Figure 2A . HEK293 cells treated with non - targeting ( Ctrl ) or FIP200 siRNA were co - transfected with EGFP - LC3 and either empty vector control ( EV ) , Myc - C9orf72S or Myc - C9orf72L . 24 h post transfection cells were treated with vehicle or 100 nM BafA1 for 6 h . Samples were lysed and subjected to SDS - PAGE and immunoblot . Autophagy levels were determined by immunoblot for EGFP - LC3 - I and II . Expression of Myc - C9orf72 was confirmed using anti - Myc ( * indicates a nonspecific band ) . FIP200 knockdown was confirmed using anti - FIP200 antibodies . α - tubulin was used as loading control . B . HeLa cells treated with non - targeting ( Ctrl ) or FIP200 siRNA were co - transfected with empty vector ( EV ) , Myc - C9orf72S or Myc - C9orf72L ( red ) and EGFP - LC3 ( green ) to label autophagosomes . As positive control EV transfected cells were treated for 3 h with Torin1 ( 250 nM ) . Autophagy was quantified as the number of EGFP - LC3 positive autophagosomes per cell from 3 independent experiments ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test : * * * * P ≤ 0 . 0001 , N ( cells ) = Ctrl / EV : 73 ; FIP200 / EV : 76 ; Ctrl / EV / Torin1 : 73 , FIP200 / EV / Torin1 : 70 ; Ctrl / C9orf72L : 71 ; FIP200 / C9orf72L : 74 ; Ctrl / C9orf72S : 72 ; FIP200 / C9orf72S : 72 ) . Scale bar = 20 µm . FIP200 knockdown was confirmed by immunoblot ( Appendix Fig S2 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2A. HEK293 cells treated with non-targeting (Ctrl) or FIP200 siRNA were co-transfected with EGFP-LC3 and either empty vector control (EV), Myc-C9orf72S or Myc-C9orf72L. 24 h post transfection cells were treated with vehicle or 100 nM BafA1 for 6 h. Samples were lysed and subjected to SDS-PAGE and immunoblot. Autophagy levels were determined by immunoblot for EGFP-LC3-I and II. Expression of Myc-C9orf72 was confirmed using anti-Myc (* indicates a nonspecific band). FIP200 knockdown was confirmed using anti-FIP200 antibodies. α-tubulin was used as loading control.B. HeLa cells treated with non-targeting (Ctrl) or FIP200 siRNA were co-transfected with empty vector (EV), Myc-C9orf72S or Myc-C9orf72L (red) and EGFP-LC3 (green) to label autophagosomes. As positive control EV transfected cells were treated for 3 h with Torin1 (250 nM). Autophagy was quantified as the number of EGFP-LC3 positive autophagosomes per cell from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test: **** P ≤ 0.0001, N (cells) = Ctrl/EV: 73; FIP200/EV: 76; Ctrl/EV/Torin1: 73, FIP200/EV/Torin1: 70; Ctrl/C9orf72L: 71; FIP200/C9orf72L: 74; Ctrl/C9orf72S: 72; FIP200/C9orf72S: 72). Scale bar = 20 µm. FIP200 knockdown was confirmed by immunoblot (Appendix Fig S2).


