( c ) Time - lapse imaging showing DCFP1 puncta formation on parkin - labelled mitochondria . A HeLa cell expressing KR - dMito , EGFP - DFCP1 ( green ) and EBFP2 - parkin ( pseudo - coloured red ) were 559 nm illuminated , leading to the generation of parkin - labelled mitochondria tubules . Time - lapse imaging ( 1 frame per min ) revealed that DFCP1 puncta ( green ) formed transiently at discrete sites on parkin - labelled mitochondrial tubules ( red ) . White arrows mark two DFCP1 puncta ( ER - associated omegasomes ) formation / disassembly events directly on parkin - labelled tubules . Scale bar , 5 μm .

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(c) Time-lapse imaging showing DCFP1 puncta formation on parkin-labelled mitochondria. A HeLa cell expressing KR-dMito, EGFP-DFCP1 (green) and EBFP2-parkin (pseudo-coloured red) were 559 nm illuminated, leading to the generation of parkin-labelled mitochondria tubules. Time-lapse imaging (1 frame per min) revealed that DFCP1 puncta (green) formed transiently at discrete sites on parkin-labelled mitochondrial tubules (red). White arrows mark two DFCP1 puncta (ER-associated omegasomes) formation/disassembly events directly on parkin-labelled tubules. Scale bar, 5 μm.


( d ) A COS - 7 cell expressing EBFP2 - parkin ( cyan ) , AcGFP - sec61β ( green ) and TagRFP - DFCP1 ( red ) were treated with 10 μM CCCP for 4 h and imaged . Omegasomes ( DFCP1 ) appeared where ER ( sec61β ) and impaired mitochondria ( parkin ) overlap . Scale bar , 10 μm .

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(d) A COS-7 cell expressing EBFP2-parkin (cyan), AcGFP-sec61β (green) and TagRFP-DFCP1 (red) were treated with 10 μM CCCP for 4 h and imaged. Omegasomes (DFCP1) appeared where ER (sec61β) and impaired mitochondria (parkin) overlap. Scale bar, 10 μm.


( e ) Histogram of the lifetime of individual DFCP1 foci on parkin - labelled mitochondria in CCCP - treated HeLa cells expressing TagRFP - DFCP1 , EGFP - LC3B and EBFP2 - parkin .

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(e) Histogram of the lifetime of individual DFCP1 foci on parkin-labelled mitochondria in CCCP-treated HeLa cells expressing TagRFP-DFCP1, EGFP-LC3B and EBFP2-parkin.


( f ) Wortmannin treatments delayed the initial LC3 recruitment times onto parkin - labelled mitochondria in HeLa cells expressing KR - dMito , EGFP - LC3B and EBFP2 - parkin . Each ' + ' marks the times measured in individual HeLa cells ( the red ' □ ' ( square ) denotes the average from five cells ) . P = 0 . 0172 ( Student ' s t - test ) .

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

(f) Wortmannin treatments delayed the initial LC3 recruitment times onto parkin-labelled mitochondria in HeLa cells expressing KR-dMito, EGFP-LC3B and EBFP2-parkin. Each '+' marks the times measured in individual HeLa cells (the red '□' (square) denotes the average from five cells). P=0.0172 (Student's t-test).


( a ) A COS - 7 cell expressing EBFP2 - Ub ( red ) , AcGFP - sec61β ( green ) and mcherry - parkin ( cyan ) was treated with 10 μM CCCP for 1 h and imaged . Impaired mitochondria ( parkin ) displayed preferentially ubiquitinated foci ( white arrows ) at selected regions that overlap with the ER ( sec61β ) .

(a) A COS-7 cell expressing EBFP2-Ub (red), AcGFP-sec61β (green) and mcherry-parkin (cyan) was treated with 10 μM CCCP for 1 h and imaged. Impaired mitochondria (parkin) displayed preferentially ubiquitinated foci (white arrows) at selected regions that overlap with the ER (sec61β).


( b ) HeLa cells expressing KR - dMito ( red ) , EGFP - LC3 ( green ) and EBFP2 - parkin ( cyan ) were illuminated with 559 nm at the white dotted square regions , generating parkin - labelled mitochondria ( bottom right , magnified view of the light - activated regions ) . ( c ) Magnified view of the photo - inactivated region in b 80 min after illumination ( post - stained with anti - ubiquitin antibody ) . LC3 and ubiquitin colocalized at discrete sites along parkin - labelled mitochondria ( white arrows ) .

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(b) HeLa cells expressing KR-dMito (red), EGFP-LC3 (green) and EBFP2-parkin (cyan) were illuminated with 559 nm at the white dotted square regions, generating parkin-labelled mitochondria (bottom right, magnified view of the light-activated regions). (c) Magnified view of the photo-inactivated region in b 80 min after illumination (post-stained with anti-ubiquitin antibody). LC3 and ubiquitin colocalized at discrete sites along parkin-labelled mitochondria (white arrows).


( d ) A MG - 132 treated HeLa cell expressing KR - dMito , EBFP2 - parkin ( green ) and EGFP - LC3B ( red ) was 559 nm illuminated , leading to the appearance of parkin - labelled mitochondria ( white arrows one to three indicated structures , top panels ) . The parkin - labelled structures did not fragment nor became engulfed by LC3 structures ( bottom panels ) in the presence MG - 132 . Scale bars ( all panels ) , 5 μm .

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

(d) A MG-132 treated HeLa cell expressing KR-dMito, EBFP2-parkin (green) and EGFP-LC3B (red) was 559 nm illuminated, leading to the appearance of parkin-labelled mitochondria (white arrows one to three indicated structures, top panels). The parkin-labelled structures did not fragment nor became engulfed by LC3 structures (bottom panels) in the presence MG-132. Scale bars (all panels), 5 μm.


A . Confocal images of mitochondrial morphology in wild - type ( WT ) and Drp1 - / - 293T cells transfected with empty vector ( left panel ) and Myc - hFis1 ( right panel ) , stained with MitoTracker ( red ) followed by immunostaining with anti - Myc antibody ( green ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Confocal images of mitochondrial morphology in wild-type (WT) and Drp1-/- 293T cells transfected with empty vector (left panel) and Myc-hFis1 (right panel), stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green).


B . Quantitative analyses of fragmented mitochondria size ( mean area ( μm2 ) per mitochondrion ) after Myc - hFis1 overexpression in WT and Drp1 - / - 293T cells using Image J software ( Particle analysis ) in three independent experiments . In each cell , only dispersed individual mitochondria were analyzed . The total number of mitochondria ( mito ) analyzed is indicated for each condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantitative analyses of fragmented mitochondria size (mean area (μm2) per mitochondrion) after Myc-hFis1 overexpression in WT and Drp1-/- 293T cells using Image J software (Particle analysis) in three independent experiments. In each cell, only dispersed individual mitochondria were analyzed. The total number of mitochondria (mito) analyzed is indicated for each condition.


C . Quantitative analysis of mean mitochondria number per cell in WT and Drp1 - / - 293T cells overexpressing Myc - hFis1 using Image J software ( Particle analysis ) in 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

C. Quantitative analysis of mean mitochondria number per cell in WT and Drp1-/- 293T cells overexpressing Myc-hFis1 using Image J software (Particle analysis) in three independent experiments.


E . Percentages of cells with indicated mitochondrial morphologies in WT and Drp1 - / - 293T cells transfected with empty vector ( control ) , Myc - hFis1 and either Myc - or GFP - tagged mutants as indicated in 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

E. Percentages of cells with indicated mitochondrial morphologies in WT and Drp1-/- 293T cells transfected with empty vector (control), Myc-hFis1 and either Myc- or GFP-tagged mutants as indicated in three independent experiments.


A . Confocal images of mitochondrial morphology in WT 293T cells treated with scrambled siRNA ( control ) and Dyn2 siRNA as indicated , followed by transfection with empty vector ( left panel ) and Myc - hFis1 ( right three panels ) as indicated . Cells were stained with MitoTracker ( red ) followed by immunostaining with anti - Myc antibody ( green ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Confocal images of mitochondrial morphology in WT 293T cells treated with scrambled siRNA (control) and Dyn2 siRNA as indicated, followed by transfection with empty vector (left panel) and Myc-hFis1 (right three panels) as indicated. Cells were stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green).


B . Western blot of Dyn2 , Myc - hFis1 , Drp1 and GAPDH in WT 293T cells collected from the experiments in ( A ) .

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

B. Western blot of Dyn2, Myc-hFis1, Drp1 and GAPDH in WT 293T cells collected from the experiments in (A).


C . Percentages of cells with indicated mitochondrial morphologies in WT 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated , followed by transfection with empty vector or Myc - hFis1 in 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

C. Percentages of cells with indicated mitochondrial morphologies in WT 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated, followed by transfection with empty vector or Myc-hFis1 in three independent experiments.


D . Confocal images of mitochondrial morphology in Drp1 - / - 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated , followed by transfection with empty vector ( left panel ) and Myc - hFis1 ( right three panels ) . Cells were stained with MitoTracker ( red ) followed by immunostaining with anti - Myc antibody ( green ) . Representative examples of tubular , fragmented and tubular cluster phenotypes are indicated .

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

D. Confocal images of mitochondrial morphology in Drp1-/- 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated, followed by transfection with empty vector (left panel) and Myc-hFis1 (right three panels). Cells were stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green). Representative examples of tubular, fragmented and tubular cluster phenotypes are indicated.


E . Western blot of Dyn2 , Myc - hFis1 and GAPDH in Drp1 - / - 293T cells collected from the experiments in ( 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

E. Western blot of Dyn2, Myc-hFis1 and GAPDH in Drp1-/- 293T cells collected from the experiments in (D).


F . Percentages of cells with indicated mitochondrial morphologies in Drp1 - / - 293T cells treated with scrambled siRNA or Dyn2 siRNA , followed by transfection with empty vector or Myc - hFis1 as indicated in 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

F. Percentages of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells treated with scrambled siRNA or Dyn2 siRNA, followed by transfection with empty vector or Myc-hFis1 as indicated in three independent experiments.


A . Confocal images of mitochondrial morphology in Drp1 - / - 293T cells transfected with scrambled siRNA ( control ) or with hFis1 - siRNA and then stained with MitoTracker ( red ) .

