Schematic overview of the sample collection procedure for single - cell PCR ( scPCR ) . Single IHC cytoplasms from acutely dissected organs of Corti of C57BL / 6J ( Wt ) mice after hearing onset were aspirated and processed for scPCR as depicted . ( B ' ) Expression analysis of endophilins - A1 - 3 from individually isolated IHC cytoplasms using RT - PCR from a representative experimental run . Please note that for these experiments , negative bath control samples from before and after the isolation procedure were an essential requirement to ensure lack of contamination from cellular debris in bath solution . HPRT was used as a housekeeping gene .

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Schematic overview of the sample collection procedure for single-cell PCR (scPCR). Single IHC cytoplasms from acutely dissected organs of Corti of C57BL/6J (Wt) mice after hearing onset were aspirated and processed for scPCR as depicted. (B') Expression analysis of endophilins-A1-3 from individually isolated IHC cytoplasms using RT-PCR from a representative experimental run. Please note that for these experiments, negative bath control samples from before and after the isolation procedure were an essential requirement to ensure lack of contamination from cellular debris in bath solution. HPRT was used as a housekeeping gene.


Immunoblotting of tissue lysates from p15 Wt ( C57BL / 6J ) , 1 - SKO , 1 / 2 - DKO and 1 / 3 - DKO revealed protein expression of endophilins - A1 and - A2 in the murine cochlea and ensured antibody specificity . Unfortunately , none of the commercially available endophilin - A3 antibodies we tested in these experiments gave a specific signal for A3 in cochlear extracts , but rather appeared to ( also ) detect A1 . γ - adaptin was used as loading control . All antibody epitopes localize to the distinct C - terminal regions of the different endophilin - A family members . Hi , hippocampus ; Ce , cerebellum ; Co , pooled cochleae from a single individual of the indicated genotype ; Md , modioli ( micro - dissected and pooled from 10 Wt animals ) ; OC , organs of Corti ( micro - dissected and pooled from 10 Wt animals ) .

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Immunoblotting of tissue lysates from p15 Wt (C57BL/6J), 1-SKO, 1/2-DKO and 1/3-DKO revealed protein expression of endophilins-A1 and -A2 in the murine cochlea and ensured antibody specificity. Unfortunately, none of the commercially available endophilin-A3 antibodies we tested in these experiments gave a specific signal for A3 in cochlear extracts, but rather appeared to (also) detect A1. γ-adaptin was used as loading control. All antibody epitopes localize to the distinct C-terminal regions of the different endophilin-A family members. Hi, hippocampus; Ce, cerebellum; Co, pooled cochleae from a single individual of the indicated genotype; Md, modioli (micro-dissected and pooled from 10 Wt animals); OC, organs of Corti (micro-dissected and pooled from 10 Wt animals).


Ca2 + - current - voltage relationships evoked by incremental 15 ms step depolarizations from - 87 mV to + 58 mV revealed a ~ 25 % reduction of ICa in 1 / 3 DKO and TKO mice . ( A ' ) Due to the perinatal lethality of TKO mice , detailed electrophysiological characterization of TKO IHCs had to be performed on organotypically - cultured organs of Corti after 7 days in vitro ( DIV ) . C57BL / 6J ( Wt ) and 1 / 3 - DKO served as controls . Please note that all recordings from cultured IHCs were performed at [ Ca2 + ] e of 10 mM to maximize IHC exocytic performance . ( A " ) Quantification and statistical analysis of individual maximum ICa amplitudes ( Imax ) of the respective genotypes revealed a significant reduction in Imax in both endophilin mutant genotypes . ( * p = 0 . 046 , one - way ANOVA with post - hoc Tukey ' s ) .

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Ca2+-current-voltage relationships evoked by incremental 15 ms step depolarizations from -87 mV to +58 mV revealed a ~25% reduction of ICa in 1/3 DKO and TKO mice. (A') Due to the perinatal lethality of TKO mice, detailed electrophysiological characterization of TKO IHCs had to be performed on organotypically-cultured organs of Corti after 7 days in vitro (DIV). C57BL/6J (Wt) and 1/3-DKO served as controls. Please note that all recordings from cultured IHCs were performed at [Ca2+]e of 10 mM to maximize IHC exocytic performance. (A") Quantification and statistical analysis of individual maximum ICa amplitudes (Imax) of the respective genotypes revealed a significant reduction in Imax in both endophilin mutant genotypes. (*p = 0.046, one-way ANOVA with post-hoc Tukey's).


Ca2 + - current inactivation was probed by test pulses of 100 ms to the Imax potential and revealed a significantly stronger inactivation phenotype in TKO IHCs when directly compared to Wt and 1 / 3 - DKO cells . ( B ' ) Quantification and statistical analysis of the residual current at the end of the test pulse . ( * p = 0 . 046 , one - way ANOVA with post - hoc Tukey ' s ) .

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Ca2+-current inactivation was probed by test pulses of 100 ms to the Imax potential and revealed a significantly stronger inactivation phenotype in TKO IHCs when directly compared to Wt and 1/3-DKO cells. (B') Quantification and statistical analysis of the residual current at the end of the test pulse. (*p = 0.046, one-way ANOVA with post-hoc Tukey's).


Representative ICa ( upper panel ) and Cm ( lower panel ) in response to a 50 ms depolarizing step to the potential eliciting Imax . ( C ' ) Exocytic ∆ Cm and corresponding QCa elicited by depolarizations of stimulus durations from 2 - 100 ms for all respective genotypes and at [ Ca2 + ] e = 6 mM for a second set of recordings from wild - type IHCs to experimentally approximate the decreased ICa observed in the endophilin mutants . ( C " ) Magnification of the initial , short depolarizing steps ( 2 - 20 ms ) for clarity . Exocytic ∆ Cm of cultured endophilin - deficient IHCs appears strongly reduced . ( * p < 0 . 05 ; * * p < 0 . 01 ; one - way ANOVA with post - hoc Tukey ' s or non - parametric K - W with post - hoc Dunn ' s ; please also refer to Appendix Tables S2 and S3 for detailed statistical analysis ) . The reduced Ca2 + - efficiency of exocytosis ( ∆ Cm / QCa ) in endophilin - deficient IHCs indicates that diminished Ca2 + - influx cannot fully account for the reduction of exocytosis . ( * p < 0 . 05 ; * * p < 0 . 01 ; one - way ANOVA with post - hoc Tukey ' s or non - parametric K - W with post - hoc Dunn ' s ; please also refer to Appendix Table S4 for detailed statistical analysis ) .

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Representative ICa (upper panel) and Cm (lower panel) in response to a 50 ms depolarizing step to the potential eliciting Imax. (C') Exocytic ∆Cm and corresponding QCa elicited by depolarizations of stimulus durations from 2-100 ms for all respective genotypes and at [Ca2+]e = 6 mM for a second set of recordings from wild-type IHCs to experimentally approximate the decreased ICa observed in the endophilin mutants. (C") Magnification of the initial, short depolarizing steps (2-20 ms) for clarity. Exocytic ∆Cm of cultured endophilin-deficient IHCs appears strongly reduced. (*p < 0.05; **p < 0.01; one-way ANOVA with post-hoc Tukey's or non-parametric K-W with post-hoc Dunn's; please also refer to Appendix Tables S2 and S3 for detailed statistical analysis). The reduced Ca2+-efficiency of exocytosis (∆Cm/QCa) in endophilin-deficient IHCs indicates that diminished Ca2+-influx cannot fully account for the reduction of exocytosis. (*p < 0.05; **p < 0.01; one-way ANOVA with post-hoc Tukey's or non-parametric K-W with post-hoc Dunn's; please also refer to Appendix Table S4 for detailed statistical analysis).


Ca2 + - current - voltage relationships in response to 10 ms step - depolarizations . ( A ' ) The peak of Ca2 + - influx was significantly reduced in endophilin 1 - SKO ( * * p = 0 . 0024 ) and 1 / 3 - DKO ( * * * p < 0 . 0001 ) when compared to Wt . ( Wt n = 30 / N = 20 ; 1 - SKO n = 15 / N = 9 ; 1 / 3 - DKO n = 39 / N = 20 ; Non - parametric K - W with post - hoc Dunn ' s correction ) .

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Ca2+-current-voltage relationships in response to 10 ms step-depolarizations. (A') The peak of Ca2+-influx was significantly reduced in endophilin 1-SKO (**p = 0.0024) and 1/3-DKO (***p < 0.0001) when compared to Wt. (Wt n = 30/ N = 20; 1-SKO n = 15/ N = 9; 1/3-DKO n = 39/ N = 20; Non-parametric K-W with post-hoc Dunn's correction).


Quantification of CtBP2 - labeled ribbons revealed a number of approximately 14 - 15 ribbon synapses per IHC in all groups , that remained comparable across all genotypes ( Wt n = 12 / N = 9 ; 1 - SKO n = 6 / N = 4 ; 1 / 3 - DKO n = 8 / N = 6 ; one - way ANOVA , p = 0 . 717 ) .

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Quantification of CtBP2-labeled ribbons revealed a number of approximately 14-15 ribbon synapses per IHC in all groups, that remained comparable across all genotypes (Wt n = 12/ N = 9; 1-SKO n = 6/ N = 4; 1/3-DKO n = 8/ N = 6; one-way ANOVA, p = 0.717).


Quantification of the cross - sectional area of CaV1 . 3 immunofluorescence revealed approx . 34 % smaller clusters in 1 - SKOs and 24 % smaller clusters in 1 / 3 - DKOs . ( Wt n = 153 / N = 2 ; 1 - SKO n = 102 / N = 2 ; 1 / 3 - DKO n = 441 / N = 4 ; * * * p < 0 . 0001 ; K - W and Dunn ' s post - hoc test ) .

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Quantification of the cross-sectional area of CaV1.3 immunofluorescence revealed approx. 34 % smaller clusters in 1-SKOs and 24 % smaller clusters in 1/3-DKOs. (Wt n = 153/ N = 2; 1-SKO n = 102/ N = 2; 1/3-DKO n = 441/ N = 4; ***p < 0.0001; K-W and Dunn's post-hoc test).


Representative maximum projections of confocal sections from organs of Corti of p15 Wt , 1 - SKO , and 1 / 3 - DKO mice stained for CtBP2 to label synaptic ribbons . Please note that CtBP2 expression is also found in the nucleus ( nuclei are highlighted by dashed circles in the individual panels ) . Scale bars : 10 µm .

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Representative maximum projections of confocal sections from organs of Corti of p15 Wt, 1-SKO, and 1/3-DKO mice stained for CtBP2 to label synaptic ribbons. Please note that CtBP2 expression is also found in the nucleus (nuclei are highlighted by dashed circles in the individual panels). Scale bars: 10 µm.


