Tom20 an integral MOM proteins; Aco1, soluble matrix protein. m, mature protein.


( H ) Mcp3 is a MOM . Mitochondria isolated from expressing HA - Mcp3 were treated with proteinase K ( PK , 100 µg / ml ) in absence or presence of detergent Triton X - 100 ( TX ) . Samples were precipitated with TCA analysed by SDS - PAGE immunodecoration with antibodies against HA - tag or indicated mitochondrial . Tom20 , a MOM exposed to cytosol ; Dld1 , a located in IMS .

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

(H) Mcp3 is a MOM protein. Mitochondria isolated from cells expressing HA-Mcp3 were treated with proteinase K (PK, 100 µg/ml) in the absence or presence of the detergent Triton X-100 (TX). Samples were precipitated with TCA and analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag or the indicated mitochondrial proteins. Tom20, a MOM protein exposed to the cytosol; Dld1, a protein located in the IMS.