Figure 3A , B , C . Cell lysates of HEK293 cells co - transfected with FLAG - FIP200 ( A ) and either empty vector control , Myc - C9orf72S or Myc - C9orf72L were subjected to immunoprecipitation with anti - Myc ( A and B ) antibodies . Immune pellets were probed for Myc - C9orf72 ( A and B ) , FLAG - FIP200 ( A ) on immunoblots . A , B , C . Cell lysates of HEK293 cells co - transfected with HA - ULK1 ( B ) and either empty vector control , Myc - C9orf72S or Myc - C9orf72L were subjected to immunoprecipitation with anti - Myc ( A and B ) antibodies . Immune pellets were probed for Myc - C9orf72 ( A and B ) , HA - ULK1 ( B ) on immunoblots . A , B , C . Cell lysates of HEK293 cells co - transfected with Myc - ATG13 ( C ) and either empty vector control , FLAG - C9orf72S or FLAG - C9orf72L were subjected to immunoprecipitation with anti - FLAG ( C ) antibodies . Immune pellets were probed for FLAG - C9orf72 ( C ) , Myc - ATG13 ( C ) on immunoblots . D , E . Cell lysates of HEK293 cells transfected with empty vector control , Myc - C9orf72S or Myc - C9orf72L were subjected to immunoprecipitation with anti - Myc antibodies Immune pellets were probed for Myc - C9orf72 and endogenous FIP200 , ULK1 , and ATG13 . There are multiple alternatively spliced forms of ATG13 ( Jung et al . , 2009 ; Alers et al . , 2011 ) ; ATG13 * is most likely a smaller alternative spliced form of ATG13 that is enriched by interaction with C9orf72 . F , G , H . HeLa cells transfected with Flag - FIP200 and Myc - C9orf72 were fixed and processed for PLA analysis . Transfections were laced with mVenus to enable identification of transfected cells for analysis where required ( green ) . PLA signals ( red ) were counted per cell ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test , * * * * P ≤ 0 . 0001 ; N ( cells ) = ( F ) C9orf72S : 22 ; C9orf72L : 18 ; EV + FIP200 : 18 ; C9orf72S + FIP200 : 18 ; C9orf72L + FIP200 : 17 . Scale bar = 10 µm ; see also Fig EV2 . F , G , H . HeLa cells transfected with HA - ULK1 and Myc - C9orf72 were fixed and processed for PLA analysis . Transfections were laced with mVenus to enable identification of transfected cells for analysis where required ( green ) . PLA signals ( red ) were counted per cell ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test , * * * * P ≤ 0 . 0001 ; N ( cells ) = ( G ) C9orf72S : 21 ; C9orf72L : 20 ; EV + ULK1 : 20 ; C9orf72S + ULK1 : 22 ; C9orf72L + ULK1 : 20 . Scale bar = 10 µm ; see also Fig EV2 . F , G , H . HeLa cells transfected with Myc - ATG13 and EGFP - C9orf72 were fixed and processed for PLA analysis . Transfections were laced with mVenus to enable identification of transfected cells for analysis where required ( green ) . PLA signals ( red ) were counted per cell ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test , * * * * P ≤ 0 . 0001 ; N ( cells ) = ( H ) C9orf72S : 11 ; C9orf72L : 10 ; EV + ATG13 : 11 ; C9orf72S + ATG13 : 11 ; C9orf72L + ATG13 : 11 ) . Scale bar = 10 µm ; see also Fig EV2 . I , J , K . 35S - radiolabeled recombinant FIP200 - 6xHis segments ( I ) were added to GST , GST - C9orf72S and GST - C9orf72L immobilized on glutathione - coated beads . 35S - radiolabelled recombinant proteins were visualized by phosphoimager ( top panels ) . Coomassie - stained GST , GST - C9orf72S and GST - C9orf72L in the pull - down samples are shown ( bottom panels ) . The identity of the Coomassie protein bands was confirmed by mass spectrometry ( # = E . coli DnaK Chaperonin ; * = E . coli 60kD Chaperonin ; Appendix Fig S2 ) . I , J , K . 35S - radiolabeled recombinant Myc - ATG13 ( J ) were added to GST , GST - C9orf72S and GST - C9orf72L immobilized on glutathione - coated beads . 35S - radiolabelled recombinant proteins were visualized by phosphoimager ( top panels ) . Coomassie - stained GST , GST - C9orf72S and GST - C9orf72L in the pull - down samples are shown ( bottom panels ) . The identity of the Coomassie protein bands was confirmed by mass spectrometry ( # = E . coli DnaK Chaperonin ; * = E . coli 60kD Chaperonin ; Appendix Fig S2 ) . I , J , K . 35S - radiolabeled recombinant HA - ULK1 ( K ) were added to GST , GST - C9orf72S and GST - C9orf72L immobilized on glutathione - coated beads . 