B . HeLa treated with non - targeting ( Ctrl ) or C9orf72 siRNA were transfected with mCherry - FIP200 . 24 h post transfection were treated for 3 h with Torin1 ( 250 nM ) or vehicle ( Ctrl ) . Translocation of ULK1 was quantified as number of mCherry - FIP200 positive per from 3 independent experiments ( Mean ± ; one - way ANOVA with Fisher ' s LSD , ns : not significant , * * * * P ≤ 0 . 0001 ; N ( ) = Ctrl / Ctrl : 65 ; Ctrl / Torin1 : 60 ; C9orf72 / Ctrl : 54 ; C9orf72 / Torin1 : 49 ) . C9orf72 knockdown was determined by RT - qPCR ( Appendix Fig S2 ) . Scale bar = 10 µm .

O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O B-GENEPROD O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

B. HeLa cells treated with non-targeting (Ctrl) or C9orf72 siRNA were transfected with mCherry-FIP200. 24 h post transfection cells were treated for 3 h with Torin1 (250 nM) or vehicle (Ctrl). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per cell from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, ns: not significant, **** P ≤ 0.0001; N (cells) = Ctrl/Ctrl: 65; Ctrl/Torin1: 60; C9orf72/Ctrl: 54; C9orf72/Torin1: 49). C9orf72 knockdown was determined by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm.


C . cortical neurons ( DIV5 / 6 ) were transfected with EmGFP non - targeting ( Ctrl ) or C9orf72 miRNA ( green ) mCherry - FIP200 ( red ) ; for rescue experiments were additionally transfected with mCerulean - tagged C9orf72s C9orf72L ( cyan ) . 3 days post transfection neurons were treated for 3 h with Torin1 ( 250 nM ) or vehicle ( Ctrl ) . Translocation of ULK1 was quantified as number of mCherry - FIP200 positive per soma from 2 independent experiments ( Mean ± ; one - way ANOVA with Fisher ' s LSD , ns : not significant , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ; N ( ) = Ctrl miRNA / Ctrl : 134 ; Ctrl miRNA / Torin1 : 125 ; C9orf72 miRNA / Ctrl : 101 ; C9orf72 miRNA / Torin1 : 78 ; C9orf72 miRNA + C9orf72L + C9orf72S : 41 ; C9orf72 miRNA + C9orf72L + C9orf72S / Torin1 : 39 ) . Scale bar = 5 µm .

O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O B-GENEPROD O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O

C. Primary cortical neurons (DIV5/6) were transfected with EmGFP non-targeting (Ctrl) or C9orf72 miRNA (green) and mCherry-FIP200 (red); for rescue experiments the cells were additionally transfected with mCerulean-tagged C9orf72s and C9orf72L (cyan). 3 days post transfection neurons were treated for 3 h with Torin1 (250 nM) or vehicle (Ctrl). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per soma from 2 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, ns: not significant, *** P ≤ 0.001, **** P ≤ 0.0001; N (cells) = Ctrl miRNA/Ctrl: 134; Ctrl miRNA/Torin1: 125; C9orf72 miRNA/Ctrl: 101; C9orf72 miRNA/Torin1: 78; C9orf72 miRNA+C9orf72L+C9orf72S: 41; C9orf72 miRNA+C9orf72L+C9orf72S/Torin1: 39). Scale bar = 5 µm.


D . HeLa were co - transfected with mCherry - FIP200 ( red ) empty vector ( EV ) , FLAG - C9orf72L or FLAG - C9orf72S ( green ) . As positive control EV transfected were treated for 3 h with Torin1 ( 250 nM ) . Translocation of ULK1 was quantified as number of mCherry - FIP200 positive per from 3 independent experiments ( Mean ± ; one - way ANOVA with Fisher ' s LSD , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ) ; N ( ) = EV : 47 , EV + Torin1 : 31 , C9orf72L : 46 , C9orf72S : 45 ) . Scale bar = 10 µm .

O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O

D. HeLa cells were co-transfected with mCherry-FIP200 (red) and empty vector (EV), FLAG-C9orf72L or FLAG-C9orf72S (green). As positive control EV transfected cells were treated for 3 h with Torin1 (250 nM). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per cell from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, *** P ≤ 0.001, **** P ≤ 0.0001); N (cells) = EV: 47, EV + Torin1: 31, C9orf72L: 46, C9orf72S: 45). Scale bar = 10 µm.


A , B . HeLa ( A ) or SH - SY5Y neuroblastoma ( B ) treated with non - targeting ( Ctrl ) or Rab1a siRNA were co - transfected with mCherry - FIP200 ( red ) empty vector ( EV ) , Myc - C9orf72L or Myc - C9orf72S ( green ) . As positive control EV transfected were treated for 3 h with Torin1 ( 250 nM ) . Translocation of ULK1 was quantified as number of mCherry - FIP200 positive per ( A , HeLa , Mean ± ; one - way ANOVA with Fisher ' s LSD ; ns , not significant ; * * * * P ≤ 0 . 0001 ; N ( from 3 independent experiments ) = Ctrl / EV : 81 ; Ctrl / EV / Torin1 : 48 ; Ctrl / C9orf72L : 86 ; Ctrl / C9orf72S : 48 ; Rab1a / EV : 79 ; Rab1a / EV / Torin1 : 68 ; Rab1a / C9orf72L : 78 ; Rab1a / C9orf72S : 52 ; B , SH - SY5Y : Mean ± ; one - way ANOVA with Fisher ' s LSD ; ns , not significant ; * * P ≤ 0 . 01 ; * * * * P ≤ 0 . 0001 ; N ( from 2 independent experiments ) = Ctrl / EV : 70 ; Ctrl / EV / Torin1 : 56 ; Ctrl / C9orf72L : 45 ; Ctrl / C9orf72S : 43 ; Rab1a / EV : 63 ; Rab1a / EV / Torin1 : 55 ; Rab1a / C9orf72L : 44 ; Rab1a / C9orf72S : 37 ) . Rab1a knockdown was confirmed by RT - qPCR ( Appendix Fig S2 ) . Scale bar = 10 µm .

O O O O B-CELL_LINE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

A, B. HeLa cells (A) or SH-SY5Y neuroblastoma cells (B) treated with non-targeting (Ctrl) or Rab1a siRNA were co-transfected with mCherry-FIP200 (red) and empty vector (EV), Myc-C9orf72L or Myc-C9orf72S (green). As positive control EV transfected cells were treated for 3 h with Torin1 (250 nM). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per cell (A, HeLa, Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; **** P ≤ 0.0001; N (cells from 3 independent experiments) = Ctrl/EV: 81; Ctrl/EV/Torin1: 48; Ctrl/C9orf72L: 86; Ctrl/C9orf72S: 48; Rab1a/EV: 79; Rab1a/EV/Torin1: 68; Rab1a/C9orf72L: 78; Rab1a/C9orf72S: 52; B, SH-SY5Y: Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; ** P ≤ 0.01; **** P ≤ 0.0001; N (cells from 2 independent experiments) = Ctrl/EV: 70; Ctrl/EV/Torin1: 56; Ctrl/C9orf72L: 45; Ctrl/C9orf72S: 43; Rab1a/EV: 63; Rab1a/EV/Torin1: 55; Rab1a/C9orf72L: 44; Rab1a/C9orf72S: 37). Rab1a knockdown was confirmed by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm.


C . SH - SY5Y neuroblastoma treated with non - targeting ( Ctrl ) or Rab1a siRNA were co - transfected with EGFP - LC3 ( green ) empty vector ( EV ) , Myc - C9orf72L or Myc - C9orf72S ( red ) . As positive control EV transfected were treated for 3 h with Torin1 ( 250 nM ) . Autophagosomes were quantified as number of EGFP - LC3 positive per ( Mean ± ; one - way ANOVA with Fisher ' s LSD ; ns , not significant ; * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ; N ( from 2 independent experiments ) = Ctrl / EV : 102 ; Ctrl / EV / Torin1 : 92 ; Ctrl / C9orf72L : 97 ; Ctrl / C9orf72S : 76 ; Rab1a / EV : 102 ; Rab1a / EV / Torin1 : 107 ; Rab1a / C9orf72L : 101 ; Rab1a / C9orf72S : 87 ) . Rab1a knockdown was confirmed by RT - qPCR ( Appendix Fig S2 ) . Scale bar = 10 µm .

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

C. SH-SY5Y neuroblastoma cells treated with non-targeting (Ctrl) or Rab1a siRNA were co-transfected with EGFP-LC3 (green) and empty vector (EV), Myc-C9orf72L or Myc-C9orf72S (red). As positive control EV transfected cells were treated for 3 h with Torin1 (250 nM). Autophagosomes were quantified as the number of EGFP-LC3 positive puncta per cell (Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; * P ≤ 0.05, *** P ≤ 0.001, **** P ≤ 0.0001; N (cells from 2 independent experiments) = Ctrl/EV: 102; Ctrl/EV/Torin1: 92; Ctrl/C9orf72L: 97; Ctrl/C9orf72S: 76; Rab1a/EV: 102; Rab1a/EV/Torin1: 107; Rab1a/C9orf72L: 101; Rab1a/C9orf72S: 87). Rab1a knockdown was confirmed by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm.


B . lysates of HEK293 co - transfected with Myc - Rab1a either empty vector , FLAG - C9orf72S or FLAG - C9orf72L were subjected to immunoprecipitation with anti - FLAG antibody . Bound was eluted from beads using excess FLAG peptide . Immune eluates were probed for FLAG - C9orf72 Myc - Rab1a on immunoblots . input of FLAG - C9orf72 Myc - Rab1a in transfected are shown ( Inputs ) . * indicates remaining Myc - Rab1a signal after reprobing for FLAG - C9orf72 .

