( F ) Representative western blot of DLL4 levels in scr or miR - 30c transfected TREx cells at 0 , 24 and 48 hours post induction with GAPDH as a loading control . Densitometry analysis of n = 3 Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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(F) Representative western blot of DLL4 levels in scr or miR-30c transfected TREx cells at 0, 24 and 48 hours post induction with GAPDH as a loading control. Densitometry analysis of n=3 Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( J ) Representative western blot of DLL4 levels in scr or miR - 30c antagomiR transfected TREx cells at 0 , 24 and 48 hours post induction with GAPDH as a loading control . Densitometry analysis of n = 3 . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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(J) Representative western blot of DLL4 levels in scr or miR-30c antagomiR transfected TREx cells at 0, 24 and 48 hours post induction with GAPDH as a loading control. Densitometry analysis of n=3. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( B ) Representative western blot of DLL4 levels in scr and DLL4 KD TREx cells with GAPDH as a loading control , densitometry analysis is n = 3 . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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(B) Representative western blot of DLL4 levels in scr and DLL4 KD TREx cells with GAPDH as a loading control, densitometry analysis is n=3. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( C ) Representative western blot of ORF65 levels at 0 , 24 and 48 hours post induction in scr and DLL4 KD TREx cells , GAPDH was used as a loading control and densitometry analysis performed on n = 3 . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

(C) Representative western blot of ORF65 levels at 0, 24 and 48 hours post induction in scr and DLL4 KD TREx cells, GAPDH was used as a loading control and densitometry analysis performed on n=3. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


A . Histograms of spindle angle of GFP - H2B U2OS cells transfected with either GAPDH or Spindly siRNA and filmed on rectangular micropatterns . Cells were filmed 48 hours post - transfection .

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A. Histograms of spindle angle of GFP-H2B U2OS cells transfected with either GAPDH or Spindly siRNA and filmed on rectangular micropatterns. Cells were filmed 48 hours post-transfection.


B . Mitotic timing of control - depleted U2OS cells or CLIP - 170 - depleted cells . CLIP - 170 - depeleted cells are split in two categories ; ' aligned ' and ' misaligned ' , relating to the observed chromosome misalignment

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B. Mitotic timing of control-depleted U2OS cells or CLIP-170-depleted cells. CLIP-170-depeleted cells are split in two categories; 'aligned' and 'misaligned', relating to the observed chromosome misalignment phenotype


C . The histograms of spindle angles for the two different categories of CLIP - 170 - depleted U2OS cells ( Left graph with alignment defect , right graph without alignment defect ) .

C. The histograms of spindle angles for the two different categories of CLIP-170-depleted U2OS cells (Left graph with alignment defect, right graph without alignment defect).


D and E . Mitotic timing and spindle angle histograms of control ( GAPDH ) - depleted U2OS cells and CENP - E - depleted cells .

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D and E. Mitotic timing and spindle angle histograms of control (GAPDH)-depleted U2OS cells and CENP-E-depleted cells.


A . HeLa cell stably expressing GFP - LGN and transiently expressing RFP - H2B was treated with noscapine to induce chromosome misalignments . Cells were imaged every 5 minutes . The time is relative to NEB . The red arrows indicate cortical regions where the chromosomes are in close proximity to the cell boundaries , accompanied by the displacement of LGN from the cortex .

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A. HeLa cell stably expressing GFP-LGN and transiently expressing RFP-H2B was treated with noscapine to induce chromosome misalignments. Cells were imaged every 5 minutes. The time is relative to NEB. The red arrows indicate cortical regions where the chromosomes are in close proximity to the cell boundaries, accompanied by the displacement of LGN from the cortex.


B . Similar assay as described in supplementary figure 2D was carried out in the presence of a small molecule inhibitor against Aurora - B ( ZM447439 ) in combination with the proteasome inhibitor MG - 132 to keep cells arrested in mitosis and in B . with an inhibitor against Plk1 ( BI2536 ) . Drugs were added at the same timing as Hoechst , 30 minutes prior to imaging .

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B. Similar assay as described in supplementary figure 2D was carried out in the presence of a small molecule inhibitor against Aurora-B (ZM447439) in combination with the proteasome inhibitor MG-132 to keep cells arrested in mitosis and in B. with an inhibitor against Plk1 (BI2536). Drugs were added at the same timing as Hoechst, 30 minutes prior to imaging.


A . Immunofluorescence images and quantifications of KT - localized Plk1 in U2OS cells after PBIP1 depletion . Cells were either Mock transfected or with PBIP1 siRNA for 72 hours . Cells were treated with nocodazole for 1 hour and fixed . Cells were stained with the centromere marker ACA , Plk1 and DAPI .

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A. Immunofluorescence images and quantifications of KT-localized Plk1 in U2OS cells after PBIP1 depletion. Cells were either Mock transfected or with PBIP1 siRNA for 72 hours. Cells were treated with nocodazole for 1 hour and fixed. Cells were stained with the centromere marker ACA, Plk1 and DAPI.


B . Representative images and histograms showing the effect of chromosome position on cortical LGN enrichment after 72 hours of Mock and PBIP1 depletion .

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B. Representative images and histograms showing the effect of chromosome position on cortical LGN enrichment after 72 hours of Mock and PBIP1 depletion.


C . Live cellimages and histograms of GFP - H2B U2OS cells Mock transfected or with siPBIP1 filmed on rectangular micropatterns in the presence of a CENP - E inhibitor . The spindle angles for both conditions were scored at 32 minutes after NEB .

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C. Live cellimages and histograms of GFP-H2B U2OS cells Mock transfected or with siPBIP1 filmed on rectangular micropatterns in the presence of a CENP-E inhibitor. The spindle angles for both conditions were scored at 32 minutes after NEB.


. D . The total spindle angle displacement within the first hour of mitosis was determined by measuring the spindle angle at each time point and calculating the sum of the Δ spindle angle between each time points .

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. D. The total spindle angle displacement within the first hour of mitosis was determined by measuring the spindle angle at each time point and calculating the sum of the Δ spindle angle between each time points.


C . RADX interacts with ssDNA . Extracts of U2OS cells stably expressing GFP - RADX WT or mutants were incubated with biotin - coupled ssDNA oligo immobilized on Streptavidin beads , washed extensively and immunoblotted with GFP and RPA1 antibodies .

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C. RADX interacts with ssDNA. Extracts of U2OS cells stably expressing GFP-RADX WT or mutants were incubated with biotin-coupled ssDNA oligo immobilized on Streptavidin beads, washed extensively and immunoblotted with GFP and RPA1 antibodies.


D . ssDNA - bound Streptavidin beads incubated with cell extracts as in ( C ) were washed with buffer containing indicated salt concentrations prior to immunoblotting .

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D. ssDNA-bound Streptavidin beads incubated with cell extracts as in (C) were washed with buffer containing indicated salt concentrations prior to immunoblotting.


E . Lysates of untransfected HCT116 cells were incubated with immobilized ssDNA or dsDNA probes as in ( C ) and immunoblotted with RADX and RPA1 antibodies .

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E. Lysates of untransfected HCT116 cells were incubated with immobilized ssDNA or dsDNA probes as in (C) and immunoblotted with RADX and RPA1 antibodies.


F . Immunoblot analysis of RADX expression in human cell lines . See also Fig EV1B .

F. Immunoblot analysis of RADX expression in human cell lines. See also Fig EV1B.


G . Representative images from in situ proximity ligation assays ( PLAs ) in BrdU - labeled U2OS and GFP / GFP - RADX cell lines ( Fig EV1E , F ) using GFP and BrdU antibodies under native conditions . Scale bar , 10 μm .

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G. Representative images from in situ proximity ligation assays (PLAs) in BrdU-labeled U2OS and GFP/GFP-RADX cell lines (Fig EV1E,F) using GFP and BrdU antibodies under native conditions. Scale bar, 10 μm.


I . Soluble and chromatin - enriched fractions of U2OS cell lines stably expressing GFP - RADX alleles were immunoblotted with indicated antibodies .

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I. Soluble and chromatin-enriched fractions of U2OS cell lines stably expressing GFP-RADX alleles were immunoblotted with indicated antibodies.


A . Immunoblot analysis of chromatin fractions of U2OS cells transfected with indicated siRNAs and treated or not with HU .

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A. Immunoblot analysis of chromatin fractions of U2OS cells transfected with indicated siRNAs and treated or not with HU.


B . U2OS cells treated with HU in the presence or absence of ATR inhibitor ( ATRi ) were fractionated and immunoblotted with indicated antibodies . Relative RADX levels on chromatin were quantified and normalized to histone H3 . See also Fig EV2A .

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B. U2OS cells treated with HU in the presence or absence of ATR inhibitor (ATRi) were fractionated and immunoblotted with indicated antibodies. Relative RADX levels on chromatin were quantified and normalized to histone H3. See also Fig EV2A.


C . Representative images of U2OS cells stably expressing indicated GFP - RADX alleles that were subjected to laser microirradiation , fixed with methanol / acetone immediately afterwards and immunostained with γH2AX antibody .

C. Representative images of U2OS cells stably expressing indicated GFP-RADX alleles that were subjected to laser microirradiation, fixed with methanol/acetone immediately afterwards and immunostained with γH2AX antibody.


D . As in ( C ) , except cells were fixed with paraformaldehyde 20 min after DNA damage infliction and immunostained with PCNA antibody .

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D. As in (C), except cells were fixed with paraformaldehyde 20 min after DNA damage infliction and immunostained with PCNA antibody.


E . Representative images from PLAs with GFP and RPA2 antibodies in U2OS and U2OS / GFP - RADX cell lines labeled with EdU for 30 min and then fixed or exposed to HU or CPT for 4 h before fixation .

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E. Representative images from PLAs with GFP and RPA2 antibodies in U2OS and U2OS/GFP-RADX cell lines labeled with EdU for 30 min and then fixed or exposed to HU or CPT for 4 h before fixation.


A . Immunoblot analysis of HCT116 cells transfected with indicated RADX siRNAs and RADX knockout cell lines ( RADXΔ ) .