O O O O O O O O O O O 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. Confocal images of mitochondrial morphology in Drp1-/- 293T cells transfected with scrambled siRNA (control) or with hFis1-siRNA and then stained with MitoTracker (red).


B . Confocal images of mitochondrial morphology in Drp1 - / - 293T cells transfected with either Mfn1 - Myc , Mfn2 - Myc or OPA1 - Myc , stained with MitoTracker ( red ) followed by immunostaining with anti - Myc antibody ( green ) .

O O O O O O O O O O O O O O O O O O O O O O O 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. Confocal images of mitochondrial morphology in Drp1-/- 293T cells transfected with either Mfn1-Myc, Mfn2-Myc or OPA1-Myc, stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green).


C . Percentages ( mean ± s . e . m . ) of cells with indicated mitochondrial morphologies in Drp1 - / - 293T cells transfected with either scrambled siRNA ( Ctr ) , hFis1 siRNA , Mfn1 - Myc , Mfn2 - Myc or OPA1 - Myc in three independent experiments for each condition ( n represents the number of cells analyzed ) .

C. Percentages (mean ± s.e.m.) of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells transfected with either scrambled siRNA (Ctr), hFis1 siRNA, Mfn1-Myc, Mfn2-Myc or OPA1-Myc in three independent experiments for each condition (n represents the number of cells analyzed).


D . hFis1 interacts with Mfn1 , Mfn2 and OPA1 as well as Drp1 , but not with Dyn2 at endogenous levels following chemical crosslinking . Wild - type ( WT ) and Drp1 - / - 293T cells were in vivo crosslinked with 1 % formaldehyde ( FA ) and cell lysates were used for co - immunoprecipitation ( IP ) with Protein G beads bound to rabbit normal IgG ( negative control ) or rabbit anti - hFis1 antibody as indicated , followed by immunoblotting with indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. hFis1 interacts with Mfn1, Mfn2 and OPA1 as well as Drp1, but not with Dyn2 at endogenous levels following chemical crosslinking. Wild-type (WT) and Drp1-/- 293T cells were in vivo crosslinked with 1% formaldehyde (FA) and cell lysates were used for co-immunoprecipitation (IP) with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by immunoblotting with indicated antibodies.


hFis1 binds to Mfn1 , Mfn2 and OPA1 at endogenous levels also in the absence of chemical crosslinking . Cell lysates prepared from WT 293T ( E ) cells without chemical crosslinking were used for co - immunoprecipitation ( IP ) with Protein G beads bound to rabbit normal IgG ( negative control ) or rabbit anti - hFis1 antibody as indicated , followed by Western blotting with indicated antibodies .

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

hFis1 binds to Mfn1, Mfn2 and OPA1 at endogenous levels also in the absence of chemical crosslinking. Cell lysates prepared from WT 293T (E) cells without chemical crosslinking were used for co-immunoprecipitation (IP) with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by Western blotting with indicated antibodies.


F . hFis1 binds to Mfn1 , Mfn2 and OPA1 at endogenous levels also in the absence of chemical crosslinking . Cell lysates prepared from HeLa ( F ) cells without chemical crosslinking were used for co - immunoprecipitation ( IP ) with Protein G beads bound to rabbit normal IgG ( negative control ) or rabbit anti - hFis1 antibody as indicated , followed by Western blotting with indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. hFis1 binds to Mfn1, Mfn2 and OPA1 at endogenous levels also in the absence of chemical crosslinking. Cell lysates prepared from HeLa (F) cells without chemical crosslinking were used for co-immunoprecipitation (IP) with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by Western blotting with indicated antibodies.


G , H . Interaction of hFis1 with Mfn1 / 2 and with OPA1 are independent events . WT 293T cells were treated with control , OPA1 ( G ) or Mfn1 plus Mfn2 ( H ) siRNA , followed by in vivo crosslinking with 1 % FA . Cell lysates were used for co - IP with Protein G beads bound to rabbit normal IgG ( negative control ) or rabbit anti - hFis1 antibody as indicated , followed by immunoblotting with indicated antibodies .

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G, H. Interaction of hFis1 with Mfn1/2 and with OPA1 are independent events. WT 293T cells were treated with control, OPA1 (G) or Mfn1 plus Mfn2 (H) siRNA, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by immunoblotting with indicated antibodies.


I . Interactions between Mfn1 / 2 and OPA1 occur independent of hFis1 . WT 293T cells were treated with control or hFis1 siRNA , followed by in vivo crosslinking with 1 % FA . Cell lysates were used for co - IP with Protein G beads bound to mouse normal IgG ( negative control ) or mouse anti - OPA1 antibody as indicated , followed by immunoblotting with indicated antibodies .

I. Interactions between Mfn1/2 and OPA1 occur independent of hFis1. WT 293T cells were treated with control or hFis1 siRNA, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to mouse normal IgG (negative control) or mouse anti-OPA1 antibody as indicated, followed by immunoblotting with indicated antibodies.


J . Interaction between Mfn1 and Mfn2 is not affected by hFis1 overexpression . 293T cells were transfected with empty vector or Myc - hFis1 , followed by in vivo crosslinking with 1 % FA . Cell lysates were used for co - IP with Protein G beads bound to mouse normal IgG or mouse anti - Mfn1 antibody , followed by immunoblotting with indicated antibodies .

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

J. Interaction between Mfn1 and Mfn2 is not affected by hFis1 overexpression. 293T cells were transfected with empty vector or Myc-hFis1, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to mouse normal IgG or mouse anti-Mfn1 antibody, followed by immunoblotting with indicated antibodies.


B . Interaction of different hFis1 mutants with Mfns and OPA1 . hFis1 - / - 293T cells were transfected with the full - length and the truncated hFis1 mutants as indicated , followed by in vivo crosslinking with 1 % FA . Cell lysates were used for co - IP with anti - Myc or anti - GFP agarose beads and the immunoprecipitates were analyzed by Western blotting with indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Interaction of different hFis1 mutants with Mfns and OPA1. hFis1-/- 293T cells were transfected with the full-length and the truncated hFis1 mutants as indicated, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with anti-Myc or anti-GFP agarose beads and the immunoprecipitates were analyzed by Western blotting with indicated antibodies.


A , B . Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG - based fusion assay in the presence or absence of exogenous hFis1 . WT 293T cells stably expressing mitoGFP or mitoRFP were co - cultured , subsequently transfected with empty vector ( control ) or with Myc - hFis1 as indicated and then treated with PEG for cell fusion . Mitochondrial fusion is indicated by co - localization of mitoGFP and mitoRFP ( i . e . yellow mitochondria ) . Left panel : empty vector transfected ; right panel : Myc - hFis1transfected . Insets represent magnification of the boxed areas in upper panel ( A ) . Quantitative analysis of the extent of mitochondrial fusion in individual hybrid cells was performed by the Pearson ' s correlation coefficient ( PCC ) in three independent experiments for each condition and data summarized in ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of exogenous hFis1. WT 293T cells stably expressing mitoGFP or mitoRFP were co-cultured, subsequently transfected with empty vector (control) or with Myc-hFis1 as indicated and then treated with PEG for cell fusion. Mitochondrial fusion is indicated by co-localization of mitoGFP and mitoRFP (i.e. yellow mitochondria). Left panel: empty vector transfected; right panel: Myc-hFis1transfected. Insets represent magnification of the boxed areas in upper panel (A). Quantitative analysis of the extent of mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient (PCC) in three independent experiments for each condition and data summarized in (B).


C , D . Representative confocal images of mitochondrial fusion in WT 293T cells assessed by the mito - PAGFP - based fusion assay in the presence or absence of exogenous hFis1 . WT 293T cells co - transfected with mito - PAGFP ( 0 . 5 µg ) , mito - DsRed ( 0 . 2 µg ) , and either empty vector ( 0 . 5 µg , the upper panel ) or hFis1 ( 0 . 5 µg , the lower panel ) were photoactivated in a small region of interest ( ROI ) ( white circle , 3 µm diameter ) in preactivation images of mitochondria seen by mito - DsRed ( red ) . After photoactivation , sequential Z - stack images with photoactivated GFP ( green ) and mitochondrial marker ( mito - DsRed ) were collected at indicated time points using series of Z - sections from the top to the cell bottom with intervals between sections set to 0 . 5 - 0 . 75 μm ( C ) . Mitochondrial fusion was quantified by analyzing changes in fluorescence intensity of photoactivated mito - PAGFP in ROIs at 40 sec , 15 , 30 , 45 min after photoactivation . The dilution rates ( percentage ) of the GFP fluorescence intensity at different time points were normalized by the fluorescence intensity at 40 sec after photoactivation ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative confocal images of mitochondrial fusion in WT 293T cells assessed by the mito-PAGFP-based fusion assay in the presence or absence of exogenous hFis1. WT 293T cells co-transfected with mito-PAGFP (0.5 µg), mito-DsRed (0.2 µg), and either empty vector (0.5 µg, the upper panel) or hFis1 (0.5 µg, the lower panel) were photoactivated in a small region of interest (ROI) (white circle, 3 µm diameter) in preactivation images of mitochondria seen by mito-DsRed (red). After photoactivation, sequential Z-stack images with photoactivated GFP (green) and mitochondrial marker (mito-DsRed) were collected at indicated time points using series of Z-sections from the top to the cell bottom with intervals between sections set to 0.5-0.75 μm (C). Mitochondrial fusion was quantified by analyzing changes in fluorescence intensity of photoactivated mito-PAGFP in ROIs at 40 sec, 15, 30, 45 min after photoactivation. The dilution rates (percentage) of the GFP fluorescence intensity at different time points were normalized by the fluorescence intensity at 40 sec after photoactivation (D).


E , F . Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG - based fusion assay in the presence or absence of endogenous hFis1 . Both WT 293T cells stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA ( control siRNA ) or hFis1 - siRNA as indicated and then co - cultured and fused using PEG treatment . Left panel : control siRNA transfected ; right panel : hFis1 - siRNA transfected ( E ) . Quantitative analysis of the mitochondrial fusion in individual hybrid cells was performed by the Pearson ' s correlation coefficient in three independent experiments and data summarized in ( F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of endogenous hFis1. Both WT 293T cells stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA (control siRNA) or hFis1-siRNA as indicated and then co-cultured and fused using PEG treatment. Left panel: control siRNA transfected; right panel: hFis1-siRNA transfected (E). Quantitative analysis of the mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient in three independent experiments and data summarized in (F).


