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 .

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

D-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 ) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, 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 ) and fractional activation of ICa - channels ( B ) between OtofI515T / I515T IHCs ( n = 16 ) and IHCs of Otof + / + littermates ( n = 13 ) .

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

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 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 .

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

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 .

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

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 .

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

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

F, 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 ) and Otof + / + littermates ( black , n = 39 ) were not significantly different ( p = 0 . 83 , Kolmogorov - Smirnov test ) .

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

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 ± 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 .

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

E, 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 .

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

F, 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 ) .

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

G, 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 ) .

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

H, 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 ) .

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

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

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 temperature .

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

A, 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, 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 ) and OtofI515T / I515T ( red ) IHCs . Individual cells recorded at indicated temperatures ( light circles ) , mean ± SEM for perforated patch clamp experiments ( t - test ) .

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

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 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 ) .

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

F,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 .

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

H-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H-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 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 .

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 ) 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 ) .

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

D-G, 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 .

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 .

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

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 .

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

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

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 and 39oC . Scale bar 100nm .

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

A-D, 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 .

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

F-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-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 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 .

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

(A) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 - 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 .

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

(A) 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

(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 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 ) .

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

(D) 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 and mmm1Δ cells were transformed and analysed as described in ( D ) .

(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 and mdm34Δ cells were transformed and 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 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 .

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

(A) 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

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

(E) 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 ∆ 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ∆ ( ∆ ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

(D) 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 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 ) .

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

(E) 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 . ) 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 .

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

(F) 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 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 .

(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 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 .

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

(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.


( I ) Mcp3 behaves like an outer membrane protein in density gradient centrifugation . Mitochondrial vesicles isolated from cells over - expressing HA - Mcp3 were subjected to sucrose density gradient centrifugation . Fractions of the gradient were collected and analysed by SDS - PAGE and immunodecoration with antibodies against the indicated proteins . The processed form of HA - Mcp3 is shown . Right panel : The intensities of the various bands were quantified and depicted . The sum of all intensities for each protein was set to 100 % .

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(I) Mcp3 behaves like an outer membrane protein in density gradient centrifugation. Mitochondrial vesicles isolated from cells over-expressing HA-Mcp3 were subjected to sucrose density gradient centrifugation. Fractions of the gradient were collected and analysed by SDS-PAGE and immunodecoration with antibodies against the indicated proteins. The processed form of HA-Mcp3 is shown. Right panel: The intensities of the various bands were quantified and depicted. The sum of all intensities for each protein was set to 100%.


( J ) Mcp3 lacking either transmembrane domain fails to rescue loss of Mdm10 . Wild - type cells carrying the empty plasmid ( wt + ) or mdm10 ∆ cells transformed with the empty plasmid ( ) or plasmid encoding Mdm10 , Mcp3 , or Mcp3 lacking TMD1 or TMD2 ( MDM10 , MCP3 , ΔTMD1 , ΔTMD2 ) 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 .

(J) Mcp3 lacking either transmembrane domain fails to rescue loss of Mdm10. Wild-type cells carrying the empty plasmid (wt+) or mdm10∆ cells transformed with the empty plasmid () or plasmid encoding Mdm10, Mcp3, or Mcp3 lacking TMD1 or TMD2 (MDM10, MCP3, ΔTMD1, ΔTMD2) 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.


( K ) Mcp3 lacking either of the two transmembrane domains is unstable . Crude mitochondria were obtained from wild - type cells containing an empty plasmid ( Ø ) or mcp3Δ cells ( Δ ) transformed with plasmid encoding Mcp3 or Mcp3 lacking TMD1 or TMD2 ( MCP3 , ΔTMD1 , ΔTMD2 ) . Samples were analysed by SDS - PAGE and immunodecoration with antibodies against Mcp3 and Tom20 as loading control .

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(K) Mcp3 lacking either of the two transmembrane domains is unstable. Crude mitochondria were obtained from wild-type cells containing an empty plasmid (Ø) or mcp3Δ cells (Δ) transformed with plasmid encoding Mcp3 or Mcp3 lacking TMD1 or TMD2 (MCP3, ΔTMD1, ΔTMD2). Samples were analysed by SDS-PAGE and immunodecoration with antibodies against Mcp3 and Tom20 as loading control.


( A ) Isolated mitochondria were incubated with radiolabelled Mcp3 precursor protein for the indicated time periods . After import mitochondria were reisolated and analysed by SDS - PAGE and autoradiography . The arrowhead marks an additional band of the size of the cleaved N - terminus of Mcp3 . p and m , precursor and mature forms , respectively .

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(A) Isolated mitochondria were incubated with radiolabelled Mcp3 precursor protein for the indicated time periods. After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. The arrowhead marks an additional band of the size of the cleaved N-terminus of Mcp3. p and m, precursor and mature forms, respectively.