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A. Immunoblot analysis of HCT116 cells transfected with indicated RADX siRNAs and RADX knockout cell lines (RADXΔ).


C . Cells in ( A ) were labeled with consecutive pulses of CldU and IdU as shown in ( B ) . Replication fork speeds were calculated as length of labeled track divided by pulse time ( bars , mean ; n = 400 fibers , pooled from two independent experiments , per condition ) .

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C. Cells in (A) were labeled with consecutive pulses of CldU and IdU as shown in (B). Replication fork speeds were calculated as length of labeled track divided by pulse time (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


D . Bidirectional replication fork symmetry was calculated as percentage of shorter divided by longer tracks from ( C ) . Concordance is 100 % , representing equal rates of bidirectional elongation for both daughter forks ( bars , mean ; n = 50 bidirectional forks , pooled from two independent experiments , per condition ) .

D. Bidirectional replication fork symmetry was calculated as percentage of shorter divided by longer tracks from (C). Concordance is 100%, representing equal rates of bidirectional elongation for both daughter forks (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition).


E . Proportion of stalled forks ( CldU - only tracks ) among DNA fibers from ( C ) ( bars , mean ; 200 fibers analyzed per condition ; n = 2 independent experiments ) .

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E. Proportion of stalled forks (CldU-only tracks) among DNA fibers from (C) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


F . Proportion of new origins ( IdU - only tracks ) among DNA fibers in ( C ) ( bars , mean ; 20 fields of view quantified per condition ; n = 2 independent experiments ) .

F. Proportion of new origins (IdU-only tracks) among DNA fibers in (C) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).


H . Proportion of stalled forks ( CldU - only tracks ) among DNA fibers from cells labeled as in ( G ) ( bars , mean ; 200 fibers analyzed per condition ; n = 2 independent experiments ) .

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H. Proportion of stalled forks (CldU-only tracks) among DNA fibers from cells labeled as in (G) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


I . Proportion of restarted forks ( CldU - and IdU - positive tracks ) among DNA fibers in ( G ) ( bars , mean ; 200 fibers analyzed per condition ; n = 2 independent experiments ) .

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I. Proportion of restarted forks (CldU- and IdU-positive tracks) among DNA fibers in (G) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


A . Replication fork speeds in U2OS or U2OS / GFP - RADX cells transfected with control ( − ) or RADX ( # 6 ) siRNA targeting the 3 ' UTR and labeled with CldU and IdU as in Fig 3B ( bars , mean ; n = 400 fibers , pooled from two independent experiments , per condition ) .

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A. Replication fork speeds in U2OS or U2OS/GFP-RADX cells transfected with control (−) or RADX(#6) siRNA targeting the 3'UTR and labeled with CldU and IdU as in Fig 3B (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


B . Fork symmetry in cells treated as in ( A ) ( bars , mean ; n = 50 bidirectional forks , pooled from two independent experiments , per condition ) .

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B. Fork symmetry in cells treated as in (A) (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition).


C . Proportion of stalled forks ( CldU - only tracks ) among DNA fibers in ( A ) ( bars , mean ; 200 fibers analyzed per condition ; n = 2 independent experiments ) .

C. Proportion of stalled forks (CldU-only tracks) among DNA fibers in (A) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


D . Proportion of new origins ( IdU - only tracks ) among DNA fibers in ( A ) ( bars , mean ; 20 fields of view quantified per condition ; n = 2 independent experiments ) .

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D. Proportion of new origins (IdU-only tracks) among DNA fibers in (A) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).


E . Parental U2OS and derivative lines stably expressing GFP - RADX alleles or all three RPA isoforms at near - endogenous levels ( U2OS / Super - RPA ) ( Fig EV4A ) were transfected with control ( − ) or RADX siRNA . Replication fork speeds were determined as in ( A ) ( bars , mean ; n = 400 fibers , pooled from two independent experiments , per condition ) .

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E. Parental U2OS and derivative lines stably expressing GFP-RADX alleles or all three RPA isoforms at near-endogenous levels (U2OS/Super-RPA) (Fig EV4A) were transfected with control (−) or RADX siRNA. Replication fork speeds were determined as in (A) (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


F . Replication fork speeds in HCT116 cells with indicated genotypes transfected with control ( − ) , RPA1 ( 0 . 2 nM concentration [ 17 ] ) or RAD51 siRNAs ( Fig EV4B ) and labeled with CldU and IdU as in Fig 3B ( bars , mean ; n = 400 fibers , pooled from two independent experiments , per condition ) .

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F. Replication fork speeds in HCT116 cells with indicated genotypes transfected with control (−), RPA1 (0.2 nM concentration [17]) or RAD51 siRNAs (Fig EV4B) and labeled with CldU and IdU as in Fig 3B (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


G . Fork symmetry among DNA fibers in ( F ) ( bars , mean ; n = 50 bidirectional forks , pooled from two independent experiments , per condition ) .

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G. Fork symmetry among DNA fibers in (F) (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition).


H . New origin firing among DNA fibers in ( F ) ( bars , mean ; 20 fields of view quantified per condition ; n = 2 independent experiments ) .

H. New origin firing among DNA fibers in (F) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).


B . Replication fork degradation ( IdU / CldU ratio ) in U2OS cells transfected with indicated siRNAs and processed as in ( A ) ( bars , mean ; n = 200 fibers , pooled from two independent experiments , per condition ) .

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B. Replication fork degradation (IdU/CldU ratio) in U2OS cells transfected with indicated siRNAs and processed as in (A) (bars, mean; n=200 fibers, pooled from two independent experiments, per condition).


C . As in ( B ) , except fork degradation was analyzed in U2OS / GFP - RADX cell lines .

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C. As in (B), except fork degradation was analyzed in U2OS/GFP-RADX cell lines.


D . U2OS or U2OS / GFP - RADX cells transfected with indicated siRNAs and exposed to HU for 4 h were co - immunostained with RPA1 and γ - H2AX antibodies and analyzed by QIBC . Proportion of cells displaying maximal RPA chromatin loading accompanied by H2AX hyperphosphorylation ( Fig EV5A ) , reflecting replication fork catastrophe [ 17 ] , is indicated ( bars , mean ; n = 5 independent experiments ( ≥ 3000 cells analyzed per condition ) ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , unpaired t - test ) .

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D. U2OS or U2OS/GFP-RADX cells transfected with indicated siRNAs and exposed to HU for 4 h were co-immunostained with RPA1 and γ-H2AX antibodies and analyzed by QIBC. Proportion of cells displaying maximal RPA chromatin loading accompanied by H2AX hyperphosphorylation (Fig EV5A), reflecting replication fork catastrophe [17], is indicated (bars, mean; n=5 independent experiments (≥3000 cells analyzed per condition); *P≤0.05,**P≤0.01,***P≤0.001,****P≤0.0001, unpaired t-test).


E . As in ( D ) , but using U2OS or U2OS / Super - RPA cells exposed or not to HU ( n = 6 independent experiments ) . See also Fig EV5B .

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E. As in (D), but using U2OS or U2OS/Super-RPA cells exposed or not to HU (n=6 independent experiments). See also Fig EV5B.


F . Clonogenic survival of U2OS cells transfected with indicated siRNAs and subjected to different CPT doses for 24 h ( mean ± SEM ; n = 3 independent experiments ) . LQ ( Linear Quadratic ) model was fitted to the fractional survival data , using non - linear least square method . Overlap between confidence intervals of the fitting coefficients was used to evaluate the statistical difference between cell survival after siRADX and siCTRL treatment ( * No overlap of 95 % confidence interval with siCTRL ) .

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F. Clonogenic survival of U2OS cells transfected with indicated siRNAs and subjected to different CPT doses for 24 h (mean±SEM;n=3 independent experiments). LQ (Linear Quadratic) model was fitted to the fractional survival data, using non-linear least square method. Overlap between confidence intervals of the fitting coefficients was used to evaluate the statistical difference between cell survival after siRADX and siCTRL treatment (*No overlap of 95% confidence interval with siCTRL).


G . As in ( F ) , except cells were treated with indicated doses of HU ( mean ± SEM ; n = 3 independent experiments ) .

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G. As in (F), except cells were treated with indicated doses of HU (mean±SEM;n=3 independent experiments).


H . As in ( F ) , using U2OS and U2OS / Super - RPA cells ( mean ± SEM ; n = 3 independent experiments ) .

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H. As in (F), using U2OS and U2OS/Super-RPA cells (mean±SEM;n=3 independent experiments).


A - B Time dependent effects of ingesting 0 , 0 . 1 , 1 , 5 , 10 , 30 , and 50 mM vitamin C without any further nutrition on the survival of wild - type control flies ( w1118 ) ( A ) males ( B ) females , n = 6 . Kaplan - Meier survival analyses were performed to compare the survival rates of agar only and fed conditions at different concentrations of vitamin

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A-B Time dependent effects of ingesting 0, 0.1, 1, 5, 10, 30, and 50 mM vitamin C without any further nutrition on the survival of wild-type control flies (w1118) (A) males (B) females, n = 6. Kaplan-Meier survival analyses were performed to compare the survival rates of agar only and fed conditions at different concentrations of vitamin C


F Electrophysiology mapping of 31 sensilla with control to 50 mM vitamin C , n = 10 - 18 .

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F Electrophysiology mapping of 31 sensilla with control to 50 mM vitamin C, n = 10-18.


G Tip recordings from the L4 and L6 sensilla of specific GRN - ablated flies with 50 mM vitamin C . + / - indicates the presence or absence of the transgene , respectively , n = 10 - 13 .

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G Tip recordings from the L4 and L6 sensilla of specific GRN-ablated flies with 50 mM vitamin C. +/- indicates the presence or absence of the transgene, respectively, n = 10-13.


C Genetically recovered flies of Ir25a2 and Ir76b1 driven by their own GAL4 or Gr64f - GAL4 with the wild - type cDNA transgene . + / - indicate the presence or absence of the transgene , respectively , n = 10 - 15 .