G , H . Representative confocal images of mitochondrial fusion in WT 293T cells assessed by the mito - PAGFP - based fusion assay in the presence or absence of endogenous hFis1 . Scrambled siRNA or hFis1 siRNA treated cells were co - transfected with mito - PAGFP and mito - DsRed and were subsequently photoactivated ( G ) as described in ( C ) . Mitochondrial fusion was quantified ( H ) as described in ( 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

G, H. Representative confocal images of mitochondrial fusion in WT 293T cells assessed by the mito-PAGFP-based fusion assay in the presence or absence of endogenous hFis1. Scrambled siRNA or hFis1 siRNA treated cells were co-transfected with mito-PAGFP and mito-DsRed and were subsequently photoactivated (G) as described in (C). Mitochondrial fusion was quantified (H) as described in (D).


A , B . Representative confocal images of mitochondrial fusion in Drp1 - / - 293T polykaryons in cultures subjected to the PEG - based fusion assay in the presence or absence of exogenous hFis1 . Drp1 - / - cells stably expressing mitoGFP or mitoRFP were co - cultured , subsequently transfected with empty vector ( control ) or Myc - hFis1 as indicated and then stimulated with PEG treatment for cell fusion , followed by immunostaining with anti - Myc antibody ( green ) . Mitochondrial fusion was indicated by co - localization of mitoGFP and mitoRFP ( i . e . yellow mitochondria ) . Left panel : empty vector transfected ; right panel : Myc - hFis1 transfected . Insets represent magnification of the boxed areas in upper panel ( A ) . Quantitative analyses for measuring the extent of mitochondrial fusion in individual hybrid cells were performed by the Pearson ' s correlation coefficient ( PCC ) in three independent experiments and summarized in ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative confocal images of mitochondrial fusion in Drp1-/- 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of exogenous hFis1. Drp1-/- cells stably expressing mitoGFP or mitoRFP were co-cultured, subsequently transfected with empty vector (control) or Myc-hFis1 as indicated and then stimulated with PEG treatment for cell fusion, followed by immunostaining with anti-Myc antibody (green). Mitochondrial fusion was indicated by co-localization of mitoGFP and mitoRFP (i.e. yellow mitochondria). Left panel: empty vector transfected; right panel: Myc-hFis1 transfected. Insets represent magnification of the boxed areas in upper panel (A). Quantitative analyses for measuring the extent of mitochondrial fusion in individual hybrid cells were performed by the Pearson's correlation coefficient (PCC) in three independent experiments and summarized in (B).


C , D . Representative confocal images of mitochondrial fusion in Drp1 - / - 293T cells assessed by the mito - PAGFP - based fusion assay in the presence or absence of exogenous hFis1 . Drp1 - / - cells co - transfected with mito - PAGFP , mito - DsRed , and either empty vector ( the upper panel ) or hFis1 ( the lower panel ) were photoactivated in a small region of interest ( ROI ) ( white circle , 3 µm diameter ) as indicated in preactivation images of mitochondria seen by mito - DsRed ( red ) . After photoactivation , Z - stack images with photoactivated GFP ( green ) and mitochondrial marker ( mito - DsRed ) were collected at times 40 sec , 15 , 30 , 45 min as indicated ( C ) . Mitochondrial fusion was quantified by analysis of changes in the fluorescence intensity of photoactivated mito - PAGFP in ROIs at 40 sec , 15 , 30 , 45 min after photoactivation . The dilution rates ( percentage ) of the GFP fluorescence intensity at different time points were normalized by the fluorescence intensity at 40 sec after photoactivation ( 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 O O O O O O O O O O O O O O O O O O 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. Representative confocal images of mitochondrial fusion in Drp1-/- 293T cells assessed by the mito-PAGFP-based fusion assay in the presence or absence of exogenous hFis1. Drp1-/- cells co-transfected with mito-PAGFP, mito-DsRed, and either empty vector (the upper panel) or hFis1 (the lower panel) were photoactivated in a small region of interest (ROI) (white circle, 3 µm diameter) as indicated in preactivation images of mitochondria seen by mito-DsRed (red). After photoactivation, Z-stack images with photoactivated GFP (green) and mitochondrial marker (mito-DsRed) were collected at times 40 sec, 15, 30, 45 min as indicated (C). Mitochondrial fusion was quantified by analysis of changes in the fluorescence intensity of photoactivated mito-PAGFP in ROIs at 40 sec, 15, 30, 45 min after photoactivation. The dilution rates (percentage) of the GFP fluorescence intensity at different time points were normalized by the fluorescence intensity at 40 sec after photoactivation (D).


E , F . Representative confocal images of mitochondrial fusion in Drp1 - / - 293T polykaryons in cultures subjected to the PEG - based fusion assay in the presence or absence of endogenous hFis1 . Drp1 - / - cells stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA ( control siRNA ) or with hFis1 - siRNA as indicated and then co - cultured and fused by PEG treatment . Left panel : control siRNA transfected ; right panel : hFis1 - siRNA transfected ( E ) . Quantitative analysis of extent of mitochondrial fusion in individual hybrid cells was performed by the Pearson ' s correlation coefficient ( PCC ) in three independent experiments and summarized in ( F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative confocal images of mitochondrial fusion in Drp1-/- 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of endogenous hFis1. Drp1-/- cells stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA (control siRNA) or with hFis1-siRNA as indicated and then co-cultured and fused by PEG treatment. Left panel: control siRNA transfected; right panel: hFis1-siRNA transfected (E). Quantitative analysis of extent of mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient (PCC) in three independent experiments and summarized in (F).


G , H . Representative confocal images of mitochondrial fusion in Drp1 - / - 293T cells monitored by the mito - PAGFP - based fusion assay in the presence or absence of endogenous hFis1 . Scrambled siRNA or hFis1 siRNA treated cells were co - transfected with mito - PAGFP and mito - DsRed and were subsequently photoactivated ( G ) as described in ( C ) . Mitochondrial fusion was quantified by analysis of changes in the fluorescence intensity of photoactivated mito - PAGFP ( H ) as described in ( 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

G, H. Representative confocal images of mitochondrial fusion in Drp1-/- 293T cells monitored by the mito-PAGFP-based fusion assay in the presence or absence of endogenous hFis1. Scrambled siRNA or hFis1 siRNA treated cells were co-transfected with mito-PAGFP and mito-DsRed and were subsequently photoactivated (G) as described in (C). Mitochondrial fusion was quantified by analysis of changes in the fluorescence intensity of photoactivated mito-PAGFP (H) as described in (D).


A , B . GTP hydrolysis activities of Mfn1 , Mfn2 , Mfn2K109A and OPA1 in the presence or absence of recombinant GST - hFis1 or GST ( negative control ) were determined by measuring the concentration of free phosphate ( Pi ) released from GTP . Myc - tagged Mfn1 , Mfn2 , Mfn2K109A and OPA1 were transiently expressed separately in 293T cells and immunopurified by anti - Myc agarose beads . GTPase activities of immunopurified Myc - tagged Mfn1 , Mfn2 , Mfn2K109A and OPA1 were determined after treatment with or without recombinant GST - hFis1 or GST protein ( A ) . The input levels of immunopurified Mfn1 - Myc , Mfn2 - Myc and OPA1 - Myc ( left panel ) as well as recombinant GST - hFis1 ( ~ 42 kDa ) and GST ( ~ 26 kDa ) ( right panel ) were assessed by immunoblotting with indicated antibodies ( B ) .

O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. GTP hydrolysis activities of Mfn1, Mfn2, Mfn2K109A and OPA1 in the presence or absence of recombinant GST-hFis1 or GST (negative control) were determined by measuring the concentration of free phosphate (Pi) released from GTP. Myc-tagged Mfn1, Mfn2, Mfn2K109A and OPA1 were transiently expressed separately in 293T cells and immunopurified by anti-Myc agarose beads. GTPase activities of immunopurified Myc-tagged Mfn1, Mfn2, Mfn2K109A and OPA1 were determined after treatment with or without recombinant GST-hFis1 or GST protein (A). The input levels of immunopurified Mfn1-Myc, Mfn2-Myc and OPA1-Myc (left panel) as well as recombinant GST-hFis1 (~42 kDa) and GST (~26 kDa) (right panel) were assessed by immunoblotting with indicated antibodies (B).


C , D . GTP hydrolysis activities of Drp1 in the presence or absence of recombinant GST - hFis1 or GST ( negative control ) were determined as described in ( A ) . Untagged WT Drp1 and Drp1Q34A were transiently expressed in Drp1 - / - 293T cells and immunopurified by protein G beads pre - incubated with Drp1 antibody . GTPase activities of immunopurified Drp1 and Drp1Q34A were determined after treatment with or without recombinant hFis1 or GST protein ( C ) . The input levels of immunopurified Drp1 and Drp1Q34A were assessed by immunoblotting with anti - Drp1 antibody ( 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

C, D. GTP hydrolysis activities of Drp1 in the presence or absence of recombinant GST-hFis1 or GST (negative control) were determined as described in (A). Untagged WT Drp1 and Drp1Q34A were transiently expressed in Drp1-/- 293T cells and immunopurified by protein G beads pre-incubated with Drp1 antibody. GTPase activities of immunopurified Drp1 and Drp1Q34A were determined after treatment with or without recombinant hFis1 or GST protein (C). The input levels of immunopurified Drp1 and Drp1Q34A were assessed by immunoblotting with anti-Drp1 antibody (D).