( B ) Import of Mcp3 is dependent on the receptor Tom70 . Mitochondria isolated from wild - type or tom70 / 71Δ cells were incubated with radiolabelled Mcp3 or pSu9 - DHFR ( as control ) for the indicated time periods . After import mitochondria were reisolated and analysed by SDS - PAGE and autoradiography . p and m , precursor and mature forms , respectively . I , 20 % of radiolabelled precursor protein used in each import reaction . Bands corresponding to the mature ( m ) form were quantified . Import into wild - type mitochondria after 15 min was set to 100 % . The mean with standard deviations is depicted ( n = 3 ; SD ) .

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(B) Import of Mcp3 is dependent on the receptor Tom70. Mitochondria isolated from wild-type or tom70/71Δ cells were incubated with radiolabelled Mcp3 or pSu9-DHFR (as control) for the indicated time periods. After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. p and m, precursor and mature forms, respectively. I, 20% of radiolabelled precursor protein used in each import reaction. Bands corresponding to the mature (m) form were quantified. Import into wild-type mitochondria after 15 min was set to 100%. The mean with standard deviations is depicted (n=3; SD).


( C ) Reduced steady state levels of HA - Mcp3 in cells lacking Tom70 . Crude mitochondria isolated from the indicated cells were analysed by SDS - PAGE and immunodecoration with antibodies against the HA - tag , AAC as control substrate of Tom70 , and Fis1 as a loading control . HA - Mcp3 levels were quantified in relation to Fis1 levels and the levels in WT cells were set to 100 % . The bar diagram shows the mean with standard deviation ( n = 3 ; SD ; * , p < 0 . 05 ) .

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(C) Reduced steady state levels of HA-Mcp3 in cells lacking Tom70. Crude mitochondria isolated from the indicated cells were analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag, AAC as control substrate of Tom70, and Fis1 as a loading control. HA-Mcp3 levels were quantified in relation to Fis1 levels and the levels in WT cells were set to 100%. The bar diagram shows the mean with standard deviation (n=3; SD; *, p < 0.05).


( D ) Import of Mcp3 is dependent on Tom40 . Mitochondria isolated from wild - type and tom4025 cells were incubated with radiolabelled Mcp3 for the indicated time periods . Next , mitochondria were reisolated and analysed by SDS - PAGE and autoradiography as described in ( B ) . As a control Ugo1 was imported into the same mitochondria . Organelles were reisolated and analysed by BN - PAGE and autoradiography . The band corresponding to the Ugo1 dimer at around 150 kDa was quantified . The mean with standard deviation is shown ( n = 3 ; SD ) .

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(D) Import of Mcp3 is dependent on Tom40. Mitochondria isolated from wild-type and tom4025 cells were incubated with radiolabelled Mcp3 for the indicated time periods. Next, mitochondria were reisolated and analysed by SDS-PAGE and autoradiography as described in (B). As a control Ugo1 was imported into the same mitochondria. Organelles were reisolated and analysed by BN-PAGE and autoradiography. The band corresponding to the Ugo1 dimer at around 150 kDa was quantified. The mean with standard deviation is shown (n=3; SD).


( E ) Steady state levels of HA - Mcp3 are lower in cells harbouring a mutant TOM40 allele . Crude mitochondria from wild - type and tom40 - 25 cells were obtained and analysed as described in ( C ) . Immunodecoration was performed with antibodies against the HA - tag , Tom40 , and Fis1 as loading control .

(E) Steady state levels of HA-Mcp3 are lower in cells harbouring a mutant TOM40 allele. Crude mitochondria from wild-type and tom40-25 cells were obtained and analysed as described in (C). Immunodecoration was performed with antibodies against the HA-tag, Tom40, and Fis1 as loading control.


( A ) Mcp3 precursor binds in vitro to cytosolic domains of TOM receptors . Radiolabelled Mcp3 was incubated with recombinant GST alone or GST fused to the cytosolic domains of Tom70 , Tom20 or Tom22 prebound to glutathione beads or with unloaded beads . After washing bound material was eluted with sample buffer . Proteins were analysed by SDS - PAGE , blotting onto a membrane and autoradiography . To demonstrate equal amounts of GST fusion proteins the membrane was stained with Ponceau S . I , input 5 % of precursor used .

(A) Mcp3 precursor binds in vitro to cytosolic domains of TOM receptors. Radiolabelled Mcp3 was incubated with recombinant GST alone or GST fused to the cytosolic domains of Tom70, Tom20 or Tom22 prebound to glutathione beads or with unloaded beads. After washing bound material was eluted with sample buffer. Proteins were analysed by SDS-PAGE, blotting onto a membrane and autoradiography. To demonstrate equal amounts of GST fusion proteins the membrane was stained with Ponceau S. I, input 5% of precursor used.


( B ) Mcp3 precursor binds to Tom22 in organello . Radiolabelled Mcp3 was incubated with mitochondria isolated from WT His - Tom22 containing cells . Mitochondria were pre - incubated with CCCP ( 40 µM ) to halt import . After lysis with β - dodecyl maltoside samples were incubated with Ni - NTA beads . After washing bound proteins were eluted with sample buffer and analysed by SDS - PAGE and autoradiography . I , 5 % of 35S - Mcp3 used ; pel . , 2 % insoluble material after clarifying spin ; sup . , 2 % of material incubated with Ni - NTA ; n . b . 2 % of non - bound material after binding to Ni - NTA ; eluate , 100 % of bound material .

