( D ) The extent of Ubqln4 co ‐ immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln4 , Ubqln4ΔUBL and Ubqln4ΔUBA . The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT . Each bar represents the average and s . e . m . of four independent experiments . * P ‐ value = 0 . 0003 and * * P ‐ value = 0 . 0001 .

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(D) The extent of Ubqln4 co‐immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln4, Ubqln4ΔUBL and Ubqln4ΔUBA. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average and s.e.m. of four independent experiments. *P‐value=0.0003 and **P‐value=0.0001.


( E ) 293 cells were co ‐ transfected with GFP ‐ S ‐ Ubqln1 and HA ‐ Ubqln4 , HA ‐ Ubqln4 ΔUBA or HA ‐ Ubqln4 ΔUBA I55A ( I55 in the UBL domain of Ubqln4 was mutated to A ) . GFP was immunoprecipitated and analysed by western blot using anti ‐ GFP and anti ‐ HA antibodies .

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(E) 293 cells were co‐transfected with GFP‐S‐Ubqln1 and HA‐Ubqln4, HA‐Ubqln4 ΔUBA or HA‐Ubqln4 ΔUBA I55A (I55 in the UBL domain of Ubqln4 was mutated to A). GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies.


( F ) The extent of Ubqln4 co ‐ immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln4 was calculated for WT Ubqln4 , Ubqln4ΔUBA and Ubqln4ΔUBA I55A . The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT . Each bar represents the average of two independent experiments . GFP , green fluorescent protein ; HA , haemagglutinin ; IP , immunoprecipitation ; UBA , ubiquitin ‐ associated ; UBL , ubiquitin ‐ like ; Ubqln , Ubiquilin ; WCL , whole ‐ cell lysate ; WT , wild ‐ type .

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(F) The extent of Ubqln4 co‐immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln4 was calculated for WT Ubqln4, Ubqln4ΔUBA and Ubqln4ΔUBA I55A. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average of two independent experiments. GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; UBA, ubiquitin‐associated; UBL, ubiquitin‐like; Ubqln, Ubiquilin; WCL, whole‐cell lysate; WT, wild‐type.


( A ) Confocal images of Hela cells transfected with mCherry ‐ GFP ‐ LC3 and also transfected with control , Ubqln1 or Ubqln4 siRNA . The cells were either untreated or were transferred to starvation medium for 8 h .

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(A) Confocal images of Hela cells transfected with mCherry‐GFP‐LC3 and also transfected with control, Ubqln1 or Ubqln4 siRNA. The cells were either untreated or were transferred to starvation medium for 8 h.


( B ) Quantification of the number of mCherry / GFP double ‐ positive and mCherry single ‐ positive puncta per cell in ( A ) . Each bar represents the average and s . e . m . of 30 cells per condition in three independent experiments . The number of mCherry single ‐ positive puncta of Ubqln1 and Ubqln4 siRNA was compared with that of control siRNA . * P ‐ value = 0 . 0475 and * * P ‐ value = 0 . 0373 . GFP , green fluorescent protein ; LC3 , microtubule ‐ associated protein light chain 3 ; Ubqln , Ubiquilin .

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(B) Quantification of the number of mCherry/GFP double‐positive and mCherry single‐positive puncta per cell in (A). Each bar represents the average and s.e.m. of 30 cells per condition in three independent experiments. The number of mCherry single‐positive puncta of Ubqln1 and Ubqln4 siRNA was compared with that of control siRNA. *P‐value=0.0475 and **P‐value=0.0373. GFP, green fluorescent protein; LC3, microtubule‐associated protein light chain 3; Ubqln, Ubiquilin.


( B ) Structures of S . cerevisiae Hrr251 - 394 K38R bound to CK1 - 7 ( Chijiwa et al , 1989 ; Xu et al , 1996 ) and C . glabrata Hrr251 - 403 K38R bound to ADP , with domains colored as in ( A ) . Bound PO4 - / SO4 - ions are shown as spheres . See Figure EV2A for additional C . glabrataHrr25 structures , and Figure EV3A for SO4 - ion electron density .

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(B) Structures of S. cerevisiae Hrr251-394 K38R bound to CK1-7 (Chijiwa et al, 1989; Xu et al, 1996) and C. glabrata Hrr251-403 K38R bound to ADP, with domains colored as in (A). Bound PO4-/SO4- ions are shown as spheres. See Figure EV2A for additional C. glabrataHrr25 structures, and Figure EV3A for SO4- ion electron density.