2D STED images of IHC ribbon synapses stained for CtBP2 ( magenta ) and CaV1 . 3 ( green ) . CaV1 . 3 Ca2 + - channels remain clustered at AZs of 1 - SKO and 1 / 3 - DKO IHCs . Scale bars : 1 µm .

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2D STED images of IHC ribbon synapses stained for CtBP2 (magenta) and CaV1.3 (green). CaV1.3 Ca2+-channels remain clustered at AZs of 1-SKO and 1/3-DKO IHCs. Scale bars: 1 µm.


Representative Ca2 + - currents ( upper panel ) and exocytic membrane capacitance increments ( ∆ Cm ; lower panel ) in response to a 200 ms depolarizing step to - 14 mV .

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Representative Ca2+-currents (upper panel) and exocytic membrane capacitance increments (∆Cm; lower panel) in response to a 200 ms depolarizing step to -14 mV.


Exocytic ∆ Cm ( top ) and corresponding Ca2 + - current integrals QCa ( bottom ) of Wt and endophilin - deficient IHCs in response to voltage steps from - 84 to - 14 mV of variable stimulus duration ( 5 to 200 ms ) . In 1 / 3 - DKO IHCs , our data indicate reduced sustained exocytosis to a strong depolarization ( 200 ms ; * p = 0 . 0470 , K - W with Dunn ' s post - hoc test ; please also refer to Appendix Tables S5 and S6 for detailed statistical analysis ) . Inset : Initial ∆ Cm showed comparable RRP exocytosis in endophilin - deficient IHCs .

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Exocytic ∆Cm (top) and corresponding Ca2+-current integrals QCa (bottom) of Wt and endophilin-deficient IHCs in response to voltage steps from -84 to -14 mV of variable stimulus duration (5 to 200 ms). In 1/3-DKO IHCs, our data indicate reduced sustained exocytosis to a strong depolarization (200 ms; *p = 0.0470, K-W with Dunn's post-hoc test; please also refer to Appendix Tables S5 and S6 for detailed statistical analysis). Inset: Initial ∆Cm showed comparable RRP exocytosis in endophilin-deficient IHCs.


Ca2 + - efficiency to drive exocytosis was reduced for strong depolarizations ( 200 ms ) in 1 / 3 - DKO IHCs ; * p = 0 . 0323 , K - W with Dunn ' s post - hoc test ; please also refer to Appendix Table S7 for detailed statistical analysis .

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Ca2+-efficiency to drive exocytosis was reduced for strong depolarizations (200 ms) in 1/3-DKO IHCs; *p = 0.0323, K-W with Dunn's post-hoc test; please also refer to Appendix Table S7 for detailed statistical analysis.


Confocal maximum projections of otoferlin - immunolabeled organs of Corti from p15 Wt and 1 / 3 - DKO mice illustrated with an intensity - coded look - up table where brighter colors indicate higher intensity . Scale bars : 5 µm .

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Confocal maximum projections of otoferlin-immunolabeled organs of Corti from p15 Wt and 1/3-DKO mice illustrated with an intensity-coded look-up table where brighter colors indicate higher intensity. Scale bars: 5 µm.


Otoferlin levels were reduced by approx . 28 . 4 ± 3 . 5 % in 1 / 3 - DKO IHCs compared to Wt IHCs ( relative intensities normalized to Wt ; Wt n = 31 images ( 229 IHCs ) / N = 5 organs of Corti ( 3 animals ) ; 1 / 3 - DKO n = 20 images ( 196 IHCs ) / N = 5 organs of Corti ( 4 animals ) ; unpaired student ' s t - test ; * * * p < 0 . 001 ; box plots illustrate the median with the interquartile range , whiskers indicate 10 - 90 % of data points , the squares present the respective mean value ) .

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Otoferlin levels were reduced by approx. 28.4 ± 3.5 % in 1/3-DKO IHCs compared to Wt IHCs (relative intensities normalized to Wt; Wt n = 31 images (229 IHCs)/ N = 5 organs of Corti (3 animals); 1/3-DKO n = 20 images (196 IHCs)/ N = 5 organs of Corti (4 animals); unpaired student's t-test; ***p < 0.001; box plots illustrate the median with the interquartile range, whiskers indicate 10-90 % of data points, the squares present the respective mean value).


Normalized otoferlin intensity line profiles through single IHCs of the respective genotypes at a longitudinal central plane through the nucleus from apical ( 0 . 0 ) to basal ( 1 . 0 ) revealed similar distribution patterns of otoferlin in IHCs of Wt and 1 / 3 - DKO and an overall reduction of otoferlin levels throughout the entire cell in 1 / 3 - DKOs . Traces indicate the mean pixel intensity ± SEM ; Wt n = 123 / N = 3 ; 1 / 3 - DKO n = 124 / N = 3 .

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Normalized otoferlin intensity line profiles through single IHCs of the respective genotypes at a longitudinal central plane through the nucleus from apical (0.0) to basal (1.0) revealed similar distribution patterns of otoferlin in IHCs of Wt and 1/3-DKO and an overall reduction of otoferlin levels throughout the entire cell in 1/3-DKOs. Traces indicate the mean pixel intensity ± SEM; Wt n = 123/ N = 3; 1/3-DKO n = 124/ N = 3.


Interaction of otoferlin and endophilin - A1 detected by co - IP in HeLa cells co - expressing GFP - otoferlin and RFP - endophilin - A1 . Otoferlin - GFP was immunoprecipitated ( IP ) by GFP - Trap beads , and blots were probed with a KO - validated anti - endophilin - A1 antibody .

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Interaction of otoferlin and endophilin-A1 detected by co-IP in HeLa cells co-expressing GFP-otoferlin and RFP-endophilin-A1. Otoferlin-GFP was immunoprecipitated (IP) by GFP-Trap beads, and blots were probed with a KO-validated anti-endophilin-A1 antibody.


Exogenously overexpressed GFP - otoferlin was immunoprecipitated using GFP - Trap beads and incubated with purified endophilin - A1 ( pA1 ) . IP was then followed by immunoblotting with an anti - endophilin - A1 antibody .

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Exogenously overexpressed GFP-otoferlin was immunoprecipitated using GFP-Trap beads and incubated with purified endophilin-A1 (pA1). IP was then followed by immunoblotting with an anti-endophilin-A1 antibody.


Endocytosis was assessed by determining the decrease in Cm during 20 s post - depolarization in perforated patch recordings from both Wt , 1 / 2 - DKO and 1 / 3 - DKO IHCs . The two kinetic components are presented as average ∆ Cm recordings after ( A ) 20 ms ( Wt n = 12 / N = 8 ; 1 / 2 - DKO n = 13 / N = 7 ; 1 / 3 - DKO n = 13 / N = 10 ) and ( B ) 200 ms ( Wt n = 12 / N = 8 ; 1 / 2 - DKO n = 13 / N = 6 ; 1 / 3 - DKO n = 14 / N = 10 ) depolarization stimuli .

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Endocytosis was assessed by determining the decrease in Cm during 20 s post-depolarization in perforated patch recordings from both Wt, 1/2-DKO and 1/3-DKO IHCs. The two kinetic components are presented as average ∆Cm recordings after (A) 20 ms (Wt n = 12/ N = 8; 1/2-DKO n = 13/ N = 7; 1/3-DKO n = 13/ N = 10) and (B) 200 ms (Wt n = 12/ N = 8; 1/2-DKO n = 13/ N = 6; 1/3-DKO n = 14/ N = 10) depolarization stimuli.


Average time of return to baseline obtained by fitting a linear function to the Cm - data following a 20 ms depolarization .

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Average time of return to baseline obtained by fitting a linear function to the Cm-data following a 20 ms depolarization.


Average slope of the linear component of endocytosis for 20 ms and 200 ms depolarizing pulses , obtained from fitting Cm - data for 20 s after cessation of the 20 ms depolarizations or for the last 10 s of the Cm - recording for 200 ms depolarizations . ( * p < 0 . 05 ; one - way ANOVA with post - hoc Tukey ' s or non - parametric K - W with post - hoc Dunn ' s ) .

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Average slope of the linear component of endocytosis for 20 ms and 200 ms depolarizing pulses, obtained from fitting Cm-data for 20 s after cessation of the 20 ms depolarizations or for the last 10 s of the Cm- recording for 200 ms depolarizations. (*p < 0.05; one-way ANOVA with post-hoc Tukey's or non-parametric K-W with post-hoc Dunn's).


Average amplitude of the exponential component obtained from fits to Cm - data following 200 ms depolarization ( nexp fit = 11 for Wt , 10 for 1 / 2 - DKO and 11 for 1 / 3 - DKO ; x data points in ( E ) correspond to IHCs with no exponential component in Cm ) . correspond to statistically identified outliers .

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Average amplitude of the exponential component obtained from fits to Cm-data following 200 ms depolarization (nexp fit = 11 for Wt, 10 for 1/2-DKO and 11 for 1/3-DKO; x data points in (E) correspond to IHCs with no exponential component in Cm). correspond to statistically identified outliers.


time constant of the exponential component obtained from fits to Cm - data following 200 ms

time constant of the exponential component obtained from fits to Cm-data following 200 ms depolarization


Representative electron micrograph illustrating the region of interest for analysis within a radius of r = 1 µm from the ribbon center ( exemplary for a 1 / 3 - DKO specimen ) . Graphical aids indicate small vesicles , ELVs and different subpopulations of coated structures quantified in ( B - D ) . Scale bar : 500 nm .

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Representative electron micrograph illustrating the region of interest for analysis within a radius of r = 1 µm from the ribbon center (exemplary for a 1/3-DKO specimen). Graphical aids indicate small vesicles, ELVs and different subpopulations of coated structures quantified in (B-D). Scale bar: 500 nm.


While an increased number of cytosolic vesicles can be observed at 1 - SKO AZs , reduced numbers of small vesicles are present in a 1 µm radius around the ribbon of 1 / 2 - DKOs . RA - SVs were excluded from this quantification .

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While an increased number of cytosolic vesicles can be observed at 1-SKO AZs, reduced numbers of small vesicles are present in a 1 µm radius around the ribbon of 1/2-DKOs. RA-SVs were excluded from this quantification.


Accumulations of ELVs occur in 1 / 2 - DKO and 1 / 3 - DKO IHCs , as measured by the relative cumulative ELV area per section .

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Accumulations of ELVs occur in 1/2-DKO and 1/3-DKO IHCs, as measured by the relative cumulative ELV area per section.