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(B) Mcp3 precursor binds to Tom22 in organello. Radiolabelled Mcp3 was incubated with mitochondria isolated from WT His-Tom22 containing cells. Mitochondria were pre-incubated with CCCP (40 µM) to halt import. After lysis with β-dodecyl maltoside samples were incubated with Ni-NTA beads. After washing bound proteins were eluted with sample buffer and analysed by SDS-PAGE and autoradiography. I, 5% of 35S-Mcp3 used; pel., 2% insoluble material after clarifying spin; sup., 2% of material incubated with Ni-NTA; n.b. 2% of non-bound material after binding to Ni-NTA; eluate, 100% of bound material.


( C ) Radiolabelled Mcp3 was incubated with organelles described in ( B ) . After import mitochondria were solubilized in digitonin , the lysate was cleared by centrifugation and incubated with or without an antibody against the His - tag ( α - His ) . Samples were analysed by BN - PAGE , autoradiography and afterwards immunodecoration with an antibody against Tom40 . Bands shifted by the anti - His antibody are indicated by arrowhead .

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(C) Radiolabelled Mcp3 was incubated with organelles described in (B). After import mitochondria were solubilized in digitonin, the lysate was cleared by centrifugation and incubated with or without an antibody against the His-tag (α-His). Samples were analysed by BN-PAGE, autoradiography and afterwards immunodecoration with an antibody against Tom40. Bands shifted by the anti-His antibody are indicated by arrowhead.


( A ) Import of Mcp3 is dependent on the TIM23 complex . Mitochondria isolated from wild - type and tim23ts cells were incubated with radiolabelled Mcp3 for the indicated time periods . Further treatment and analysis ( n = 3 ; SD ) was as described for Fig . 5D .

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(A) Import of Mcp3 is dependent on the TIM23 complex. Mitochondria isolated from wild-type and tim23ts cells were incubated with radiolabelled Mcp3 for the indicated time periods. Further treatment and analysis (n=3; SD) was as described for Fig. 5D.


( B ) Steady state levels of HA - Mcp3 are lower in cells harbouring a temperature sensitive TIM23 allele . Crude mitochondria were obtained at the non - permissive temperature from WT or tim23ts cells containing a plasmid expressing HA - Mcp3 . Samples were analysed by SDS - PAGE and immunodecoration with antibodies against the HA - tag , the matrix proteins Mge1 and Yah1 as typical TIM23 substrates , Ugo1 and Fis1 as TIM23 - independent substrates . HA - Mcp3 levels were quantified in relation to Fis1 levels . Levels in WT cells were set to 100 % . The bar diagram shows the mean with standard deviation of six independent experiments ( n = 6 ; SD ; * * , p < 0 . 01 ) .

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(B) Steady state levels of HA-Mcp3 are lower in cells harbouring a temperature sensitive TIM23 allele. Crude mitochondria were obtained at the non-permissive temperature from WT or tim23ts cells containing a plasmid expressing HA-Mcp3. Samples were analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag, the matrix proteins Mge1 and Yah1 as typical TIM23 substrates, Ugo1 and Fis1 as TIM23-independent substrates. HA-Mcp3 levels were quantified in relation to Fis1 levels. Levels in WT cells were set to 100%. The bar diagram shows the mean with standard deviation of six independent experiments (n=6; SD; **, p < 0.01).


( C ) Import and processing of Mcp3 is dependent on the mitochondrialmembrane potential . Isolated mitochondria were incubated with radiolabelled Mcp3 or pSu9 - DHFR as control for the indicated time periods or for 15 minutes in the presence of either CCCP or o - phenantroline and EDTA ( o - Phen . / EDTA ) . After import mitochondria were reisolated and analysed by SDS - PAGE and autoradiography . L + MPP : radiolabelled proteins were incubated with recombinant MPP .

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(C) Import and processing of Mcp3 is dependent on the mitochondrialmembrane potential. Isolated mitochondria were incubated with radiolabelled Mcp3 or pSu9-DHFR as control for the indicated time periods or for 15 minutes in the presence of either CCCP or o-phenantroline and EDTA (o-Phen./EDTA). After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. L+MPP: radiolabelled proteins were incubated with recombinant MPP.


( D ) Mcp3 is processed by Imp1 / 2 . Single deletion strains of known mitochondrial proteases and peptidases were transformed with a plasmid expressing HA - Mcp3 . Crude mitochondria were isolated and analysed by SDS - PAGE and immunodecoration with antibodies against the indicated proteins and the HA - epitope . Cox2 , a known substrate of IMP ; Tom70 , a mitochondrial outer membrane protein .