C Genetically recovered flies of Ir25a2 and Ir76b1 driven by their own GAL4 or Gr64f-GAL4 with the wild-type cDNA transgene. +/- indicate the presence or absence of the transgene, respectively, n = 10-15.


D Genetically recovered flies of Gr5a , Gr61a , Gr64b , Gr64c , and Gr64e mutants driven by their own GAL4 or Gr64f - GAL4 . + / - indicate the presence or absence of the transgene , respectively , n = 10 - 21 .

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D Genetically recovered flies of Gr5a, Gr61a, Gr64b, Gr64c, and Gr64e mutants driven by their own GAL4 or Gr64f-GAL4. +/- indicate the presence or absence of the transgene, respectively, n = 10-21.


D Behavioral rescue of Gr5a ∆ 5 , Gr61a1 , Gr64bLEXA , Gr64cLEXA , and Gr64eLEXA deficits in the vitamin C attraction via the expression of the respective UAS transgenes under the control of their specific GAL4s or Gr64f - GAL4 , n = 6 - 8 .

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D Behavioral rescue of Gr5a∆5, Gr61a1, Gr64bLEXA, Gr64cLEXA, and Gr64eLEXA deficits in the vitamin C attraction via the expression of the respective UAS transgenes under the control of their specific GAL4s or Gr64f-GAL4, n = 6-8.


( C ) Western blot showing that Hel2 ( 1 - 315 ) is defective in RQC but not in NGD . The arrest products derived from the R ( CGN ) 12 reporter in ltn1 ∆ cells expressing truncated Hel2 mutant protein were detected with an anti - GFP antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot showing that Hel2(1-315) is defective in RQC but not in NGD. The arrest products derived from the R(CGN)12 reporter in ltn1∆ cells expressing truncated Hel2 mutant protein were detected with an anti-GFP antibody.


( D ) Northern blot showing the 5 ' NGD - IM derived from the R ( CGN ) 12 reporter in ski2 ∆ cells expressing the indicated Hel2 mutant proteins . 5 ' NGD - IMs were detected with a DIG - labelled GFP probe .

O O O O O O O O O O O O O O O O O O O O O O O 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) Northern blot showing the 5' NGD-IM derived from the R(CGN)12 reporter in ski2∆ cells expressing the indicated Hel2 mutant proteins. 5' NGD-IMs were detected with a DIG-labelled GFP probe.


( E ) Primer extension mapping of 5 ' ends of 3 ' NGD - intermediates in Hel2 - WT or Hel2 ( 1 - 315 ) mutant cells at nucleotide resolution . . The primer extension samples were analysed using 5 % TBE - Urea - PAGE and detected by fluorescence . Non - specific reverse transcription ( ReTr ) products are indicated by asterisks .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primer extension mapping of 5' ends of 3' NGD-intermediates in Hel2-WT or Hel2(1-315) mutant cells at nucleotide resolution. . The primer extension samples were analysed using 5% TBE-Urea-PAGE and detected by fluorescence. Non-specific reverse transcription (ReTr) products are indicated by asterisks.


( A ) Northern blot analysis demonstrating that K63 - linked ubiquitination was required for an endonucleolytic cleavage by R ( CGN ) 12 . The ski2 ∆ UB - WT and ski2 ∆ ub - K63R cells were transformed with the R ( CGN ) 12 reporter , and total RNA samples were separated by 2 % agarose / MOPS gel . 5 ' NGD - IMs in the cells were detected as in Fig 1D .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Northern blot analysis demonstrating that K63-linked ubiquitination was required for an endonucleolytic cleavage by R(CGN)12. The ski2∆UB-WT and ski2∆ub-K63R cells were transformed with the R(CGN)12 reporter, and total RNA samples were separated by 2% agarose/MOPS gel. 5'NGD-IMs in the cells were detected as in Fig 1D.


( B ) Western blot showing that K63 - linked ubiquitination was required for RQC . Protein samples from UB - WT , UB - WT ltn1 ∆ , ub - K63R , ub - K63R ltn1 ∆ cells expressing the GFP - R ( CGN ) 12 - HIS3 reporter were subjected to Western blot analysis using an anti - GFP antibody to detect the arrest products . Note the accumulation of RQC - specific CAT - tails in lane 2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot showing that K63-linked ubiquitination was required for RQC. Protein samples from UB-WT, UB-WT ltn1∆, ub-K63R, ub-K63R ltn1∆ cells expressing the GFP-R(CGN)12-HIS3 reporter were subjected to Western blot analysis using an anti-GFP antibody to detect the arrest products. Note the accumulation of RQC-specific CAT-tails in lane 2.


( C ) Western blot analysis demonstrating polyubiquitination of uS10 : Affinity - tagged Hel2 - Flag - TEV - ProteinA ( FTP ) was co - purified with ribosomes harbouring HA - tagged wild type uS10 ( uS10 - 3HA ribosomes ) or uS10 mutated in its ubiquitination sites ( K6 / 8R ) . Western blots of whole protein extracts were performed using an anti - HA antibody to detect polyubiquitinated uS10 - 3HA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis demonstrating polyubiquitination of uS10: Affinity-tagged Hel2-Flag-TEV-ProteinA (FTP) was co-purified with ribosomes harbouring HA-tagged wild type uS10 (uS10-3HA ribosomes) or uS10 mutated in its ubiquitination sites (K6/8R). Western blots of whole protein extracts were performed using an anti-HA antibody to detect polyubiquitinated uS10-3HA.


( D ) Western blot analysis showing that uS10 - polyubiquitination in Hel2 - bound ribosomes was mainly K63 - linked : Affinity tagged Hel2 ( Hel2 - ProteinA - TEV - His6 ; PTH - Hel2 ) was co - purified with ribosomes from a strain expressing wild type ubiquitin ( UB - WT ) or K63R mutant ubiquitin ( ub - K63R ) and HA - tagged uS10 ( uS10 - 3HA ) . Western blot analysis of whole protein extracts was performed using anti - HA antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis showing that uS10-polyubiquitination in Hel2-bound ribosomes was mainly K63-linked: Affinity tagged Hel2(Hel2-ProteinA-TEV-His6; PTH-Hel2) was co-purified with ribosomes from a strain expressing wild type ubiquitin (UB-WT) or K63R mutant ubiquitin (ub-K63R) and HA-tagged uS10 (uS10-3HA). Western blot analysis of whole protein extracts was performed using anti-HA antibody.


( E ) Western blot of in vitro polyubiquitination assays of uS10 . Reactions were performed with the indicated components including His - tagged ubiquitin and several ubiquitin mutants . Polyubiquitinated HA - tagged uS10 ( uS10 - 3HA ) was detected by Western blot analysis using an anti - HA antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot of in vitro polyubiquitination assays of uS10. Reactions were performed with the indicated components including His-tagged ubiquitin and several ubiquitin mutants. Polyubiquitinated HA-tagged uS10 (uS10-3HA) was detected by Western blot analysis using an anti-HA antibody.


( F - H ) Northern blot analysis of NGD - cleavage sites in the absence of uS10 ubiquitination or RQT complex components : The full - length GFP - R ( CGN ) 12 - FLAG - HIS3 mRNA and 5 ' NGD - intermediates ( 5 ' NGD - IM ) or 3 ' NGD - intermediates ( 3 ' NGD - IM ) were detected in the indicated mutant cells by Northern blotting with DIG - labelled probes . 5 ' NGD - intermediates were detected by DIG - labelled GFP probe and 3 ' NGD - intermediates were detected by the DIG - labelled HIS3 probe . SCR1 was used as loading control . FL = full - length . Note the upstream shift of NGD cleavage sites in lanes F4 , G4 , H6 , and H8 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) Northern blot analysis of NGD-cleavage sites in the absence of uS10 ubiquitination or RQT complex components: The full-length GFP-R(CGN)12-FLAG-HIS3 mRNA and 5' NGD-intermediates (5' NGD-IM) or 3' NGD-intermediates (3' NGD-IM) were detected in the indicated mutant cells by Northern blotting with DIG-labelled probes. 5' NGD-intermediates were detected by DIG-labelled GFP probe and 3' NGD-intermediates were detected by the DIG-labelled HIS3 probe. SCR1 was used as loading control. FL = full-length. Note the upstream shift of NGD cleavage sites in lanes F4, G4, H6, and H8.


( I ) Mapping of 5 ' ends of 3 ' NGD - intermediates as demonstrated in Fig 1E . Non - specific reverse transcription ( ReTr ) products are indicated by asterisks . Note that both uS10 ubiquitination and Slh1 / Rqt2 were required for an endonucleolytic cleavage within the road - blocked ribosome ( X1 - X4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mapping of 5' ends of 3' NGD-intermediates as demonstrated in Fig 1E. Non-specific reverse transcription (ReTr) products are indicated by asterisks. Note that both uS10 ubiquitination and Slh1/Rqt2 were required for an endonucleolytic cleavage within the road-blocked ribosome (X1-X4).


Both RQC and NGDRQC + are triggered by a ( CGA - CCG ) dicodon containing arrest sequence in vivo . ( B ) Western blot showing the arrest products derived from the GFP - X - FLAG - HIS3 reporter . Translation products were detected by western blot using an anti - GFP antibody .

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

Both RQC and NGDRQC+ are triggered by a (CGA-CCG) dicodon containing arrest sequence in vivo. (B) Western blot showing the arrest products derived from the GFP-X-FLAG-HIS3 reporter. Translation products were detected by western blot using an anti-GFP antibody.


Both RQC and NGDRQC + are triggered by a ( CGA - CCG ) dicodon containing arrest sequence in vivo . ( C ) Northern blot for the 5 ' NGD - IM derived from the ( CGA - CCG ) reporter in ski2 ∆ cells . 5 ' - NGD - IMs were detected with a DIG - labelled GFP probe . SCR1 was used as a load control .