E , F . GTP hydrolysis activities of Dyn2 in the presence or absence of recombinant GST - hFis1 or GST ( negative control ) were determined as described in ( A ) . Endogenous Dyn2 in Drp1 - / - 293T cells was immunopurified by protein G beads pre - incubated with anti - Dyn2 antibody . GTPase activity of immunopurified Dyn2 was determined after treatment with or without recombinant GST - hFis1 or GST protein ( E ) . The input levels of immunopurified Dyn2 were assessed by immunoblotting with anti - Dyn2 antibody ( F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. GTP hydrolysis activities of Dyn2 in the presence or absence of recombinant GST-hFis1 or GST (negative control) were determined as described in (A). Endogenous Dyn2 in Drp1-/- 293T cells was immunopurified by protein G beads pre-incubated with anti-Dyn2 antibody. GTPase activity of immunopurified Dyn2 was determined after treatment with or without recombinant GST-hFis1 or GST protein (E). The input levels of immunopurified Dyn2 were assessed by immunoblotting with anti-Dyn2 antibody (F).


G . Summary of the effects of pro - fusion GTPases ( Mfns and OPA1 ) on hFis1 - induced mitochondrial fragmentation in Drp1 - / - cells . Percentages ( mean ± s . e . m . ) of cells with indicated mitochondrial morphologies in Drp1 - / - 293T cells co - expressing Myc - hFis1 and either empty vector , Mfn1 - Myc , Mfn2 - Myc or OPA1 - Myc in three independent experiments . n represents the number of cells analyzed . Corresponding confocal images shown in Fig EV4 .

G. Summary of the effects of pro-fusion GTPases (Mfns and OPA1) on hFis1-induced mitochondrial fragmentation in Drp1-/- cells. Percentages (mean ± s.e.m.) of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells co-expressing Myc-hFis1 and either empty vector, Mfn1-Myc, Mfn2-Myc or OPA1-Myc in three independent experiments. n represents the number of cells analyzed. Corresponding confocal images shown in Fig EV4.


A . Ablation of OPA1 using CRISPR - Cas9 gene editing in Drp1 - / - 293T cells was confirmed by Western blotting analysis .

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

A. Ablation of OPA1 using CRISPR-Cas9 gene editing in Drp1-/- 293T cells was confirmed by Western blotting analysis.


B . Confocal images of mitochondrial morphology in Drp1 - / - ( left panel ) and Drp1 / OPA1DKO 293T ( right panel ) cells . ( Aggr / frag : aggregation / fragmentation of mitochondria ; Bulb / tubular : bulb - like / tubular mitochondria ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Confocal images of mitochondrial morphology in Drp1-/- (left panel) and Drp1/OPA1DKO 293T (right panel) cells. (Aggr/frag: aggregation/fragmentation of mitochondria; Bulb/tubular: bulb-like/tubular mitochondria).


C . Confocal images of mitochondrial morphology in Drp1 / OPA1DKO 293T cells treated with the indicated siRNAs , combinations of siRNAs or with Myc - hFis1 plasmid ( lower three panels ) compared to mitochondrial morphology in Drp1 - / - 293T cells overexpressing Myc - hFis1 or not ( upper panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Confocal images of mitochondrial morphology in Drp1/OPA1DKO 293T cells treated with the indicated siRNAs, combinations of siRNAs or with Myc-hFis1 plasmid (lower three panels) compared to mitochondrial morphology in Drp1-/- 293T cells overexpressing Myc-hFis1 or not (upper panel).


D . Percentages ( mean ± s . e . m . ) of cells with indicated mitochondrial morphologies in Drp1 - / - 293T cells transfected with empty vector or Myc - hFis1 plasmid , and in Drp1 / OPA1DKO 293T cells treated with the indicated siRNA , combinations of siRNA or Myc - hFis1 plasmid . The data were collected from three independent experiments . Total cell numbers ( n ) used for statistical analysis are indicated for each condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Percentages (mean ± s.e.m.) of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells transfected with empty vector or Myc-hFis1 plasmid, and in Drp1/OPA1DKO 293T cells treated with the indicated siRNA, combinations of siRNA or Myc-hFis1 plasmid. The data were collected from three independent experiments. Total cell numbers (n) used for statistical analysis are indicated for each condition.


E . Knockdown of the indicated proteins by siRNA in Drp1 / OPA1DKO 293T cells was confirmed by Western blotting analysis .

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

E. Knockdown of the indicated proteins by siRNA in Drp1/OPA1DKO 293T cells was confirmed by Western blotting analysis.


Expression of Mpc proteins in mitochondria assessed by SDS - PAGE and immunoblot analysis of crude mitochondrial preparations . The individual untagged Mpc1 ( Mpc1 ) , histidine - tagged Mpc1 ( Mpc1his ) , histidine - tagged Mpc3 ( Mpc3his ) or the Mpc1 / Mpc3 hetero - complex ( Mpc1 / Mpc3his ) were detected with antibodies raised against Mpc1 ( left panel ) or Mpc3 ( right panel ) and are shown with dashed arrows .

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

Expression of Mpc proteins in mitochondria assessed by SDS-PAGE and immunoblot analysis of crude mitochondrial preparations. The individual untagged Mpc1 (Mpc1), histidine-tagged Mpc1 (Mpc1his), histidine-tagged Mpc3 (Mpc3his) or the Mpc1/Mpc3 hetero-complex (Mpc1/Mpc3his) were detected with antibodies raised against Mpc1 (left panel) or Mpc3 (right panel) and are shown with dashed arrows.


Five micrograms of each affinity - purified Mpc protein were analysed by SDS - PAGE and the bands were visualised by Coomassie Blue stain . Peptide mass finger printing was used to identify the major protein

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

Five micrograms of each affinity-purified Mpc protein were analysed by SDS-PAGE and the bands were visualised by Coomassie Blue stain. Peptide mass finger printing was used to identify the major protein bands


The stability of the purified proteins was assessed via thermal denaturation by fluorescent CPM - adduct formation . The thermal denaturation profiles ( left ) were used to calculate the first derivative ( right ) , which provides the apparent melting temperature , indicated with the same colour coding .

The stability of the purified proteins was assessed via thermal denaturation by fluorescent CPM-adduct formation. The thermal denaturation profiles (left) were used to calculate the first derivative (right), which provides the apparent melting temperature, indicated with the same colour coding.


The stability of the same samples , as in panel ( D ) , was assessed by NanoDSF . The changes in the 330 nm / 350 nm ratio with temperature ( left ) were used to calculate the first derivative ( right ) .

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

The stability of the same samples, as in panel (D), was assessed by NanoDSF. The changes in the 330 nm/350 nm ratio with temperature (left) were used to calculate the first derivative (right).


Nickel - affinity - purified Mpc1 / Mpc3 hetero - complex used for SEC - MALLS analysis , showing a 1 : 1 stoichiometry of the protomers .

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

Nickel-affinity-purified Mpc1/Mpc3 hetero-complex used for SEC-MALLS analysis, showing a 1:1 stoichiometry of the protomers.


SEC - MALLS analysis of the hetero - complex . The light scattering trace for Mpc1 / Mpc3 is shown as a black line . The masses of the protein - detergent - lipid complex ( PDL , green ) , the detergent - lipid micelle ( DL , blue ) and the protein ( P , red ) are indicated . Protein fractions across the peak were assessed by SDS - PAGE and visualised by Coomassie Blue staining ( B , inset ) .

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

SEC-MALLS analysis of the hetero-complex. The light scattering trace for Mpc1/Mpc3 is shown as a black line. The masses of the protein-detergent-lipid complex (PDL, green), the detergent-lipid micelle (DL, blue) and the protein (P, red) are indicated. Protein fractions across the peak were assessed by SDS-PAGE and visualised by Coomassie Blue staining (B, inset).


Nickel - affinity - purified Mpc3 protein used for SEC - MALLS analysis .

Nickel-affinity-purified Mpc3 protein used for SEC-MALLS analysis.


SEC - MALLS analysis of Mpc3 , colour designation as in ( B ) . The protein fractions across the peak ( D , inset ) were assessed as in ( B ) .

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

SEC-MALLS analysis of Mpc3, colour designation as in (B). The protein fractions across the peak (D, inset) were assessed as in (B).


Time course of pyruvate homo - exchange by the Mpc1 / Mpc3 hetero - complex in liposomes ( n = 8 ) in comparison to empty liposomes ( n = 6 ) at a ΔpH of 1 . 6 .

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

Time course of pyruvate homo-exchange by the Mpc1/Mpc3 hetero-complex in liposomes (n=8) in comparison to empty liposomes (n=6) at a ΔpH of 1.6.


Kinetic analysis of pyruvate homo - exchange at ΔpH of 1 . 6 ( n = 3 ) . The hetero - complex was assayed for initial rates of uptake in the concentration range of 25 - 600 μM . The calculated KΜ was 299 μΜ in this experiment and 318 and 409 μΜ in two additional biological repeats .

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

Kinetic analysis of pyruvate homo-exchange at ΔpH of 1.6 (n=3). The hetero-complex was assayed for initial rates of uptake in the concentration range of 25-600 μM. The calculated KΜ was 299 μΜ in this experiment and 318 and 409 μΜ in two additional biological repeats.


Time course of pyruvate homo - exchange by the Mpc1 / Mpc3 hetero - complex in physiological pH ( n = 4 ) compared to empty liposomes ( n = 4 ) .

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

Time course of pyruvate homo-exchange by the Mpc1/Mpc3 hetero-complex in physiological pH (n=4) compared to empty liposomes (n=4).


In the absence of a ΔpH , the time course of pyruvate homo - exchange was similar for the Mpc1 / Mpc3 proteoliposomes and the empty liposomes ( n = 4 ) .

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

In the absence of a ΔpH, the time course of pyruvate homo-exchange was similar for the Mpc1/Mpc3 proteoliposomes and the empty liposomes (n=4).


Inhibition of [ 14C ] - pyruvate homo - exchange by UK5099 ( 1 - 100 μM ) , Zaprinast ( 1 - 1000 μM ) , lonidamine ( 10 - 10000 μM ) and 7ACC2 ( 5 - 500 μM ) . Data points represent the mean of three technical replicates of a typical experiment . The IC50 measurements have also been independently replicated , three times for UK5099 Zaprinast and 7ACC2 ( average IC50 9 ± 7 μM , 18 ± 8 μM and 27 ± 13 μM , respectively ) and two times for lonidamine ( average IC50 , 118 ± 24 μM ) .