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(D) Mcp3 is processed by Imp1/2. Single deletion strains of known mitochondrial proteases and peptidases were transformed with a plasmid expressing HA-Mcp3. Crude mitochondria were isolated and analysed by SDS-PAGE and immunodecoration with antibodies against the indicated proteins and the HA-epitope. Cox2, a known substrate of IMP; Tom70, a mitochondrial outer membrane protein.


( E ) C - terminally tagged Mcp3 - HA is processed by Imp1 . WT and imp1Δ cells were transformed with a plasmid expressing Mcp3 - HA and further analysis was performed as described in ( D ) .

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(E) C-terminally tagged Mcp3-HA is processed by Imp1. WT and imp1Δ cells were transformed with a plasmid expressing Mcp3-HA and further analysis was performed as described in (D).


( F ) Mcp3 is processed by Imp1 / 2 after in vitro import into mitochondria . Mitochondria isolated from WT , imp1Δ or imp2Δ strains were incubated with radiolabelled precursor of Mcp3 or pSu9 - DHFR as control . Further treatment and analysis was as described in Fig . 5A .

(F) Mcp3 is processed by Imp1/2 after in vitro import into mitochondria. Mitochondria isolated from WT, imp1Δ or imp2Δ strains were incubated with radiolabelled precursor of Mcp3 or pSu9-DHFR as control. Further treatment and analysis was as described in Fig. 5A.


( G ) Full - length Mcp3 precursor accumulates during import if IMP processing is abolished . Radiolabelled internally HA - tagged Mcp3 without ( HA - Mcp3 ) or with mutation in the Imp1 cleavage site ( D70G + HA ) were incubated with mitochondria as in ( F ) . After import samples were incubated with or without PK ( 20 µg / ml ) . Samples were analysed by SDS - PAGE and autoradiography .

(G) Full-length Mcp3 precursor accumulates during import if IMP processing is abolished. Radiolabelled internally HA-tagged Mcp3 without (HA-Mcp3) or with mutation in the Imp1 cleavage site (D70G+HA) were incubated with mitochondria as in (F). After import samples were incubated with or without PK (20 µg/ml). Samples were analysed by SDS-PAGE and autoradiography.


( H ) Mitochondrial membrane potential is required for efficient import of Mcp3 precursor into mitochondria . Isolated mitochondria were incubated with radiolabelled Mcp3 in the presence or absence of CCCP . After import mitochondria were incubated with PK as in ( G ) and analysed by SDS - PAGE and autoradiography . I , 20 % of radiolabelled precursor protein used in each import reaction ; The arrowhead marks an additional band of the size of the cleaved N - terminus of Mcp3 .

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(H) Mitochondrial membrane potential is required for efficient import of Mcp3 precursor into mitochondria. Isolated mitochondria were incubated with radiolabelled Mcp3 in the presence or absence of CCCP. After import mitochondria were incubated with PK as in (G) and analysed by SDS-PAGE and autoradiography. I, 20% of radiolabelled precursor protein used in each import reaction; The arrowhead marks an additional band of the size of the cleaved N-terminus of Mcp3.


( A ) Import of Mcp3 is mediated by the MIM complex . Mitochondria isolated from WT , mim1Δ , or mim2Δ cells were incubated with radiolabelled Mcp3 for the indicated time periods . After import mitochondria were reisolated and analysed by SDS - PAGE and autoradiography . Bands corresponding to the mature ( m ) form were quantified . Import after 15 min into wild - type mitochondria was set to 100 % . The mean with standard deviations is depicted ( n = 3 ; SD ) . I , 20 % of radiolabelled precursor protein used in each import

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(A) Import of Mcp3 is mediated by the MIM complex. Mitochondria isolated from WT, mim1Δ, or mim2Δ cells were incubated with radiolabelled Mcp3 for the indicated time periods. After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. Bands corresponding to the mature (m) form were quantified. Import after 15 min into wild-type mitochondria was set to 100%. The mean with standard deviations is depicted (n=3; SD). I, 20% of radiolabelled precursor protein used in each import reaction


B ) Steady state levels of over - expressed HA - Mcp3 are reduced in cells lacking MIM subunits . Crude mitochondria were obtained from WT cells containing an empty plasmid ( Ø ) and from wild - type , mim1Δ or mim2Δ cells containing a plasmid encoded HA - Mcp3 . Samples were analysed by SDS - PAGE and immunodecoration with antibodies against the HA - tag , Tom20 as MIM substrate and Fis1 as a loading control . HA - Mcp3 levels were quantified in relation to Fis1 levels . Levels in wild - type cells were set to 100 % . The bar diagram shows the mean with standard deviation ( n = 3 ; SD ; * , p < 0 . 05 ; * * , p < 0 . 01 ) .

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B) Steady state levels of over-expressed HA-Mcp3 are reduced in cells lacking MIM subunits. Crude mitochondria were obtained from WT cells containing an empty plasmid (Ø) and from wild-type, mim1Δ or mim2Δ cells containing a plasmid encoded HA-Mcp3. Samples were analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag, Tom20 as MIM substrate and Fis1 as a loading control. HA-Mcp3 levels were quantified in relation to Fis1 levels. Levels in wild-type cells were set to 100%. The bar diagram shows the mean with standard deviation (n=3; SD; *, p < 0.05; **, p < 0.01).