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

Both RQC and NGDRQC+ are triggered by a (CGA-CCG) dicodon containing arrest sequence in vivo. (C) Northern blot for the 5' NGD-IM derived from the (CGA-CCG) reporter in ski2∆ cells. 5'-NGD-IMs were detected with a DIG-labelled GFP probe. SCR1 was used as a load control.


( D ) Western blot of test translations using the ( CGA - CCG ) dicodon stalling mRNA reporter shown in ( A ) . The mRNA reporter was added to a yeast in vitro translation extract obtained from a ski2 ∆ uS10 - 3HA strain . Expression of the translation products ( free His - and V5 tagged truncated uL4 protein and the same protein attached to tRNA ) was visualized with an anti - V5 antibody .

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(D) Western blot of test translations using the (CGA-CCG) dicodon stalling mRNA reporter shown in (A). The mRNA reporter was added to a yeast in vitro translation extract obtained from a ski2∆uS10-3HA strain. Expression of the translation products (free His- and V5 tagged truncated uL4 protein and the same protein attached to tRNA) was visualized with an anti-V5 antibody.


( E ) Sucrose gradient fractions ( top ) and western blot analysis ( bottom ) of the ( CGA - CCG ) dicodon - stalled RNC samples . After the translation reaction , the RNCs were affinity - purified as indicated in ( C ) . The eluate was loaded on a 10 - 50 % sucrose gradient and fractionated . Each collected fraction was analysed using anti - HA antibody to detect uS10 - HA . Note that disomes are preferentially polyubiquitinated over monosomes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Sucrose gradient fractions (top) and western blot analysis (bottom) of the (CGA-CCG) dicodon-stalled RNC samples. After the translation reaction, the RNCs were affinity-purified as indicated in (C). The eluate was loaded on a 10-50 % sucrose gradient and fractionated. Each collected fraction was analysed using anti-HA antibody to detect uS10-HA. Note that disomes are preferentially polyubiquitinated over monosomes.


( A ) Western blot analysis after overexpression of Hel2 in strains expressing HA - tagged ribosomal proteins using an anti - HA antibody . Note the increase of the polyubiquitinated eS7 as well as uS10 and uS3 .

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(A) Western blot analysis after overexpression of Hel2 in strains expressing HA-tagged ribosomal proteins using an anti-HA antibody. Note the increase of the polyubiquitinated eS7 as well as uS10 and uS3.


( C ) Western blot analysis showing the role of Hel2 and Not4 in eS7A ubiquitination : Both copies of eS7 ( eS7A and eS7B ) were deleted and transformed with a plasmid containing HA - tagged eS7A ( eS7 - WT ) or eS7A mutated in the four potential ubiquitination sites ( 4KR ) . Whole protein extracts we obtained from eS7AΔeS7BΔ cells or cells with an additional deletion in E3 ligases Hel2 and Not4 ( hel2Δ and not4Δ ) . Note that the four mutated lysine residues were responsible for Not4 - dependent monoubiquitination .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis showing the role of Hel2 and Not4 in eS7A ubiquitination: Both copies of eS7 (eS7A and eS7B) were deleted and transformed with a plasmid containing HA-tagged eS7A (eS7-WT) or eS7A mutated in the four potential ubiquitination sites (4KR). Whole protein extracts we obtained from eS7AΔeS7BΔ cells or cells with an additional deletion in E3 ligases Hel2 and Not4 (hel2Δ and not4Δ). Note that the four mutated lysine residues were responsible for Not4-dependent monoubiquitination.


( D ) Western blot showing that Not4 was required for Hel2 - mediated polyubiquitination of eS7 in Hel2 - bound ribosomal complexes : Cells expressing HA - tagged eS7A or the eS7 - 4KR mutant , as well as PTH - tagged Hel2 in eS7AΔeS7BΔ and eS7AΔeS7BΔnot4Δ background were probed for eS7 - ubiquitination . Either cell lysates or affinity - purified Hel2 - ribosome complexes were used . The levels of the ubiquitinated eS7A in the lysates and in the affinity - purified samples with PTH - Hel2 were determined by Western blot analysis using an anti - HA antibody to detect eS7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot showing that Not4 was required for Hel2-mediated polyubiquitination of eS7 in Hel2-bound ribosomal complexes: Cells expressing HA-tagged eS7A or the eS7-4KR mutant, as well as PTH-tagged Hel2 in eS7AΔeS7BΔ and eS7AΔeS7BΔnot4Δ background were probed for eS7-ubiquitination. Either cell lysates or affinity-purified Hel2-ribosome complexes were used. The levels of the ubiquitinated eS7A in the lysates and in the affinity-purified samples with PTH-Hel2 were determined by Western blot analysis using an anti-HA antibody to detect eS7.


( E ) Western blotting of in vitro ubiquitination assays of eS7A . The reactions were performed with the indicated components including His - tagged ubiquitin . Ribosomes were purified from Hel2Δ cells expressing HA - tagged eS7A . Polyubiquitinated HA - tagged eS7A ( eS7A - 3HA ) was detected by Western blot analysis using an anti - HA antibody . We observed Hel2 - mediated polyubiquitination of the monoubiquitinated eS7A .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blotting of in vitro ubiquitination assays of eS7A. The reactions were performed with the indicated components including His-tagged ubiquitin. Ribosomes were purified from Hel2Δ cells expressing HA-tagged eS7A. Polyubiquitinated HA-tagged eS7A (eS7A-3HA) was detected by Western blot analysis using an anti-HA antibody. We observed Hel2-mediated polyubiquitination of the monoubiquitinated eS7A.


( F ) Western blot analysis of eS7A in vitro ubiquitination assays showing that Hel2 - mediated polyubiquitination of eS7 was mainly K63 - linked and required Not4 - dependent monoubiquitination : These assays were performed similarly as described in ( E ) except yeast strains were lacking Not4 ( not4Δ ) . Reactions were performed with the indicated components including His - tagged ubiquitin and several ubiquitin mutants and eS7A - ubiquitination was monitored using an anti - HA antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of eS7A in vitro ubiquitination assays showing that Hel2-mediated polyubiquitination of eS7 was mainly K63-linked and required Not4-dependent monoubiquitination: These assays were performed similarly as described in (E) except yeast strains were lacking Not4 (not4Δ). Reactions were performed with the indicated components including His-tagged ubiquitin and several ubiquitin mutants and eS7A-ubiquitination was monitored using an anti-HA antibody.


( A - C ) Northern blots probing for the 5 ' NGD - IM in mutant cells expressing the R ( CGN ) 12 reporter . ( A ) 5 ' NGD - IM detection in not4 ∆ ski2 ∆ , hel2 ∆ ski2 ∆ , slh1 ∆ ski2 ∆ , not4 ∆ slh1 ∆ ski2 ∆ . Quantification of full length and the 5 ' NGD - IM relative to the loading control ( SCR1 ) is given below . Note the size difference of 5 ' - NGD IMs resulting from NGDRQC + and NGDRQC - and that the shorter 5 ' - NGD intermediate ( representing intermediates of the NGDRQC - pathway ) was reduced in not4 ∆ slh1 ∆ ski2 ∆ mutant cells .

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

(A-C) Northern blots probing for the 5' NGD-IM in mutant cells expressing the R(CGN)12 reporter. (A) 5' NGD-IM detection in not4∆ski2∆, hel2∆ski2∆, slh1∆ski2∆, not4∆ slh1∆ski2∆. Quantification of full length and the 5'NGD-IM relative to the loading control (SCR1) is given below. Note the size difference of 5'-NGD IMs resulting from NGDRQC+ and NGDRQC- and that the shorter 5'-NGD intermediate (representing intermediates of the NGDRQC- pathway) was reduced in not4∆slh1∆ski2∆ mutant cells.


Northern blots probing for the 5 ' NGD - IM in mutant cells expressing the R ( CGN ) 12 reporter . ( B ) Northern blot as in ( A ) except uS10 - WTski2 ∆ or uS10 - K6 / 8Rski2 ∆ mutant cells with or without a deletion of Not4 ( not4 ∆ ) were used . Note that the shorter 5 ' - NGD intermediates are also reduced in ski2 ∆ not4 ∆ uS10 - K6 / 8R cells .

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

Northern blots probing for the 5' NGD-IM in mutant cells expressing the R(CGN)12 reporter. (B) Northern blot as in (A) except uS10-WTski2∆ or uS10-K6/8Rski2∆ mutant cells with or without a deletion of Not4 (not4∆) were used. Note that the shorter 5'-NGD intermediates are also reduced in ski2∆not4∆uS10-K6/8R cells.


Northern blots probing for the 5 ' NGD - IM in mutant cells expressing the R ( CGN ) 12 reporter . ( C ) Cells expressing HA - tagged eS7A or the eS7A - 4KR mutant in a ski2ΔeS7AΔeS7BΔ or ski2ΔeS7AΔeS7BΔslh1Δ backgrounds were probed for 5 ' - NGD intermediates by Northern blotting . Note that the four mutated lysine residues in eS7 were responsible for NGDRQC - in the R ( CGN ) 12 reporter .

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

Northern blots probing for the 5' NGD-IM in mutant cells expressing the R(CGN)12 reporter. (C) Cells expressing HA-tagged eS7A or the eS7A-4KR mutant in a ski2ΔeS7AΔeS7BΔ or ski2ΔeS7AΔeS7BΔslh1Δ backgrounds were probed for 5'-NGD intermediates by Northern blotting. Note that the four mutated lysine residues in eS7 were responsible for NGDRQC- in the R(CGN)12 reporter.


( D ) Western blot analysis showing that K83 and K84 of eS7A are main target sites for Hel2 and Not4 - mediated ubiquitination : Similarly , as in Fig 6C , eS7AΔeS7BΔ mutant cells were used and wild type and single mutants of HA - tagged eS7A were expressed . Ubiquitinated eS7 - HA was monitored using an anti - HA antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis showing that K83 and K84 of eS7A are main target sites for Hel2 and Not4-mediated ubiquitination: Similarly, as in Fig 6C, eS7AΔeS7BΔ mutant cells were used and wild type and single mutants of HA-tagged eS7A were expressed. Ubiquitinated eS7-HA was monitored using an anti-HA antibody.