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

Inhibition of [14C]-pyruvate homo-exchange by UK5099 (1-100 μM), Zaprinast (1-1000 μM), lonidamine (10-10000 μM) and 7ACC2 (5-500 μM). Data points represent the mean of three technical replicates of a typical experiment. The IC50 measurements have also been independently replicated, three times for UK5099 Zaprinast and 7ACC2 (average IC50 9 ± 7 μM, 18 ± 8 μM and 27 ± 13 μM, respectively) and two times for lonidamine (average IC50, 118 ± 24 μM).


[ 14C ] - pyruvate homo - exchange inhibition by the TZDs , pioglitazone and rosiglitazone ( n = 6 ) .

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

[14C]-pyruvate homo-exchange inhibition by the TZDs, pioglitazone and rosiglitazone (n=6).


Time course of pyruvate homo - exchange in proteoliposomes at a ΔpH of 1 . 6 was compared for Mpc3 ( n = 4 ) , the Mpc1 / Mpc3 hetero - complex ( n = 6 ) and empty liposomes ( n = 4 ) .

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

Time course of pyruvate homo-exchange in proteoliposomes at a ΔpH of 1.6 was compared for Mpc3 (n=4), the Mpc1/Mpc3 hetero-complex (n=6) and empty liposomes (n=4).


Time course of pyruvate homo - exchange at a ΔpH of 1 . 6 was compared for Mpc1 ( n = 4 ) , Mpc1 / Mpc3 hetero - complex ( n = 4 ) and empty liposomes ( n = 4 ) .

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

Time course of pyruvate homo-exchange at a ΔpH of 1.6 was compared for Mpc1 (n=4), Mpc1/Mpc3 hetero-complex (n=4) and empty liposomes (n=4).


Thermostability analysis via cpm . In detergent solution , Zaprinast ( 250 μM ) increased the thermostability of the Mpc1 / Mpc3 hetero - complex ( black lines ) ( n = 3 ) but not of Mpc3 ( blue lines ) ( n = 3 ) .

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

Thermostability analysis via cpm. In detergent solution, Zaprinast (250 μM) increased the thermostability of the Mpc1/Mpc3 hetero-complex (black lines) (n=3) but not of Mpc3 (blue lines) (n=3).


Thermostability analysis via nanoDSF . In detergent solution , Zaprinast ( 250 μM ) increased the thermostability of the Mpc1 / Mpc3 hetero - complex ( black lines ) ( n = 3 ) but not of Mpc1 ( orange lines ) ( n = 3 ) .

Thermostability analysis via nanoDSF. In detergent solution, Zaprinast (250 μM) increased the thermostability of the Mpc1/Mpc3 hetero-complex (black lines) (n=3) but not of Mpc1 (orange lines) (n=3).


Fluorescence was observed in the transformed N . benthamiana leaf epidermal cells , which results from complementation of the N - terminal part of the YFP fused with ATG8a ( ATG8a - N - YFP ) by the C - terminal part of the YFP fused with NBR1 ( NBR1 - C - YFP ) . No fluorescence was observed when ATG8a - N - YFP was coexpressed with unfused C - YFP or with mNBR1 - C - YFP or when unfused N - YFP was coexpressed with NBR1 - C - YFP . YFP epifluorescence images , bright - field images and overlay images of the same cells are shown .

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

Fluorescence was observed in the transformed N. benthamiana leaf epidermal cells, which results from complementation of the N-terminal part of the YFP fused with ATG8a (ATG8a-N-YFP) by the C-terminal part of the YFP fused with NBR1 (NBR1-C-YFP). No fluorescence was observed when ATG8a-N-YFP was coexpressed with unfused C-YFP or with mNBR1-C-YFP or when unfused N-YFP was coexpressed with NBR1-C-YFP. YFP epifluorescence images, bright-field images and overlay images of the same cells are shown.


Five weeks - old Arabidopsis wild - type Col - 0 plants were placed in the 22 ° C and 45 ° C growth chambers and total RNA was isolated from leaf samples collected at indicated times . Transcript levels were determined using real - time qRT - PCR . Error bars indicate SE ( n = 3 ) .

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

Five weeks-old Arabidopsis wild-type Col-0 plants were placed in the 22°C and 45°C growth chambers and total RNA was isolated from leaf samples collected at indicated times. Transcript levels were determined using real-time qRT-PCR. Error bars indicate SE (n = 3).


( A ) Four - weeks old transgenic wild - type Col - 0 ( WT ) and atg7 - 2 mutant plants expressing GFP - ATG8a were treated with ( 45 ° C ) or without ( 22 ° C ) heat shock for 3 h and then placed at room temperature for 0 . 5 h . The leaves were visualized by fluorescence confocal microscopy of GFP signal . ( B ) Numbers of punctate GFP - ATG8a spots representing autophagosomes per 10 , 000 µm2 section . Means and SE were calculated from three experiments . According to Duncan ' s multiple range test ( P = 0 . 05 ) , means do not differ significantly if they are indicated with the same letter . Bar = 20 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Four-weeks old transgenic wild-type Col-0 (WT) and atg7-2 mutant plants expressing GFP-ATG8a were treated with (45°C) or without (22°C) heat shock for 3 h and then placed at room temperature for 0.5 h. The leaves were visualized by fluorescence confocal microscopy of GFP signal. (B) Numbers of punctate GFP-ATG8a spots representing autophagosomes per 10,000 µm2 section. Means and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter. Bar = 20 µm.


( Top panel ) Four - weeks old transgenic wild - type Col - 0 ( WT ) , atg7 - 2 and nbr1 - 1 mutant plants expressing GFP - NBR1 were treated with ( 45 ° C ) or without ( 22 ° C ) heat shock for 3 h and then placed at room temperature for 0 . 5 h . The leaves were visualized by fluorescence confocal microscopy of GFP signal . ( Bottom panel ) Numbers of punctate GFP - NBR1 spots per 10 , 000 µm2 section . Means and SE were calculated from three experiments . According to Duncan ' s multiple range test ( P = 0 . 05 ) , means do not differ significantly if they are indicated with the same letter . Bar = 20 µm .

(Top panel) Four-weeks old transgenic wild-type Col-0 (WT), atg7-2 and nbr1-1 mutant plants expressing GFP-NBR1 were treated with (45°C) or without (22°C) heat shock for 3 h and then placed at room temperature for 0.5 h. The leaves were visualized by fluorescence confocal microscopy of GFP signal. (Bottom panel) Numbers of punctate GFP-NBR1 spots per 10,000 µm2 section. Means and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter. Bar = 20 µm.


( A ) Five weeks - old Arabidopsis Col - 0 wild type ( WT ) , atg5 , atg7 and nbr1 mutant plants were placed in 22 ° C and 45 ° C growth for 10 hours and then moved to room temperature for 3 - day recovery or ( B ) sprayed with 20 µM methyl viologen ( MV ) and kept under light for two days before the picture of representative plants was taken . The experiments were repeated three times with similar results .

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

(A) Five weeks-old Arabidopsis Col-0 wild type (WT), atg5, atg7 and nbr1 mutant plants were placed in 22°C and 45°C growth for 10 hours and then moved to room temperature for 3-day recovery or (B) sprayed with 20 µM methyl viologen (MV) and kept under light for two days before the picture of representative plants was taken. The experiments were repeated three times with similar results.


( B ) Fv / Fm images . ( C ) Average values for the Fv / Fm images . Five weeks - old Arabidopsis Col - 0 wild type ( WT ) , atg5 , atg7 and nbr1 mutant plants were placed in 22 ° C and 45 ° C growth chambers . Fully expanded leaves were sampled after 10 hours in the indicated temperatures and immediately measured for electrolyte leakage and Fv / Fm . Error bars in ( A ) and ( C ) indicate SE ( n = 5 ) . According to Duncan ' s multiple range test ( P = 0 . 05 ) , means of EL or Fv / Fm do not differ significantly if they are indicated with the same letter . The experiments were repeated twice with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fv/Fm images. (C) Average values for the Fv/Fm images. Five weeks-old Arabidopsis Col-0 wild type (WT), atg5, atg7 and nbr1 mutant plants were placed in 22°C and 45°C growth chambers. Fully expanded leaves were sampled after 10 hours in the indicated temperatures and immediately measured for electrolyte leakage and Fv/Fm. Error bars in (A) and (C) indicate SE (n = 5). According to Duncan's multiple range test (P = 0.05), means of EL or Fv/Fm do not differ significantly if they are indicated with the same letter. The experiments were repeated twice with similar results.


( A ) Five weeks - old Arabidopsis Col - 0 wild type ( WT ) , atg5 , atg7 and nbr1 mutant plants were placed into a growth chamber with approximately 50 % humidity . The photograph of representative plants was taken 10 days after withholding watering . The experiment was repeated twice with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Five weeks-old Arabidopsis Col-0 wild type (WT), atg5, atg7 and nbr1 mutant plants were placed into a growth chamber with approximately 50% humidity. The photograph of representative plants was taken 10 days after withholding watering. The experiment was repeated twice with similar results.


( B ) Seven days - old seedlings Col - 0 WT , atg5 , atg7 and nbr1 grown on solid MS medium were transferred to the same medium ( control ) or the same medium containing 0 . 16 M NaCl and photographed 5 days later . The survived seedlings were scored 5 days after the transfer and the average values and SE were calculated from three experiments . According to Duncan ' s multiple range test ( P = 0 . 05 ) , means do not differ significantly if they are indicated with the same letter .

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

(B) Seven days-old seedlings Col-0 WT, atg5, atg7 and nbr1 grown on solid MS medium were transferred to the same medium (control) or the same medium containing 0.16 M NaCl and photographed 5 days later. The survived seedlings were scored 5 days after the transfer and the average values and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter.


( A ) Eight weeks old wild type ( WT ) , atg5 , atg7 and nbr1 plants grown under normal growth conditions in a growth chamber . ( B ) Four weeks old WT , atg5 , atg7 and nbr1 plants after being kept in the dark for 6 days . ( C ) Percentage of plants that survived the various lengths in dark as determined by resumption of growth . Each point represents the average of 10 plants . The experiments were repeated twice with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Eight weeks old wild type (WT), atg5, atg7 and nbr1 plants grown under normal growth conditions in a growth chamber. (B) Four weeks old WT, atg5, atg7 and nbr1 plants after being kept in the dark for 6 days. (C) Percentage of plants that survived the various lengths in dark as determined by resumption of growth. Each point represents the average of 10 plants. The experiments were repeated twice with similar results.