( C ) Mcp3 precursor binds in vitro to Mim1 . Radiolabelled Mcp3 was incubated with amylose beads harbouring recombinant MBP or MBP fused to Mim1 . After washing proteins were eluted with sample buffer . Analysis was performed by SDS - PAGE , blotting and autoradiography . To demonstrate equal amounts of MBP fusion proteins the membrane was stained with Ponceau S . I , input 5 % of precursor used ; n . b . , 5 % of unbound material ; . b , 100 % of bound material .

(C) Mcp3 precursor binds in vitro to Mim1. Radiolabelled Mcp3 was incubated with amylose beads harbouring recombinant MBP or MBP fused to Mim1. After washing proteins were eluted with sample buffer. Analysis was performed by SDS-PAGE, blotting and autoradiography. To demonstrate equal amounts of MBP fusion proteins the membrane was stained with Ponceau S. I, input 5% of precursor used; n.b., 5% of unbound material;.b, 100% of bound material.


Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and BMI Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) .

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Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and BMI Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D).


Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression , plasma alanine aminotransferase ( ALAT ) ( B ) Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) .

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Scatter plot and correlation coefficient (r) of ASK1 mRNA expression , plasma alanine aminotransferase (ALAT) (B) Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D).


Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and liver fat content ( C ) . Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) .

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Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and liver fat content (C). Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D).


ASK1 mRNA expression in patients with different NASH scores Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . * * *

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ASK1 mRNA expression in patients with different NASH scores Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D). ***


Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and ATG5 ( E ) mRNA expression . Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . * * *

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Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and ATG5 (E) mRNA expression. Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D). ***


Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and ATG7 Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) .

Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and ATG7 Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D).


Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and ATG12 mRNA expression . Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . * * *

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Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and ATG12 mRNA expression. Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D). ***


Hepatocytes were treated with BSA , Palm or Palm + Rap for 24 h . Cells were stained for lipid droplet accumulation ( Bodipy 493 / 503 , green ) and nuclei ( Hoechst , blue ) . Lipid accumulation was quantified using automated image - based analysis ( n = 4 biological replicates ) . Scale bar represents 100 µm .

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Hepatocytes were treated with BSA, Palm or Palm+Rap for 24 h. Cells were stained for lipid droplet accumulation (Bodipy 493/503, green) and nuclei (Hoechst, blue). Lipid accumulation was quantified using automated image-based analysis (n=4 biological replicates). Scale bar represents 100 µm.


Shown is one representative Western blot from two independent experiments of hepatocytes transfected with siRNA targeting ASK1 ( siASK1 ) or non - targeting siRNA control ( siCtrl ) .

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Shown is one representative Western blot from two independent experiments of hepatocytes transfected with siRNA targeting ASK1 (siASK1) or non-targeting siRNA control (siCtrl).


Lipid accumulation was quantified using automated image - based analysis in ASK1 knockdown ( siASK1 ; grey bars ) or control ( siCtrl ; black bars ) cells treated with BSA , Palm , Palm + Rap and stained as mentioned above ( n = 4 biological replicates ) .

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Lipid accumulation was quantified using automated image-based analysis in ASK1 knockdown (siASK1; grey bars) or control (siCtrl; black bars) cells treated with BSA, Palm, Palm+Rap and stained as mentioned above (n=4 biological replicates).


Shown is one representative Western blot from two independent experiments and quantification of LC3 - II / I and p62 protein levels in siCtrl ( n = 4 biological replicates ) or siASK1 ( n = 4 biological replicates ) cells treated with Palm .

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Shown is one representative Western blot from two independent experiments and quantification of LC3-II/I and p62 protein levels in siCtrl (n=4 biological replicates) or siASK1 (n=4 biological replicates) cells treated with Palm.


Colocalization of LC3 - II punctate ( red ; arrow ) with lipid ( BODIPY 493 / 503 , green ) and nuclei ( Hoechst , blue ) in siCtrl or siASK1 cells treated with BSA , Palm or Palm + Rap . Scale bar represents 100 µm .

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Colocalization of LC3-II punctate (red; arrow) with lipid (BODIPY 493/503, green) and nuclei (Hoechst, blue) in siCtrl or siASK1 cells treated with BSA, Palm or Palm+Rap. Scale bar represents 100 µm.


Colocalization of LC3 - II punctate with lipid droplets ( BODIPY 493 / 503 , green ) was quantified in hepatocytes transfected with siRNA targeting ASK1 ( siASK1 ; grey bars ) or non - targeting siRNA control ( siCtrl ; black bars ) and treated with BSA or BSA + Baf for 24 hours ( n = 4 biological replicates ) .

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Colocalization of LC3-II punctate with lipid droplets (BODIPY 493/503, green) was quantified in hepatocytes transfected with siRNA targeting ASK1 (siASK1; grey bars) or non-targeting siRNA control (siCtrl; black bars) and treated with BSA or BSA+Baf for 24 hours (n=4 biological replicates).