( E ) Northern Blot analysis probing for the levels of 5 ' NGD - IM derived from the R ( CGN ) 12 reporter expressed in eS7AΔeS7BΔski2Δ cells and HA - tagged eS7 or eS7 mutants . We observed , that ubiquitination at K83 or K84 of eS7A is mainly responsible for NGDRQC - .

(E) Northern Blot analysis probing for the levels of 5' NGD-IM derived from the R(CGN)12 reporter expressed in eS7AΔeS7BΔski2Δ cells and HA-tagged eS7 or eS7 mutants. We observed, that ubiquitination at K83 or K84 of eS7A is mainly responsible for NGDRQC-.


( F ) The levels of polyubiquitinated eS7A were increased by the overproduction of wild - type Hel2 ( FL ) but neither Hel2ΔRING nor Hel2 ( 1 - 315 ) mutants . eS7A - 3HAhel2Δ mutant cells harbouring the indicated plasmids expressing wild - type Hel2 ( FL ) , Hel2ΔRING or Hel2 ( 1 - 315 ) mutant proteins were harvested . Protein samples were analysed by Western blotting with an anti - HA ( Top panel ) or anti - FLAG antibody ( Bottom panel ) .

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(F) The levels of polyubiquitinated eS7A were increased by the overproduction of wild-type Hel2 (FL) but neither Hel2ΔRING nor Hel2(1-315) mutants. eS7A-3HAhel2Δ mutant cells harbouring the indicated plasmids expressing wild-type Hel2 (FL), Hel2ΔRING or Hel2(1-315) mutant proteins were harvested. Protein samples were analysed by Western blotting with an anti-HA (Top panel) or anti-FLAG antibody (Bottom panel).


( G ) Hel2 ( 1 - 315 ) polyubiquitinates eS7A but not uS10 . Western blotting after an in vitro ubiquitination assay using the Hel2 - ( 1 - 315 ) mutant and purified ribosomes containing HA - tagged uS10 .

O O O O O O O O O O O O O O O O O O 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) Hel2(1-315) polyubiquitinates eS7A but not uS10. Western blotting after an in vitro ubiquitination assay using the Hel2-(1-315) mutant and purified ribosomes containing HA-tagged uS10.


Decreased Drp1S616 phosphorylation in PINK1 - null HEK293 cells Lysates of PINK1 - null ( PINK1KO ) and parkin - null ( parkinKO ) HEK293 cells ( a ) and their matched wildtype controls ( WT ) were immuno - detected with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) , phospho ( Ser637 ) - Drp1 ( pS637 ) or Drp1 . β - actin was detected as a loading control . Quantitation of pS616 / Drp1 for each experiment is shown Two independent PINK1KO and parkinKO HEK293 cell lines ( 1 , 2 ) were shown . Student ' s test . * p < 0 . 05 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Decreased Drp1S616 phosphorylation in PINK1-null HEK293 cells Lysates of PINK1-null (PINK1KO) and parkin-null (parkinKO) HEK293 cells (a) and their matched wildtype controls (WT) were immuno-detected with antibodies against either phospho (Ser616)-Drp1 (pS616), phospho (Ser637)-Drp1 (pS637) or Drp1. β-actin was detected as a loading control. Quantitation of pS616/Drp1 for each experiment is shown Two independent PINK1KO and parkinKO HEK293 cell lines (1, 2) were shown. Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


Decreased Drp1S616 phosphorylation in PINK1 - null MEF cells Lysates of PINK1 - null ( PINK1KO ) and parkin - null ( parkinKO ) MEF cells ( c ) and their matched wildtype controls ( WT ) were immuno - detected with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) , phospho ( Ser637 ) - Drp1 ( pS637 ) or Drp1 . β - actin was detected as a loading control . Quantitation of pS616 / Drp1 for each experiment is shown Student ' s test . * p < 0 . 05 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent 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 O O O O O O O O O O O O O O O O O O O O O O O O

Decreased Drp1S616 phosphorylation in PINK1-null MEF cells Lysates of PINK1-null (PINK1KO) and parkin-null (parkinKO) MEF cells (c) and their matched wildtype controls (WT) were immuno-detected with antibodies against either phospho (Ser616)-Drp1 (pS616), phospho (Ser637)-Drp1 (pS637) or Drp1. β-actin was detected as a loading control. Quantitation of pS616/Drp1 for each experiment is shown Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


Decreased Drp1S616 phosphorylation in PINK1 - null mouse substantial nigra tissues . Lysates of PINK1 - null ( PINK1KO ) and parkin - null ( parkinKO ) mouse substantial nigra tissues ( e ) , and their matched wildtype controls ( WT ) were immuno - detected with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) , phospho ( Ser637 ) - Drp1 ( pS637 ) or Drp1 . β - actin was detected as a loading control . Quantitation of pS616 / Drp1 for each experiment is shown Two independent PINK1KO and parkinKO substantial nigra from two separate PINK1KO and parkinKO mice ( 1 , 2 ) were shown . Student ' s test . * p < 0 . 05 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Decreased Drp1S616 phosphorylation in PINK1-null mouse substantial nigra tissues. Lysates of PINK1-null (PINK1KO) and parkin-null (parkinKO) mouse substantial nigra tissues (e), and their matched wildtype controls (WT) were immuno-detected with antibodies against either phospho (Ser616)-Drp1 (pS616), phospho (Ser637)-Drp1 (pS637) or Drp1. β-actin was detected as a loading control. Quantitation of pS616/Drp1 for each experiment is shown Two independent PINK1KO and parkinKO substantial nigra from two separate PINK1KO and parkinKO mice (1, 2) were shown. Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( g , h ) . Overexpression of PINK1 , not PINK1 kinase mutants , reverses Drp1S616 phosphorylation in PINK1KO HEK293 cells . Lysates of PINK1KO and PINK1WT control ( WT ) HEK293 cells expressing PINK1 variants were immuno - detected with an anti - phospho ( Ser616 ) - Drp1 antibody ( pS616 ) , an anti - Drp1 antibody ( Drp1 ) and an anti - PINK1 antibody ( PINK1 ) . Cells with mock transfection ( Blank ) and transfected with an empty plasmid ( 3 . 1 ) were included as controls ( g ) . Quantitation of pS616 / Drp1 is shown ( h ) . PINK1 : wildtype PINK1 ; D384N : PINK1 kinase - dead mutant ; G309D , pathogenic PINK1 mutant ; ∆ 110 : PINK1 mito - target deficient mutant . Student ' s test . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(g, h). Overexpression of PINK1, not PINK1 kinase mutants, reverses Drp1S616 phosphorylation in PINK1KO HEK293 cells. Lysates of PINK1KO and PINK1WT control (WT) HEK293 cells expressing PINK1 variants were immuno-detected with an anti-phospho(Ser616)-Drp1 antibody (pS616), an anti-Drp1 antibody (Drp1) and an anti-PINK1 antibody (PINK1). Cells with mock transfection (Blank) and transfected with an empty plasmid (3.1) were included as controls (g). Quantitation of pS616/Drp1 is shown (h). PINK1: wildtype PINK1; D384N: PINK1 kinase-dead mutant; G309D, pathogenic PINK1 mutant; ∆110: PINK1 mito-target deficient mutant. Student's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( i , j ) . PINK1 deficiency does not affect expression and kinase activity of CDK1 and ERK1 / 2 . Lysates of PINK1KO and PINK1WT control ( WT ) HEK293 cells were immuno - detected with an anti - phospho ( Ser616 ) - Drp1 antibody ( pS616 ) , an anti - Drp1 antibody ( Drp1 ) , an anti - PINK1 antibody ( PINK1 ) , an anti - CDK1 antibody ( CDK1 ) , an anti - p44 / p42 MAPK antibody ( ERK1 / 2 ) , an anti - phospho ( Thr161 ) - CDK1 antibody ( pThr161 ) , and an anti - phospho ( Thr202 / 204 ) - p44 / p42 MAPK antibody ( pThr202 / 204 ) . β - actin was detected as a loading control ( i ) . Quantitation of CDK1Thr161 phosphorylation ( pThr161 ) and ERK1 / 2Thr202 / 204 phosphorylation ( pThr202 / 204 ) was shown ( j ) . Student ' s test . ns : no significance . Data was presented as mean ± SEM of three independent 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(i, j). PINK1 deficiency does not affect expression and kinase activity of CDK1 and ERK1/2. Lysates of PINK1KO and PINK1WT control (WT) HEK293 cells were immuno-detected with an anti-phospho(Ser616)-Drp1 antibody (pS616), an anti-Drp1 antibody (Drp1), an anti-PINK1 antibody (PINK1), an anti-CDK1 antibody(CDK1), an anti-p44/p42 MAPK antibody (ERK1/2), an anti-phospho(Thr161)-CDK1 antibody (pThr161), and an anti-phospho(Thr202/204)-p44/p42 MAPK antibody (pThr202/204). β-actin was detected as a loading control (i). Quantitation of CDK1Thr161 phosphorylation (pThr161) and ERK1/2Thr202/204 phosphorylation (pThr202/204) was shown (j). Student's test. ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( l , m ) . PINK1 phosphorylates Drp1S616 in vitro . Recombinant TcPINK1 was incubated with either GST - Drp1518 - 736 ( WT ) or GST - Drp1518 - 736 , S616A ( S616A ) in the presence ( + ) / absence ( - ) of ATP . After heat inactivation , reactions were treated with ( + ) / without ( - ) shrimp alkaline phosphatase ( SAP ) . Phosphorylation of Drp1S616 ( pS616 ) was immuno - detected ( l , upper panel ) . Total Drp1 ( l , middle panel ) and protein loading stained with Coomassie blue staining ( l , lower panel ) were shown . Quantitation of pS616 / Drp1 ratio was shown ( m ) . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 . Data was presented as mean ± SEM of three independent 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 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 O O O O O O O O O O O O O O O O O O

(l, m). PINK1 phosphorylates Drp1S616 in vitro. Recombinant TcPINK1 was incubated with either GST-Drp1518-736 (WT) or GST-Drp1518-736, S616A (S616A) in the presence (+)/absence (-) of ATP. After heat inactivation, reactions were treated with (+)/without (-) shrimp alkaline phosphatase (SAP). Phosphorylation of Drp1S616 (pS616) was immuno-detected (l, upper panel). Total Drp1 (l, middle panel) and protein loading stained with Coomassie blue staining (l, lower panel) were shown. Quantitation of pS616/Drp1 ratio was shown (m). One-way ANOVA followed with Tukey's test. ***p<0.001. Data was presented as mean ± SEM of three independent experiments.