35S - radiolabelled recombinant proteins were visualized by phosphoimager ( top panels ) . Coomassie - stained GST , GST - C9orf72S and GST - C9orf72L in the pull - down samples are shown ( bottom panels ) . The identity of the Coomassie protein bands was confirmed by mass spectrometry ( # = E . coli DnaK Chaperonin ; * = E . coli 60kD Chaperonin ; Appendix Fig S2 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3A, B, C. Cell lysates of HEK293 cells co-transfected with FLAG-FIP200 (A) and either empty vector control, Myc-C9orf72S or Myc-C9orf72L were subjected to immunoprecipitation with anti-Myc (A and B) antibodies. Immune pellets were probed for Myc-C9orf72 (A and B), FLAG-FIP200 (A) on immunoblots.A, B, C. Cell lysates of HEK293 cells co-transfected with HA-ULK1 (B) and either empty vector control, Myc-C9orf72S or Myc-C9orf72L were subjected to immunoprecipitation with anti-Myc (A and B) antibodies. Immune pellets were probed for Myc-C9orf72 (A and B), HA-ULK1 (B) on immunoblots.A, B, C. Cell lysates of HEK293 cells co-transfected with Myc-ATG13 (C) and either empty vector control, FLAG-C9orf72S or FLAG-C9orf72L were subjected to immunoprecipitation with anti-FLAG (C) antibodies. Immune pellets were probed for FLAG-C9orf72 (C), Myc-ATG13 (C) on immunoblots.D, E. Cell lysates of HEK293 cells transfected with empty vector control, Myc-C9orf72S or Myc-C9orf72L were subjected to immunoprecipitation with anti-Myc antibodies Immune pellets were probed for Myc-C9orf72 and endogenous FIP200, ULK1, and ATG13. There are multiple alternatively spliced forms of ATG13 (Jung et al., 2009; Alers et al., 2011); ATG13* is most likely a smaller alternative spliced form of ATG13 that is enriched by interaction with C9orf72.F, G, H. HeLa cells transfected with Flag-FIP200 and Myc-C9orf72 were fixed and processed for PLA analysis. Transfections were laced with mVenus to enable identification of transfected cells for analysis where required (green). PLA signals (red) were counted per cell (Mean ± SEM; one-way ANOVA with Fisher's LSD test, **** P ≤ 0.0001; N (cells) = (F) C9orf72S: 22; C9orf72L: 18; EV+FIP200: 18; C9orf72S+FIP200: 18; C9orf72L+FIP200: 17. Scale bar = 10 µm; see also Fig EV2.F, G, H. HeLa cells transfected with HA-ULK1 and Myc-C9orf72 were fixed and processed for PLA analysis. Transfections were laced with mVenus to enable identification of transfected cells for analysis where required (green). PLA signals (red) were counted per cell (Mean ± SEM; one-way ANOVA with Fisher's LSD test, **** P ≤ 0.0001; N (cells) = (G) C9orf72S: 21; C9orf72L: 20; EV+ULK1: 20; C9orf72S+ULK1: 22; C9orf72L+ULK1: 20. Scale bar = 10 µm; see also Fig EV2.F, G, H. HeLa cells transfected with Myc-ATG13 and EGFP-C9orf72 were fixed and processed for PLA analysis. Transfections were laced with mVenus to enable identification of transfected cells for analysis where required (green). PLA signals (red) were counted per cell (Mean ± SEM; one-way ANOVA with Fisher's LSD test, **** P ≤ 0.0001; N (cells) = (H) C9orf72S: 11; C9orf72L: 10; EV+ ATG13: 11; C9orf72S+ATG13: 11; C9orf72L+ATG13: 11). Scale bar = 10 µm; see also Fig EV2.I, J, K. 35S-radiolabeled recombinant FIP200-6xHis segments (I) were added to GST, GST-C9orf72S and GST-C9orf72L immobilized on glutathione-coated beads. 35S-radiolabelled recombinant proteins were visualized by phosphoimager (top panels). Coomassie-stained GST, GST-C9orf72S and GST-C9orf72L in the pull-down samples are shown (bottom panels). The identity of the Coomassie protein bands was confirmed by mass spectrometry (# = E. coli DnaK Chaperonin; * = E. coli 60kD Chaperonin; Appendix Fig S2).I, J, K. 35S-radiolabeled recombinant Myc-ATG13 (J) were added to GST, GST-C9orf72S and GST-C9orf72L immobilized on glutathione-coated beads. 35S-radiolabelled recombinant proteins were visualized by phosphoimager (top panels). Coomassie-stained GST, GST-C9orf72S and GST-C9orf72L in the pull-down samples are shown (bottom panels). The identity of the Coomassie protein bands was confirmed by mass spectrometry (# = E. coli DnaK Chaperonin; * = E. coli 60kD Chaperonin; Appendix Fig S2).I, J, K. 35S-radiolabeled recombinant HA-ULK1 (K) were added to GST, GST-C9orf72S and GST-C9orf72L immobilized on glutathione-coated beads. 35S-radiolabelled recombinant proteins were visualized by phosphoimager (top panels). Coomassie-stained GST, GST-C9orf72S and GST-C9orf72L in the pull-down samples are shown (bottom panels). The identity of the Coomassie protein bands was confirmed by mass spectrometry (# = E. coli DnaK Chaperonin; * = E. coli 60kD Chaperonin; Appendix Fig S2).