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

B. Cell lysates of HEK293 cells co-transfected with Myc-Rab1a and either empty vector, FLAG-C9orf72S or FLAG-C9orf72L were subjected to immunoprecipitation with anti-FLAG antibody. Bound protein was eluted from beads using excess FLAG peptide. Immune eluates were probed for FLAG-C9orf72 and Myc-Rab1a on immunoblots. The input levels of FLAG-C9orf72 and Myc-Rab1a in the transfected cells are shown (Inputs).* indicates remaining Myc-Rab1a signal after reprobing for FLAG-C9orf72.


C . lysates of HEK293 transfected with Myc - C9orf72S or Myc - C9orf72L were subjected to immunoprecipitation with anti - Myc antibody . resulting immune pellet was probed for endogenous Rab1a .

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

C. Cell lysates of HEK293 cells transfected with Myc-C9orf72S or Myc-C9orf72L were subjected to immunoprecipitation with anti-Myc antibody. The resulting immune pellet was probed for endogenous Rab1a.


D . 35S - radiolabelled recombinant Myc - Rab1a loaded with vehicle , GDP or GMP - PNP was added to GST , GST - C9orf72S GST - C9orf72L immobilized on glutathione - coated beads . 35S - radiolabelled recombinant Myc - Rab1a was visualized by autoradiography ( top panel ) . Coomassie - stained GST , GST - C9orf72S GST - C9orf72L in pull - down samples are shown ( bottom panel ) . identity of Coomassie bands was confirmed by mass spectrometry ( # = E . coli DnaK Chaperonin ; * = E . coli 60kD Chaperonin ; Appendix Fig S3 ) . Relative binding of Rab1a to C9orf72 was quantified from 3 independent experiments ( Mean ± ; one - way ANOVA with Fisher ' s LSD ; ns , not significant ; * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ) .

O O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD I-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 35S-radiolabelled recombinant Myc-Rab1a protein loaded with vehicle, GDP or GMP-PNP was added to GST, GST-C9orf72S and GST-C9orf72L immobilized on glutathione-coated beads. 35S-radiolabelled recombinant Myc-Rab1a protein was visualized by autoradiography (top panel). Coomassie-stained GST, GST-C9orf72S and GST-C9orf72L in the pull-down samples are shown (bottom panel). The identity of the Coomassie protein bands was confirmed by mass spectrometry (# = E. coli DnaK Chaperonin; * = E. coli 60kD Chaperonin; Appendix Fig S3). Relative binding of Rab1a to C9orf72 was quantified from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).


E . Increasing volumes of 35S - radiolabelled recombinant Myc - C9orf72L were incubated with equal amounts of GST - Rab1a immobilized on glutathione - coated beads in an equilibrium binding experiment . 8 µl of 35S - radiolabelled recombinant Myc - C9orf72L was incubated with GST as a negative control . Bound 35S - radiolabelled Myc - C9orf72L was visualized by phosphoimager . Coomassie stained GST - Rab1a GST in pull down samples are shown . Densitometry of amount of 35S - radiolabelled recombinant Myc - C9orf72L bound to GST - Rab1a in different binding reactions was used to fit an equilibrium binding hyperbola ( R2 = 0 . 94 ) .

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

E. Increasing volumes of 35S-radiolabelled recombinant Myc-C9orf72L protein were incubated with equal amounts of GST-Rab1a immobilized on glutathione-coated beads in an equilibrium binding experiment. 8 µl of 35S-radiolabelled recombinant Myc-C9orf72L protein was incubated with GST as a negative control. Bound 35S-radiolabelled Myc-C9orf72L protein was visualized by phosphoimager. Coomassie stained GST-Rab1a and GST in the pull down samples are shown. Densitometry analysis of the amount of 35S-radiolabelled recombinant Myc-C9orf72L protein bound to GST-Rab1a in the different binding reactions was used to fit an equilibrium binding hyperbola (R2=0.94).


A . HEK293 treated with non - targeting ( Ctrl ) or C9orf72 siRNA were transfected with HA - ULK1 , Myc - Rab1a or HA - ULK1 + Myc - Rab1a as indicated . Transfections were laced with mVenus to enable identification of transfected for ( green ) . Transfected were probed with anti - HA anti - Myc antibodies processed for PLA . PLA proximity signals ( red ) per were determined from 3 independent experiments ( Mean ± ; one - way ANOVA with Fisher ' s LSD , * * * * P ≤ 0 . 0001 ) ; N ( ) = Ctrl / HA - ULK1 : 125 , Ctrl / Myc - Rab1a : 149 , Ctrl / HA - ULK1 + Myc - Rab1a : 163 , C9orf72 / HA - ULK1 : 136 , C9orf72 / Myc - Rab1a : 133 , C9orf72 / HA - ULK1 + Myc - Rab1a : 155 ) . Scale bar = 20µm .

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

A. HEK293 cells treated with non-targeting (Ctrl) or C9orf72 siRNA were transfected with HA-ULK1, Myc-Rab1a or HA-ULK1+Myc-Rab1a as indicated. Transfections were laced with mVenus to enable identification of transfected cells for analysis (green). Transfected cells were probed with anti-HA and anti-Myc antibodies and processed for PLA. PLA proximity signals (red) per cell were determined from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, **** P ≤ 0.0001); N (cells) = Ctrl/HA-ULK1: 125, Ctrl/Myc-Rab1a: 149, Ctrl/HA-ULK1+Myc-Rab1a: 163, C9orf72/HA-ULK1: 136, C9orf72/Myc-Rab1a: 133, C9orf72/HA-ULK1+Myc-Rab1a: 155). Scale bar = 20µm.


B . HeLa treated with non - targeting ( Ctrl ) or C9orf72 siRNA were co - transfected with mCherry - FIP200 ( red ) empty vector ( EV ) or Myc - Rab1a ( Q70L ) ( green ) . Translocation of ULK1 was quantified as number of mCherry - FIP200 positive per from 4 independent experiments ( Mean ± ; one - way ANOVA with Fisher ' s LSD , * P ≤ 0 . 05 , * * * * P ≤ 0 . 0001 ) ; N ( ) = Ctrl / EV : 101 ; Ctrl / Q70L : 106 ; C9orf72 / Q70L : 42 . C9orf72 knockdown was determined by RT - qPCR ( Appendix Fig S2 ) . Scale bar = 10 µm .

O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

B. HeLa cells treated with non-targeting (Ctrl) or C9orf72 siRNA were co-transfected with mCherry-FIP200 (red) and empty vector (EV) or Myc-Rab1a(Q70L) (green). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per cell from 4 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, * P ≤ 0.05, **** P ≤ 0.0001); N (cells) = Ctrl/EV: 101; Ctrl/Q70L: 106; C9orf72/Q70L: 42. C9orf72 knockdown was determined by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm.


C . HeLa treated with non - targeting ( Ctrl ) or C9orf72 siRNA were co - transfected with EGFP - LC3 ( green ) empty vector ( EV ) or Myc - Rab1a ( Q70L ) ( red ) . Autophagosomes were quantified as number of EGFP - LC3 positive per ( Mean ± ; one - way ANOVA with Fisher ' s LSD , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 ) ; N ( ) = Ctrl / EV : 44 ; Ctrl / Q70L : 32 ; C9orf72 / Q70L : 47 . C9orf72 knockdown was determined by RT - qPCR ( Appendix Fig S2 ) . Scale bar = 10 µm .

O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

C. HeLa cells treated with non-targeting (Ctrl) or C9orf72 siRNA were co-transfected with EGFP-LC3 (green) and empty vector (EV) or Myc-Rab1a(Q70L) (red). Autophagosomes were quantified as the number of EGFP-LC3 positive puncta per cell (Mean ± SEM; one-way ANOVA with Fisher's LSD test, ** P ≤ 0.01, **** P ≤ 0.0001); N (cells) = Ctrl/EV: 44; Ctrl/Q70L: 32; C9orf72/Q70L: 47. C9orf72 knockdown was determined by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm.


A . HeLa treated with non - targeting ( Ctrl ) or C9orf72 siRNA were immunostained for endogenous p62 . Accumulation of p62 was quantified by counting p62 positive per from 3 independent experiments ( Mean ± ; unpaired t - , * * * * P ≤ 0 . 0001 ) ; N ( ) = Ctrl : 74 , C9orf72 : 55 ) . C9orf72 knockdown was confirmed by RT - qPCR ( Appendix Fig S2 ) . Scale bar = 10 µm .

O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

A. HeLa cells treated with non-targeting (Ctrl) or C9orf72 siRNA were immunostained for endogenous p62. Accumulation of p62 was quantified by counting p62 positive puncta per cell from 3 independent experiments (Mean ± SEM; unpaired t-test, **** P ≤ 0.0001); N (cells) = Ctrl: 74, C9orf72: 55). C9orf72 knockdown was confirmed by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm.


B . cortical neurons ( DIV5 ) were transduced with 4TU / EmGFP non - targeting control miRNA ( Ctrl ) or C9orf72 miRNA ( green ) ; for rescue experiments were additionally transduced with 4TU / mVenus - tagged C9orf72s C9orf72L ( verified by immunoblot , Appendix Fig S2 ) . Neurons were immunostained for endogenous p62 3 days post transduction . Accumulation of p62 was quantified by counting p62 positive per soma from 2 independent experiments ( Mean ± ; one - way ANOVA with Fisher ' s LSD , * * P ≤ 0 . 01 ; N ( ) = Ctrl miRNA : 80 ; C9orf72 miRNA : 80 ; C9orf72 miRNA + C9orf72L + C9orf72S : 75 ) . Scale bar = 10 µm .