( A ) Responses of Col - 0 wild type ( WT ) , atg5 , atg7 and nbr1 plants to Botrytis . Wild - type and mutant plants were inoculated by spraying with spore suspension at a density of 2 . 5 × 105 spores ml − 1 , and kept at high humidity . Pictures of representative plants were taken at 4 and 5 days post inoculation ( dpi ) . ( B ) Quantitative real - time PCR analysis of the B . cinerea ActA ( BcActA ) transcript levels in infected Arabidopsis plants at 5 dpi . The experiments were repeated twice with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Responses of Col-0 wild type (WT), atg5, atg7 and nbr1 plants to Botrytis. Wild-type and mutant plants were inoculated by spraying with spore suspension at a density of 2.5×105 spores ml−1, and kept at high humidity. Pictures of representative plants were taken at 4 and 5 days post inoculation (dpi). (B) Quantitative real-time PCR analysis of the B. cinerea ActA (BcActA) transcript levels in infected Arabidopsis plants at 5 dpi. The experiments were repeated twice with similar results.


( A ) Diagrams of wild - type and mutant NBR1 proteins . The conserved W and I residues in the LIR motif between the two UBA domains were changed to A residues in the NBR1W661A / I664A ( mNBR1 ) mutant protein . ( B ) Five weeks - old Arabidopsis Col - 0 wild type ( WT ) , nbr1 and transgenic nbr1 plants expressing the wild - type NBR1 ( lines 3 and 6 ; see Figure S2 ) or the NBR1W661A / I664A ( mNBR1 ) ( lines 2 and 6 ; see Figure S2 ) mutant gene were placed in a 45 ° C growth chambers for 10 hours and then moved to room temperature for 3 - day recovery before the picture as taken . The experiment was repeated three times with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Diagrams of wild-type and mutant NBR1 proteins. The conserved W and I residues in the LIR motif between the two UBA domains were changed to A residues in the NBR1W661A/I664A (mNBR1) mutant protein. (B) Five weeks-old Arabidopsis Col-0 wild type (WT), nbr1 and transgenic nbr1 plants expressing the wild-type NBR1 (lines 3 and 6; see Figure S2) or the NBR1W661A/I664A (mNBR1) (lines 2 and 6; see Figure S2) mutant gene were placed in a 45°C growth chambers for 10 hours and then moved to room temperature for 3-day recovery before the picture as taken. The experiment was repeated three times with similar results.


Leaf tissues from wild type ( WT ) and atg7 expressing NBR1 - TAP were collected at indicated hours ( h ) under 45 ° C and prepared for soluble and insoluble proteins as described in Materials and Methods . Proteins from the first supernatants ( S ) and last pellets ( P ) were subjected to SDS - PAGES and probed with a peroxidase - conjugated anti - peroxidase antibody for detection of NBR1 - TAP . Protein samples prepared from non - transgenic Col - 0 wild - type plants were included as controls .

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

Leaf tissues from wild type (WT) and atg7 expressing NBR1-TAP were collected at indicated hours (h) under 45°C and prepared for soluble and insoluble proteins as described in Materials and Methods. Proteins from the first supernatants (S) and last pellets (P) were subjected to SDS-PAGES and probed with a peroxidase-conjugated anti-peroxidase antibody for detection of NBR1-TAP. Protein samples prepared from non-transgenic Col-0 wild-type plants were included as controls.


( A ) Accumulation of insoluble proteins . Leaf tissues from wild - type ( WT ) , atg7 and nbr1 mutants collected at indicated hours ( h ) under 45 ° C for preparation of total , soluble and insoluble proteins as described in Materials and Methods . Total proteins in the starting homogenates and insoluble proteins in the last pellets were determined the percentages of insoluble proteins to total proteins were calculated . ( B ) Profiles of soluble and insoluble proteins . Proteins from the first supernatants ( S ) and last pellets ( P ) were subjected to SDS - PAGES and stained with Coomassie brilliant blue . Major proteins accumulated in the pellets from heat - stressed atg7 mutant plants were indicated by arrows .

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

(A) Accumulation of insoluble proteins. Leaf tissues from wild-type (WT), atg7 and nbr1 mutants collected at indicated hours (h) under 45°C for preparation of total, soluble and insoluble proteins as described in Materials and Methods. Total proteins in the starting homogenates and insoluble proteins in the last pellets were determined the percentages of insoluble proteins to total proteins were calculated. (B) Profiles of soluble and insoluble proteins. Proteins from the first supernatants (S) and last pellets (P) were subjected to SDS-PAGES and stained with Coomassie brilliant blue. Major proteins accumulated in the pellets from heat-stressed atg7 mutant plants were indicated by arrows.


Leaf tissues from wild type ( WT ) , atg7 and nbr1 mutants were collected at indicated hours ( h ) under 45 ° C and prepared for soluble and insoluble proteins as described in Materials and Methods . Proteins from the first supernatants ( S ) and last pellets ( P ) were subjected to SDS - PAGES and probed with anti - ubiquitin monoclonal antibody . The experiment was repeated three times with similar results .

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

Leaf tissues from wild type (WT), atg7 and nbr1 mutants were collected at indicated hours (h) under 45°C and prepared for soluble and insoluble proteins as described in Materials and Methods. Proteins from the first supernatants (S) and last pellets (P) were subjected to SDS-PAGES and probed with anti-ubiquitin monoclonal antibody. The experiment was repeated three times with similar results.


Bovine heart mitochondria were incubated at the indicated pH in absence or presence of 200 μM DPC , treated with 1 % ( w / v ) digitonin and F - ATP synthase was immunoprecipitated with an anti - F - ATP synthase Ab followed by 15 % SDS - PAGE . β subunit and CyPD content were detected by Western blotting . The ratio between CyPD and the corresponding β subunit is reported in the lower part of the panel , referring to pH 7 . 4 values as 100 % . Data are an average ± s . e . of 3 independent experiments .

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

Bovine heart mitochondria were incubated at the indicated pH in absence or presence of 200 μM DPC, treated with 1% (w/v) digitonin and F-ATP synthase was immunoprecipitated with an anti-F-ATP synthase Ab followed by 15% SDS-PAGE. β subunit and CyPD content were detected by Western blotting. The ratio between CyPD and the corresponding β subunit is reported in the lower part of the panel, referring to pH 7.4 values as 100%. Data are an average ± s.e. of 3 independent experiments.


B , C EDTA - SMP were solubilized at the indicated pH values , extracted with 1 % ( w / v ) digitonin and subjected to BN - PAGE in order to separate dimers ( Vd ) and monomers ( Vm ) , which were identified by Coomassie blue ( panel B ) or in - gel activity staining ( panel C ) and analyzed by densitometry , which is reported in the bottom part of each panel . Values report the dimer / monomer ratio , where the ratio at pH 7 . 4 was taken as 100 % . Data are an average ± s . e . of 3 independent experiments . The p - values calculated with the Student t test are shown * p ≤ 0 . 05 * * * p ≤ 0 . 001 .

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

B,C EDTA-SMP were solubilized at the indicated pH values, extracted with 1% (w/v) digitonin and subjected to BN-PAGE in order to separate dimers (Vd) and monomers (Vm), which were identified by Coomassie blue (panel B) or in-gel activity staining (panel C) and analyzed by densitometry, which is reported in the bottom part of each panel. Values report the dimer/monomer ratio, where the ratio at pH 7.4 was taken as 100%. Data are an average ± s.e. of 3 independent experiments. The p-values calculated with the Student t test are shown * p≤0.05 *** p≤0.001.


D Sequence of peptides 95 - 113 of OSCP before ( left panel ) and after reaction with DPC ( right panel ) obtained from tryptic digests of the SDS - PAGE band corresponding to the expected molecular mass of OSCP in immunoprecipitated F - ATP synthase . Fragments of the series b and y identified in the LC - MS / MS analysis are indicated on the sequence of the peptides . H112 and H112CeT are indicated in red boldface . Ions y4 - y17 show a mass shift of + 72 Da in the modified peptide .

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

D Sequence of peptides 95-113 of OSCP before (left panel) and after reaction with DPC (right panel) obtained from tryptic digests of the SDS-PAGE band corresponding to the expected molecular mass of OSCP in immunoprecipitated F-ATP synthase. Fragments of the series b and y identified in the LC-MS/MS analysis are indicated on the sequence of the peptides. H112 and H112CeT are indicated in red boldface. Ions y4-y17 show a mass shift of +72 Da in the modified peptide.


Permeabilized HEK293T cells expressing wild type ( WT ) or edited ( H112Q or H112Y ) OSCP subunit were suspended in KSCN - based media at pH 7 . 4 ( left traces ) or pH 6 . 5 ( right traces ) and PTP opening was measured as the decrease in absorbance at 540 nm . Where indicated ( arrows ) 0 . 1 mM Ca2 + in the absence of further additions ( black traces ) or in the presence of 0 . 5 mM DPC ( red traces ) or 2 µM CsA ( blue traces ) was added . The bottom panel reports the difference between the absorbance readings before the addition of Ca2 + and at the end of the swelling phase . Data are mean ± s . e . of 5 experiments ( triplicate samples ) for Ca2 + alone ( black bars ) and Ca2 + + DPC ( red bars ) and of 3 experiments ( duplicate samples ) for Ca2 + + CsA ( blue bars ) . Solid bars , pH 7 . 4 , hatched bars , pH 6 .