( n , o ) . Label - free relative quantitative mass spectrum analysis of Drp1S616 phosphorylation . Representative spectra for the Drp1 phospho - peptide ( S616 ) SKPIPMPA ( p ) SPQK and non - phospho - peptide SKPIPMPA ( p ) SPQK . Spectra obtained for peptides from PINK1WT HEK293 cells are shown ( n ) . The relative ratios of the phospho - peptides containing phosphor - Ser616 to peptides containing Ser616 from PINK1WT and PINK1KO HEK293 samples were calculated and plotted ( o ) .

(n, o). Label-free relative quantitative mass spectrum analysis of Drp1S616 phosphorylation. Representative spectra for the Drp1 phospho-peptide (S616) SKPIPMPA(p)SPQK and non-phospho-peptide SKPIPMPA(p)SPQK. Spectra obtained for peptides from PINK1WT HEK293 cells are shown (n). The relative ratios of the phospho-peptides containing phosphor-Ser616 to peptides containing Ser616 from PINK1WT and PINK1KO HEK293 samples were calculated and plotted (o).


( p , q ) . Km and Kcat for PINK1 - mediated phosphorylation of ubiquitin and Drp1 . In vitro kinase assays were performed with different concentrations of ubiquitin ( p ) or Drp1 ( q ) with TcPINK1 at fixed ATP concentration ( 500 μM ) . Results were analyzed on phos ‐ tag gels and modeled to the Michaelis - Menten equation . The given graphs represent global fits to data collected from 3 sets of reactions for both Ub and Drp1 performed independently . Km : michaelis constant . Kcat : catalytic constant . Bars represent the mean ± SEM .

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

(p, q). Km and Kcat for PINK1-mediated phosphorylation of ubiquitin and Drp1. In vitro kinase assays were performed with different concentrations of ubiquitin (p) or Drp1 (q) with TcPINK1 at fixed ATP concentration (500 μM). Results were analyzed on phos‐tag gels and modeled to the Michaelis-Menten equation. The given graphs represent global fits to data collected from 3 sets of reactions for both Ub and Drp1 performed independently. Km: michaelis constant. Kcat: catalytic constant. Bars represent the mean ± SEM.


( a , b ) . Decreased Drp1S616 phosphorylation in PINK1 - null primary neurons . Lysates of DIV6 neurons generated from PINK1KO and PINK1WT control mice were immune - detected with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) or Drp1 . Tuj1 was detected as a loading control ( a ) . Quantitation of pS616 / Drp1 for each experiment is shown ( b ) . Student ' s test . * * p < 0 . 01 . Data was presented as mean ± SEM of three independent 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

(a, b). Decreased Drp1S616 phosphorylation in PINK1-null primary neurons. Lysates of DIV6 neurons generated from PINK1KO and PINK1WT control mice were immune-detected with antibodies against either phospho (Ser616)-Drp1 (pS616) or Drp1. Tuj1 was detected as a loading control (a). Quantitation of pS616/Drp1 for each experiment is shown (b). Student's test. **p<0.01. Data was presented as mean ± SEM of three independent experiments.


( c , d ) . TEM analysis of mitochondria in mouse substantial nigra . Sections of substantial nigra from 18 - month - old PINK1KO and PINK1WT control mice were analyzed under TEM ( c , left panels , scale bar = 2 µm ) . Amplified images of mitochondria from dot line frames gated region are shown ( c , right panels , scale bar = 1 µm ) . Asterisks indicate abnormal elongated mitochondria . A quantitative analysis of mitochondria with perimeter > 2µm is shown ( d ) . Over 300 identifiable mitochondria ( cristae and / or double membrane ) per mice were randomly and blindly selected for perimeter measurement . Student ' s test . * p < 0 . 05 . Data was presented as mean ± SEM of three independent 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c, d). TEM analysis of mitochondria in mouse substantial nigra. Sections of substantial nigra from 18-month-old PINK1KO and PINK1WT control mice were analyzed under TEM (c, left panels, scale bar=2 µm). Amplified images of mitochondria from dot line frames gated region are shown (c, right panels, scale bar=1 µm). Asterisks indicate abnormal elongated mitochondria. A quantitative analysis of mitochondria with perimeter >2µm is shown (d). Over 300 identifiable mitochondria (cristae and/or double membrane) per mice were randomly and blindly selected for perimeter measurement. Student's test. *p<0.05. Data was presented as mean ± SEM of three independent experiments.


( e , f ) . Overexpression of Drp1WT , but not phosphorylation mutant Drp1S616A , suppresses mitochondrial fusion in dendrites of PINK1KO primary neurons . ( e ) . DIV4 neurons generated from PINK1 WT control or PINK1KO mice were co - transfected mito - GFP with either pcDNA3 . 1 ( 3 . 1 ) or Drp1WT or Drp1S616A . Neurons were immuno - detected with an antibody against myc - tag ( Myc ) ( red , to detect exogenous Drp1 ) . Mito - GFP : green , to detect mitochondria . Representative images were shown , scale bar = 25 μm . Magnified dendritic shafts gated by a dot line frame are shown ( Zoom , right panels , scale bar = 5 μm ) . ( f ) . Quantification of Mito - index in dendrites of neurons from experiments of ( e ) . 10 neurons were randomly selected and analyzed in each experiment . One - way ANOVA followed with Tukey ' s test . * p < 0 . 05 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, f). Overexpression of Drp1WT, but not phosphorylation mutant Drp1S616A, suppresses mitochondrial fusion in dendrites of PINK1KO primary neurons. (e). DIV4 neurons generated from PINK1 WT control or PINK1KO mice were co-transfected mito-GFP with either pcDNA3.1 (3.1) or Drp1WT or Drp1S616A. Neurons were immuno-detected with an antibody against myc-tag (Myc) (red, to detect exogenous Drp1). Mito-GFP: green, to detect mitochondria. Representative images were shown, scale bar=25 μm. Magnified dendritic shafts gated by a dot line frame are shown (Zoom, right panels, scale bar=5 μm).(f). Quantification of Mito-index in dendrites of neurons from experiments of (e). 10 neurons were randomly selected and analyzed in each experiment. One-way ANOVA followed with Tukey's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( a - c ) . Drp1 is required for mitochondrial fission induced by PINK1 . Drp1 - null HeLa cells ( Drp1KO ) and their wildtype controls ( Drp1WT ) were co - transfected Δ110 - PINK1 - GFP - FKBP with FRB - MTS . Cells were induced with 250 nM rapalog for 2 hours to activate PINK1 kinase , followed by immunodetecting mitochondria ( TOM20 , red ) and Δ110 - PINK1 - GFP - FKBP / FRB - MTS ( Δ110 - PINK1 , green ) . Nuclei were labeled with DAPI ( blue ) . Cells treated with solvent ( DMSO ) were used as a treatment control . Representative images were shown ( a , upper panel , scale bar = 25 μm ) . Higher magnification images are also included ( a , lower panel , scale bar = 10 μm ) . Mitochondrial morphology in different transfections was quantified . ( b , c ) . Student ' s test . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments . For each condition , > 100 cells were analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-c). Drp1 is required for mitochondrial fission induced by PINK1. Drp1-null HeLa cells (Drp1KO) and their wildtype controls (Drp1WT) were co-transfected Δ110-PINK1-GFP-FKBP with FRB-MTS. Cells were induced with 250 nM rapalog for 2 hours to activate PINK1 kinase, followed by immunodetecting mitochondria (TOM20, red) and Δ110-PINK1-GFP-FKBP /FRB-MTS (Δ110-PINK1, green). Nuclei were labeled with DAPI (blue). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown (a, upper panel, scale bar=25 μm). Higher magnification images are also included (a, lower panel, scale bar=10 μm). Mitochondrial morphology in different transfections was quantified. (b, c). Student's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( d - f ) . Drp1S616 phosphorylation is required for mitochondrial fission induced by PINK1 . Drp1KO HeLa cells were co - transfected Δ110 - PINK1 - GFP - FKBP / FRB - MTS with either a plasmid encoding Drp1WT or a Drp1 dephosphorylation mimic mutant Drp1S616A . Cells were treated with 250 nM rapalog for 2 hours to activate PINK1 kinase , followed by immunodetecting mitochondria ( TOM20 , blue ) , exogenous Drp1 ( myc , red ) , Δ110 - PINK1 - GFP - FKBP / FRB - MTS ( PINK1 - FKBP , green ) . Cells treated with solvent ( DMSO ) were used as a treatment control . Representative images were shown , scale bar = 25 μm ( d ) . Higher magnification images are also included ( d , Zoom , scale bar = 5 μm ) . Mitochondrial morphology in different transfections was quantified ( e , f ) . Student ' s test . * * p < 0 . 01 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments . For each condition , > 100 cells were analyzed .