Increased overall number of coated structures in endophilin mutants . ( D ' ) Prominent accumulation of coated pits can be observed in 1 / 2 - DKO and 1 / 3 - DKO IHCs alongside a shift towards more coated vacuoles ( d > 70 nm ) in 1 / 2 - DKO IHCs and 1 / 3 - DKO IHCs .

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Increased overall number of coated structures in endophilin mutants. (D') Prominent accumulation of coated pits can be observed in 1/2-DKO and 1/3-DKO IHCs alongside a shift towards more coated vacuoles (d > 70 nm) in 1/2-DKO IHCs and 1/3-DKO IHCs.


Representative electron micrographs of random ultrathin sections from IHC ribbon synapses of p15 mutants lacking different endophilin - A alleles . Scale bars : 200 nm .

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Representative electron micrographs of random ultrathin sections from IHC ribbon synapses of p15 mutants lacking different endophilin-A alleles. Scale bars: 200 nm.


Reduced numbers of RA - SVs in IHCs of endophilin mutants with the strongest reduction observed in 1 / 2 - DKO mice . One - way ANOVA followed by Tukey ' s post - hoc test ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Reduced ratio of RA - SVs in the distal half of the ribbon over RA - SVs in its proximal half in 1 / 2 - DKO and 1 / 3 - DKO IHCs . K - W followed by Dunn ' s test ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

Reduced numbers of RA-SVs in IHCs of endophilin mutants with the strongest reduction observed in 1/2-DKO mice. One-way ANOVA followed by Tukey's post-hoc test; *p < 0.05; **p < 0.01; ***p < 0.001. Reduced ratio of RA-SVs in the distal half of the ribbon over RA-SVs in its proximal half in 1/2-DKO and 1/3-DKO IHCs. K-W followed by Dunn's test; **p < 0.01; ***p < 0.001.


Reduced numbers of MP - SVs ( D ) in IHCs of endophilin mutants with the strongest reduction observed in 1 / 2 - DKO mice . One - way ANOVA followed by Tukey ' s post - hoc test ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

Reduced numbers of MP-SVs (D) in IHCs of endophilin mutants with the strongest reduction observed in 1/2-DKO mice. One-way ANOVA followed by Tukey's post-hoc test; *p < 0.05; **p < 0.01; ***p < 0.001.


Significantly reduced numbers of RA - SVs in 1 / 2 - DKO IHCs ( one - way ANOVA followed by Tukey ' s post - hoc test ; * p = 0 . 0229 ) .

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

Significantly reduced numbers of RA-SVs in 1/2-DKO IHCs (one-way ANOVA followed by Tukey's post-hoc test; *p = 0.0229).


Significantly reduced numbers of MP - SVs in 1 / 2 - DKO and a strong trend ( p = 0 . 0511 ) towards less MP - SVs in 1 / 3 - DKO IHCs ( one - way ANOVA followed by Tukey ' s post - hoc test ; * p = 0 . 0138 ) .

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

Significantly reduced numbers of MP-SVs in 1/2-DKO and a strong trend (p = 0.0511) towards less MP-SVs in 1/3-DKO IHCs (one-way ANOVA followed by Tukey's post-hoc test; *p = 0.0138).


Increased SV diameter in 1 / 2 - DKO , but unchanged SV diameter in 1 / 3 - DKOs ( one - way ANOVA followed by Tukey ' s post - hoc test ; * p = 0 . 0154 ) .

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

Increased SV diameter in 1/2-DKO, but unchanged SV diameter in 1/3-DKOs (one-way ANOVA followed by Tukey's post-hoc test; *p = 0.0154).


Accumulation of ELVs with a minimal distance to the ribbon of less than 200 nm in 1 / 2 - DKOs ( one - way ANOVA followed by Tukey ' s post - hoc test ; * * * p = 0 . 0002 ) .

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

Accumulation of ELVs with a minimal distance to the ribbon of less than 200 nm in 1/2-DKOs (one-way ANOVA followed by Tukey's post-hoc test; ***p = 0.0002).


Representative electron micrographs of Wt and 1 / 2 - DKO specimen indicate accumulations of ELVs and high numbers of coated pits forming from the ELVs in ribbon proximity ( radius r = 1 µm from the center of the ribbon ) in the DKO after 15 min stimulation with extracellular solution supplemented with 50 mM K + . Scale bars : 200 nm .

Representative electron micrographs of Wt and 1/2-DKO specimen indicate accumulations of ELVs and high numbers of coated pits forming from the ELVs in ribbon proximity (radius r = 1 µm from the center of the ribbon) in the DKO after 15 min stimulation with extracellular solution supplemented with 50 mM K+. Scale bars: 200 nm.


Significantly reduced numbers of RA - SVs in IHCs of 1 / 2 - DKO compared to Wt after stimulation , as quantified using random ultrathin sections of ribbon - occupied AZs from several IHCs per genotype . Mann - Whitney test ; * * * p < 0 . 001 .

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

Significantly reduced numbers of RA-SVs in IHCs of 1/2-DKO compared to Wt after stimulation, as quantified using random ultrathin sections of ribbon-occupied AZs from several IHCs per genotype. Mann-Whitney test; ***p < 0.001.


Numbers of MP - SVs were slightly reduced in stimulated Wt compared to stimulated 1 / 2 - DKOs , where SV numbers closely matched the ones of unstimulated 1 / 2 - DKO . Mann - Whitney test ; * p < 0 . 05 .

Numbers of MP-SVs were slightly reduced in stimulated Wt compared to stimulated 1/2-DKOs, where SV numbers closely matched the ones of unstimulated 1/2-DKO. Mann-Whitney test; *p < 0.05.


Reduced numbers of small vesicles are present in a 1 µm radius around the ribbon of 1 / 2 - DKOs compared to Wt after stimulation . RA - SVs were excluded from this quantification . Student ' s t - test ; * * * p < 0 . 001 .

Reduced numbers of small vesicles are present in a 1 µm radius around the ribbon of 1/2-DKOs compared to Wt after stimulation. RA-SVs were excluded from this quantification. Student's t-test; ***p < 0.001.


Stimulated 1 / 2 - DKOs exhibited accumulations of ELVs in comparison to stimulated Wt . Mann - Whitney test ; * * * p < 0 . 001 .

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

Stimulated 1/2-DKOs exhibited accumulations of ELVs in comparison to stimulated Wt. Mann-Whitney test; ***p < 0.001.


Increased overall number of coated structures in stimulated endophilin 1 / 2 - DKOs . ( F ' ) Both , coated vesicles and coated pits forming at ELVs were significantly increased in 1 / 2 - DKO after stimulation . Mann - Whitney test ; * * p < 0 . 01 ; * * * p < 0 . 001 .

Increased overall number of coated structures in stimulated endophilin 1/2-DKOs. (F') Both, coated vesicles and coated pits forming at ELVs were significantly increased in 1/2-DKO after stimulation. Mann-Whitney test; **p < 0.01; ***p < 0.001.


( A ) CHO cells were grown in a control medium ( see Materials and methods ) or starved for 3 or 13 h , after which they were incubated in 50 μM DHE and visualized as detailed in Materials and methods .

(A) CHO cells were grown in a control medium (see Materials and methods) or starved for 3 or 13 h, after which they were incubated in 50 μM DHE and visualized as detailed in Materials and methods.


( B ) CHO cells were grown as in ( A ) , treated with 5 nM MitoTracker Red for 30 min at 37 ° C , washed , treated with 30 μM DCFDA and visualized as detailed in Materials and methods .

(B) CHO cells were grown as in (A), treated with 5 nM MitoTracker Red for 30 min at 37°C, washed, treated with 30 μM DCFDA and visualized as detailed in Materials and methods.


( C ) HeLa cells grown in 96 ‐ well plates were deprived of serum ( αMEM ) , completely starved ( EBSS ) or maintained in a control medium ( α - MEM , 10 % FCS ) for 2 h , after which they were treated with DCFDA as in ( B ) and subsequently analyzed in a fluorimeter , as explained in Materials and methods .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells grown in 96‐well plates were deprived of serum (αMEM), completely starved (EBSS) or maintained in a control medium (α-MEM, 10% FCS) for 2 h, after which they were treated with DCFDA as in (B) and subsequently analyzed in a fluorimeter, as explained in Materials and methods.


( D ) Data collected from the fluorometric measurements were analyzed as detailed in Materials and methods .

(D) Data collected from the fluorometric measurements were analyzed as detailed in Materials and methods.


( A ) Left panel : HeLa cells were starved for 2 h in the presence or absence of either 100 nM wortmannin or 10 mM 3MA , after which they were treated with DCFDA and analyzed by a fluorimeter as described above . Right panel : the rate of degradation of long ‐ lived proteins was measured in cells incubated in either α ‐ MEM medium or EBSS medium in the absence or presence of 100 nM wortmannin or 10 mM 3MA . Values are represented as the means ± s . d . of three separate determinations .

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

(A) Left panel: HeLa cells were starved for 2 h in the presence or absence of either 100 nM wortmannin or 10 mM 3MA, after which they were treated with DCFDA and analyzed by a fluorimeter as described above. Right panel: the rate of degradation of long‐lived proteins was measured in cells incubated in either α‐MEM medium or EBSS medium in the absence or presence of 100 nM wortmannin or 10 mM 3MA. Values are represented as the means±s.d. of three separate determinations.


( B ) WT MEFs or Atg5 ( − / − ) MEFs from two separate clones were starved for 2 h before treatment with DCFDA and visualization or fluorometric analysis .

O O O O O O O O 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) WT MEFs or Atg5 (−/−) MEFs from two separate clones were starved for 2 h before treatment with DCFDA and visualization or fluorometric analysis.


( A ) Upper panel : CHO cells stably transfected with GFP ‐ GATE ‐ 16 were preincubated in the presence or absence of 10 mM NAC or 1000 μ / ml catalase for 10 min before starvation for 2 h in the presence or absence of these drugs , or grown in a control medium containing the drugs for 2 h . The cells were then fixed , permeabilized and stained with anti ‐ GFP monoclonal antibodies . Representative images are shown . Lower panel : HEK 293 cells were transfected with GFP ‐ GATE ‐ 16 . At 24 h post ‐ transfection , the cells were treated with NAC or catalase and starved as explained above , lysed in Ripa buffer and 100 ‐ g of each lysate was separated on 10 % SDS - PAGE and subsequently analyzed with anti ‐ GFP antibodies to detect the transfected GATE ‐ 16 and anti ‐ tubulin antibodies as control . The data were quantified using NIH image program and are depicted as the percentage of lipidated protein from the total GATE ‐ 16 . ( * ) indicates non ‐ lipidated and ( * * ) indicates lipidated GFP ‐ GATE ‐ 16 .