Lipid accumulation was quantified using automated image - based analysis in ASK1 knockdown ( siASK1 ; grey bars ) or control ( siCtrl ; black bars ) cells treated with BSA , Palm or Palm + Baf and stained as mentioned above ( n = 4 biological replicates ) . Values are expressed as mean ± SEM . *

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Lipid accumulation was quantified using automated image-based analysis in ASK1 knockdown (siASK1; grey bars) or control (siCtrl; black bars) cells treated with BSA, Palm or Palm+Baf and stained as mentioned above (n=4 biological replicates). Values are expressed as mean ± SEM. *


Representative images of liver sections stained with H & E from mice fed a HFD for 20 weeks . Scale bar represents 100 µm .

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Representative images of liver sections stained with H&E from mice fed a HFD for 20 weeks. Scale bar represents 100 µm.


Liver triglyceride ( TG ) content of mice fed a chow ( ASK1F / F n = 6 mice ; ASK1Δhep n = 6 mice ) or HFD ( ASK1F / F n = 14 mice ; ASK1Δhep n = 15 mice ) .

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Liver triglyceride (TG) content of mice fed a chow (ASK1F/F n=6 mice; ASK1Δhep n=6 mice) or HFD (ASK1F/F n=14 mice; ASK1Δhep n=15 mice).


Relative mRNA expression of perilipin ( Plin ) and very low - density lipoprotein receptor ( Vldlr ) in liver of mice fed a HFD for 20 weeks ( ASK1F / F n = 6 mice ; ASK1Δhep n = 7 mice ( Vldlr ) or n = 8 mice ( Plin ) ) .

Relative mRNA expression of perilipin (Plin) and very low-density lipoprotein receptor (Vldlr) in liver of mice fed a HFD for 20 weeks (ASK1F/F n=6 mice; ASK1Δhep n=7 mice (Vldlr) or n=8 mice (Plin)).


Relative mRNA expression of genes involved in inflammation in liver of mice fed a HFD for 20 weeks ( ASK1F / F n = 6 mice ; ASK1Δhep n = 7 mice ( F4 / 80 , Mcp1 ) or ASK1Δhep n = 8 mice ( Il - 6 ) ) .

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Relative mRNA expression of genes involved in inflammation in liver of mice fed a HFD for 20 weeks (ASK1F/F n=6 mice; ASK1Δhep n=7 mice (F4/80, Mcp1) or ASK1Δhep n=8 mice (Il-6)).


Relative mRNA expression of collagen , type I , alpha 1 ( Col1a1 ) ( ASK1F / F n = 11 mice ; ASK1Δhep n = 15 mice ) and transforming growth factor beta 1 ( Tgfβ1 ) ( ASK1F / F n = 6 mice ; ASK1Δhep n = 8 mice ) in liver of HFD - fed mice .

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Relative mRNA expression of collagen, type I, alpha 1 (Col1a1) (ASK1F/F n=11 mice; ASK1Δhep n=15 mice) and transforming growth factor beta 1 (Tgfβ1) (ASK1F/F n=6 mice; ASK1Δhep n=8 mice) in liver of HFD-fed mice.


Representative images of liver sections stained with Sirius Red ( scale bar represents 100 µm )

Representative images of liver sections stained with Sirius Red (scale bar represents 100 µm)


quantification of Sirius Red positive area in liver of mice fed a HFD ( ASK1F / F n = 8 mice ; ASK1Δhep n = 8 mice ) .

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quantification of Sirius Red positive area in liver of mice fed a HFD (ASK1F/F n=8 mice; ASK1Δhep n=8 mice).


Representative images of liver sections stained with α - SMA from mice fed a HFD for 20 weeks . Scale bar represents 100 µm .

Representative images of liver sections stained with α-SMA from mice fed a HFD for 20 weeks. Scale bar represents 100 µm.


Shown is one representative Western blot from two independent experiments and quantification of hepatic COL1A1 protein levels in mice fed a HFD for 20 weeks ( ASK1F / F n = 8 mice ; ASK1Δhep n = 8 mice ) .

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Shown is one representative Western blot from two independent experiments and quantification of hepatic COL1A1 protein levels in mice fed a HFD for 20 weeks (ASK1F/F n=8 mice; ASK1Δhep n=8 mice).


Liver triglyceride ( TG ) content ( ASK1F / F n = 7 mice ; ASK1Δhep n = 7 mice ) and quantification of Sirius Red positive area in livers ( ASK1F / F n = 7 mice ; ASK1Δhep n = 7 mice ) of 15 months old chow - fed mice . Values are expressed as mean ± SEM . #

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Liver triglyceride (TG) content (ASK1F/F n=7 mice; ASK1Δhep n=7 mice) and quantification of Sirius Red positive area in livers (ASK1F/F n=7 mice; ASK1Δhep n=7 mice) of 15 months old chow-fed mice. Values are expressed as mean ± SEM. #


Shown is one representative Western blots from two independent experiments and quantification of respective protein levels in mice fed a HFD for 20 weeks ( ASK1F / F n = 8 mice ; ASK1Δhep n = 8 mice ) .