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

B. Primary cortical neurons (DIV5) were transduced with 4TU/cell EmGFP non-targeting control miRNA (Ctrl) or C9orf72 miRNA (green); for rescue experiments the cells were additionally transduced with 4TU/cell mVenus-tagged C9orf72s and C9orf72L (verified by immunoblot, Appendix Fig S2). Neurons were immunostained for endogenous p62 3 days post transduction. Accumulation of p62 was quantified by counting p62 positive puncta per soma from 2 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, ** P ≤ 0.01; N (cells) = Ctrl miRNA: 80; C9orf72 miRNA: 80; C9orf72 miRNA+C9orf72L+C9orf72S: 75). Scale bar = 10 µm.


A , B . Two C9ALS / FTD iNeuron cultures ( 183 201 ) their matching controls ( 170 155 ) were treated with vehicle ( Ctrl ) or bafilomycin A1 ( 100 nM ; 6 h - BafA1 ) processed for immunoblot detection of LC3 . LC3 - II were normalized to α - Tubulin ( Mean ± ; one - way ANOVA with Fisher ' s LSD ; ns , not significant ; * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * * P ≤ 0 . 0001 ; Ctrl 209 / Pat 201 , n = 4 ; Ctrl 155 / Pat 183 , n = 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 B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B. Two C9ALS/FTD iNeuron cultures (183 and 201) and their matching controls (170 and 155) were treated with vehicle (Ctrl) or bafilomycin A1 (100 nM; 6 h - BafA1) and processed for immunoblot detection of LC3. LC3-II levels were normalized to α-Tubulin (Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; * P ≤ 0.05; ** P ≤ 0.01; **** P ≤ 0.0001; Ctrl 209/Pat 201, n=4; Ctrl 155/Pat 183, n=6).


( A ) Wild - type B were treated with 5 μg / mL of anti - kappa antibody ( a - k chain , top ) or 1 µM LatrunculinA ( LatA , bottom ) . Intracellular calcium flux was measured by flow cytometry in presence of Src - inhibitor ( PP2 ) , Syk - inhibitor ( BAY 613606 ) or vehicle control .

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

(A) Wild-type primary B cells were treated with 5 μg/mL of anti-kappa antibody (a-k chain, top) or 1 µM LatrunculinA (LatA, bottom). Intracellular calcium flux was measured by flow cytometry in the presence of Src-inhibitor (PP2), Syk-inhibitor (BAY 613606) or vehicle control.


( B ) Wild - type B were treated with vehicle control ( - ) , 5 μg / mL anti - kappa antibody or 1 µM LatA for indicated time . were lysed analysed by SDS - PAGE followed by immunoblotting with phospho - Lyn ( Y507 ) actin as loading control . of phosphorylated , normalized to actin , was referred to respective unstimulated sample , set as 1 .

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

(B) Wild-type primary B cells were treated with vehicle control (-), 5 μg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-Lyn (Y507) and actin as loading control. The intensity of phosphorylated proteins, normalized to actin, was referred to the respective unstimulated sample, set as 1.


( C D ) Wild - type B were treated with vehicle control ( - ) , 5 µg / mL anti - kappa antibody or 1 µM LatA for indicated time . were lysed analysed by SDS - PAGE followed by immunoblotting with phospho - Syk ( pSyk ) , phospho - CD19 ( pCD19 ) , phospho - Akt ( pAkt ) , phospho - ERK ( pERK ) total ERK as loading control . of phosphorylated , normalized to ERK , was referred to respective unstimulated sample , set as 1 .

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

(C and D) Wild-type primary B cells were treated with vehicle control (-), 5 µg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-Syk (pSyk), phospho-CD19 (pCD19), phospho-Akt (pAkt), phospho-ERK (pERK) and total ERK as loading control. The intensity of phosphorylated proteins, normalized to ERK, was referred to the respective unstimulated sample, set as 1.


( E F ) Wild - type B were treated with vehicle control ( - ) , 5 µg / mL anti - kappa antibody or 1 µM LatA for indicated time . were lysed analysed by SDS - PAGE followed by immunoblotting with phospho - PLCγ2 ( pPLCγ2 ) total ERK as loading control . of phosphorylated , normalized to ERK , was referred to respective unstimulated sample , set as 1 .

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

(E and F) Wild-type primary B cells were treated with vehicle control (-), 5 µg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-PLCγ2 (pPLCγ2) and total ERK as loading control. The intensity of phosphorylated proteins, normalized to ERK, was referred to the respective unstimulated sample, set as 1.


( G H ) Wild - type B were treated with vehicle control ( - ) , 5 µg / mL anti - kappa antibody or 1 µM LatA for indicated time . were lysed analysed by SDS - PAGE followed by immunoblotting with phospho - CD22 ( pCD22 ) , phospho - SHP1 ( pSHP1 ) , CD22 or total ERK as loading controls . of phosphorylated , normalized to CD22 or ERK , was referred to respective unstimulated sample , set as 1 .

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

(G and H) Wild-type primary B cells were treated with vehicle control (-), 5 µg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-CD22 (pCD22), phospho-SHP1 (pSHP1), and CD22 or total ERK as loading controls. The intensity of phosphorylated proteins, normalized to CD22 or ERK, was referred to the respective unstimulated sample, set as 1.


( I ) Wild - type Cd22 - / - B were treated with 5 µg / mL anti - kappa antibody or 1 µM LatA intracellular calcium flux was measured by flow cytometry .

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

(I) Wild-type and Cd22-/- primary B cells were treated with 5 µg/mL anti-kappa antibody or 1 µM LatA and intracellular calcium flux was measured by flow cytometry.


( J ) Wild - type Cd22Y2 , 5 , 6F B were treated with 5 µg / mL anti - kappa antibody or 1 µM LatA intracellular calcium flux was measured by flow cytometry .

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

(J) Wild-type and Cd22Y2,5,6F primary B cells were treated with 5 µg/mL anti-kappa antibody or 1 µM LatA and intracellular calcium flux was measured by flow cytometry.


( K L ) Wild - type , Cd22 - / - Cd22Y2 , 5 , 6F B were treated with vehicle control ( - ) or 1 µM LatA for indicated time . were lysed analysed by SDS - PAGE followed by immunoblotting with phospho - CD19 ( pCD19 ) , phospho - Akt ( pAkt ) , phospho - ERK ( pERK ) actin as loading control . of phosphorylated , normalized to actin , was referred to unstimulated sample of wild type , set as 1 .

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

(K and L) Wild-type, Cd22-/- and Cd22Y2,5,6F primary B cells were treated with vehicle control (-) or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-CD19 (pCD19), phospho-Akt (pAkt), phospho-ERK (pERK) and actin as loading control. The intensity of phosphorylated proteins, normalized to actin, was referred to the unstimulated sample of the wild type cells, set as 1.


( A ) Determining number density of CD22 , CD19 , IgM IgD on surface of wild - type B . were stained with PE - conjugated antibody against CD22 ( blue profile ) , CD19 ( red profile ) , IgM ( red profile ) or IgD ( red profile ) their profiles compared to those of standard beads ( grey profiles ) , which each possess a well - defined number of binding sites ( st0 = blank , st1 - st4 = increasing number of binding sites ) against antibody . For IgM , where range of expression is large , low high 25 percentiles were calculated as in Mattila et al . 2013 shown in brackets . Data are representative of three independent experiments .

O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Determining the number and density of CD22, CD19, IgM and IgD on the surface of wild-type primary B cells. Cells were stained with PE-conjugated antibody against CD22 (blue profile), CD19 (red profile), IgM (red profile) or IgD (red profile) and their fluorescence profiles compared to those of standard beads (grey profiles), which each possess a well-defined number of binding sites (st0=blank, st1-st4=increasing number of binding sites) against the primary antibody. For IgM, where the range of expression is large, the low and high 25 percentiles were calculated as in Mattila et al. 2013 and shown in brackets.Data are representative of three independent experiments.


( B - E ) Dual - colour SIM of CD22 , IgM , IgD CD19 . Wild - type B were fixed , stained with Atto633 or 488 - conjugated antibody against CD22 , IgM or IgD Alexa647 - conjugated antibody against CD19 settled onto nonstimulatory coverslips . were then embedded in agarose , imaged with SIM analysed . ( B ) SIM of CD22 , IgM , IgD CD19 . ( C ) Cross - correlation function . ( D ) Pearson correlation coefficient . ( E ) Cluster calculated from autocorrelation .

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

(B-E) Dual-colour SIM analysis of CD22, IgM, IgD and CD19. Wild-type primary B cells were fixed, stained with Atto633 or 488-conjugated antibody against CD22, IgM or IgD and Alexa647-conjugated antibody against CD19 and settled onto nonstimulatory coverslips. Cells were then embedded in agarose, imaged with SIM and analysed.(B) SIM images of CD22, IgM, IgD and CD19.(C) Cross-correlation function.(D) Pearson correlation coefficient.(E) Cluster size calculated from autocorrelation analysis.


Figure 3 . CD22 is highly clustered on surface of resting B ( A - C ) dSTORM of CD22 , CD19 , IgM IgD . Wild - type B were stained with Alexa647 - conjugated antibody against CD22 , Cy5 - conjugated antibody against CD19 or IgD , or Cy5 - conjugated Fab fragments against IgM settled onto nonstimulatory coverslips . were then fixed , imaged with dSTORM analysed . ( A ) 2D ( top middle ) pseudo 3D ( bottom ) dSTORM were reconstructed from single molecule localizations . dashed square is shown in magnification ( middle ) pseudo 3D ( bottom ) . ( B C ) of distribution of CD22 , CD19 , IgM IgD with H function Hopkins index . Error bars ( Hopkins index ) thin lines ( H function ) denote mean ± . Data are pooled from at least three experiments per receptor . * P < 0 . 001 , * * P < 0 . 0001 ( Student ' s t - ) .