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

(d-f). Drp1S616 phosphorylation is required for mitochondrial fission induced by PINK1. Drp1KO HeLa cells were co-transfected Δ110-PINK1-GFP-FKBP /FRB-MTS with either a plasmid encoding Drp1WT or a Drp1 dephosphorylation mimic mutant Drp1S616A. Cells were treated with 250 nM rapalog for 2 hours to activate PINK1 kinase, followed by immunodetecting mitochondria (TOM20, blue), exogenous Drp1 (myc, red), Δ110-PINK1-GFP-FKBP /FRB-MTS (PINK1-FKBP, green). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown, scale bar=25 μm (d). Higher magnification images are also included (d, Zoom, scale bar=5 μm). Mitochondrial morphology in different transfections was quantified (e, f). Student's test. **p<0.01, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( g , h ) . Targeting PINK1 kinase domain to OMM increases mitochondrial localization of phosphorylated Drp1S616 . HEK293 cells co - transfected FRB - MTS with Δ110 - PINK1 - GFP - FKBP were treated with 250 nM rapalog ( Rapalog ) . After 2hours treatment , cytosol protion ( cyto ) and mitochondria ( Mito ) were fractionated and immunobloted with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) , Drp1 ( Drp1 ) , or GFP ( PINK1 ) . Tom20 and β - actin were detected as mitochondrial loading control and cytosol loading control , respectively ( g ) . Quantitation of pS616 / Drp1 in various conditions was shown ( h ) . Student ' s test . * * p < 0 . 01 , ns : no significance . Data was presented as mean ± SEM of three independent 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 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 O O O O O O O O O O O O O O O O O O

(g, h). Targeting PINK1 kinase domain to OMM increases mitochondrial localization of phosphorylated Drp1S616. HEK293 cells co-transfected FRB-MTS with Δ110-PINK1-GFP-FKBP were treated with 250 nM rapalog (Rapalog). After 2hours treatment, cytosol protion (cyto) and mitochondria (Mito) were fractionated and immunobloted with antibodies against either phospho (Ser616)-Drp1 (pS616), Drp1 (Drp1), or GFP (PINK1). Tom20 and β-actin were detected as mitochondrial loading control and cytosol loading control, respectively (g). Quantitation of pS616/Drp1 in various conditions was shown (h). Student's test. **p<0.01, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( i , j ) . Targeting PINK1 kinase domain to the OMM increases mitochondrial localization of Drp1 . HeLa cells co - transfected FRB - MTS with Δ110 - PINK1 - GFP - FKBP ( FKBP - PINK1 ) were treated with 250 nM rapalog ( Rapalog ) for 2 hours to activate PINK1 kinase . Cells treated with solvent ( DMSO ) were included as a control . Cells were immunostained with antibodies against either Tom20 , Drp1 and GFP ( PINK1 - FKBP ) . Representative images were shown , scale bar = 2 μm ( i ) . Higher magnification images from gated box are shown ( bottom panels ) . Arrows indicate Drp1 puncta . Mitochondria - associated ( yellow ) and non - associated Drp1 ( Red ) are shown , scale bar = 1 μm . Mitochondria - associated Drp1 puncta were analyzed by using an ImageJ plugin - Coloc 2 and expressed as the Manders ' Colocalization Coefficients ( MCC ) ( j ) . Three different regions from each cell and > 15 cells for each experimental group were analyzed . Student ' s test . * * p < 0 . 01 . Data was presented as mean ± SEM of three independent 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(i, j). Targeting PINK1 kinase domain to the OMM increases mitochondrial localization of Drp1. HeLa cells co-transfected FRB-MTS with Δ110-PINK1-GFP-FKBP (FKBP-PINK1) were treated with 250 nM rapalog (Rapalog) for 2 hours to activate PINK1 kinase. Cells treated with solvent (DMSO) were included as a control. Cells were immunostained with antibodies against either Tom20, Drp1 and GFP (PINK1-FKBP). Representative images were shown, scale bar=2 μm (i). Higher magnification images from gated box are shown (bottom panels). Arrows indicate Drp1 puncta. Mitochondria-associated (yellow) and non-associated Drp1(Red) are shown, scale bar=1 μm. Mitochondria-associated Drp1 puncta were analyzed by using an ImageJ plugin-Coloc 2 and expressed as the Manders' Colocalization Coefficients (MCC) (j). Three different regions from each cell and >15 cells for each experimental group were analyzed. Student's test. **p<0.01. Data was presented as mean ± SEM of three independent experiments.


( k , l ) . PINK1 regulates S616 and S637 phosphorylation of Drp1 via independent mechanisms . DRP1KO HEK293 cells co - transfected FRB - MTS / Δ110 - PINK1 - GFP - FKBP ( PINK1WT ) with either myc - Drp1WT or myc - Drp1S616A were treated with 250 nM rapalog ( Rapalog ) for 2 hours to activate PINK1 kinase . Cells treated with solvent ( DMSO ) were included as a control . Cell lysates were immunoblotting with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) , phospho - ( Ser637 ) - Drp1 ( pS637 ) , Drp1 and GFP ( to detect PINK1 - FKBP ) ( k ) . β - actin was detected as a loading control . Quantitation of pS637 / Drp1 in various conditions was shown ( l ) . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(k, l). PINK1 regulates S616 and S637 phosphorylation of Drp1 via independent mechanisms. DRP1KO HEK293 cells co-transfected FRB-MTS/Δ110-PINK1-GFP-FKBP (PINK1WT) with either myc-Drp1WT or myc-Drp1S616A were treated with 250 nM rapalog (Rapalog) for 2 hours to activate PINK1 kinase. Cells treated with solvent (DMSO) were included as a control. Cell lysates were immunoblotting with antibodies against either phospho (Ser616)-Drp1 (pS616), phospho-(Ser637)-Drp1 (pS637), Drp1 and GFP (to detect PINK1-FKBP) (k). β-actin was detected as a loading control. Quantitation of pS637/Drp1 in various conditions was shown (l). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( a - e ) . ATG5 is dispensable for PINK1 - mediated mitochondrial fission . MEF cells derived from ATG5 - null ( ATG5KO ) and their wildtype control mice ( ATG5WT ) were co - transfected Δ110 - PINK1 - GFP - FKBP / FRB - MTS with either PINK1WT or a kinase - dead mutant PINK1D384N . Cells were induced with 250 nM rapalog ( Rapalog ) for 2 hours to activate PINK1 kinase , followed by immunodetecting mitochondria ( TOM20 , red ) and Δ110 - PINK1 - GFP - FKBP / FRB - MTS ( PINK1 - FKBP , green ) . Cells treated with solvent ( DMSO ) were used as a treatment control . Representative images are shown , scale bar = 25 μm ( a ) . Higher magnification images are also included ( panels beneath the large cell images , scale bar = 10 μm ) . Mitochondrial morphology in different transfections was quantified ( b - e ) . Student ' s test . * p < 0 . 05 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments . For each condition , > 100 cells were analyzed .

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

(a-e). ATG5 is dispensable for PINK1-mediated mitochondrial fission. MEF cells derived from ATG5-null (ATG5KO) and their wildtype control mice (ATG5WT) were co-transfected Δ110-PINK1-GFP-FKBP /FRB-MTS with either PINK1WT or a kinase-dead mutant PINK1D384N. Cells were induced with 250 nM rapalog (Rapalog) for 2 hours to activate PINK1 kinase, followed by immunodetecting mitochondria (TOM20, red) and Δ110-PINK1-GFP-FKBP /FRB-MTS (PINK1-FKBP, green). Cells treated with solvent (DMSO) were used as a treatment control. Representative images are shown, scale bar=25 μm (a). Higher magnification images are also included (panels beneath the large cell images, scale bar=10 μm). Mitochondrial morphology in different transfections was quantified (b-e). Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( f - i ) . Drp1 is dispensable for recruitment of parkin to mitochondria . Drp1WT and Drp1KO HeLa cells were transfected with GFP - parkin . Cells were treated with 20 μM CCCP for 2 hours ( f ) followed by immunodetecting mitochondria ( TOM20 , red ) and parkin ( GFP - parkin , green ) . Nuclei were labeled with DAPI ( blue ) . Cells treated with solvent ( DMSO ) were used as a treatment control . Representative images were shown , scale bar = 25 μm . Cell with mitochondrial parkin ( % ) in different experimental group was quantified ( g , i ) . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments . For each condition , > 100 cells were analyzed .

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

(f-i). Drp1 is dispensable for recruitment of parkin to mitochondria. Drp1WT and Drp1KO HeLa cells were transfected with GFP-parkin. Cells were treated with 20 μM CCCP for 2 hours (f) followed by immunodetecting mitochondria (TOM20, red) and parkin (GFP-parkin, green). Nuclei were labeled with DAPI (blue). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown, scale bar=25 μm. Cell with mitochondrial parkin (%) in different experimental group was quantified (g, i). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( f - i ) . Drp1 is dispensable for recruitment of parkin to mitochondria . Drp1WT and Drp1KO HeLa cells were transfected with GFP - parkin . Cells were treated with 150 μM actinonin for 6 hours ( h ) , followed by immunodetecting mitochondria ( TOM20 , red ) and parkin ( GFP - parkin , green ) . Nuclei were labeled with DAPI ( blue ) . Cells treated with solvent ( DMSO ) were used as a treatment control . Representative images were shown , scale bar = 25 μm . Cell with mitochondrial parkin ( % ) in different experimental group was quantified ( g , i ) . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments . For each condition , > 100 cells were analyzed .

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

(f-i). Drp1 is dispensable for recruitment of parkin to mitochondria. Drp1WT and Drp1KO HeLa cells were transfected with GFP-parkin. Cells were treated with 150 μM actinonin for 6 hours (h), followed by immunodetecting mitochondria (TOM20, red) and parkin (GFP-parkin, green). Nuclei were labeled with DAPI (blue). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown, scale bar=25 μm. Cell with mitochondrial parkin (%) in different experimental group was quantified (g, i). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( a , b ) . Drp1WT and Drp1S616D , but not Drp1S616A , rescue PINK1 - null induced crushed thorax in Drosophila . Representative thorax images of PINK1WT and PINK1 - deficiency ( PINK1KO ) Drosophila expressing either mhc - gal4 ( Mhc / + ) , mhc - gal4 driven human Drp1wt ( Mhc > hDrp1WT ) or mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) are shown . Images were taken either under light microscopy ( a , upper panels , yellow arrowhead indicates the collapsed throax ) or SEM ( b , lower panels ) . Quantitation of crushed thorax is shown ( b ) . > 200 flies for each experiment are analyzed . Scale bar = 200µm . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 . Data was presented as mean ± SEM of three independent experiments . For each condition , > 200 flies were quantified .