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

(A) Upper panel: CHO cells stably transfected with GFP‐GATE‐16 were preincubated in the presence or absence of 10 mM NAC or 1000 μ/ml catalase for 10 min before starvation for 2 h in the presence or absence of these drugs, or grown in a control medium containing the drugs for 2 h. The cells were then fixed, permeabilized and stained with anti‐GFP monoclonal antibodies. Representative images are shown. Lower panel: HEK 293 cells were transfected with GFP‐GATE‐16. At 24 h post‐transfection, the cells were treated with NAC or catalase and starved as explained above, lysed in Ripa buffer and 100 ‐g of each lysate was separated on 10% SDS-PAGE and subsequently analyzed with anti‐GFP antibodies to detect the transfected GATE‐16 and anti‐tubulin antibodies as control. The data were quantified using NIH image program and are depicted as the percentage of lipidated protein from the total GATE‐16. (*) indicates non‐lipidated and (**) indicates lipidated GFP‐GATE‐16.


( B ) CHO cells treated with NAC or catalase and starved as detailed in ( A ) were incubated with DCFDA and visualized by a confocal microscope or analyzed by a fluorimeter as in Figure 1 .

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

(B) CHO cells treated with NAC or catalase and starved as detailed in (A) were incubated with DCFDA and visualized by a confocal microscope or analyzed by a fluorimeter as in Figure 1.


( C ) The rate of degradation of long ‐ lived proteins was measured in CHO cells incubated in either α ‐ MEM medium or EBSS medium , or following pretreatment with 10 mM NAC for 10 min or with 1000 μ / ml catalase overnight . A representative experiment is shown .

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

(C) The rate of degradation of long‐lived proteins was measured in CHO cells incubated in either α‐MEM medium or EBSS medium, or following pretreatment with 10 mM NAC for 10 min or with 1000 μ/ml catalase overnight. A representative experiment is shown.


( A ) CHO cells ( for mammalian Atg8s ) or cells of S . cerevisiae ( for ScAtg8 ) were incubated in starvation medium ( EBSS or SD ‐ N , respectively ) for different time periods as indicated before isolation of RNA by the Tri ‐ reagent as explained in Materials and methods . For each gene , data obtained from non ‐ starved cells were set to an arbitrary value of 1 , and results from the corresponding starved cells were normalized accordingly . Results represent the means ± s . d . of three separate experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CHO cells (for mammalian Atg8s) or cells of S. cerevisiae (for ScAtg8) were incubated in starvation medium (EBSS or SD‐N, respectively) for different time periods as indicated before isolation of RNA by the Tri‐reagent as explained in Materials and methods. For each gene, data obtained from non‐starved cells were set to an arbitrary value of 1, and results from the corresponding starved cells were normalized accordingly. Results represent the means±s.d. of three separate experiments.


( B ) HEK 293 cells were grown in control medium or starved for 2 h in EBSS in the absence or presence of 100 nM bafilomycin A1 . Lysates ( 100 μg ) obtained in Ripa buffer were run on 12 % SDS - PAGE and analyzed by Western blot , using anti ‐ GATE ‐ 16 or anti ‐ tubulin antibodies .

O O 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 O O O O O O O O O O O O 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) HEK 293 cells were grown in control medium or starved for 2 h in EBSS in the absence or presence of 100 nM bafilomycin A1. Lysates (100 μg) obtained in Ripa buffer were run on 12% SDS-PAGE and analyzed by Western blot, using anti‐GATE‐16 or anti‐tubulin antibodies.


( C ) CHO cells were grown in a control medium in the presence or absence of 1 mM H2O2 for 1 h , or starved for 3 or 13 h . Lysates ( 10 μg ) obtained in Ripa buffer were incubated with recombinant His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) in 50 KT reaction buffer ( 25 mM Tris , pH 7 . 4 , 50 mM KCl ) at 30 ° C for 45 min , in the presence or absence of 1 mM DTT . The reaction was stopped by addition of sample buffer and boiling , after which the samples were resolved on 15 % SDS - PAGE and subsequently analyzed by Western blot , using anti ‐ His monoclonal antibodies . The experiment was repeated six times ; a representative blot is shown .

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

(C) CHO cells were grown in a control medium in the presence or absence of 1 mM H2O2 for 1 h, or starved for 3 or 13 h. Lysates (10 μg) obtained in Ripa buffer were incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer (25 mM Tris, pH 7.4, 50 mM KCl) at 30°C for 45 min, in the presence or absence of 1 mM DTT. The reaction was stopped by addition of sample buffer and boiling, after which the samples were resolved on 15% SDS-PAGE and subsequently analyzed by Western blot, using anti‐His monoclonal antibodies. The experiment was repeated six times; a representative blot is shown.


( D ) Left panel : HeLa cells transfected with Myc ‐ GATE ‐ 16 ‐ HA or with an empty vector as control were labeled with [ 35S ] methionine for 10 min and lysed immediately or chased for 1 h before lysis in Ripa buffer . Lysates were immunoprecipitated using anti ‐ Myc antibodies and the immunoprecipitates were resolved on 15 % SDS - PAGE . Middle panel : HeLa cells transfected with Myc ‐ GATE ‐ 16 ‐ HA were kept in control medium or starved for 30 min or 13 h in EBSS before labeling with [ 35S ] methionine for 10 min , immediate lysis , analysis by SDS - PAGE and quantification ( right panel ) using NIH image program . Values are presented as the average percentage of unprimed form out of the total GATE ‐ 16 . ( * ) in all sections of this figure indicates non ‐ cleaved His6 ‐ GATE ‐ 16 ‐ HA or Myc ‐ GATE ‐ 16 ‐ HA and ( * * ) indicates cleaved His6 ‐ GATE ‐ 16 or Myc ‐ GATE ‐ 16 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left panel: HeLa cells transfected with Myc‐GATE‐16‐HA or with an empty vector as control were labeled with [35S]methionine for 10 min and lysed immediately or chased for 1 h before lysis in Ripa buffer. Lysates were immunoprecipitated using anti‐Myc antibodies and the immunoprecipitates were resolved on 15% SDS-PAGE. Middle panel: HeLa cells transfected with Myc‐GATE‐16‐HA were kept in control medium or starved for 30 min or 13 h in EBSS before labeling with [35S]methionine for 10 min, immediate lysis, analysis by SDS-PAGE and quantification (right panel) using NIH image program. Values are presented as the average percentage of unprimed form out of the total GATE‐16. (*) in all sections of this figure indicates non‐cleaved His6‐GATE‐16‐HA or Myc‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16 or Myc‐GATE‐16.


( A ) Cleavage activity was tested by incubation of recombinant His6 ‐ HsAtg4A ( 0 . 1 μg ) and His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) in 50 KT reaction buffer at 30 ° C for 45 min in the presence of indicated concentrations of DTT followed by Western blot analysis , using anti ‐ His monoclonal antibodies . The resulting bands from three separate experiments were quantified with a densitometer using the Bio ‐ Rad Multi ‐ Analyst program and are presented as the average percentage of cleaved form out of the total GATE ‐ 16 . ( * ) indicates non ‐ cleaved His6 ‐ GATE ‐ 16 ‐ HA and ( * * ) indicates cleaved His6 ‐ GATE ‐ 16 .

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

(A) Cleavage activity was tested by incubation of recombinant His6‐HsAtg4A (0.1 μg) and His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer at 30°C for 45 min in the presence of indicated concentrations of DTT followed by Western blot analysis, using anti‐His monoclonal antibodies. The resulting bands from three separate experiments were quantified with a densitometer using the Bio‐Rad Multi‐Analyst program and are presented as the average percentage of cleaved form out of the total GATE‐16. (*) indicates non‐cleaved His6‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16.


( B ) His6 ‐ HsAtg4A was incubated in the presence of 200 μM DTT at 4 ° C for 10 min . Reduced His6 ‐ HsAtg4A ( 0 . 1 μg ) was then incubated in 50 KT ( to obtain 15 μM DTT ) with the indicated concentrations of H2O2 at 25 ° C for 5 min , after which recombinant His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) was added and incubation proceeded at 30 ° C for 45 min . Reaction mixtures from three separate experiments were analyzed and are presented as explained 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) His6‐HsAtg4A was incubated in the presence of 200 μM DTT at 4°C for 10 min. Reduced His6‐HsAtg4A (0.1 μg) was then incubated in 50 KT (to obtain 15 μM DTT) with the indicated concentrations of H2O2 at 25°C for 5 min, after which recombinant His6‐GATE‐16‐HA (0.3 μg) was added and incubation proceeded at 30°C for 45 min. Reaction mixtures from three separate experiments were analyzed and are presented as explained in (A).


( C ) His6 ‐ HsAtg4A ( 0 . 1 μg ) was incubated with recombinant His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) after the following procedures : no treatment ( lane 1 ) ; pretreatment with 200 μM DTT at 25 ° C for 5 min ( lane 2 ) ; treatment with 200 μM DTT followed by treatment with 1 mM H2O2 at 25 ° C for 5 min ( lane 3 ) ; and treatment with 200 μM DTT followed by treatment with 1 mM H2O2 for 5 min and then 2 mM DTT ( lane 4 ) . Reaction mixtures were analyzed by Western blot using anti ‐ His monoclonal antibodies .

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

(C) His6‐HsAtg4A (0.1 μg) was incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) after the following procedures: no treatment (lane 1); pretreatment with 200 μM DTT at 25°C for 5 min (lane 2); treatment with 200 μM DTT followed by treatment with 1 mM H2O2 at 25°C for 5 min (lane 3); and treatment with 200 μM DTT followed by treatment with 1 mM H2O2 for 5 min and then 2 mM DTT (lane 4). Reaction mixtures were analyzed by Western blot using anti‐His monoclonal antibodies.


( A ) Recombinant His6 ‐ HsAtg4AWT , His6 ‐ HsAtg4AC77A or His6 ‐ HsAtg4AC81S ( 0 . 1 μg ) was incubated with His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) in 50 KT reaction buffer at 30 ° C for 45 min in the presence or absence of 1 mM DTT . Reaction mixtures were analyzed by Western blot using anti ‐ His monoclonal antibodies . ( * ) indicates non ‐ cleaved His6 ‐ GATE ‐ 16 ‐ HA and ( * * ) indicates cleaved His6 ‐ GATE ‐ 16 .

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

(A) Recombinant His6‐HsAtg4AWT, His6‐HsAtg4AC77A or His6‐HsAtg4AC81S (0.1 μg) was incubated with His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer at 30°C for 45 min in the presence or absence of 1 mM DTT. Reaction mixtures were analyzed by Western blot using anti‐His monoclonal antibodies. (*) indicates non‐cleaved His6‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16.