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Shown is one representative Western blots from two independent experiments and quantification of respective protein levels in mice fed a HFD for 20 weeks (ASK1F/F n=8 mice; ASK1Δhep n=8 mice).


Representative immunofluorescence images of liver sections stained for LC3 - II punctate ( red ) and nuclei ( DAPI , blue ) in mice . Scale bar represents 100 µm .

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Representative immunofluorescence images of liver sections stained for LC3-II punctate (red) and nuclei (DAPI, blue) in mice. Scale bar represents 100 µm.


Primary hepatocytes isolated from chow - fed ASK1F / F ( ) and ASK1Δhep ( ) mice were treated with BSA , Palm , Palm + Rap or Palm + Rap + Baf for 24 h . Cells were stained for lipid droplet accumulation ( Bodipy 493 / 503 , green ) and nuclei ( Hoechst , blue ) . Lipid accumulation was quantified using automated image - based analysis ( n = 4 biological replicates ) .

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Primary hepatocytes isolated from chow-fed ASK1F/F () and ASK1Δhep () mice were treated with BSA, Palm, Palm+Rap or Palm+Rap+Baf for 24 h. Cells were stained for lipid droplet accumulation (Bodipy 493/503, green) and nuclei (Hoechst, blue). Lipid accumulation was quantified using automated image-based analysis (n=4 biological replicates).


Colocalization of LC3 - II punctate ( red ) with lipid ( BODIPY 493 / 503 , green ) and nuclei ( Hoechst , blue ) in primary hepatocytes treated with BSA , Palm , Palm + Rap or Palm + Rap + Baf . Scale bar represents 100 µm . Values are expressed as mean ± SEM .

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Colocalization of LC3-II punctate (red) with lipid (BODIPY 493/503, green) and nuclei (Hoechst, blue) in primary hepatocytes treated with BSA, Palm, Palm+Rap or Palm+Rap+Baf. Scale bar represents 100 µm. Values are expressed as mean ± SEM.


Western blots and quantification of respective protein levels in liver of HFD - fed mice treated with or without ASK1 inhibitor ( n = 4 mice per treatment ) .

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Western blots and quantification of respective protein levels in liver of HFD-fed mice treated with or without ASK1 inhibitor (n=4 mice per treatment).


Representative images of liver sections stained with H & E ( scale bar represents 100 µm )

Representative images of liver sections stained with H&E (scale bar represents 100 µm)


liver triglyceride ( TG ) content of HFD - fed mice treated with or without ASK1 inhibitor ( n = 8 mice per treatment ) .

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liver triglyceride (TG) content of HFD-fed mice treated with or without ASK1 inhibitor (n=8 mice per treatment).


Representative images of liver sections stained with H & E from mice fed a HFD for 20 weeks . Scale bar represents 100 µm .

Representative images of liver sections stained with H&E from mice fed a HFD for 20 weeks. Scale bar represents 100 µm.


Liver triglyceride ( TG ) content ( ASK1F / F n = 11 mice ; ASK1 + hep n = 10 mice ) in livers of mice fed a HFD for 20 weeks .

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Liver triglyceride (TG) content (ASK1F/F n=11 mice; ASK1+hep n=10 mice) in livers of mice fed a HFD for 20 weeks.


Western blots and quantification of protein levels of respective targets in liver of HFD - fed mice ( n = 8 mice per genotype for pJNK and ATG12 , n = 4 mice per genotype for pBCN1 ) .

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Western blots and quantification of protein levels of respective targets in liver of HFD-fed mice (n=8 mice per genotype for pJNK and ATG12, n=4 mice per genotype for pBCN1).


Quantification of Sirius Red positive area ( ASK1F / F n = 11 mice ; ASK1 + hep n = 9 mice ) in livers of CCl4 - injected mice .

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Quantification of Sirius Red positive area (ASK1F/F n=11 mice; ASK1+hep n=9 mice) in livers of CCl4-injected mice.


Relative mRNA expression ( ASK1F / F n = 13 mice ; ASK1 + hep n = 14 mice ) of respective genes involved in inflammation and fibrosis in liver of CCl4 - injected mice . Values are expressed as mean ± SEM . #

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Relative mRNA expression (ASK1F/F n=13 mice; ASK1+hep n=14 mice) of respective genes involved in inflammation and fibrosis in liver of CCl4-injected mice. Values are expressed as mean ± SEM. #


Relative mRNA expression ( ASK1F / F n = 13 mice ; ASK1 + hep n = 14 mice ) of respective genes involved in inflammation and fibrosis in liver of CCl4 - injected mice . Values are expressed as mean ± SEM .

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Relative mRNA expression (ASK1F/F n=13 mice; ASK1+hep n=14 mice) of respective genes involved in inflammation and fibrosis in liver of CCl4-injected mice. Values are expressed as mean ± SEM.