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

Figure 3. CD22 is highly clustered on the surface of resting B cells(A-C) dSTORM analysis of CD22, CD19, IgM and IgD. Wild-type primary B cells were stained with Alexa647-conjugated antibody against CD22, Cy5-conjugated antibody against CD19 or IgD, or Cy5-conjugated Fab fragments against IgM and settled onto nonstimulatory coverslips. Cells were then fixed, imaged with dSTORM and analysed.(A) 2D (top and middle) and pseudo 3D (bottom) dSTORM images were reconstructed from single molecule localizations. The white dashed square is shown in magnification (middle) and pseudo 3D (bottom).(B and C) Quantification of the distribution of CD22, CD19, IgM and IgD with H function and Hopkins index. Error bars (Hopkins index) and thin lines (H function) denote mean ± SEM. Data are pooled from at least three experiments per receptor.*P < 0.001, **P < 0.0001 (Student's t-test).


( A - C ) Single - particle - tracking of CD22 , CD19 , IgM IgD . Wild - type B were stained with Atto633 - conjugated Fab fragments against CD22 , CD19 , IgM IgD , settled onto nonstimulatory coverslips imaged . Single particle trajectories were then analysed . ( A ) Trajectories of CD22 showing diffusion of single particles over 6 s . ( B ) Magnified region from ( A ) traces one particle ( current position indicated with dashed circle , trajectory in red ) over 6 s . ( C ) Diffusion coefficients of 300 representative CD22 , CD19 , IgM IgD particles . Bars indicate median . Data are pooled from at least three experiments per receptor . * P < 0 . 0001 ( Student ' s t - ) .

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

(A-C) Single-particle-tracking of CD22, CD19, IgM and IgD. Wild-type primary B cells were stained with Atto633-conjugated Fab fragments against CD22, CD19, IgM and IgD, settled onto nonstimulatory coverslips and imaged. Single particle trajectories were then analysed.(A) Trajectories of CD22 showing diffusion of single particles over 6 s.(B) Magnified region from (A) traces one particle (current position indicated with white dashed circle, trajectory in red) over 6 s.(C) Diffusion coefficients of 300 representative CD22, CD19, IgM and IgD particles. Bars indicate the median. Data are pooled from at least three experiments per receptor. *P < 0.0001 (Student's t-test).


( D - F ) Single - particle - tracking of CD22 in LifeAct - GFP - expressing B . were labelled with Atto633 - conjugated Fab fragments against CD22 , settled onto nonstimulatory coverslips imaged . Single particle trajectories were then analysed . ( D ) Trajectories of CD22 ( left ) showing diffusion of single particles over 6 s overlaid with a still image of F - actin ( middle ) . Areas of high F - actin density ( grey boundaries ) with trajectories of CD22 classified as being inside ( red ) outside ( blue ) of F - actin rich areas . ( E ) Diffusion coefficients of CD22 particles from ( D ) whose trajectories were classed as inside ( in ) or outside ( out ) of actin rich areas . overall diffusion is also shown ( ) . Bars numbers are median . Data are pooled from two different experiments . * P < 0 . 0001 ( Student ' s t - ) . ( F ) Correlation . For each position in each CD22 track , actin was measured compared to same number of random positions on same . Values from different were normalized so that average in F - actin rich regions corresponds to a value of one average for F - actin low regions to zero .

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

(D-F) Single-particle-tracking of CD22 in primary LifeAct-GFP-expressing B cells. Cells were labelled with Atto633-conjugated Fab fragments against CD22, settled onto nonstimulatory coverslips and imaged. Single particle trajectories were then analysed.(D) Trajectories of CD22 (left) showing diffusion of single particles over 6 s and overlaid with a still image of F-actin intensity (middle). Areas of high F-actin density (grey line boundaries) with trajectories of CD22 classified as being inside (red) and outside (blue) of F-actin rich areas.(E) Diffusion coefficients of CD22 particles from (D) whose trajectories were classed as inside (in) or outside (out) of actin rich areas. The overall diffusion is also shown (all). Bars and numbers are the median. Data are pooled from two different experiments. *P < 0.0001 (Student's t-test).(F) Correlation analysis. For each position in each CD22 track, the actin intensity was measured and compared to the same number of random positions on the same cell. Values from different cells were normalized so that the average intensity in F-actin rich regions corresponds to a value of one and the average for F-actin low regions to zero.


( G ) Wild - type B were labelled with Atto633 - conjugated Fab fragments against CD22 , settled onto nonstimulatory coverslips treated with vehicle control ( DMSO ) or 1µM LatA . Single particle trajectories were then analysed . Bars numbers indicate median . Data are pooled from two experiments . * P < 0 . 0001 ( Student ' s t - ) .

O O O O O O B-CELL_TYPE O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O 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) Wild-type primary B cells were labelled with Atto633-conjugated Fab fragments against CD22, settled onto nonstimulatory coverslips and treated with vehicle control (DMSO) or 1µM LatA. Single particle trajectories were then analysed. Bars and numbers indicate the median. Data are pooled from two experiments.*P < 0.0001 (Student's t-test).


( H - J ) Wild - type B were stained with Alexa647 - conjugated antibody against CD22 settled onto nonstimulatory coverslips . were then fixed , imaged with dSTORM analysed . ( H ) 2D ( top ) pseudo 3D ( bottom ) dSTORM were reconstructed from single molecule localizations . dashed square is shown in pseudo 3D ( bottom ) . ( I - J ) of distribution of CD22 Hopkins index with H function . Error bars ( Hopkins index ) thin lines ( H function ) denote mean ± . Data are pooled from two different experiments .

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

(H-J) Wild-type primary B cells were stained with Alexa647-conjugated antibody against CD22 and settled onto nonstimulatory coverslips. Cells were then fixed, imaged with dSTORM and analysed.(H) 2D (top) and pseudo 3D (bottom) dSTORM images were reconstructed from single molecule localizations. The white dashed square is shown in pseudo 3D (bottom).(I-J) Quantification of the distribution of CD22 Hopkins index and with H function. Error bars (Hopkins index) and thin lines (H function) denote mean ± SEM.Data are pooled from two different experiments.


( K ) Flow cytometric of F - actin by intracellular phalloidin of unstimulated or LatA treated wild - type B for indicated of time .

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

(K) Flow cytometric analysis of F-actin by intracellular phalloidin staining of unstimulated or LatA treated wild-type B cells for the indicated length of time.


( L ) Flow cytometric of CD22 surface expression in resting or stimulated B with 10µg / ml anti - µ chain ( Jackson ImmunoResearch ) , 1µM or 10µM LatA for 5 15 minutes . Error bars denote mean ± SD .

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

(L) Flow cytometric analysis of CD22 surface expression in resting or stimulated B cells with 10µg/ml anti-µ chain (Jackson ImmunoResearch), 1µM or 10µM LatA for 5 and 15 minutes. Error bars denote mean ± SD.


( A - B ) Wild - type CD45 - deficient B were stained with Alexa647conjugated antibody against CD22 settled onto nonstimulatory coverslips . were then fixed , imaged with dSTORM analysed . ( A ) 2D ( top ) pseudo 3D ( bottom ) dSTORM were reconstructed from single molecule localizations . dashed square is shown in pseudo 3D ( bottom ) . ( B ) Error bars ( Hopkins index ) thin lines ( H function ) denote mean ± . Data are pooled from three experiments .

O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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) Wild-type and CD45-deficient primary B cells were stained with Alexa647conjugated antibody against CD22 and settled onto nonstimulatory coverslips. Cells were then fixed, imaged with dSTORM and analysed.(A) 2D (top) and pseudo 3D (bottom) dSTORM images were reconstructed from single molecule localizations. The white dashed square is shown in pseudo 3D (bottom).(B) Error bars (Hopkins index) and thin lines (H function) denote mean ± SEM. Data are pooled from three experiments.


( C - E ) Wild - type CD45 - deficient B were labelled with Atto633 - conjugated Fab fragments against CD22 or IgM , settled onto nonstimulatory coverslips imaged . Single particle trajectories were then analysed . ( C ) Trajectories of CD22 in wild - type ( left ) CD45 - deficient ( right ) showing diffusion of single particles over 6 s . ( D ) Diffusion coefficients of 300 representative CD22 IgM particles . Bars numbers indicate median . Data are pooled from three experiments . ( E ) Confinement of CD22 IgM . Bars numbers indicate median .

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

(C-E) Wild-type and CD45-deficient primary B cells were labelled with Atto633-conjugated Fab fragments against CD22 or IgM, settled onto nonstimulatory coverslips and imaged. Single particle trajectories were then analysed.(C) Trajectories of CD22 in wild-type (left) and CD45-deficient cells (right) showing diffusion of single particles over 6 s.(D) Diffusion coefficients of 300 representative CD22 and IgM particles. Bars and numbers indicate the median. Data are pooled from three experiments.(E) Confinement analysis of CD22 and IgM. Bars and numbers indicate the median.


( F ) Two - population of CD22 diffusion . Diffusion coefficient were plotted on a logarithmic scale fitted to two Gaussian - shaped curves to account for slower ( grey ) faster ( grey ) diffusing populations . Wild - type ( blue circles , left ) CD45 - deficient ( blue squares , left middle ) B were compared in an overlay ( right middle ) their proportions plotted ( right ) .

O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O

(F) Two-population analysis of CD22 diffusion. Diffusion coefficient were plotted on a logarithmic scale and fitted to two Gaussian-shaped curves to account for slower (light grey) and faster (dark grey) diffusing populations. Wild-type (blue line and circles, left) and CD45-deficient (blue line and squares, left middle) primary B cells were compared in an overlay (right middle) and their proportions plotted (right).


( G H ) CD45 - deficient B were labelled with Atto633 - conjugated Fab fragments against CD22 or IgM , settled onto nonstimulatory coverslips , treated with vehicle control ( DMSO ) or 1µM LatA imaged . Single particle trajectories were then analysed diffusion coefficients calculated . ( G ) Diffusion coefficients of 300 representative CD22 IgM particles . Bars numbers indicate median . ( H ) Confinement . Bars numbers indicate median .