( B ) His6 ‐ HsAtg4AC81S ( 0 . 1 μg ) was incubated with recombinant His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) following the same procedure as detailed in Figure 5C . Reaction mixtures were analyzed and are presented as explained 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

(B) His6‐HsAtg4AC81S (0.1 μg) was incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) following the same procedure as detailed in Figure 5C. Reaction mixtures were analyzed and are presented as explained in (A).


( A ) CHO cells stably expressing GFP ‐ GATE ‐ 16 were transiently transfected with HsAtg4AWT ‐ Myc ‐ His6 or HsAtg4AC81S ‐ Myc ‐ His6 . At 24 h post ‐ transfection , cells were starved for 2 . 5 h after which they were fixed , permeabilized and incubated with anti ‐ Myc monoclonal antibodies . Upper panel : typical images of cells transfected with the above constructs , as visualized using a confocal microscope ; representative autophagosomes are indicated by arrows . Lower panel : quantification of the average number of autophagosomes per cell in the different transfectants . Images of the fixed cells were visualized using a Nikon eclipse TE300 fluorescent microscope and used for quantification of the number of autophagosomes per cell . The results presented are the means ± s . d . of a total of 100 cells from three separate experiments . ( * ) indicates significance at P0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CHO cells stably expressing GFP‐GATE‐16 were transiently transfected with HsAtg4AWT‐Myc‐His6 or HsAtg4AC81S‐Myc‐His6. At 24 h post‐transfection, cells were starved for 2.5 h after which they were fixed, permeabilized and incubated with anti‐Myc monoclonal antibodies. Upper panel: typical images of cells transfected with the above constructs, as visualized using a confocal microscope; representative autophagosomes are indicated by arrows. Lower panel: quantification of the average number of autophagosomes per cell in the different transfectants. Images of the fixed cells were visualized using a Nikon eclipse TE300 fluorescent microscope and used for quantification of the number of autophagosomes per cell. The results presented are the means±s.d. of a total of 100 cells from three separate experiments. (*) indicates significance at P0.001.


( B ) Left panel : HEK 293 cells were cotransfected with GFP ‐ GATE ‐ 16 and each of the Atg4A constructs mentioned in ( A ) . At 40 h post ‐ transfection , the cells were starved for 2 . 5 h , lysed in Ripa buffer and 100 μg of each lysate was loaded to 10 % SDS - PAGE and subsequently analyzed with anti ‐ GFP antibodies to detect the transfected GATE ‐ 16 , anti ‐ Myc antibodies to detect the transfected HsAtg4A and anti ‐ tubulin antibodies as control . ( * ) indicates non ‐ lipidated GFP ‐ GATE ‐ 16 and ( * * ) indicates lipidated GFP ‐ GATE ‐ 16 . Right panel : results from three separate experiments , as detailed in the left panel , were analyzed using NIH image program and quantified as follows : the amount of lipidated GFP ‐ GATE ‐ 16 out of the total of GFP ‐ GATE ‐ 16 was calculated for each mutant in each experiment . The value obtained for HsAtg4AWT in each experiment was set to 100 % and the relative lipidation in cells transfected with the mutant was calculated accordingly .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left panel: HEK 293 cells were cotransfected with GFP‐GATE‐16 and each of the Atg4A constructs mentioned in (A). At 40 h post‐transfection, the cells were starved for 2.5 h, lysed in Ripa buffer and 100 μg of each lysate was loaded to 10% SDS-PAGE and subsequently analyzed with anti‐GFP antibodies to detect the transfected GATE‐16, anti‐Myc antibodies to detect the transfected HsAtg4A and anti‐tubulin antibodies as control. (*) indicates non‐lipidated GFP‐GATE‐16 and (**) indicates lipidated GFP‐GATE‐16. Right panel: results from three separate experiments, as detailed in the left panel, were analyzed using NIH image program and quantified as follows: the amount of lipidated GFP‐GATE‐16 out of the total of GFP‐GATE‐16 was calculated for each mutant in each experiment. The value obtained for HsAtg4AWT in each experiment was set to 100% and the relative lipidation in cells transfected with the mutant was calculated accordingly.


( A ) HeLa cells were transiently transfected with GFP - HsAtg4BWT or GFP ‐ HsAtg4BC78S . At 24 h post ‐ transfection , cells were starved for 2 . 5 h in the presence of 4 mM H2O2 after which they were fixed , permeabilized and incubated with anti ‐ LC3 polyclonal antibodies . Cells were visualized ( upper panel ) and quantified ( lower panel ) as explained in Figure 8 . Representative autophagosomes are indicated in the upper panel by arrows . The results presented in the lower panel are the means ± s . d . of a total of 100 cells from three separate experiments . ( * ) indicates significance at P0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were transiently transfected with GFP-HsAtg4BWT or GFP‐HsAtg4BC78S. At 24 h post‐transfection, cells were starved for 2.5 h in the presence of 4 mM H2O2 after which they were fixed, permeabilized and incubated with anti‐LC3 polyclonal antibodies. Cells were visualized (upper panel) and quantified (lower panel) as explained in Figure 8. Representative autophagosomes are indicated in the upper panel by arrows. The results presented in the lower panel are the means±s.d. of a total of 100 cells from three separate experiments. (*) indicates significance at P0.001.


( B ) Left panel : HeLa cells transfected and treated as in ( A ) were lysed in Ripa buffer and 40 μg of each lysate was loaded to 12 % SDS - PAGE and subsequently analyzed with anti ‐ LC3 antibodies and anti ‐ GFP antibodies to detect the transfected HsAtg4B and anti ‐ tubulin antibodies as control . ( * ) indicates non ‐ lipidated LC3 and ( * * ) indicates lipidated LC3 . Right panel : results from three separate experiments , as detailed in the left panel , were analyzed as explained in Figure 8B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left panel: HeLa cells transfected and treated as in (A) were lysed in Ripa buffer and 40 μg of each lysate was loaded to 12% SDS-PAGE and subsequently analyzed with anti‐LC3 antibodies and anti‐GFP antibodies to detect the transfected HsAtg4B and anti‐tubulin antibodies as control. (*) indicates non‐lipidated LC3 and (**) indicates lipidated LC3. Right panel: results from three separate experiments, as detailed in the left panel, were analyzed as explained in Figure 8B.


( A - E ) GFP - pho8Δ60 - expressing wild - type ( WT ) , atg5Δ , atg7Δ , and atg5Δpep4Δ cells were starved ( A ) or treated with AmphoB ( B - E ) and subjected to western blotting . Cells were treated with 2 . 5 µg / ml AmphoB for the indicated time ( B ) or with the indicated doses for 24 hr ( C - E ) . Generation of free GFP was a marker of proteolysis .

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(A-E) GFP-pho8Δ60-expressing wild-type (WT), atg5Δ, atg7Δ, and atg5Δpep4Δ cells were starved (A) or treated with AmphoB (B-E) and subjected to western blotting. Cells were treated with 2.5 µg/ml AmphoB for the indicated time (B) or with the indicated doses for 24 hr (C-E). Generation of free GFP was a marker of proteolysis.


( F ) GFP - pho8Δ60 - and Pep4 - mRFP - expressing atg5Δ cells were cultured with ( AmphoB ) or without ( NT ) AmphoB ( 2 . 5 µg / ml , 24 hr ) , and localization of GFP - pho8Δ60 and Pep4 - mRFP was observed by confocal microscopy . Arrowheads indicate the cells in which GFP - pho8Δ60 was delivered to the vacuoles . Scale bars = 2 µm .

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(F) GFP-pho8Δ60- and Pep4-mRFP-expressing atg5Δ cells were cultured with (AmphoB) or without (NT) AmphoB (2.5 µg/ml, 24 hr), and localization of GFP-pho8Δ60 and Pep4-mRFP was observed by confocal microscopy. Arrowheads indicate the cells in which GFP-pho8Δ60 was delivered to the vacuoles. Scale bars = 2 µm.


( G , H ) Accumulation of autophagic body ( AB ) - like structures by AmphoB in atg5Δpep4Δ cells . pep4Δ and atg5Δpep4Δ cells were starved for 3 hr or treated with AmphoB ( 2 . 5 µg / ml , 24 hr ) . Scale bars = 2 µm . Cells containing AB - like structures were counted under phase - contrast microscopy . Representative images ( G ) and the percentage of cells with AB - like structures ( H ) are shown . * p < 0 . 01 vs the value of pep4Δ cells . ( I - K ) Induction of AB - like structures in atg5Δpep4Δ cells treated with AmphoB ( 2 . 5 µg / ml , 24 hr ) . Representative images of quick - frozen replicas ( I ) and thin section of the frozen - substituted material ( J ) are shown .

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(G, H) Accumulation of autophagic body (AB)-like structures by AmphoB in atg5Δpep4Δ cells. pep4Δ and atg5Δpep4Δ cells were starved for 3 hr or treated with AmphoB (2.5 µg/ml, 24 hr). Scale bars = 2 µm. Cells containing AB-like structures were counted under phase-contrast microscopy. Representative images (G) and the percentage of cells with AB-like structures (H) are shown. *p < 0.01 vs the value of pep4Δ cells. (I-K) Induction of AB-like structures in atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml, 24 hr). Representative images of quick-frozen replicas (I) and thin section of the frozen-substituted material (J) are shown.


( I ) AB - like structures accumulated in the vacuole . Scale bar = 0 . 5 µm . ( J ) AB - like structures containing lipid particles and mitochondria accumulated in the cross - section of the vacuole . An autophagosome ( AP ) - like double - membrane compartment containing a ribosome and Golgigranule is present in the cytosol . Scale bar = 0 . 5 µm . A magnified photo is available in Appendix Fig . S4B . ( K ) The cells containing AB - like structures were counted under electron microscopy [ no treatment ( NT ) : n = 20 , AmphoB : n = 192 ] . * p < 0 . 01 vs the value of NT .

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

(I) AB-like structures accumulated in the vacuole. Scale bar = 0.5 µm. (J) AB-like structures containing lipid particles and mitochondria accumulated in the cross-section of the vacuole. An autophagosome (AP)-like double-membrane compartment containing a ribosome and Golgigranule is present in the cytosol. Scale bar = 0.5 µm. A magnified photo is available in Appendix Fig. S4B. (K) The cells containing AB-like structures were counted under electron microscopy [no treatment (NT): n = 20, AmphoB: n = 192]. * p < 0.01 vs the value of NT.