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

(a, b). Drp1WT and Drp1S616D, but not Drp1S616A, rescue PINK1-null induced crushed thorax in Drosophila. Representative thorax images of PINK1WT and PINK1-deficiency (PINK1KO) Drosophila expressing either mhc-gal4 (Mhc/+), mhc-gal4 driven human Drp1wt (Mhc>hDrp1WT) or mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D) are shown. Images were taken either under light microscopy (a, upper panels, yellow arrowhead indicates the collapsed throax) or SEM (b, lower panels). Quantitation of crushed thorax is shown (b). > 200 flies for each experiment are analyzed. Scale bar=200µm. One-way ANOVA followed with Tukey's test. ***p<0.001. Data was presented as mean ± SEM of three independent experiments. For each condition, >200 flies were quantified.


( c ) Drp1WT and Drp1S616D , but not Drp1S616A , restore abnormal ATP production in PINK1 - null flies . ATP production was measured using muscle lysates generated from PINK1WT flies expressing mhc - gal4 ( Mhc / + ) ( as a control ) and PINK1KO flies expressing either mhc - gal4 ( Mhc / + ) , mhc - gal4 driven human Drp1wt ( Mhc > hDrp1wt ) or mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments .

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

(c) Drp1WT and Drp1S616D, but not Drp1S616A, restore abnormal ATP production in PINK1-null flies. ATP production was measured using muscle lysates generated from PINK1WT flies expressing mhc-gal4 (Mhc/+) (as a control) and PINK1KO flies expressing either mhc-gal4 (Mhc/+), mhc-gal4 driven human Drp1wt (Mhc>hDrp1wt) or mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


Drp1WT and Drp1S616D , but not Drp1S616A , rescue mitochondrial structures in PINK1KO flies . Representative images of Mito - GFP ( green ) labeled mitochondria ( d ) from PINK1WT flies expressing mhc - gal4 ( Mhc / + ) ( as a control ) and PINK1KO flies expressing either mhc - gal4 ( Mhc / + ) , Mhc - gal4 driven human Drp1wt ( Mhc > hDrp1wt ) or Mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) are shown . Scale bar = 10 μm ( d ) Average mitochondrial size was quantified , > 3 flies / group and 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . One - way ANOVA followed with Tukey ' s test . * p < 0 . 05 , * * * p < 0 . 001 . Data was presented as mean ± SEM of three independent experiments ( e ) .

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

Drp1WT and Drp1S616D, but not Drp1S616A, rescue mitochondrial structures in PINK1KO flies. Representative images of Mito-GFP (green) labeled mitochondria (d) from PINK1WT flies expressing mhc-gal4 (Mhc/+) (as a control) and PINK1KO flies expressing either mhc-gal4 (Mhc/+), Mhc-gal4 driven human Drp1wt (Mhc>hDrp1wt) or Mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D) are shown. Scale bar=10 μm (d) Average mitochondrial size was quantified, >3 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat. One-way ANOVA followed with Tukey's test. *p<0.05, ***p<0.001. Data was presented as mean ± SEM of three independent experiments (e).


Drp1WT and Drp1S616D , but not Drp1S616A , rescue mitochondrial structures in PINK1KO flies . Representative images of TEM analysis ( f ) of IFMs from PINK1WT flies expressing mhc - gal4 ( Mhc / + ) ( as a control ) and PINK1KO flies expressing either mhc - gal4 ( Mhc / + ) , Mhc - gal4 driven human Drp1wt ( Mhc > hDrp1wt ) or Mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) are shown . Scale bar = 1 μm ( f ) . Average mitochondrial size was quantified , > 3 flies / group and 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . Normal and abnormal mitochondrial number per mm2 were quantified . > 3 flies / group and 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . Bars represent the mean ± SEM of three independent experiments ( g ) .

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

Drp1WT and Drp1S616D, but not Drp1S616A, rescue mitochondrial structures in PINK1KO flies. Representative images of TEM analysis (f) of IFMs from PINK1WT flies expressing mhc-gal4 (Mhc/+) (as a control) and PINK1KO flies expressing either mhc-gal4 (Mhc/+), Mhc-gal4 driven human Drp1wt (Mhc>hDrp1wt) or Mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D) are shown. Scale bar= 1 μm (f). Average mitochondrial size was quantified, >3 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat. Normal and abnormal mitochondrial number per mm2 were quantified. >3 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat. Bars represent the mean ± SEM of three independent experiments (g).


( h , i ) . Drp1WT and Drp1S616D , but not Drp1S616A , suppress cell death in PINK1KO flies . Representative TUNEL staining ( Red ) images of IFM sections from PINK1WT and PINK1KO flies expressing either Mhc - gal4 ( Mhc / + ) , Mhc - gal4 driven human Drp1wt ( Mhc > hDrp1WT ) or Mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) are shown . Nuclei were counter - stained with DAPI ( blue ) , scale bar = 10 μm . Apoptotic cells ( % ) in different genotype flies were quantified ( i ) . > 5 flies / group and 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 . Data was presented as mean ± SEM of three independent 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). Drp1WT and Drp1S616D, but not Drp1S616A, suppress cell death in PINK1KO flies. Representative TUNEL staining (Red) images of IFM sections from PINK1WT and PINK1KO flies expressing either Mhc-gal4 (Mhc/+), Mhc-gal4 driven human Drp1wt (Mhc>hDrp1WT) or Mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D) are shown. Nuclei were counter-stained with DAPI (blue), scale bar=10 μm. Apoptotic cells (%) in different genotype flies were quantified (i). >5 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat.One-way ANOVA followed with Tukey's test. ***p<0.001. Data was presented as mean ± SEM of three independent experiments.


( j ) . Expression and phosphorylation of hDrp1 in Drosophila . Fly muscle lysates generated from PINK1WT or PINK1KO flies expressing mhc - gal4 driven hDrp1WT ( WT ) and hDrp1S616A ( S616A ) were immunoblotted with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) ( to detect phosphorylated hDrp1S616 ) , Drp1 ( to detect both endogenous and exogenous Drp1 ) , myc - tag ( to detect exogenous Drp1 ) . α - tubulin was detected as a loading control . Flies expressing mhc - gal4 ( - ) were included as an expression control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). Expression and phosphorylation of hDrp1 in Drosophila. Fly muscle lysates generated from PINK1WT or PINK1KO flies expressing mhc-gal4 driven hDrp1WT (WT) and hDrp1S616A (S616A) were immunoblotted with antibodies against either phospho(Ser616)-Drp1(pS616) (to detect phosphorylated hDrp1S616), Drp1 (to detect both endogenous and exogenous Drp1), myc-tag (to detect exogenous Drp1). α-tubulin was detected as a loading control. Flies expressing mhc-gal4 (-) were included as an expression control.


( a , b ) . Drp1 rescues PINK1KO induced collapsed thorax phenotype in dATG7 - deficient Drosophila . Representative SEM images of thorax from PINK1WT , ( upper panels ) and PINK1KO ( lower panels ) flies combined with dATP7 deficiency ( dATG7KO ) or their controls ( dATG7WT ) , followed by expressing either mhc - gal4 ( Mhc / + ) alone , or Mhc - gal4 driven expression of human Drp1 ( Mhc > hDrp1WT ) , mhc - gal4 driven expression of human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven expression of human Drp1S616D ( Mhc > hDrp1S616D ) are shown ( a ) . Quantitation of crushed thorax in flies with indicated genotypes is presented ( b ) . > 100 flies for each experiment are analyzed . Scale bar = 200µm . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 . Data was presented as mean ± SEM of three independent 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a, b). Drp1 rescues PINK1KO induced collapsed thorax phenotype in dATG7-deficient Drosophila. Representative SEM images of thorax from PINK1WT, (upper panels) and PINK1KO (lower panels) flies combined with dATP7 deficiency (dATG7KO) or their controls (dATG7WT), followed by expressing either mhc-gal4 (Mhc/+) alone, or Mhc-gal4 driven expression of human Drp1 (Mhc>hDrp1WT), mhc-gal4 driven expression of human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven expression of human Drp1S616D (Mhc>hDrp1S616D) are shown (a). Quantitation of crushed thorax in flies with indicated genotypes is presented (b). > 100 flies for each experiment are analyzed. Scale bar=200µm. One-way ANOVA followed with Tukey's test. ***p<0.001. Data was presented as mean ± SEM of three independent experiments.


( c , d ) Drp1 rescues PINK1KO induced mitochondrial defects in dATG7KO Drosophila . Representative TEM images of indirect flight muscle from PINK1WT ( upper panels ) and PINK1KO ( lower panels ) flies combined with dATG7KO or their controls ( dATG7WT ) , followed by expressing either mhc - gal4 ( Mhc / + ) alone , or Mhc - gal4 driven expression of human Drp1 ( Mhc > hDrp1WT ) , mhc - gal4 driven expression of human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven expression of human Drp1S616D ( Mhc > hDrp1S616D ) are shown ( c ) . Scale bar = 1 μm . Normal and abnormal mitochondrial number per mm2 from each experimental group were and quantified . > 3 flies / group and 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . Bars represent the mean ± SEM of three independent experiments ( d ) .

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

(c, d) Drp1 rescues PINK1KO induced mitochondrial defects in dATG7KO Drosophila. Representative TEM images of indirect flight muscle from PINK1WT (upper panels) and PINK1KO (lower panels) flies combined with dATG7KO or their controls (dATG7WT), followed by expressing either mhc-gal4 (Mhc/+) alone, or Mhc-gal4 driven expression of human Drp1(Mhc>hDrp1WT), mhc-gal4 driven expression of human Drp1S616A(Mhc>hDrp1S616A) or mhc-gal4 driven expression of human Drp1S616D(Mhc>hDrp1S616D) are shown (c). Scale bar=1 μm. Normal and abnormal mitochondrial number per mm2 from each experimental group were and quantified. >3 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat. Bars represent the mean ± SEM of three independent experiments (d).