( C ) Overlay of the kinase domain of C . glabrataHrr25 ( Apo form , crystallized with 1 . 2 M PO4 - ) with ratCK1δ crystallized in the presence of tungstate ions ( WO4 ; PDB ID 1CKJ , ( Longenecker et al , 1996 ) ) See Figure EV2B for detailed views of ion binding .

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(C) Overlay of the kinase domain of C. glabrataHrr25 (Apo form, crystallized with 1.2 M PO4-) with ratCK1δ crystallized in the presence of tungstate ions (WO4; PDB ID 1CKJ, (Longenecker et al, 1996)) See Figure EV2B for detailed views of ion binding.


( D ) Stereo view of CK1 - 7 binding to S . cerevisiae Hrr25 . Bound drug is positioned identically to a previous structure of S . pombe Cki1 bound to CK1 - 7 ( Xu et al , 1996 ) .

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(D) Stereo view of CK1-7 binding to S. cerevisiae Hrr25. Bound drug is positioned identically to a previous structure of S. pombe Cki1 bound to CK1-7 (Xu et al, 1996).


( E ) Stereo view of ADP binding to C . glabrata Hrr25 ( formate structure ; SO4 - structure is equivalent , but the GxGxxG motif is disordered in that structure ) . All active - site residues shown in panels ( D ) and ( E ) are conserved between S . cerevisiae and C . glabrata Hrr25 .

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(E) Stereo view of ADP binding to C. glabrata Hrr25 (formate structure; SO4- structure is equivalent, but the GxGxxG motif is disordered in that structure). All active-site residues shown in panels (D) and (E) are conserved between S. cerevisiae and C. glabrata Hrr25.


( A ) Two views of C . glabrata Hrr25 ( Apo form ) with domains colored as in Figure 1 , showing bound PO4 - ions and the previously - identified Mam1 - binding residues His25 and Glu34 ( Petronczki et al , 2006 ) .

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(A) Two views of C. glabrata Hrr25 (Apo form) with domains colored as in Figure 1, showing bound PO4- ions and the previously-identified Mam1-binding residues His25 and Glu34 (Petronczki et al, 2006).


( B ) Two views , roughly equivalent to the views in ( A ) , of the Hrr25 central domain , colored as a rainbow according to the schematic at top . Dotted lines in the schematic indicate disordered loops . See Figure EV1A for sequence alignment of this domain .

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(B) Two views, roughly equivalent to the views in (A), of the Hrr25 central domain, colored as a rainbow according to the schematic at top. Dotted lines in the schematic indicate disordered loops. See Figure EV1A for sequence alignment of this domain.


( B ) Overall structure of the S . cerevisiae Hrr251 - 394 K38R : Mam187 - 191 complex , colored as in ( A ) . View is equivalent to Figure 2B .

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(B) Overall structure of the S. cerevisiae Hrr251-394 K38R:Mam187-191 complex, colored as in (A). View is equivalent to Figure 2B.


( C ) Close - up view of Mam187 - 191 , colored as in the schematic in ( A ) . ( D ) Close - up of the variant zinc knuckle motif of Mam1 , with zinc anomalous difference electron density shown at 3 σ ( gray ) and 10 σ ( red ) .

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(C) Close-up view of Mam187-191, colored as in the schematic in (A). (D) Close-up of the variant zinc knuckle motif of Mam1, with zinc anomalous difference electron density shown at 3 σ (gray) and 10 σ (red).


( E ) Geometry of zinc binding in Mam1 . Zn - S bonds and the Zn - N bond were restrained to ~ 2 . 3 Å and ~ 2 . 0 Å , respectively , during refinement .

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(E) Geometry of zinc binding in Mam1. Zn-S bonds and the Zn-N bond were restrained to ~2.3 Å and ~2.0 Å, respectively, during refinement.


( A ) The Hrr25 : Mam1 complex with Mam1 shown as orange ribbons , and Hrr25 shown as a white surface with Mam1 - binding interface # 1 shown in green and # 2 in magenta .

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(A) The Hrr25:Mam1 complex with Mam1 shown as orange ribbons, and Hrr25 shown as a white surface with Mam1-binding interface #1 shown in green and #2 in magenta.


( B ) Stereo view of interface # 1 , which involves hydrophobic interactions by Mam1 L92 , L97 , L100 , and I104 , and a hydrogen - bond network between Mam1 Q101 / E108 / R131 and Hrr25 residues 12 - 15 . ( C ) Stereo view of interface # 2 . Hrr25 residues H25 and E34 participate in a buried hydrogen - bond network with Mam1 residues R149 and Y158 , and are surrounded by hydrophobic residues on both proteins . For clarity , Mam1 helix 4 , on which R149 , Y153 , and Y158 are located , is not shown .