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

Permeabilized HEK293T cells expressing wild type (WT) or edited (H112Q or H112Y) OSCP subunit were suspended in KSCN-based media at pH 7.4 (left traces) or pH 6.5 (right traces) and PTP opening was measured as the decrease in absorbance at 540 nm. Where indicated (arrows) 0.1 mM Ca2+ in the absence of further additions (black traces) or in the presence of 0.5 mM DPC (red traces) or 2 µM CsA (blue traces) was added. The bottom panel reports the difference between the absorbance readings before the addition of Ca2+ and at the end of the swelling phase. Data are mean ± s.e. of 5 experiments (triplicate samples) for Ca2+ alone (black bars) and Ca2+ + DPC (red bars) and of 3 experiments (duplicate samples) for Ca2+ + CsA (blue bars). Solid bars, pH 7.4, hatched bars, pH 6.5


Mitochondria were prepared from cells , suspended in sucrose buffer at pH 7 . 4 ( solid bars ) or 6 . 5 ( hatched bars ) in the absence ( black bars ) or presence ( blue bars ) of 1 . 6 µM CsA , extracted with 1 % ( w / v ) digitonin and immunoprecipitated with an F - ATP synthase antibody . The immunoprecipitates were subjected to 12 % SDS - PAGE followed by Western blotting for CyPD and α subunit of F - ATP synthase . Each immunodetected band was analyzed by densitometry , the ratio between CyPD and α subunit was measured and expressed relative to the ratio obtained in absence of CsA at pH 7 . 4 , which was taken as 100 % . Data are average ± s . e . of 6 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Mitochondria were prepared from cells, suspended in sucrose buffer at pH 7.4 (solid bars) or 6.5 (hatched bars) in the absence (black bars) or presence (blue bars) of 1.6 µM CsA, extracted with 1% (w/v) digitonin and immunoprecipitated with an F-ATP synthase antibody. The immunoprecipitates were subjected to 12% SDS-PAGE followed by Western blotting for CyPD and α subunit of F-ATP synthase. Each immunodetected band was analyzed by densitometry, the ratio between CyPD and α subunit was measured and expressed relative to the ratio obtained in absence of CsA at pH 7.4, which was taken as 100%. Data are average ± s.e. of 6 independent experiments.


HEK293T cells were incubated in a balanced salt solution containing 2 - deoxyglucose at pH 7 . 4 ( solid bars ) or 6 . 5 ( hatched bars ) and anoxia induced by incubation in a BD GasPak EZ Anaerobe Gas - generating Pouch System ( BD Biosciences ) . Cell death was measured based on LDH release ( average ± s . e . from 8 independent experiments ) . In all panels the p values calculated with the Student t test are shown ( * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) .

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

HEK293T cells were incubated in a balanced salt solution containing 2-deoxyglucose at pH 7.4 (solid bars) or 6.5 (hatched bars) and anoxia induced by incubation in a BD GasPak EZ Anaerobe Gas-generating Pouch System (BD Biosciences). Cell death was measured based on LDH release (average ± s.e. from 8 independent experiments). In all panels the p values calculated with the Student t test are shown (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).


Representative current traces recorded at V ( pipette ) + 10 mV from mitoplasts derived from wild type ( WT ) or H112Q OSCP mutation - harboring 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

Representative current traces recorded at V(pipette) +10 mV from mitoplasts derived from wild type (WT) or H112Q OSCP mutation-harboring cells.


Channel activity ( top ) and corresponding amplitude histograms ( bottom ) recorded in wild type mitochondria before ( WT pH 7 . 3 ) and after acidification to pH 6 . 5 ( WT pH 6 . 5 ) . Amplitude histograms were obtained from 120 second - long current traces for the corresponding pH values and the panels are from the same experiment .

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

Channel activity (top) and corresponding amplitude histograms (bottom) recorded in wild type mitochondria before (WT pH 7.3) and after acidification to pH 6.5 (WT pH 6.5). Amplitude histograms were obtained from 120 second-long current traces for the corresponding pH values and the panels are from the same experiment.


Recordings as in panel B from mitoplasts of cells harboring the OSCP H112Q replacement . Left panel , pH 7 . 3 ; middle panel , 440 seconds after acidification to pH 6 , 5 ; right panel , 30 seconds after the addition of 5 mM Ba2 + at pH 6 . 5 . Amplitude histograms were obtained from 100 second - long segments and the panels are from the same experiment . Note that both in panels B and C well - visible subconductance states can be detected in the amplitude histograms ( peaks between open levels o2 and o3 on left panels ) . Gaussian fitting has been obtained using the Origin Program set . In all panels c , closed ; o1 , o2 , o3 open states with discrete subconductance .

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

Recordings as in panel B from mitoplasts of cells harboring the OSCP H112Q replacement. Left panel, pH 7.3; middle panel, 440 seconds after acidification to pH 6,5; right panel, 30 seconds after the addition of 5 mM Ba2+ at pH 6.5. Amplitude histograms were obtained from 100 second-long segments and the panels are from the same experiment. Note that both in panels B and C well-visible subconductance states can be detected in the amplitude histograms (peaks between open levels o2 and o3 on left panels). Gaussian fitting has been obtained using the Origin Program set. In all panels c, closed; o1, o2, o3 open states with discrete subconductance.


The left panel depicts a cryo - EM based atomic model of monomeric bovine F - ATP synthase ( pdb 5ara ) ( excluding subunits e , f , g , DAPIT , 6 . 8PL and A6L ) with OSCP ( cyan ) and subunit b ( yellow ) highlighted on the structure of F - ATP synthase in the lower part of the panel . The region H112 - E115 is shown as van der Waals spheres ( inflated for better visibility ) . The backbone of the stretch T130 - K135 is shown in red van der Waals spheres . The top part of the panel shows the results of restrained molecular dynamics simulations of the isolated lateral stalk ( OSCP and subunits b , d , F6 ) displaying a large number of conformations in this region . Snapshots taken at 1 ns intervals along the last 15 ns of the trajectory are shown for the fragment 112 - HRGE - 115 for the deprotonated ( HIS ) and the protonated form ( HIS + ) . Only for protonated H112 a salt bridge with a well defined turn or helix arrangement at H112 - E115 is found in a large number of MD conformations . The larger conformational variability of the deprotonated form can also be appreciated . The right panel reports a sequence comparison of OSCP across 16 out of 197 species ( HRGE is boxed in red ; Sauria sequences are in the thin black box ) . Conservation , quality of the aligment and consensus at each position in the alignment of all vertebrate sequences in RefSeq , excluding Sauria and most divergent sequences , are described in the documentation of the program JalView [ 67 ] .

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The left panel depicts a cryo-EM based atomic model of monomeric bovine F-ATP synthase (pdb 5ara) (excluding subunits e, f, g, DAPIT, 6.8PL and A6L) with OSCP (cyan) and subunit b (yellow) highlighted on the structure of F-ATP synthase in the lower part of the panel. The region H112-E115 is shown as van der Waals spheres (inflated for better visibility). The backbone of the stretch T130-K135 is shown in red van der Waals spheres. The top part of the panel shows the results of restrained molecular dynamics simulations of the isolated lateral stalk (OSCP and subunits b, d, F6) displaying a large number of conformations in this region. Snapshots taken at 1 ns intervals along the last 15 ns of the trajectory are shown for the fragment 112-HRGE-115 for the deprotonated (HIS) and the protonated form (HIS+). Only for protonated H112 a salt bridge with a well defined turn or helix arrangement at H112-E115 is found in a large number of MD conformations. The larger conformational variability of the deprotonated form can also be appreciated. The right panel reports a sequence comparison of OSCP across 16 out of 197 species (HRGE is boxed in red; Sauria sequences are in the thin black box). Conservation, quality of the aligment and consensus at each position in the alignment of all vertebrate sequences in RefSeq, excluding Sauria and most divergent sequences, are described in the documentation of the program JalView [67].


( A ) Relative expression of the BBSome subunits in the indicated organs and T cells measured by qPCR . CT values of the BBS genes were normalized to the geometric mean of the CT values of reference genes Gapdh , Tubb2a , and Eef1a1 . The expression levels are normalized to those of brain ( = 1 ) . Mean + SD . Three biological replicates ( the result of each biological replicate is a median of three technical replicates ) .

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(A) Relative expression of the BBSome subunits in the indicated organs and T cells measured by qPCR. CT values of the BBS genes were normalized to the geometric mean of the CT values of reference genes Gapdh, Tubb2a, and Eef1a1. The expression levels are normalized to those of brain (=1). Mean+SD. Three biological replicates (the result of each biological replicate is a median of three technical replicates).


( B ) Immunoblot analysis of BBS4 expression in T and B lymphocytes isolated from the lymph nodes and spleen of C57Bl / 6 mouse . β - actin staining served as a loading control .

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(B) Immunoblot analysis of BBS4 expression in T and B lymphocytes isolated from the lymph nodes and spleen of C57Bl/6 mouse. β-actin staining served as a loading control.


( D ) Immunoblot analysis of BBS4 expression in testes and thymi lysates of Bbs4 + / + ( WT ) , Bbs4GT / + , and Bbs4GT / GT mice . β - actin staining serves as a loading control . A representative experiment out of three biological replicates in total is shown .

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(D) Immunoblot analysis of BBS4 expression in testes and thymi lysates of Bbs4+/+ (WT), Bbs4GT/+, and Bbs4GT/GT mice. β-actin staining serves as a loading control. A representative experiment out of three biological replicates in total is shown.


( E ) Immunoblot analysis of BBS4 expression in the brain lysates of Bbs4 + / + , Bbs4GT / GT , and Bbs4KO / KO mice . β - actin staining served as a loading control . A representative experiment out of two biological replicates is shown .

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(E) Immunoblot analysis of BBS4 expression in the brain lysates of Bbs4+/+, Bbs4GT/GT, and Bbs4KO/KO mice. β-actin staining served as a loading control. A representative experiment out of two biological replicates is shown.


( F ) Hematoxylin and eosin staining of sections of seminiferous tubules from ~ 4 weeks old Bbs4 + / + ( n = 2 mice ) and Bbs4GT / GT ( n = 4 ) males . Scale bar , 100 µm . Representative images are shown .

(F) Hematoxylin and eosin staining of sections of seminiferous tubules from ~4 weeks old Bbs4+/+ (n=2 mice) and Bbs4GT/GT (n=4) males. Scale bar, 100 µm. Representative images are shown.


( G ) Genotypic ratio of Bbs4 + / + , Bbs4GT / + or Bbs4KO / + ( Het ) , and Bbs4GT / GT ( GT ) or Bbs4KO / KO ( KO ) offspring at weaning from mating of Bbs4GT / + ( n = 145 pups ) or Bbs4 + / KO ( n = 168 pups ) parents . Binomial test was used for statistical comparison of the observed distribution to the expected Mendelian ratio .