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

(G and H) CD45-deficient primary B cells were labelled with Atto633-conjugated Fab fragments against CD22 or IgM, settled onto nonstimulatory coverslips, treated with vehicle control (DMSO) or 1µM LatA and imaged. Single particle trajectories were then analysed and diffusion coefficients calculated.(G) Diffusion coefficients of 300 representative CD22 and IgM particles. Bars and numbers indicate the median.(H) Confinement analysis. Bars and numbers indicate the median.


( I J ) CD45 - deficient B were stained with Alexa 647 - conjugated antibody against CD22 , settled onto nonstimulatory coverslips treated with vehicle control or sialidase . were then fixed , imaged with dSTORM analysed . ( I ) 2D ( top ) pseudo 3D ( bottom ) dSTORM were reconstructed from single molecule localizations . dashed square is shown in pseudo 3D ( bottom ) . ( J ) of distribution of CD22 with Hopkins index H function . Error bars ( Hopkins index ) thin lines ( H function ) denote mean ± . Data are pooled from two experiments . * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 0001 ( Student ' s t - ) .

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

(I and J) CD45-deficient primary B cells were stained with Alexa 647-conjugated antibody against CD22, settled onto nonstimulatory coverslips and treated with vehicle control or sialidase. Cells were then fixed, imaged with dSTORM and analysed.(I) 2D (top) and pseudo 3D (bottom) dSTORM images were reconstructed from single molecule localizations. The white dashed square is shown in pseudo 3D (bottom).(J) Quantification of the distribution of CD22 with Hopkins index and H function. Error bars (Hopkins index) and thin lines (H function) denote mean ± SEM.Data are pooled from two experiments.*P<0.05, **P<0.001, ***P<0.0001 (Student's t-test).


( K L ) Wild - type CD45 - deficient B were treated with vehicle control ( - ) or 1 µM LatA for indicated times . were lysed analysed by SDS - PAGE followed by immunoblotting with phospho - CD22 ( p - CD22 ) total CD22 as loading control . of p - CD22 , normalized to CD22 , was referred to unstimulated sample of wild - type , set as 1 .

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

(K and L) Wild-type and CD45-deficient primary B cells were treated with vehicle control (-) or 1 µM LatA for the indicated times. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-CD22 (p-CD22) and total CD22 as loading control. The intensity of p-CD22, normalized to CD22, was referred to the unstimulated sample of the wild-type cells, set as 1.


( A - E ) Wild - type Cd22R130E B were labelled with Atto 633 - conjugated Fab fragments against CD22 or IgM , settled onto nonstimulatory coverslips imaged . Single particle trajectories were then analysed . ( A ) Trajectories of CD22 in Wild - type ( left ) Cd22R130E ( right ) showing diffusion of single particles over 6 s . B ) Diffusion coefficients of 300 representative CD22 IgM particles . Bars numbers indicate median . ( C ) Confinement . Bars numbers indicate median . ( D E ) Two - population of CD22 diffusion . of CD22 diffusion plotted on a logarithmic scale fitted to two Gaussian - shaped curves to account for slower faster diffusing populations . ( D ) Wild - type ( blue circles ) Cd22R130E ( blue dotted squares middle ) B were compared in an overlay ( left ) proportions of faster diffusing populations plotted ( right ) . ( E ) Proportions of faster - diffusing populations .

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

(A-E) Wild-type and Cd22R130E primary B cells were labelled with Atto 633-conjugated Fab fragments against CD22 or IgM, settled onto nonstimulatory coverslips and imaged. Single particle trajectories were then analysed.(A) Trajectories of CD22 in Wild-type (left) and Cd22R130E cells (right) showing diffusion of single particles over 6 s.B) Diffusion coefficients of 300 representative CD22 and IgM particles. Bars and numbers indicate the median.(C) Confinement analysis. Bars and numbers indicate the median.(D and E) Two-population analysis of CD22 diffusion. Analysis of CD22 diffusion plotted on a logarithmic scale and fitted to two Gaussian-shaped curves to account for slower and faster diffusing populations.(D) Wild-type (blue line and circles) and Cd22R130E (blue dotted line and squares middle) primary B cells were compared in an overlay (left) and the proportions of the faster diffusing populations plotted (right).(E) Proportions of faster-diffusing populations.


( F G ) Wild - type Cd22R130E B were stained with Alexa 647 - conjugated antibody against CD22 settled onto nonstimulatory coverslips . were then fixed , imaged with dSTORM analysed . ( F ) 2D ( top ) pseudo 3D ( bottom ) dSTORM were reconstructed from single molecule localizations . dashed square is shown in pseudo 3D ( bottom ) . ( G ) of distribution of CD22 with Hopkins index H function . Error bars ( Hopkins index ) thin lines ( H function ) denote mean ± . Data are pooled from three experiments .

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

(F and G) Wild-type and Cd22R130E primary B cells were stained with Alexa 647-conjugated antibody against CD22 and settled onto nonstimulatory coverslips. Cells were then fixed, imaged with dSTORM and analysed.(F) 2D (top) and pseudo 3D (bottom) dSTORM images were reconstructed from single molecule localizations. The white dashed square is shown in pseudo 3D (bottom).(G) Quantification of the distribution of CD22 with Hopkins index and H function. Error bars (Hopkins index) and thin lines (H function) denote mean ± SEM. Data are pooled from three experiments.


( H ) Wild - type Cd22R130E B were treated with 1 µM LatA intracellular calcium flux was measured by flow cytometry .

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(H) Wild-type and Cd22R130E primary B cells were treated with 1 µM LatA and intracellular calcium flux was measured by flow cytometry.


( I - K ) Wild - type Cd22R130E B were treated with vehicle control ( - ) or 1 µM LatA for indicated time . were lysed analysed by SDS - PAGE followed by immunoblotting with phospho - CD19 ( p - CD19 ) , phospho - Akt ( pAkt ) , phospho - ERK ( pERK ) actin total ERK as loading controls . of phosphorylated , normalized to actin or ERK , was referred to unstimulated sample of wild type , set as 1 . Data are representative of at least two experiments .

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

(I-K) Wild-type and Cd22R130E primary B cells were treated with vehicle control (-) or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-CD19 (p-CD19), phospho-Akt (pAkt), phospho-ERK (pERK) and actin and total ERK as loading controls. The intensity of phosphorylated proteins, normalized to actin or ERK, was referred to the unstimulated sample of the wild type cells, set as 1.Data are representative of at least two experiments.


( B ) Total lysates from HEK293T transfected with Flag - OFD1 were IP with anti - myosin VI antibodies ( 1295 1296 ) an unrelated rabbit antibody ( anti - GST , as control ) . IB was performed with anti - Flag anti - myosin VI antibodies . ( C ) Total lysates from HEK293T transfected with Flag - OFD1 or Flag ( as control ) were IP with anti - Flag antibody - conjugated beads . IB was performed with anti - Flag anti - myosin VI antibodies .

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

(B) Total lysates from HEK293T transfected with Flag-OFD1 were IP with anti-myosin VI antibodies (1295 and 1296) and an unrelated rabbit antibody (anti-GST, as control). IB was performed with anti-Flag and anti-myosin VI antibodies. (C) Total lysates from HEK293T transfected with Flag-OFD1 or Flag (as control) were IP with anti-Flag antibody-conjugated beads. IB was performed with anti-Flag and anti-myosin VI antibodies.


( D ) Selected sections deriving from of A549 wild - type versus myosin VI KO . were immunogold - labelled with anti - myosin VI antibody . centrioles in are indicated with dashed circles . Scale bar , 200 nm . Top : representation of estimated localisation of myosin VI at centrioles .

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(D) Selected sections deriving from TEM analysis of A549 wild-type versus myosin VI KO cells. The cells were immunogold-labelled with anti-myosin VI antibody. The centrioles in the images are indicated with white dashed circles. Scale bar, 200 nm. Top: representation of the estimated localisation of myosin VI at the centrioles.


( F ) GST pulldown using myosin VI tail ( 835 - 1295 ) construct ( or GST alone as control ) lysates from HEK293T transfected with indicated Flag - OFD1 constructs ( or Flag alone , as control ) . IB was performed with anti - Flag antibody . Ponceau as indicated .

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(F) GST pulldown assay using myosin VI tail (835-1295) construct (or GST alone as control) and lysates from HEK293T cells transfected with the indicated Flag-OFD1 constructs (or Flag alone, as control). IB was performed with anti-Flag antibody. Ponceau staining as indicated.


D ) of OFD1 at mother or daughter centrioles . Results are expressed as fold change with respect to mock average . Bars represent mean ± SD . Mother centrioles : Mock , n = 148 ; MyoVI KD , n = 199 , from four independent experiments . Daughter centrioles : Mock , n = 101 ; MyoVI KD , n = 148 , from three independent experiments . * * * * P < 0 , 0001 by Mann - Whitney .

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

D) Quantification of OFD1 intensity at the mother or daughter centrioles. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ± SD. Mother centrioles: Mock, n=148 cells; MyoVI KD, n=199 cells, from four independent experiments. Daughter centrioles: Mock, n=101 cells; MyoVI KD, n=148 cells, from three independent experiments. **** P<0,0001 by Mann-Whitney test.


( F - G ) IF of Cep164 signal . hTERT - RPE1 were transfected with siRNA against myosin VI / or OFD1 . Four days after transfection , were treated with nocodazole ( 1 hour , 6 μg / ml ) immunostained with anti - OFD1 , anti - centrin1 anti - Cep164 antibodies . ( F ) Representative . Scale bar , 1 μm . ( G ) of Cep164 at mother centrioles . Results are expressed as fold change with respect to mock average . Bars represent mean ± SD . Mock , n = 147 ; OFD1 KD , n = 146 ; MyoVI KD , n = 150 ; MyoVI + OFD1 KD , n = 151 , from four independent experiments . * * P < 0 , 005 ; * * * * P < 0 , 0001 by Kruskal - Wallis .