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(B) Stereo view of interface #1, which involves hydrophobic interactions by Mam1 L92, L97, L100, and I104, and a hydrogen-bond network between Mam1 Q101/E108/R131 and Hrr25 residues 12-15. (C) Stereo view of interface #2. Hrr25 residues H25 and E34 participate in a buried hydrogen-bond network with Mam1 residues R149 and Y158, and are surrounded by hydrophobic residues on both proteins. For clarity, Mam1 helix 4, on which R149, Y153, and Y158 are located, is not shown.


( D ) Pulldown assay with in vitro - translated Mam187 - 191 ( wild - type and alanine mutants ) and Hrr251 - 394 K38R . Mam1 mutations also disrupt Hrr25 binding when the two are co - expressed in E . coli ( Figure EV6B ) . Bottom : Sporeviability of MAM1 mutant strains ( see Table EV2 for strains ; strains tested are KC549 , KC560 , KC566 , KC552 , KC554 , KC556 , and KC558 ; 46 - 48 tetrads were dissected for each strain ) . " - - " indicates that this mutation was not tested for sporeviability .

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(D) Pulldown assay with in vitro-translated Mam187-191 (wild-type and alanine mutants) and Hrr251-394 K38R. Mam1 mutations also disrupt Hrr25 binding when the two are co-expressed in E. coli (Figure EV6B). Bottom: Sporeviability of MAM1 mutant strains (see Table EV2 for strains; strains tested are KC549, KC560, KC566, KC552, KC554, KC556, and KC558; 46-48 tetrads were dissected for each strain). "--" indicates that this mutation was not tested for sporeviability.


( A - C ) Ribbon views of the S . cerevisiae Hrr251 - 394 : Mam187 - 191 structure ( form 1 ) , CK1 - 7 bound S . cerevisiae Hrr251 - 394 , and ADP - bound C . glabrata Hrr251 - 403 ( formate condition ) , with Hrr25 colored according to main - chain B - factor from low ( purple ) to cyan ( high ) . For each , coloring is normalized to correspond to the average of the 20 lowest ( purple ) or highest ( cyan ) main - chain B - factors in Hrr25 . Mam1 ( panel A ) is colored in light gray .

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(A-C) Ribbon views of the S. cerevisiae Hrr251-394:Mam187-191 structure (form 1), CK1-7 bound S. cerevisiae Hrr251-394, and ADP-bound C. glabrata Hrr251-403 (formate condition), with Hrr25 colored according to main-chain B-factor from low (purple) to cyan (high). For each, coloring is normalized to correspond to the average of the 20 lowest (purple) or highest (cyan) main-chain B-factors in Hrr25. Mam1 (panel A) is colored in light gray.


( D ) Overall main - chain B - factors for the N - lobe ( residues 1 - 85 ) of four structures of Hrr25 ( both C . glabrata and S . cerevisiae ) and the three crystallographically - unique views of the S . cerevisiae Hrr25 : Mam1 complex . Values are normalized to the average main - chain B - factor for the entire Hrr25 chain ( dotted line at 1 . 0 ) . P - value = 0 . 004 ( student ' s T - test ) . See Figure EV4 for residue - by - residue B - factor plots for each structure .

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(D) Overall main-chain B-factors for the N-lobe (residues 1-85) of four structures of Hrr25 (both C. glabrata and S. cerevisiae) and the three crystallographically-unique views of the S. cerevisiae Hrr25:Mam1 complex. Values are normalized to the average main-chain B-factor for the entire Hrr25 chain (dotted line at 1.0). P-value=0.004 (student's T-test). See Figure EV4 for residue-by-residue B-factor plots for each structure.


( A ) ADP - Glo ATPase assay showing stimulation of S . cerevisiae Hrr251 - 394 ( blue circles ) , Hrr251 - 394 K38R ( red diamonds ) , Hrr25 full - length : Mam187 - 191 ( black triangles ) , Hrr251 - 394 : Mam187 - 191 ( orange squares ) , and Hrr251 - 394 : Mam187 - 191 R131A ( green triangles ) by bovine casein . See Figure EV6A for SDS - PAGE analysis of purified proteins .

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(A) ADP-Glo ATPase assay showing stimulation of S. cerevisiae Hrr251-394 (blue circles), Hrr251-394 K38R (red diamonds), Hrr25 full-length:Mam187-191 (black triangles), Hrr251-394:Mam187-191 (orange squares), and Hrr251-394:Mam187-191 R131A (green triangles) by bovine casein. See Figure EV6A for SDS-PAGE analysis of purified proteins.