Figure 4A . HEK293 cells were transfected with non - targeting ( Ctrl ) or C9orf72 siRNA . Cells were treated with rapamycin for 6 h to induce autophagy . Activation of ULK1 was determined on immunoblots using phospho - ULK1 ( Ser757 ) , total ULK1 and GAPDH Abs ( loading control ) . 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 . 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4A. HEK293 cells were transfected with non-targeting (Ctrl) or C9orf72 siRNA. Cells were treated with rapamycin for 6 h to induce autophagy. Activation of ULK1 was determined on immunoblots using phospho-ULK1 (Ser757), total ULK1 and GAPDH Abs (loading control).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. 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 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.


Figure 5A , 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5A, 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 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.


Figure 6B . 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 . 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 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6B. 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. 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 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 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).


Figure 7A . 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7A. 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 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 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.


Figure 8A . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 8A. 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. 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.


Figure 9A , 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 9A, 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).


Figure 1 ( 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 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 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 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 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 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 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1(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 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 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 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 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 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 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 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.


Figure 2 ( 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2(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 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 3Figure 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3Figure 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).


Figure 5 ( 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 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 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 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 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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 5(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 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 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 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 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 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.


Figure 6 ( 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 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 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 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 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6(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 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 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 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 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 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.


Figure 7 ( 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 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 and Cd22R130E primary B cells were treated with 1 µM LatA and intracellular calcium flux was measured by flow cytometry . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7(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 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 and Cd22R130E primary B cells were treated with 1 µM LatA and intracellular calcium flux was measured by flow cytometry.(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.


Figure 1 ( 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 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1(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 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 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.


Figure 2D ) 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2D) 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 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.


Figure 3 ( 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 images of cells treated with the indicated p53 and myosin VI siRNAs . Scale bar , 200 μm . ( 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 ) 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(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 images of cells treated with the indicated p53 and myosin VI siRNAs. Scale bar, 200 μm.(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) 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.


Figure 4 ( 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 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4(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 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 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.


Figure 5 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5(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.


Figure 6 ( 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 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6(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 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 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.


Figure 1A 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 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 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 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 and prk4 after infiltration into leaves . Infiltration with xanthine buffer ( X ) was used as a control .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1A 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 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 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 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 and prk4 after infiltration into leaves. Infiltration with xanthine buffer (X) was used as a control.


Figure 2B 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2B 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 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.