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(G) Genotypic ratio of Bbs4+/+, Bbs4GT/+ or Bbs4KO/+ (Het), and Bbs4GT/GT (GT) or Bbs4KO/KO (KO) offspring at weaning from mating of Bbs4GT/+ (n=145 pups) or Bbs4+/KO (n=168 pups) parents. Binomial test was used for statistical comparison of the observed distribution to the expected Mendelian ratio.


( H ) Bbs4 + / + and Bbs4GT / GT female littermates at 20 weeks of age . Representative litter out of seven in total . ( I ) Bbs4 + / + and Bbs4KO / KO female littermates at 20 weeks of age . Representative litter out of five in total .

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(H) Bbs4+/+ and Bbs4GT/GT female littermates at 20 weeks of age. Representative litter out of seven in total. (I) Bbs4+/+ and Bbs4KO/KO female littermates at 20 weeks of age. Representative litter out of five in total.


( J ) Growth curves of Bbs4 - deficient mice , mean ± SD is shown . Females : Bbs4 + / + ( n = 12 mice ) , Bbs4GT / GT ( n = 12 ) , Bbs4KO / KO ( n = 6 ) . Males : Bbs4 + / + ( n = 6 mice ) , Bbs4GT / GT ( n = 7 ) , Bbs4KO / KO ( n = 5 ) .

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(J) Growth curves of Bbs4-deficient mice, mean ± SD is shown. Females: Bbs4+/+ (n=12 mice), Bbs4GT/GT (n=12), Bbs4KO/KO (n=6). Males: Bbs4+/+ (n=6 mice), Bbs4GT/GT (n=7), Bbs4KO/KO (n=5).


( K ) Leptin concentration in blood plasma taken from young adult ( 7 - 8 weeks ) or mid - age ( 14 - 20 weeks ) mice . Young adult mice : Bbs4 + / + ( n = 4 mice ) , Bbs4KO / KO ( n = 3 ) , analyzed in two independent experiments . Mean + SEM . Mid - age adult mice : Bbs4 + / + ( n = 7 mice ) , Bbs4KO / KO ( n = 5 ) , Bbs4GT / GT ( n = 3 ) , analyzed in 4 independent experiments . Kruskal - Wallis tests was used for the statistical analysis . Mean + SEM .

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(K) Leptin concentration in blood plasma taken from young adult (7-8 weeks) or mid-age (14-20 weeks) mice. Young adult mice: Bbs4+/+ (n=4 mice), Bbs4KO/KO (n=3), analyzed in two independent experiments. Mean+SEM. Mid-age adult mice: Bbs4+/+ (n=7 mice), Bbs4KO/KO (n=5), Bbs4GT/GT (n=3), analyzed in 4 independent experiments. Kruskal-Wallis tests was used for the statistical analysis. Mean+SEM.


Cells isolated from the bone marrow ( BM ) of 18 - 25 weeks old Bbs4 + / + ( WT ) , Bbs4GT / GT , and Bbs4KO / KO mice were analyzed by flow cytometry . Number of analyzed mice ( n ) is indicated . Six ( Bbs4GT / GT strain and Bbs4WT / WT controls ) or eight ( Bbs4KO / KO strain and Bbs4WT / WT controls ) independent experiments were performed . ( A ) Percentage of B220low positive cells in the bone marrow . Up : Bbs4 + / + n = 9 , Bbs4GT / GT n = 10 . Down : Bbs4 + / + ( n = 11 ) , Bbs4KO / KO ( n = 10 ) . Statistical significance was calculated using two - tailed Mann - Whitney test . Medians are shown .

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Cells isolated from the bone marrow (BM) of 18-25 weeks old Bbs4+/+ (WT), Bbs4GT/GT, and Bbs4KO/KO mice were analyzed by flow cytometry. Number of analyzed mice (n) is indicated. Six (Bbs4GT/GT strain and Bbs4WT/WT controls) or eight (Bbs4KO/KO strain and Bbs4WT/WT controls) independent experiments were performed. (A) Percentage of B220low positive cells in the bone marrow . Up: Bbs4+/+ n=9, Bbs4GT/GT n=10. Down: Bbs4+/+ (n =11), Bbs4KO/KO (n=10). Statistical significance was calculated using two-tailed Mann-Whitney test. Medians are shown.


Cells isolated from the bone marrow ( BM ) of 18 - 25 weeks old Bbs4 + / + ( WT ) , Bbs4GT / GT , and Bbs4KO / KO mice were analyzed by flow cytometry . Number of analyzed mice ( n ) is indicated . Six ( Bbs4GT / GT strain and Bbs4WT / WT controls ) or eight ( Bbs4KO / KO strain and Bbs4WT / WT controls ) independent experiments were performed . ( B ) Percentage of B - cell precursors ( IgM - IgD - ) in the bone marrow . Gated on viable B220 + cells . Up : Bbs4 + / + ( n = 9 ) , Bbs4GT / GT ( n = 10 ) . Down : Bbs4 + / + ( n = 11 ) , Bbs4KO / KO ( n = 10 ) . Statistical significance was calculated using two - tailed Mann - Whitney test . Medians are shown .

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Cells isolated from the bone marrow (BM) of 18-25 weeks old Bbs4+/+ (WT), Bbs4GT/GT, and Bbs4KO/KO mice were analyzed by flow cytometry. Number of analyzed mice (n) is indicated. Six (Bbs4GT/GT strain and Bbs4WT/WT controls) or eight (Bbs4KO/KO strain and Bbs4WT/WT controls) independent experiments were performed. (B) Percentage of B-cell precursors (IgM- IgD-) in the bone marrow. Gated on viable B220+ cells. Up: Bbs4+/+ (n=9), Bbs4GT/GT (n=10). Down: Bbs4+/+ (n =11), Bbs4KO/KO (n=10). Statistical significance was calculated using two-tailed Mann-Whitney test. Medians are shown.


Cells isolated from the bone marrow ( BM ) of 18 - 25 weeks old Bbs4 + / + ( WT ) , Bbs4GT / GT , and Bbs4KO / KO mice were analyzed by flow cytometry . Number of analyzed mice ( n ) is indicated . Six ( Bbs4GT / GT strain and Bbs4WT / WT controls ) or eight ( Bbs4KO / KO strain and Bbs4WT / WT controls ) independent experiments were performed . ( C ) Percentage of pre - B cells ( CD43 - CD24high ) in the bone marrow of Bbs4 + / + ( n = 11 ) and Bbs4KO / KO mice ( n = 10 ) . Gated on viable B220 + IgM - IgD - cells . Statistical significance was calculated using two - tailed Mann - Whitney test . Medians are shown .

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Cells isolated from the bone marrow (BM) of 18-25 weeks old Bbs4+/+ (WT), Bbs4GT/GT, and Bbs4KO/KO mice were analyzed by flow cytometry. Number of analyzed mice (n) is indicated. Six (Bbs4GT/GT strain and Bbs4WT/WT controls) or eight (Bbs4KO/KO strain and Bbs4WT/WT controls) independent experiments were performed. (C) Percentage of pre-B cells (CD43- CD24high) in the bone marrow of Bbs4+/+ (n=11) and Bbs4KO/KO mice (n=10). Gated on viable B220+ IgM- IgD- cells. Statistical significance was calculated using two-tailed Mann-Whitney test. Medians are shown.


Cells isolated from the spleens ( SPL ) of 18 - 25 weeks old Bbs4 + / + ( WT ) , Bbs4GT / GT , and Bbs4KO / KO mice were analyzed by flow cytometry . Number of analyzed mice ( n ) is indicated . Six ( Bbs4GT / GT strain and Bbs4WT / WT controls ) or eight ( Bbs4KO / KO strain and Bbs4WT / WT controls ) independent experiments were performed . ( D ) Percentage of late mature ( IgM - IgD + ) B cells in the spleen of Bbs4 + / + ( n = 11 ) and Bbs4KO / KO mice ( n = 10 ) Gated on viable CD19 + cells . Statistical significance was calculated using two - tailed Mann - Whitney test . Medians are shown .

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Cells isolated from the spleens (SPL) of 18-25 weeks old Bbs4+/+ (WT), Bbs4GT/GT, and Bbs4KO/KO mice were analyzed by flow cytometry. Number of analyzed mice (n) is indicated. Six (Bbs4GT/GT strain and Bbs4WT/WT controls) or eight (Bbs4KO/KO strain and Bbs4WT/WT controls) independent experiments were performed. (D) Percentage of late mature (IgM- IgD+) B cells in the spleen of Bbs4+/+ (n=11) and Bbs4KO/KO mice (n=10) Gated on viable CD19+ cells. Statistical significance was calculated using two-tailed Mann-Whitney test. Medians are shown.


Cells isolated from the spleens ( SPL ) of 18 - 25 weeks old Bbs4 + / + ( WT ) , Bbs4GT / GT , and Bbs4KO / KO mice were analyzed by flow cytometry . Number of analyzed mice ( n ) is indicated . Six ( Bbs4GT / GT strain and Bbs4WT / WT controls ) or eight ( Bbs4KO / KO strain and Bbs4WT / WT controls ) independent experiments were performed . ( E ) An alternative analysis of the experiment shown in ( D ) . Geometric mean fluorescence intensity ( MFI ) of BV421 ( IgM ) on IgD + B cells was determined . Mean of MFI values for each genotype per experiment was quantified , and obtained values were normalized to Bbs4 + / + ( = 1 ) for each experiment . Two - tailed One Sample Wilcoxon Signed Rank Test was used for the statistical analysis .

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Cells isolated from the spleens (SPL) of 18-25 weeks old Bbs4+/+ (WT), Bbs4GT/GT, and Bbs4KO/KO mice were analyzed by flow cytometry. Number of analyzed mice (n) is indicated. Six (Bbs4GT/GT strain and Bbs4WT/WT controls) or eight (Bbs4KO/KO strain and Bbs4WT/WT controls) independent experiments were performed. (E) An alternative analysis of the experiment shown in (D). Geometric mean fluorescence intensity (MFI) of BV421 (IgM) on IgD+ B cells was determined. Mean of MFI values for each genotype per experiment was quantified, and obtained values were normalized to Bbs4+/+ (=1) for each experiment. Two-tailed One Sample Wilcoxon Signed Rank Test was used for the statistical analysis.