( A - D ) Generation of Golgi stacks by AmphoB . Stacked Golgicisternae are observed in quick - frozen replicas ( A - C ) and substituted thin section ( D ) of atg5Δpep4Δ cells treated with AmphoB ( 2 . 5 µg / ml ) for the indicated time . ( A ) Scale bar = 1 µm . ( B ) The magnified image shows the area indicated by the square in ( A ) . Scale bar = 0 . 5 µm . ( C , D ) A representative Golgi stack with four cisternae is shown . Scale bar = 0 . 5 µm .

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(A-D) Generation of Golgi stacks by AmphoB. Stacked Golgicisternae are observed in quick-frozen replicas (A-C) and substituted thin section (D) of atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml) for the indicated time. (A) Scale bar = 1 µm. (B) The magnified image shows the area indicated by the square in (A). Scale bar = 0.5 µm. (C, D) A representative Golgi stack with four cisternae is shown. Scale bar = 0.5 µm.


( E - G ) Generation of AP - like double - membrane compartment from the stacked Golgi . A quick - frozen replica ( E ) and a substituted thin section ( F , G ) are shown . Scale bars = 0 . 5 µm . The distal stacks are extended , curved , and form AP - like structure ( E , F ) . Mitochondria are encircled by the Golgi membrane ( F ) . ( G ) The AP - like structure was separated away from Golgi stacks .

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(E-G) Generation of AP-like double-membrane compartment from the stacked Golgi. A quick-frozen replica (E) and a substituted thin section (F, G) are shown. Scale bars = 0.5 µm. The distal stacks are extended, curved, and form AP-like structure (E, F). Mitochondria are encircled by the Golgi membrane (F). (G) The AP-like structure was separated away from Golgi stacks.


( H , I ) The scene that AP - like structure was fused with vacuole . A magnified image of ( H ) is shown in ( I ) . In atg5Δpep4Δ cells treated with AmphoB ( 2 . 5 µg / ml , 24 hr ) , outer membrane of AP - like structure ( red arrows ) was fused with vacuolar membrane ( blue arrows ) . Inner membranes of AP - like structure ( yellow arrowheads ) were entered into the vacuoles to become AB - like structure . Green arrows indicate the membrane fusion site . Scale bars = 0 . 5 µm ( H ) and 0 . 2 µm ( I ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, I) The scene that AP-like structure was fused with vacuole. A magnified image of (H) is shown in (I). In atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml, 24 hr), outer membrane of AP-like structure (red arrows) was fused with vacuolar membrane (blue arrows). Inner membranes of AP-like structure (yellow arrowheads) were entered into the vacuoles to become AB-like structure. Green arrows indicate the membrane fusion site. Scale bars = 0.5 µm (H) and 0.2 µm (I).


( J ) The number of elongated single cisternae ( green bars ) , Golgi stacks ( red bars ) , and AB - like structures ( blue bars ) was counted over time using electron microscopy images . Error bars indicate s . e . m . ( 3 hr : n = 21 , 6 hr : n = 20 , 12 hr : n = 193 , 24 hr : n = 192 ) .

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(J) The number of elongated single cisternae (green bars), Golgi stacks (red bars), and AB-like structures (blue bars) was counted over time using electron microscopy images. Error bars indicate s.e.m. (3 hr: n = 21, 6 hr: n = 20, 12 hr: n = 193, 24 hr: n = 192).


( K ) Translocation of the trans - Golgi protein to the vacuolar membrane induced by AmphoB treatment . GFP - Sft2 - expressing atg5Δ cells ( trans - Golgi marker protein ) were incubated with or without AmphoB ( 2 . 5 µg / ml , 24 hr ) , and localization of GFP - Sft2 was assessed by confocal microscopy . Arrowhead indicates the GFP signal on the vacuolar membrane . Scale bars = 2 µm .

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(K) Translocation of the trans-Golgi protein to the vacuolar membrane induced by AmphoB treatment. GFP-Sft2-expressing atg5Δ cells (trans-Golgi marker protein) were incubated with or without AmphoB (2.5 µg/ml, 24 hr), and localization of GFP-Sft2 was assessed by confocal microscopy. Arrowhead indicates the GFP signal on the vacuolar membrane. Scale bars = 2 µm.


( L - O ) Grh1 and Gvp36 involvement in AmphoB - induced autophagy - like proteolysis . ( L , M ) Representative electron microscopy images of grh1Δatg5Δpep4Δ ( L ) and gvp36Δatg5Δpep4Δ cells ( M ) treated with AmphoB ( 2 . 5 µg / ml , 24 hr ) . AP - like and AB - like structures were absent . ( L ) A unilamellar Golgi cisterna was observed in the cytoplasm ( dotted square ) . Scale bar = 1 µm . ( M ) Formation of a Golgi stack , but not Golgi curvature , was observed ( dotted square ) . Bar = 0 . 5 µm .

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(L-O) Grh1 and Gvp36 involvement in AmphoB-induced autophagy-like proteolysis. (L, M) Representative electron microscopy images of grh1Δatg5Δpep4Δ (L) and gvp36Δatg5Δpep4Δ cells (M) treated with AmphoB (2.5 µg/ml, 24 hr). AP-like and AB-like structures were absent. (L) A unilamellar Golgi cisterna was observed in the cytoplasm (dotted square). Scale bar = 1 µm. (M) Formation of a Golgi stack, but not Golgi curvature, was observed (dotted square). Bar = 0.5 µm.


( L - O ) Grh1 and Gvp36 involvement in AmphoB - induced autophagy - like proteolysis . ( N ) Indicated cells were treated with AmphoB for 24 hr and the number of cells containing AB - like structures was counted by phase - contrast microscopy . * p < 0 . 01 vs the value of atg5Δpep4Δ cells .

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

(L-O) Grh1 and Gvp36 involvement in AmphoB-induced autophagy-like proteolysis. (N) Indicated cells were treated with AmphoB for 24 hr and the number of cells containing AB-like structures was counted by phase-contrast microscopy. *p < 0.01 vs the value of atg5Δpep4Δ cells.


( L - O ) Grh1 and Gvp36 involvement in AmphoB - induced autophagy - like proteolysis . ( O ) GFP - pho8Δ60 - expressing atg5Δ , grh1Δatg5Δ and gvp36Δatg5Δ cells were cultured with or without AmphoB ( 2 . 5 µg / ml , 24 hr ) and subjected to western blotting for GFP . An unidentified band at 32kD ( asterisk ) is non - specific band because it was present in Pep4 - lacking grh1Δatg5Δ cells .

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(L-O) Grh1 and Gvp36 involvement in AmphoB-induced autophagy-like proteolysis. (O) GFP-pho8Δ60-expressing atg5Δ, grh1Δatg5Δ and gvp36Δatg5Δ cells were cultured with or without AmphoB (2.5 µg/ml, 24 hr) and subjected to western blotting for GFP. An unidentified band at 32kD (asterisk) is non-specific band because it was present in Pep4-lacking grh1Δatg5Δ cells.


( A ) Localization of cellular PI ( 4 ) P . GFP - 2xPHOsh2 - expressing atg5Δ , sac1 - 23 / atg5Δ , and pik1 - 83 / atg5Δ cells were incubated with or without AmphoB ( 2 . 5 µg / ml ) for 6 hr at 37 ° C , and their localization was observed by confocal microscopy . Scale bars = 2 µm .

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

(A) Localization of cellular PI(4)P. GFP-2xPHOsh2-expressing atg5Δ, sac1-23/atg5Δ, and pik1-83/atg5Δ cells were incubated with or without AmphoB (2.5 µg/ml) for 6 hr at 37 °C, and their localization was observed by confocal microscopy. Scale bars = 2 µm.


( B ) The same experiments were performed using GFP - GOLPH3 ( a GolgiPI ( 4 ) P - monitoring protein ) instead of GFP - 2xPHOsh2 . Scale bars = 2 µm .

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

(B) The same experiments were performed using GFP-GOLPH3 (a GolgiPI(4)P-monitoring protein) instead of GFP-2xPHOsh2. Scale bars = 2 µm.


( C , D ) Reduction of AmphoB - induced GOMED by the deletion of Golgi PI ( 4 ) phosphatases . ( C ) Indicated cells expressing GFP - pho8Δ60 were cultured with or without AmphoB ( 2 . 5 µg / ml , 24 hr ) and subjected to western blotting for GFP .

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(C, D) Reduction of AmphoB-induced GOMED by the deletion of Golgi PI(4) phosphatases. (C) Indicated cells expressing GFP-pho8Δ60 were cultured with or without AmphoB (2.5 µg/ml, 24 hr) and subjected to western blotting for GFP.


( C , D ) Reduction of AmphoB - induced GOMED by the deletion of Golgi PI ( 4 ) phosphatases . ( D ) The number of AP - like and AB - like structures was counted using EM images ( Fig . EV2B , C ) . ( E − H ) Induction of GOMED by the deletion of Golgi PI ( 4 ) kinase .

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(C, D) Reduction of AmphoB-induced GOMED by the deletion of Golgi PI(4) phosphatases. (D) The number of AP-like and AB-like structures was counted using EM images (Fig. EV2B, C). (E−H) Induction of GOMED by the deletion of Golgi PI(4) kinase.


( E − H ) Induction of GOMED by the deletion of Golgi PI ( 4 ) kinase . ( E ) Indicated cells were incubated at 37 ° C ( temperature shift ) for 6 hr , and cells containing AB - like structures were counted by phase - contrast microscopy . * p < 0 . 01 vs the value of atg5Δpep4Δ cells .

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(E−H) Induction of GOMED by the deletion of Golgi PI(4) kinase. (E) Indicated cells were incubated at 37 °C (temperature shift) for 6 hr, and cells containing AB-like structures were counted by phase-contrast microscopy. *p < 0.01 vs the value of atg5Δpep4Δ cells.


( E − H ) Induction of GOMED by the deletion of Golgi PI ( 4 ) kinase . ( F ) Representative electron microscopy image of pik1 - 83 / atg5Δpep4Δ cells at 3 hr after temperature shift . AP - like structure ( arrow ) and AB - like structures were generated . Scale bar = 0 . 2 µm . ( G ) The number of AP - like and AB - like structures was counted using EM images . Error bars indicate s . e . m . ( atg5Δpep4Δ cells at 37 ° C : n = 54 and pik1 - 83 / atg5Δpep4Δ cells at 37 ° C : n = 63 ) . * p < 0 . 01 vs the value of atg5Δpep4Δ cells .

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(E−H) Induction of GOMED by the deletion of Golgi PI(4) kinase. (F) Representative electron microscopy image of pik1-83/atg5Δpep4Δ cells at 3 hr after temperature shift. AP-like structure (arrow) and AB-like structures were generated. Scale bar = 0.2 µm. (G) The number of AP-like and AB-like structures was counted using EM images. Error bars indicate s.e.m. (atg5Δpep4Δ cells at 37 °C: n = 54 and pik1-83/atg5Δpep4Δ cells at 37 °C: n = 63). *p < 0.01 vs the value of atg5Δpep4Δ cells.