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(B) Stimulation of Hrr251-394, Hrr251-394:Mam187-191, and Hrr251-394:Mam187-191 R131A by ATP, measured using an enzyme-coupled ATPase assay.


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(C) ADP-Glo assay showing the effect of added CK1-7 on Hrr251-394, Hrr251-394:Mam187-191, and Hrr251-394:Mam187-191 R131A.


( D ) Km , Vmax , and IC50 values for tested enzymes . Vmax was calculated from curves in ( A ) at 100 M ATP .

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(D) Km, Vmax, and IC50 values for tested enzymes. Vmax was calculated from curves in (A) at 100 M ATP.


( A ) Phos - tag gels showing phosphorylation of purified S . cerevisiae Mtw1 complex by Hrr251 - 394 and Hrr251 - 394 : Mam187 - 191 . Asterisks denote N - terminal proteolytic cleavage products of Dsn1 , with the red asterisk denoting a previously - characterized product lacking the first 171 residues ( Hornung et al , 2011 ) .

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(A) Phos-tag gels showing phosphorylation of purified S. cerevisiae Mtw1 complex by Hrr251-394 and Hrr251-394:Mam187-191. Asterisks denote N-terminal proteolytic cleavage products of Dsn1, with the red asterisk denoting a previously-characterized product lacking the first 171 residues (Hornung et al, 2011).


Pre - enriched thymocytes from WT BALB / c thymi as in ( a ) were labeled by CFSE ( CFSE + ) , mixed with pre - enriched thymocytes from SKG thymi ( CFSE - ) , loaded with Indo - 1 , and stimulated as in ( a ) . Ratio of Indo ( Violet ) and Indo ( Blue ) was analyzed by flow cytometry . Comparison of cytoplasmic Ca2 + profiles between WT and SKG NKT1 ( c ) and NKT2 ( d ) cells in the same test tube are shown . Data are representative from one of at least three independent experiments . Graphs represent the average Ca2 + levels .

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Pre-enriched thymocytes from WT BALB/c thymi as in (a) were labeled by CFSE (CFSE+), mixed with pre-enriched thymocytes from SKG thymi (CFSE-), loaded with Indo-1, and stimulated as in (a). Ratio of Indo (Violet) and Indo (Blue) was analyzed by flow cytometry. Comparison of cytoplasmic Ca2+ profiles between WT and SKG NKT1 (c) and NKT2 (d) cells in the same test tube are shown. Data are representative from one of at least three independent experiments. Graphs represent the average Ca2+ levels.


Sorted thymic NKT1 and NKT2 cells were labeled with CellTrace Violet , and activated by anti - CD3ε / CD28 Dynabeads for 2 days , in the presence of different [ EGTA ] in the medium . CellTrace Violet / CTV signal in living cells at the end of the culture is shown . Cell proliferation was quantified as division index based on CTV signals in ( a ) .

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Sorted thymic NKT1 and NKT2 cells were labeled with CellTrace Violet, and activated by anti-CD3ε/CD28 Dynabeads for 2 days, in the presence of different [EGTA] in the medium. CellTrace Violet/CTV signal in living cells at the end of the culture is shown. Cell proliferation was quantified as division index based on CTV signals in (a).


Thymic lobes from new born ( day 0 ) BALB / c mice were cultured in vitro for 6 days . EGTA was added 48h after the start of culture ( day 2 ) , and the expression of CXCR3 ( g ) , PLZF ( h ) and PD1 ( i ) in total iNKT cells are shown .

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Thymic lobes from new born (day 0) BALB/c mice were cultured in vitro for 6 days. EGTA was added 48h after the start of culture (day 2), and the expression of CXCR3 (g), PLZF (h) and PD1 (i) in total iNKT cells are shown.


oxygen consumption rate ( OCR ) from sorted thymic T cell populations measured in a Seahorse Mitostress test ;

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oxygen consumption rate (OCR) from sorted thymic T cell populations measured in a Seahorse Mitostress test;


Confocal images of Tim 23 ( Alexa Fluor 488 , green ) and MitoTracker deep red ( Alexa Fluor 647 , red ) in freshly isolated thymic NKT1 ( c1 , 2 ) and NKT2 ( c5 , 6 ) cells using Airyscan technology . 3D images reconstructed by Imaris software in NKT1 ( c3 , 4 ) and NKT2 cells ( c7 , 8 ) . Dotted line in panel ( 5 - 8 ) indicates juxtaposition of two NKT2 cell images taken with the same resolution and magnification as NKT1 cells .