A Whole mount FISH for piwi - 1 and immunostaining for BrdU at 1 - day , 1 - week and 5 - weeks post - BrdU administration . White boxes denote the magnified regions shown in the bottom panels . Scale bars , 30 µm .

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A Whole mount FISH for piwi-1 and immunostaining for BrdU at 1-day, 1-week and 5-weeks post-BrdU administration. White boxes denote the magnified regions shown in the bottom panels. Scale bars, 30 µm.


B Quantification of the percentage of BrdU label - retaining neoblasts . Data are presented as mean ± s . d . ( n = 4 - 6 animals per time point ) .

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B Quantification of the percentage of BrdU label-retaining neoblasts. Data are presented as mean ± s.d. (n = 4-6 animals per time point).


C Double pulse experiment showing colocalization of BrdU after a 2 - week chase period with EdU after a 1 - day chase period . White boxes denote the magnified region shown on the left . Dashed lines mark the edges of the animal and white arrows mark colocalization . Scale bar , 50 µm .

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C Double pulse experiment showing colocalization of BrdU after a 2-week chase period with EdU after a 1-day chase period. White boxes denote the magnified region shown on the left. Dashed lines mark the edges of the animal and white arrows mark colocalization. Scale bar, 50 µm.


A Dissociated cells from unirradiated , 1 dpi or 7 dpi animals ( 60 Gy of irradiation ) were stained with Hoechst ( DNA ) and Pyronin Y ( RNA ) and sorted by FACS to reveal an RNAlow population . Percentages indicate the proportion of events within each gate .

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A Dissociated cells from unirradiated, 1 dpi or 7 dpi animals (60 Gy of irradiation) were stained with Hoechst (DNA) and Pyronin Y (RNA) and sorted by FACS to reveal an RNAlow population. Percentages indicate the proportion of events within each gate.


B Cell size and nucleus : cytoplasm ratio measured by CellMask ( cytoplasm ) and DAPI ( nucleus ) staining of FACS sorted cells at homeostasis ( n ≥ 47 per sample ) . Representative images are shown , with dashed lines to indicate the area measured . Scale bars , 5 µm .

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B Cell size and nucleus:cytoplasm ratio measured by CellMask (cytoplasm) and DAPI (nucleus) staining of FACS sorted cells at homeostasis (n ≥ 47 per sample). Representative images are shown, with dashed lines to indicate the area measured. Scale bars, 5 µm.


C EdU pulse - chase time course of label - retention in sorted RNAlow and S / G2 / M neoblasts ( n ≥ 3 individual experiments per time point ) . Red and blue p values indicate the statistical significance of EdU dilution from 2 dpp to 14 dpp in S / G2 / M or RNAlow neoblasts , respectively . Black p values indicate the statistical significance of EdU retention in RNAlow neoblasts compared to S / G2 / M neoblasts .

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C EdU pulse-chase time course of label-retention in sorted RNAlow and S/G2/M neoblasts (n ≥ 3 individual experiments per time point). Red and blue p values indicate the statistical significance of EdU dilution from 2 dpp to 14 dpp in S/G2/M or RNAlow neoblasts, respectively. Black p values indicate the statistical significance of EdU retention in RNAlow neoblasts compared to S/G2/M neoblasts.


D Maximum projection confocal images showing examples of cells from the beginning and end of the EdU label retention timecourse in C . Red boxes denote examples of EdU - cells . Scale bars , 5 µm .

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D Maximum projection confocal images showing examples of cells from the beginning and end of the EdU label retention timecourse in C. Red boxes denote examples of EdU- cells. Scale bars, 5 µm.


A Area measurements of cells from the RNAlow , G1 and S / G2 / M gates in intact animals and at 6 and 48 hpa ( n ≥ 28 for each sample in the RNAlow plot , n ≥ 24 in the G1 plot , n ≥ 42 in the S / G2 / M plot ) .

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A Area measurements of cells from the RNAlow, G1 and S/G2/M gates in intact animals and at 6 and 48 hpa (n ≥ 28 for each sample in the RNAlow plot, n ≥ 24 in the G1 plot, n ≥ 42 in the S/G2/M plot).


B RNAlow neoblast size in intact animals and at 48 , 72 and 96 hpa ( n ≥ 54 ) .

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B RNAlow neoblast size in intact animals and at 48, 72 and 96 hpa (n ≥ 54).


C RNAlow neoblast size in intact animals and at 48 hours following amputation , poke or incision injuries ( n ≥ 61 ) .

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C RNAlow neoblast size in intact animals and at 48 hours following amputation, poke or incision injuries (n ≥ 61).


D RNAlow neoblast size in unirradiated animals and at 1 , 2 and 3 dpi ( 12 . 5 Gy . of irradiation , n ≥ 52 ) .

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D RNAlow neoblast size in unirradiated animals and at 1, 2 and 3 dpi (12.5 Gy. of irradiation, n ≥ 52).