( E − H ) Induction of GOMED by the deletion of Golgi PI ( 4 ) kinase . ( H ) Sec7 - HA - expressing pik1 - 83 / atg5Δ cells were incubated at 37 ° C ( temperature shift ) for 3 hr , and freeze replica immunolabelling was performed as described in Methods . Sec7 - HA - positive signals ( labelled with 15 nm gold particle ) were observed on the surface membrane of AP - like structure and Golgi membrane . Scale bar = 0 . 1 µm .

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(E−H) Induction of GOMED by the deletion of Golgi PI(4) kinase. (H) Sec7-HA-expressing pik1-83/atg5Δ cells were incubated at 37 °C (temperature shift) for 3 hr, and freeze replica immunolabelling was performed as described in Methods. Sec7-HA-positive signals (labelled with 15 nm gold particle) were observed on the surface membrane of AP-like structure and Golgi membrane. Scale bar = 0.1 µm.


( I − L ) Genetic ( I , J ) and pharmacological ( K , L ) alterations in anterograde trafficking from the Golgi are required for GOMED . ( I ) Indicated cells were incubated at 37 ° C ( temperature switch ) for 3 hr , and the cells containing AB - like structures were counted by phase - contrast microscopy . * p < 0 . 01 vs the value of atg5Δpep4Δ cells .

(I−L) Genetic (I, J) and pharmacological (K, L) alterations in anterograde trafficking from the Golgi are required for GOMED. (I) Indicated cells were incubated at 37 °C (temperature switch) for 3 hr, and the cells containing AB-like structures were counted by phase-contrast microscopy. *p < 0.01 vs the value of atg5Δpep4Δ cells.


( I − L ) Genetic ( I , J ) and pharmacological ( K , L ) alterations in anterograde trafficking from the Golgi are required for GOMED . ( J ) Indicated cells were also observed using EM . Arrow indicates AP - like structure . Scale bar = 0 . 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

(I−L) Genetic (I, J) and pharmacological (K, L) alterations in anterograde trafficking from the Golgi are required for GOMED. (J) Indicated cells were also observed using EM. Arrow indicates AP-like structure. Scale bar = 0.5 µm.


Genetic ( I , J ) and pharmacological ( K , L ) alterations in anterograde trafficking from the Golgi are required for GOMED . ( K , L ) atg5Δpep4Δ cells were treated with ( CBM ) or without ( NT ) CBM ( 1 mM , 3 hr ) , and cells containing AB - like structures were counted by phase - contrast microscopy ( K ) . * p < 0 . 01 vs the value of NT .

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

Genetic (I, J) and pharmacological (K, L) alterations in anterograde trafficking from the Golgi are required for GOMED. (K, L) atg5Δpep4Δ cells were treated with (CBM) or without (NT) CBM (1 mM, 3 hr), and cells containing AB-like structures were counted by phase-contrast microscopy (K). * p < 0.01 vs the value of NT.


Genetic ( I , J ) and pharmacological ( K , L ) alterations in anterograde trafficking from the Golgi are required for GOMED . ( K , L ) atg5Δpep4Δ cells were treated with ( CBM ) or without ( NT ) CBM ( 1 mM , 3 hr ) . Cells were also observed using EM ( L ) . AB - like structures were observed . Scale bar = 0 . 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

Genetic (I, J) and pharmacological (K, L) alterations in anterograde trafficking from the Golgi are required for GOMED. (K, L) atg5Δpep4Δ cells were treated with (CBM) or without (NT) CBM (1 mM, 3 hr). Cells were also observed using EM (L). AB-like structures were observed. Scale bar = 0.5 µm.


( M − O ) Disturbance of anterograde trafficking from the Golgi to the PM in the cells with GOMED . Indicated cells expressing Hsp150 - HA ( 3 × ) were treated as follows : NT , no treatment ; starve , starvation for 6 hr ; AmphoB , 2 . 5 µg / ml for 24 hr ( M ) ; CBM , 1 mM for 24 hr ( N ) ; temperature shift , from 25 ° C to 37 ° C for 3 hr ( O ) . The cells were then lysed and examined for Hsp150 - HA ( 3 × ) expression by western blotting . Accumulation of O - glycosylated Hsp150 - HA ( 3 × ) indicated the trafficking failure from the Golgi to the PM .

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

(M−O) Disturbance of anterograde trafficking from the Golgi to the PM in the cells with GOMED. Indicated cells expressing Hsp150-HA(3×) were treated as follows: NT, no treatment; starve, starvation for 6 hr; AmphoB, 2.5 µg/ml for 24 hr (M); CBM, 1 mM for 24 hr (N); temperature shift, from 25 °C to 37 °C for 3 hr (O). The cells were then lysed and examined for Hsp150-HA(3×) expression by western blotting. Accumulation of O-glycosylated Hsp150-HA(3×) indicated the trafficking failure from the Golgi to the PM.


( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( A , B ) EM analysis revealed the formation of AP - like and AL - like structures . Atg5 KO MEFs were treated with CBM ( 2 mM ) for 3 hr . A representative low - magnification image ( A ) and high - magnification images ( B ) are shown . Scale bar = 1 µm ( A ) and 0 . 2 µm ( B ) . The arrowheads and arrows indicate AL - like and AP - like structures , respectively .

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

(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (A, B) EM analysis revealed the formation of AP-like and AL-like structures. Atg5 KO MEFs were treated with CBM (2 mM) for 3 hr. A representative low-magnification image (A) and high-magnification images (B) are shown. Scale bar = 1 µm (A) and 0.2 µm (B). The arrowheads and arrows indicate AL-like and AP-like structures, respectively.


( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( C - F ) Involvement of Golgi membranes in the generation of AL - like structures . ( C ) CLEM analysis of mCherry - syntaxin 6 - expressing Atg5 KO MEFs . Cells were treated with CBM ( 2 mM ) for 24 hr and observed using fluorescence and electron microscopy . mCherry - syntaxin 6puncta merged with the AL - like structures . Scale bar = 5 µm . " N " indicates nucleus . A magnified image of the dashed square is shown in the inset . Scale bars = 2 µm .

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

(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (C-F) Involvement of Golgi membranes in the generation of AL-like structures. (C) CLEM analysis of mCherry-syntaxin 6-expressing Atg5 KO MEFs. Cells were treated with CBM (2 mM) for 24 hr and observed using fluorescence and electron microscopy. mCherry-syntaxin 6puncta merged with the AL-like structures. Scale bar = 5 µm. "N" indicates nucleus. A magnified image of the dashed square is shown in the inset. Scale bars = 2 µm.


( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( C - F ) Involvement of Golgi membranes in the generation of AL - like structures . ( D , E ) Requirement of Grasp65 in the generation of AL - like structures . Atg5 KO and Grasp65 - silenced Atg5 KO MEFs were treated without ( NT ) or with CBM ( 2 mM ) for the indicated times followed by immunostaining with an anti - Lamp2 antibody . Representative images ( at 24 h ) are shown in ( D ) . Scale bars = 5 µm . A magnified image of the dashed square is shown in the inset . Scale bars = 1 µm . CBM induced large ring - like Lamp2 fluorescence . ( E ) Percentages of cells with large ring - like Lamp2 immunofluorescence ( mean ± s . e . m . , n = 4 ) . * p < 0 . 01 vs the value of Atg5 KO MEF .

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

(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (C-F) Involvement of Golgi membranes in the generation of AL-like structures. (D, E) Requirement of Grasp65 in the generation of AL-like structures. Atg5 KO and Grasp65-silenced Atg5 KO MEFs were treated without (NT) or with CBM (2 mM) for the indicated times followed by immunostaining with an anti-Lamp2 antibody. Representative images (at 24 h) are shown in (D). Scale bars = 5 µm. A magnified image of the dashed square is shown in the inset. Scale bars = 1 µm. CBM induced large ring-like Lamp2 fluorescence. (E) Percentages of cells with large ring-like Lamp2 immunofluorescence (mean ± s.e.m., n = 4). *p < 0.01 vs the value of Atg5 KO MEF.


( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( C - F ) Involvement of Golgi membranes in the generation of AL - like structures . ( F ) Colocalization of Lamp2 and GFP - syntaxin 6 in CBM - treated Atg5 KO MEFs . GFP - syntaxin 6 - expressing Atg5 KO MEFs were treated with ( CBM ) or without ( NT ) CBM ( 2 mM ) for 6 hr and immunostained with an anti - Lamp2 antibody . The ring - like Lamp2 fluorescence merged with the signal for syntaxin 6 . Scale bar = 5 µm . A magnified image of the dashed square is shown in the inset . Scale bars = 2 µm .

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

(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (C-F) Involvement of Golgi membranes in the generation of AL-like structures. (F) Colocalization of Lamp2 and GFP-syntaxin 6 in CBM-treated Atg5 KO MEFs. GFP-syntaxin 6-expressing Atg5 KO MEFs were treated with (CBM) or without (NT) CBM (2 mM) for 6 hr and immunostained with an anti-Lamp2 antibody. The ring-like Lamp2 fluorescence merged with the signal for syntaxin 6. Scale bar = 5 µm. A magnified image of the dashed square is shown in the inset. Scale bars = 2 µm.


( G ) The monomeric red fluorescent protein ( mRFP ) - green fluorescent protein ( GFP ) tandem protein assay revealed the induction of GOMED . Atg5 KO MEFs stably expressing tandem mRFP - GFP were incubated without ( no treatment ) or with CBM ( 2 mM ) for 24 hr . Red signals indicate acidic compartments . Lysosomes were counterstained with an anti - Lamp2 antibody ( cyan ) . Scale bars = 5 µm . Regions of interest ( ROI ) are indicated by dashed squares . Arrowheads indicate GOMED structure . Scale bars = 2 µm .

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

(G) The monomeric red fluorescent protein (mRFP)-green fluorescent protein (GFP) tandem protein assay revealed the induction of GOMED. Atg5 KO MEFs stably expressing tandem mRFP-GFP were incubated without (no treatment) or with CBM (2 mM) for 24 hr. Red signals indicate acidic compartments. Lysosomes were counterstained with an anti-Lamp2 antibody (cyan). Scale bars = 5 µm. Regions of interest (ROI) are indicated by dashed squares. Arrowheads indicate GOMED structure. Scale bars = 2 µm.


( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( H , I ) Degradation of GFP - fused proteins indicated the induction of GOMED . Atg5 KO MEFs stably expressing VSVG - GFP ( H ) or M6PR - GFP ( I ) were incubated with CBM ( 5 mM ) for the indicated times . Cell lysates were subjected to immunoblotting with the anti - GFP antibody .

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

(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (H, I) Degradation of GFP-fused proteins indicated the induction of GOMED. Atg5 KO MEFs stably expressing VSVG-GFP (H) or M6PR-GFP (I) were incubated with CBM (5 mM) for the indicated times. Cell lysates were subjected to immunoblotting with the anti-GFP antibody.


( J − M ) Genetic induction of GOMED in Atg5 KO MEFs . Atg5 KO MEFs were treated with the indicated small interfering ( si ) RNAs for 24 hr and GOMED was assessed by EM ( J - L ) . Representative images are shown in ( J , K ) . Arrows indicate AL - like structures . Scale bars = 2 µm . ( L ) The number of AP - like and AL - like structures was calculated from the EM images of cells treated with the indicated siRNAs . Error bars indicate s . e . m . ( siControl : n = 23 , siPI4k2α + siPI4k3β : n = 20 , siArfaptin1 : n = 20 ) . * p < 0 . 01 vs the value of siControl .

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

(J−M) Genetic induction of GOMED in Atg5 KO MEFs. Atg5 KO MEFs were treated with the indicated small interfering (si)RNAs for 24 hr and GOMED was assessed by EM (J-L). Representative images are shown in (J, K). Arrows indicate AL-like structures. Scale bars = 2 µm. (L) The number of AP-like and AL-like structures was calculated from the EM images of cells treated with the indicated siRNAs. Error bars indicate s.e.m. (siControl: n = 23, siPI4k2α + siPI4k3β: n = 20, siArfaptin1: n = 20). *p < 0.01 vs the value of siControl.


( J − M ) Genetic induction of GOMED in Atg5 KO MEFs . Atg5 KO MEFs were treated with the indicated small interfering ( si ) RNAs for 24 hr and GOMED was assessed by the mRFP - GFP tandem protein assay ( M ) . ( M ) The same experiments as in ( G ) were performed using cells treated with the indicated siRNAs . Representative images are shown . Arrowheads indicate GOMED structure . Scale bars = 5 µm .

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

(J−M) Genetic induction of GOMED in Atg5 KO MEFs. Atg5 KO MEFs were treated with the indicated small interfering (si)RNAs for 24 hr and GOMED was assessed by the mRFP-GFP tandem protein assay (M). (M) The same experiments as in (G) were performed using cells treated with the indicated siRNAs. Representative images are shown. Arrowheads indicate GOMED structure. Scale bars = 5 µm.


( A ) Suppression of insulin secretion in Atg5 knockout ( KO ) MIN6 cells after glucose deprivation . Atg5 KO MIN6 cells were incubated with normal culture medium ( NT ) or glucose - depleted medium for 1 hr . The amount of secreted insulin is expressed as the percentage of that of the untreated control ( NT ) ( mean ± s . e . m , n = 3 ) . * p < 0 . 01 vs the value of NT .

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

(A) Suppression of insulin secretion in Atg5 knockout (KO) MIN6 cells after glucose deprivation. Atg5 KO MIN6 cells were incubated with normal culture medium (NT) or glucose-depleted medium for 1 hr. The amount of secreted insulin is expressed as the percentage of that of the untreated control (NT) (mean ± s.e.m, n = 3). *p < 0.01 vs the value of NT.


( B , C ) EM analysis revealed the induction of GOMED structures . Atg5 KO MIN6 cells were subjected to glucose deprivation for 1 hr . Representative images are shown . The white arrow and black arrows indicate the AP - like structure and insulinvesicles , respectively . Scale bars = 0 . 2 µm . ( D ) The number of AP - like and AL - like structures was calculated from the EM images of cells left untreated ( NT ) or subjected to glucose deprivation . Error bars indicate s . e . m . ( NT : n = 17 , glucose deprivation : n = 84 ) . * p < 0 . 01 and # p < 0 . 05 vs the value of NT .

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

(B, C) EM analysis revealed the induction of GOMED structures. Atg5 KO MIN6 cells were subjected to glucose deprivation for 1 hr. Representative images are shown. The white arrow and black arrows indicate the AP-like structure and insulinvesicles, respectively. Scale bars = 0.2 µm. (D) The number of AP-like and AL-like structures was calculated from the EM images of cells left untreated (NT) or subjected to glucose deprivation. Error bars indicate s.e.m. (NT: n = 17, glucose deprivation: n = 84). *p < 0.01 and #p < 0.05 vs the value of NT.


( E ) Involvement of Golgi membrane in the generation of GOMED . Lamp1 - GFP / mCherry - Syntaxin 6 - expressing Atg5 KO MIN6 cells were subjected to glucose deprivation , and analysed by TEM and confocal microscopy . CLEM analysis revealed that AL - like structures ( arrows ) merged with Lamp1 - GFP fluorescence and Syntaxin 6 fluorescence . Scale bars = 5 µm . A magnified image of the dashed square is shown in the inset . Scale bars = 2 µm .

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

(E) Involvement of Golgi membrane in the generation of GOMED. Lamp1-GFP/mCherry-Syntaxin 6-expressing Atg5 KO MIN6 cells were subjected to glucose deprivation, and analysed by TEM and confocal microscopy. CLEM analysis revealed that AL-like structures (arrows) merged with Lamp1-GFP fluorescence and Syntaxin 6 fluorescence. Scale bars = 5 µm. A magnified image of the dashed square is shown in the inset. Scale bars = 2 µm.


( F ) GOMED - mediated degradation of ( pro ) insulin by glucose deprivation of Atg5 KO MIN6 cells . Atg5 KO MIN6 cells were incubated in glucose - depleted medium in the absence or presence of bafilomycin A1 ( 10 nM ) for the indicated times . The cell lysates were then subjected to immunoblot analysis using an anti - ( pro ) insulin antibody .

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

(F) GOMED-mediated degradation of (pro)insulin by glucose deprivation of Atg5 KO MIN6 cells. Atg5 KO MIN6 cells were incubated in glucose-depleted medium in the absence or presence of bafilomycin A1 (10 nM) for the indicated times. The cell lysates were then subjected to immunoblot analysis using an anti-(pro)insulin antibody.


( G ) Immunofluorescence analysis of ( pro ) insulin and Lamp2 . Atg5 KO MIN6 cells were left untreated ( NT ) or subjected to glucose deprivation for 1 hr in the presence of E64d ( 25 µM ) or bafilomycin A1 ( 10 nM ) , and were stained with anti - ( pro ) insulin and anti - Lamp2 ( lysosomal marker ) antibodies and observed by fluorescence microscopy . Scale bar = 1 µm . Arrowheads indicate the colocalization of ( pro ) insulin with GOMED structures .

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

(G) Immunofluorescence analysis of (pro)insulin and Lamp2. Atg5 KO MIN6 cells were left untreated (NT) or subjected to glucose deprivation for 1 hr in the presence of E64d (25 µM) or bafilomycin A1 (10 nM), and were stained with anti-(pro)insulin and anti-Lamp2 (lysosomal marker) antibodies and observed by fluorescence microscopy. Scale bar = 1 µm. Arrowheads indicate the colocalization of (pro)insulin with GOMED structures.


( A − D ) EM analysis demonstrated the induction of GOMED . Primary Atg7 KO β - cells were subjected to glucose deprivation for 1 hr . A representative AP - like structure containing insulingranule is shown in ( A ) . An AL - like structure containing insulingranules is shown in ( B ) . Scale bars = 0 . 2 µm .

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

(A−D) EM analysis demonstrated the induction of GOMED. Primary Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. A representative AP-like structure containing insulingranule is shown in (A). An AL-like structure containing insulingranules is shown in (B). Scale bars = 0.2 µm.


( A − D ) EM analysis demonstrated the induction of GOMED . Primary Atg7 KO β - cells were subjected to glucose deprivation for 1 hr . ( C , D ) GOMED structures containing Golgi vesicles . An AP - like structure containing multiple Golgi vesicles ( arrows ) ( C ) and an AL - like structure fusing with a lysosome ( arrowhead ) ( D ) are shown . Scale bar = 0 . 5 µm . A magnified image of the dashed rectangle is shown in the inset . Scale bar = 0 . 2 µm . AP - like structure contained double - membrane .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) EM analysis demonstrated the induction of GOMED. Primary Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. (C, D) GOMED structures containing Golgi vesicles. An AP-like structure containing multiple Golgi vesicles (arrows) (C) and an AL-like structure fusing with a lysosome (arrowhead) (D) are shown. Scale bar = 0.5 µm. A magnified image of the dashed rectangle is shown in the inset. Scale bar = 0.2 µm. AP-like structure contained double-membrane.


( E ) The total number of AL - like structure was calculated from the EM images of WT and Atg7 KO β - cells after glucose deprivation . Error bars indicate s . e . m . ( n = 46 − 163 ) . * p < 0 . 01 and # p < 0 . 05 vs the value of 0 min .

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

(E) The total number of AL-like structure was calculated from the EM images of WT and Atg7 KO β-cells after glucose deprivation. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min.


( F ) The total number of GOMED structure was calculated from the EM images of Atg7 KO β - cells after glucose deprivation in the absence or presence of bafilomycin A1 ( 10 nM ) . Error bars indicate s . e . m . ( n = 109 − 116 ) . * p < 0 . 01 vs the value of 60 min .

O O O O O O 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 O O O O O O O O O O O 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 total number of GOMED structure was calculated from the EM images of Atg7 KO β-cells after glucose deprivation in the absence or presence of bafilomycin A1 (10 nM). Error bars indicate s.e.m. (n = 109−116). *p < 0.01 vs the value of 60 min.


( G − J ) EM analysis showed the induction of mitophagy . WT and Atg7 KO β - cells were subjected to glucose deprivation for 1 hr . Representative images showing mitophagy in Atg7 KO β - cells are shown in ( G ) . Arrowhead ( G ) indicates mitophagy . Scale bars = 0 . 5 µm . ( H , J ) The number of structures representative of mitophagy ( H ) per cell was calculated from the EM images of β - cells after glucose depletion . Error bars indicate s . e . m . ( n = 46 − 163 ) . * p < 0 . 01 and # p < 0 . 05 vs the value of 0 min .

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

(G−J) EM analysis showed the induction of mitophagy. WT and Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. Representative images showing mitophagy in Atg7 KO β-cells are shown in (G). Arrowhead (G) indicates mitophagy. Scale bars = 0.5 µm. (H, J) The number of structures representative of mitophagy (H) per cell was calculated from the EM images of β-cells after glucose depletion. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min.