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

(F-G) IF analysis of Cep164 signal. hTERT-RPE1 cells were transfected with siRNA against myosin VI and/or OFD1. Four days after transfection, cells were treated with nocodazole (1 hour, 6 μg/ml) and immunostained with anti-OFD1, anti-centrin1 and anti-Cep164 antibodies. (F) Representative images. Scale bar, 1 μm. (G) Quantification of Cep164 intensity at the mother centrioles. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock, n=147 cells; OFD1 KD, n=146 cells; MyoVI KD, n=150 cells; MyoVI + OFD1 KD, n=151 cells, from four independent experiments. ** P<0,005; **** P<0,0001 by Kruskal-Wallis test.


( A ) Growth curve of hTERT - RPE1 transfected with myosin VI siRNA . A representative plot of three independent experiments is shown . ( B ) Growth curve of BJ - hTERT transfected with myosin VI siRNA . A representative plot of two independent experiments is shown .

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(A) Growth curve of hTERT-RPE1 cells transfected with myosin VI siRNA. A representative plot of three independent experiments is shown. (B) Growth curve of BJ-hTERT cells transfected with myosin VI siRNA. A representative plot of two independent experiments is shown.


( G ) Representative bright field of treated with indicated p53 myosin VI siRNAs . Scale bar , 200 μm .

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(G) Representative bright field images of cells treated with the indicated p53 and myosin VI siRNAs. Scale bar, 200 μm.


( I ) of OFD1 at mother or daughter centrioles . hTERT - RPE1 treated or not with Nutlin - 3 , were incubated with nocodazole for 1 hour ( 6 μg / ml ) immunostained with anti - OFD1 , anti - centrin1 anti - Cep164 antibodies . Mother centrioles were identified by coincident of centrin1 Cep164 , while daughter centrioles were centrin1 - only stained . Results are expressed as fold change with respect to mock average . Bars represent mean ± SD . Mother centrioles : Mock , n = 148 ; Nutlin - 3 , n = 169 , from four independent experiments . Daughter centrioles : Mock , n = 101 ; Nutlin - 3 , n = 122 , from three independent experiments . ns , not significant by Mann - Whitney .

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

(I) Quantification of OFD1 intensity at the mother or daughter centrioles. hTERT-RPE1 cells treated or not with Nutlin-3, were incubated with nocodazole for 1 hour (6 μg/ml) and immunostained with anti-OFD1, anti-centrin1 and anti-Cep164 antibodies. Mother centrioles were identified by the coincident staining of centrin1 and Cep164, while daughter centrioles were centrin1-only stained. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mother centrioles: Mock, n=148 cells; Nutlin-3, n=169 cells, from four independent experiments. Daughter centrioles: Mock, n=101 cells; Nutlin-3, n=122 cells, from three independent experiments. ns, not significant by Mann-Whitney test.


( J ) of Cep164 at mother centrioles in hTERT - RPE1 treated as in ( I ) . Results are expressed as fold change with respect to mock average . Bars represent mean ± SD . Mock , n = 195 ; Nutlin - 3 , n = 196 , from four independent experiments . ns , not significant by Mann - Whitney .

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(J) Quantification of Cep164 intensity at the mother centrioles in hTERT-RPE1 cells treated as in (I). Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock, n=195 cells; Nutlin-3, n=196 cells, from four independent experiments. ns, not significant by Mann-Whitney test.


( A ) Super - resolution of OFD1 localisation at centrioles . hTERT - RPE1 were transfected with siRNA against myosin VI , immunostained with anti - OFD1 anti - acetylated tubulin antibodies visualised using structured illumination microscopy ( SIM ) . Representative are shown , scale bar , 500 nm . A scheme of estimated localisation of OFD1 in two conditions is depicted on right side .

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

(A) Super-resolution analysis of OFD1 localisation at the centrioles. hTERT-RPE1 cells were transfected with siRNA against myosin VI, immunostained with anti-OFD1 and anti-acetylated tubulin antibodies and visualised using structured illumination microscopy (SIM). Representative images are shown, scale bar, 500 nm. A scheme of the estimated localisation of OFD1 in the two conditions is depicted on the right side.


( C ) dSTORM super - resolution of distal appendage markers FBF1 ODF2 in control myosin VI - depleted . To emphasise symmetry of structures , signals from nine appendages were averaged ( bottom ) . Representative are shown ; scale bar , 200 nm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O

(C) dSTORM super-resolution analysis of the distal appendage markers FBF1 and ODF2 in control and myosin VI-depleted cells. To emphasise the symmetry of the structures, the signals from all nine appendages were averaged (bottom). Representative images are shown; scale bar, 200 nm.


FRAP of centriole - associated GFP - OFD1 . hTERT - RPE stably expressing GFP - OFD1 centrin1 - dTomato were transfected with siRNA against myosin VI . After four days , were treated with nocodazole ( 1 hour , 6 μg / ml ) subjected to live - imaging . ( E ) A representative graph of one out of three experiments . For each time point , fraction of recovery of GFP - OFD1 is shown . Results are expressed as means with 95 % confidence interval . n = 12 ( Mock ) , n = 13 ( MyoVI KD ) . ( F ) of half - time of recovery ( t1 / 2 , ) of mobile fraction of GFP - OFD1 . Results are expressed as mean ± SD . n = 41 ( Mock ) , n = 31 ( MyoVI KD ) , from three independent experiments . ns , not significant ; * * * P < 0 , 0005 by Unpaired T - .

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FRAP analysis of centriole-associated GFP-OFD1. hTERT-RPE cells stably expressing GFP-OFD1 and centrin1-dTomato were transfected with siRNA against myosin VI. After four days, cells were treated with nocodazole (1 hour, 6 μg/ml) and subjected to live-cell imaging. (E) A representative graph of one out of three experiments. For each time point, the fraction of recovery of GFP-OFD1 is shown. Results are expressed as means with 95% confidence interval. n=12 cells (Mock), n=13 cells (MyoVI KD). (F) Quantification of the half-time of fluorescence recovery (t1/2,) and of the mobile fraction of GFP-OFD1. Results are expressed as mean ±SD. n=41 cells (Mock), n=31 cells (MyoVI KD), from three independent experiments. ns, not significant; *** P<0,0005 by Unpaired T-test.


( B ) IF of PCM1 signal . hTERT - RPE1 were transfected with siRNA against myosin VI immunostained with anti - PCM1 anti - Cep135 antibodies . Upper panel , representative , scale bar , 2 μm . Lower panel , of PCM1 . Results are expressed as fold change with respect to mock average . Bars represent mean ± SD . Mock , n = 96 ; MyoVI KD , n = 98cells , from two independent experiments . * * * * P < 0 , 0001 by Mann - Whitney . ( C ) IF of PCM1 signal . hTERT - RPE1 were treated with Nutlin - 3 for 24 hours immunostained with anti - PCM1 anti - Cep135 antibodies . Panels as in B . Mock , n = 96 ; Nutlin - 3 , n = 100 , from two independent experiments . * * * * P < 0 , 0001 by Mann - Whitney . ( D ) IF of PCM1 signal . hTERT - RPE1 p53 KO were transfected with siRNA against myosin VI immunostained with anti - PCM1 anti - Cep164 antibodies . Panels as in B . of PCM1 refers to a 3 μm circle around mother centriole , identified with anti - Cep164 . Mock , n = 128 ; MyoVI KD , n = 114 from three independent experiments . ns , not significant by Mann - Whitney .

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

(B) IF analysis of PCM1 signal. hTERT-RPE1 cells were transfected with siRNA against myosin VI and immunostained with anti-PCM1 and anti-Cep135 antibodies. Upper panel, representative images, scale bar, 2 μm. Lower panel, quantification of PCM1 intensity. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock, n=96 cell; MyoVI KD, n=98cells, from two independent experiments. **** P<0,0001 by Mann-Whitney test. (C) IF analysis of PCM1 signal. hTERT-RPE1 cells were treated with Nutlin-3 for 24 hours and immunostained with anti-PCM1 and anti-Cep135 antibodies. Panels as in B. Mock, n=96 cells; Nutlin-3, n=100 cells, from two independent experiments. **** P<0,0001 by Mann-Whitney test. (D) IF analysis of PCM1 signal. hTERT-RPE1 p53 KO cells were transfected with siRNA against myosin VI and immunostained with anti-PCM1 and anti-Cep164 antibodies. Panels as in B. Quantification of PCM1 intensity refers to a 3 μm circle around the mother centriole, identified with anti-Cep164 staining. Mock, n=128 cells; MyoVI KD, n=114 cells from three independent experiments. ns, not significant by Mann-Whitney test.


( B ) IF of OFD1 signal at centriolar satellites upon serum starvation . hTERT - RPE1 p53 KO were transfected with siRNA against myosin VI . After four days , were fixed ( growing ) or serum starved for 24 hours ( SS ) . were immunostained with anti - OFD1 anti - PCM1 antibodies . of OFD1 signal in covered by PCM1 was quantified normalised against of PCM1 in same . Left , representative , scale bar , 2 μm . Right , results are expressed as fold change with respect to mock average . Bars represent mean ± SD . Mock _ growing , n = 150 ; Mock _ SS , n = 149 ; MyoVI KD _ growing , n = 149 ; MyoVI KD _ SS , n = 150 , from three independent experiments . ns , not significant ; * P < 0 , 05 ; * * * * P < 0 , 0001 by Kruskal - Wallis .

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(B) IF analysis of OFD1 signal at the centriolar satellites upon serum starvation. hTERT-RPE1 p53 KO cells were transfected with siRNA against myosin VI. After four days, cells were fixed (growing) or serum starved for 24 hours (SS). Cells were immunostained with anti-OFD1 and anti-PCM1 antibodies. The intensity of OFD1 signal in the area covered by PCM1 was quantified and normalised against the intensity of PCM1 staining in the same area. Left, representative images, scale bar, 2 μm. Right, results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock_growing, n=150 cells; Mock_SS, n=149 cells; MyoVI KD_growing, n=149 cells; MyoVI KD_SS, n=150 cells, from three independent experiments. ns, not significant; * P<0,05; **** P<0,0001 by Kruskal-Wallis test.


( C ) IB of OFD1 after serum starvation in control myosin VI - depleted . hTERT - RPE1 p53 KO were transfected with siRNA against myosin VI . After four days , were serum starved for 24 hours ( SS ) . Lysates were analysed by IB with anti - OFD1 , anti - myosin VI , anti - p62 , anti - LC3 antibodies . Anti - GAPDH was used as loading control . amount of OFD1 was normalised against GAPDH signal is expressed as of OFD1 in grown in serum - starved conditions compared to grown in media containing serum . Bars represent mean ± SD . n = 5 independent experiments . * P < 0 , 05 by Kruskal - Wallis .

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(C) IB analysis of OFD1 after serum starvation in control and myosin VI-depleted cells. hTERT-RPE1 p53 KO cells were transfected with siRNA against myosin VI. After four days, cells were serum starved for 24 hours (SS). Lysates were analysed by IB with anti-OFD1, anti-myosin VI, anti-p62, and anti-LC3 antibodies. Anti-GAPDH was used as loading control. The amount of OFD1 protein was normalised against GAPDH signal and is expressed as percentage of OFD1 levels in cells grown in serum-starved conditions compared to cells grown in media containing serum. Bars represent mean ±SD. n=5 independent experiments. * P<0,05 by Kruskal-Wallis test.


( D ) IF of cilium upon serum starvation . hTERT - RPE1 p53 KO were transfected with siRNA against myosin VI . After four days , were fixed ( growing ) or serum starved for 24 hours ( SS ) . were immunostained with anti - acetylated tubulin ( to identify cilia ) , anti - Cep135 ( to identify centrioles ) DAPI . Left , representative , scale bar , 2 μm . Right , results are expressed as fold change with respect to mock average . Bars represent mean ± SD . n = 3 independent experiments . 100 - 200 / condition were counted for each experiment . * * * P < 0 . 001 by two - way ANOVA .

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(D) IF analysis of primary cilium upon serum starvation. hTERT-RPE1 p53 KO cells were transfected with siRNA against myosin VI. After four days, cells were fixed (growing) or serum starved for 24 hours (SS). Cells were immunostained with anti-acetylated tubulin (to identify the cilia), anti-Cep135 (to identify the centrioles) and DAPI. Left, representative images, scale bar, 2 μm. Right, results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. n=3 independent experiments. 100-200 cells/condition were counted for each experiment. ***P < 0.001 by two-way ANOVA test.


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A Infiltration of GRIp65-84 induced cell death similar to GRIp31-96. Bacterially produced 37 nM GST, GST‐GRIp31-96 or biochemically pure GRI‐peptides (GRIp31-96, GRIp31-51, GRIp47-68, GRIp65-84, GRIp80-96) were infiltrated into leaves of Col‐0 plants.


B Infiltration of increasing concentrations of GRIp65 - 84 into Col ‐ 0leaves , electrolyte leakage was measured after 12 h . Background ( red ) shows ion leakage from infiltration of leaves with increasing concentrations of ( inactive ) GRIp80 - 96 .

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B Infiltration of increasing concentrations of GRIp65-84 into Col‐0leaves, electrolyte leakage was measured after 12 h. Background (red line) shows ion leakage from infiltration of leaves with increasing concentrations of (inactive) GRIp80-96.


C Infiltration of leaves with 37 nM GRIp31 - 96 induced elevated ion leakage in Col ‐ 0 prk4 , but not in prk5 ‐ 1 or prk5 ‐ 2 . Infiltration with GST caused same background effect for lines .

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C Infiltration of leaves with 37 nM GRIp31-96 induced elevated ion leakage in Col‐0 and prk4, but not in prk5‐1 or prk5‐2. Infiltration with GST caused the same background effect for all lines.


D Genomic complementation of prk5 rescues insensitivity to induction of elevated ion leakage by GRIp31 - 96 .

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D Genomic complementation of prk5 rescues the insensitivity to induction of elevated ion leakage by GRIp31-96.


E Enzymatic superoxide production from xanthine / xanthine oxidase ( XXO ) induced more electrolyte leakage in gri compared to Col ‐ 0 or prk5 ‐ 1 , prk5 ‐ 2 prk4 after infiltration into leaves . Infiltration with xanthine buffer ( X ) was used as a control .

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E Enzymatic superoxide production from xanthine/xanthine oxidase (XXO) induced more electrolyte leakage in gri compared to Col‐0 or prk5‐1, prk5‐2 and prk4 after infiltration into leaves. Infiltration with xanthine buffer (X) was used as a control.


B PRK5 ‐ CFP localized to periphery in Col ‐ 0mesophyll protoplasts . C - F Co ‐ localization of PRK5 ‐ CFP PM localized CAAX ‐ YFP in Col ‐ 0mesophyll protoplasts . ( C ) overlay ; ( D ) PRK5 ‐ CFP ( 465 - 510 nm ) ; ( E ) CAAX ‐ YFP ( 521 - 587 nm ) ; ( F ) chloroplast ( 636 - 711 nm ) .

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B PRK5‐CFP localized to the cell periphery in Col‐0mesophyll protoplasts.C-F Co‐localization of PRK5‐CFP and PM localized CAAX‐YFP in Col‐0mesophyll protoplasts. (C) overlay; (D) PRK5‐CFP (465-510 nm); (E) CAAX‐YFP (521-587 nm); (F) chloroplast (636-711 nm).


H Kinase activity of PRK5 in in vitro phosphorylation assays using γ32P ‐ ATP myelin ‐ basic ( MBP ) as a substrate in presence of 10 mM MnCl2 . GST ‐ PRK5 GST ‐ PRK4 did not show kinase activity . Mutation of conserved residues in kinase subdomains VIb VII to reconstitute consensus kinase domain motif restored GST ‐ PRK5H500DA520G kinase activity . GST ‐ CRK7 was used as a positive control . Upper panel shows autoradiograph , lower panel shows Coomassie ‐ stained 15 % SDS - polyacrylamide gel .

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H Kinase activity of PRK5 in in vitro phosphorylation assays using γ32P‐ATP and myelin‐basic protein (MBP) as a substrate in the presence of 10 mM MnCl2. GST‐PRK5 and GST‐PRK4 did not show kinase activity. Mutation of conserved residues in kinase subdomains VIb and VII to reconstitute the consensus kinase domain motif restored GST‐PRK5H500DA520G kinase activity. GST‐CRK7 was used as a positive control. Upper panel shows autoradiograph, and lower panel shows the Coomassie‐stained 15% SDS-polyacrylamide gel.


A In vitro ‐ produced 35S ‐ labeled ectodomains of PRK5 ( PRK540 - 281 ) or PRK4 ( PRK440 - 279 ) were incubated with bacterially produced GST , GST ‐ GRI or GST ‐ GRI31 - 96 purified . GST ‐ GRI31 - 96 but not GST directly bound to ectodomain of PRK5 . Binding of GST ‐ GRI to PRK5 ectodomain binding of GST ‐ GRI31 - 96 GST ‐ GRI to ectodomain of PRK4 were strongly reduced . Upper part : autoradiograph , lower part : Coomassie ‐ stained 12 % SDS - polyacrylamide gel . Asterisks in Coomassie ‐ stained gel indicate GST , GST ‐ GRI GST ‐ GRI31 - 96 , respectively . Supplementary Fig S12 shows a Western blot of GST ‐ tagged .

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A In vitro‐produced 35S‐labeled ectodomains of PRK5 (PRK540-281) or PRK4 (PRK440-279) were incubated with bacterially produced GST, GST‐GRI or GST‐GRI31-96 and purified. GST‐GRI31-96 but not GST directly bound to the ectodomain of PRK5. Binding of GST‐GRI to PRK5 ectodomain and binding of GST‐GRI31-96 and GST‐GRI to the ectodomain of PRK4 were strongly reduced. Upper part: autoradiograph, lower part: Coomassie‐stained 12% SDS-polyacrylamide gel. Asterisks in the Coomassie‐stained gel indicate GST, GST‐GRI and GST‐GRI31-96, respectively. Supplementary Fig S12 shows a Western blot of the GST‐tagged proteins.


B Infiltration of 37 nM GST , GRIp31 - 96 , GRIp65 - 84 or Y ‐ GRIp65 - 84 into Col ‐ 0 or prk5 ‐ 2 leaves . Tyrosine ‐ labeled GRIp65 - 84 still induced in Col ‐ 0 but not in prk5 ‐ 2 .

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B Infiltration of 37 nM GST, GRIp31-96, GRIp65-84 or Y‐GRIp65-84 into Col‐0 or prk5‐2 leaves. Tyrosine‐labeled GRIp65-84 still induced cell death in Col‐0 but not in prk5‐2 plants.


C 125I ‐ labeled Y ‐ GRIp65 - 84 ( 0 . 46 nM ) bound specifically to Col ‐ 0membrane fractions ( gray bars ) , binding was significantly reduced in prk5 . Excess of non ‐ radioactive Y ‐ GRIp65 - 84 ( 10 μM ) reduced binding to background ( gray bars ; bars show average of three samples , triangles show data points ) .

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C 125I‐labeled Y‐GRIp65-84 (0.46 nM) bound specifically to Col‐0membrane fractions (light gray bars), the binding was significantly reduced in prk5 plants. Excess of non‐radioactive Y‐GRIp65-84 (10 μM) reduced binding to background levels (dark gray bars; all bars show the average of three samples, triangles show individual data points).


D Immunoprecipitation of PRK5 ‐ c ‐ myc , PRK4 ‐ c ‐ myc or YFP expressed in protoplasts with rabbit polyclonal anti ‐ c ‐ myc antibody , immunoblotted with mouse monoclonal anti ‐ c ‐ myc antibody .

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D Immunoprecipitation of PRK5‐c‐myc, PRK4‐c‐myc or YFP expressed in protoplasts with rabbit polyclonal anti‐c‐myc antibody, immunoblotted with mouse monoclonal anti‐c‐myc antibody.


E Binding of 125I ‐ Y ‐ GRIp65 - 84 ( 0 . 46 nM ) to immunoprecipitates ( using anti ‐ c ‐ myc antibodies ) from prk5 ‐ 2 protoplasts transfected with PRK5 ‐ c ‐ myc , PRK4 ‐ c ‐ myc or YFP , respectively . Binding was competed out with 10 μM unlabeled Y ‐ GRIp65 - 84 . Bars show average of two samples , triangles show data points . Western blot is shown in panel ( D ) .

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E Binding of 125I‐Y‐GRIp65-84 (0.46 nM) to immunoprecipitates (using anti‐c‐myc antibodies) from prk5‐2 protoplasts transfected with PRK5‐c‐myc, PRK4‐c‐myc or YFP, respectively. Binding was competed out with 10 μM unlabeled Y‐GRIp65-84. Bars show the average of two samples, and triangles show individual data points. Western blot is shown in panel (D).


F of 125I ‐ Y ‐ GRIp65 - 84 ( 0 . 46 nM ) binding competed out with increasing amounts of unlabeled Y ‐ GRIp65 - 84 to Col ‐ 0membrane extracts . Fifty inhibition ( IC50 ) occurred at 25 . 2 nM . Red shows binding average competition according to a sigmoid curve , dotted lines show 95 % confidence intervals , circles show data points .

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F Analysis of 125I‐Y‐GRIp65-84 (0.46 nM) binding competed out with increasing amounts of unlabeled Y‐GRIp65-84 to Col‐0membrane extracts. Fifty percent inhibition (IC50) occurred at 25.2 nM. Red line shows binding average competition according to a sigmoid curve, dotted lines show 95% confidence intervals, and circles show data points.


G Excess of GRIp65 - 84 ( 10 μM ) but not of GRIp31 - 96 or other peptides ( GRIp31 - 51 , GRIp47 - 68 , GRIp80 - 96 ) competed binding of 0 . 46 nM 125I ‐ Y ‐ GRIp65 - 84 to extracts from Col ‐ 0 ( bars show average of two samples , triangles show data points ) .

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G Excess of GRIp65-84 (10 μM) but not of GRIp31-96 or other peptides (GRIp31-51, GRIp47-68, GRIp80-96) competed the binding of 0.46 nM 125I‐Y‐GRIp65-84 to membrane extracts from Col‐0 (all bars show the average of two samples, triangles show individual data points).


A Recombinant AtMC9 ( rAtMC9 ) GRI25 - 168in vitro . Bacterially produced MBP ‐ GRI25 - 168 ( from 0 to 2 pmol left to right ) was incubated with 1 pmol rAtMC9 or inactive rAtMC9mut ( rAtMC9C147AC29A ) , cleavage products were analyzed by Western blot with anti ‐ MBP anti ‐ AtMC9 antibodies . Arrowheads in anti ‐ MBP blot ( from top to bottom ) indicate MBP ‐ GRI25 - 168 ( 63 . 5 kDa ) , MBPMCS ( 53 . 5 kDa ) , rAtMC9 ‐ MBP ‐ GRI25 - 168 ( 46 kDa ) . Arrowheads in anti ‐ AtMC9 blot indicate : 1 : N ‐ terminal domain + p20 + p10 subunits of AtMC9 , 2 : p20 + p10 subunits of AtMC9 , 3 : N ‐ terminal domain + p20 subunit of AtMC9 , 4 : p20 subunit of AtMC9 .

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A Recombinant AtMC9 (rAtMC9) cleaved GRI25-168in vitro. Bacterially produced MBP‐GRI25-168 (from 0 to 2 pmol left to right) was incubated with 1 pmol rAtMC9 or inactive rAtMC9mut (rAtMC9C147AC29A), and cleavage products were analyzed by Western blot with anti‐MBP and anti‐AtMC9 antibodies. Arrowheads in the anti‐MBP blot (from top to bottom) indicate MBP‐GRI25-168 (63.5 kDa), MBPMCS (53.5 kDa), rAtMC9‐cleaved MBP‐GRI25-168 (46 kDa). Arrowheads in the anti‐AtMC9 blot indicate: 1: N‐terminal domain + p20 + p10 subunits of AtMC9, 2: p20 + p10 subunits of AtMC9, 3: N‐terminal domain + p20 subunit of AtMC9, 4: p20 subunit of AtMC9.


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B Infiltration of wild‐type, prk5‐1 and atmc9‐1 leaves with 37 nM GRIp31-96, GRIp65-84, GRIp68-78 or GST. GRIp65-84 and GRIp68-78 but not GRIp31-96 were able to induce elevated ion leakage in the atmc9‐1 mutant.


D Infiltration of Col ‐ 0leaves with 37 nM of GRIp65 - 84 , GRIp68 - 78 , Y ‐ GRIp68 - 78 , GRIp31 - 96 or GST . Y ‐ GRIp68 - 78 showed similar activity in induction compared to other GRI ‐ derived peptides .

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D Infiltration of Col‐0leaves with 37 nM of GRIp65-84, GRIp68-78, Y‐GRIp68-78, GRIp31-96 or GST. Y‐GRIp68-78 showed similar activity in cell death induction compared to the other GRI‐derived peptides.


E 125I ‐ labeled Y ‐ GRIp68 - 78 ( 0 . 46 nM ) bound specifically to Col ‐ 0membrane fractions ( gray bars ) , binding was significantly reduced in prk5 ‐ 1 prk5 ‐ 2 . Excess of non ‐ radioactive Y ‐ GRIp65 - 84 ( 10 μM ) reduced binding to background ( gray bars ; bars show average of four samples , triangles show data points ) .

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E 125I‐labeled Y‐GRIp68-78 (0.46 nM) bound specifically to Col‐0membrane fractions (light gray bars), the binding was significantly reduced in prk5‐1 and prk5‐2 plants. Excess of non‐radioactive Y‐GRIp65-84 (10 μM) reduced binding to background levels (dark gray bars; all bars show the average of four samples, triangles show individual data points).


F Excess of GRIp68 - 78 GRIp65 - 84 ( 10 μM ) but not of GRIp31 - 96 or other peptides ( GRIp31 - 51 , GRIp47 - 68 , GRIp80 - 96 ) competed binding of 0 . 46 nM 125I ‐ Y ‐ GRIp68 - 78 to extracts from Col ‐ 0 ( bars show average of four samples , triangles show data points ) .

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F Excess of GRIp68-78 and GRIp65-84 (10 μM) but not of GRIp31-96 or other peptides (GRIp31-51, GRIp47-68, GRIp80-96) competed the binding of 0.46 nM 125I‐Y‐GRIp68-78 to membrane extracts from Col‐0 (all bars show average of four samples, triangles show individual data points).


G Saturation binding curve for 125I ‐ Y ‐ GRIp68 - 78 to Col ‐ 0membrane extracts . Specific binding was calculated by subtracting non ‐ specific binding from total binding . affinity of 125I ‐ Y ‐ GRIp68 - 78 to receptor ( Kd = 1 . 9 nM ) was calculated by non ‐ linear regression . Scatchard plot is shown in Supplementary Fig SI7 .

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G Saturation binding curve for 125I‐Y‐GRIp68-78 to Col‐0membrane extracts. Specific binding was calculated by subtracting non‐specific binding from the total binding. The affinity of 125I‐Y‐GRIp68-78 to the receptor (Kd = 1.9 nM) was calculated by non‐linear regression analysis. Scatchard plot is shown in Supplementary Fig SI7.


C . of HeLa transfected with plasmids encoding human GFP - Cwh43 , GFP - Cwh43 - k696fs or GFP - Cwh43 - Leu533ter fusion . To label ER , HeLa were transduced with a baculovirus encoding a fluorescently labeled RFP - calreticulin fusion containing an ER retention signal ( RFP - calreticulin - KDEL ) . To label Golgi apparatus , were immunostained for golgin - 97 ( red ) . When compared with wild type GFP - Cwh43 , GFP - Cwh43 - k696fs GFP - Cwh43 - Leu533 showed decreased association with Golgi apparatus ( but not ER ) were diffusely distributed throughout cytoplasm . Scale bar is approximately 5 µm .

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C. Fluorescence micrographs of HeLa cells transfected with plasmids encoding human GFP-Cwh43, GFP-Cwh43-k696fs or GFP-Cwh43-Leu533ter fusion proteins. To label the ER, HeLa cells were transduced with a baculovirus encoding a fluorescently labeled RFP-calreticulin fusion protein containing an ER retention signal (RFP-calreticulin-KDEL). To label the Golgi apparatus, cells were immunostained for golgin-97 (red). When compared with wild type GFP-Cwh43, GFP-Cwh43-k696fs and GFP-Cwh43-Leu533 showed decreased association with the Golgi apparatus (but not the ER) and were diffusely distributed throughout the cytoplasm. Scale bar is approximately 5 µm.