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

Confocal images of Tim 23 (Alexa Fluor 488, green) and MitoTracker deep red (Alexa Fluor 647, red) in freshly isolated thymic NKT1 (c1, 2) and NKT2 (c5, 6) cells using Airyscan technology. 3D images reconstructed by Imaris software in NKT1 (c3, 4) and NKT2 cells(c7, 8). Dotted line in panel (5-8) indicates juxtaposition of two NKT2 cell images taken with the same resolution and magnification as NKT1 cells.


Representative images of electron microscopy in NKT1 and NKT2 cells ; ( f ) individual mitochondria size and ( g ) cristae perimeters normalized to mitochondria perimeter based on EM in ( e ) .

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

Representative images of electron microscopy in NKT1 and NKT2 cells; (f) individual mitochondria size and (g) cristae perimeters normalized to mitochondria perimeter based on EM in (e).


Maximal cytoplasmic [ Ca2 + ] in single , freshly sorted NKT1 and NKT2 cells following TCR stimulation in the presence or absence of BTP2 ( 1uM ) ;

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

Maximal cytoplasmic [Ca2+] in single, freshly sorted NKT1 and NKT2 cells following TCR stimulation in the presence or absence of BTP2 (1uM);


BTP2 ( 1uM ) was added 48h after the start of culture , and the expression of Tbet in total iNKT cells is shown . Data are representative from one of three independent experiments . Graphs represent mean ± SD with symbols representing individual cells ( a ) , or individual thymic lobes ( b - f ) . * * p < 0 . 01 ; * * * * p < 0 . 0001 ; n . s . not significant ( unpaired two - tailed Student ' s t test ) .

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

BTP2 (1uM) was added 48h after the start of culture, and the expression of Tbet in total iNKT cells is shown. Data are representative from one of three independent experiments. Graphs represent mean ± SD with symbols representing individual cells (a), or individual thymic lobes (b-f). **p < 0.01; ****p < 0.0001; n.s. not significant (unpaired two-tailed Student's t test).


BTP2 ( 1uM ) was added 48h after the start of culture , and the expression of CXCR3 ( d ) , PLZF ( e ) , and PD1 ( f ) in total iNKT cells is shown . Data are representative from one of three independent experiments . Graphs represent mean ± SD with symbols representing individual cells ( a ) , or individual thymic lobes ( b - f ) . * * p < 0 . 01 ; * * * * p < 0 . 0001 ; n . s . not significant ( unpaired two - tailed Student ' s t test ) .

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

BTP2 (1uM) was added 48h after the start of culture, and the expression of CXCR3 (d), PLZF (e), and PD1 (f) in total iNKT cells is shown. Data are representative from one of three independent experiments. Graphs represent mean ± SD with symbols representing individual cells (a), or individual thymic lobes (b-f). **p < 0.01; ****p < 0.0001; n.s. not significant (unpaired two-tailed Student's t test).


Profiles showing average Ca2 + influx induced by Thapsigargin ( TG ) in freshly sorted NKT1 ( Red ) and NKT2 ( Black ) cells . The timing of TG ( 1uM ) , ionomycin ( Iono ) ( 2uM ) and CCCP ( 1uM ) addition , and extracellular [ Ca2 + ] in mM are shown .

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

Profiles showing average Ca2+ influx induced by Thapsigargin (TG) in freshly sorted NKT1 (Red) and NKT2 (Black) cells. The timing of TG (1uM), ionomycin (Iono) (2uM) and CCCP (1uM) addition, and extracellular [Ca2+] in mM are shown.


A , B . HeLa cells treated with non - targeting siRNA ( Ctrl ) or C9orf72 siRNA and transfected with mCherry - EGFP - LC3 were treated with vehicle ( Ctrl ) , Torin1 ( 250 nM ; 3 h ) , bafilomycin A1 ( 100 nM ; 6 h - BafA1 ) or combinations thereof as indicated . Autophagosomes ( green + red ) and autolysosomes ( red only ) were quantified per cell ( Mean ± SEM from 3 independent experiments ; one - way ANOVA with Fisher ' s LSD test : ns , not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 ; N ( cells ) = Ctrl / Ctrl : 120 ; Ctrl / Torin1 : 101 ; C9orf72 / Ctrl : 99 ; C9orf72 / Torin1 : 106 ; Ctrl / BafA1 : 116 ; Ctrl / Torin1 / BafA1 : 118 ; C9orf72 / BafA1 : 109 ; C9orf72 / Torin1 / BafA1 : 106 ) . Scale bar = 20 µm . C9orf72 knockdown was confirmed by RT - qPCR ( Appendix Fig S2 ) .

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

A, B. HeLa cells treated with non-targeting siRNA (Ctrl) or C9orf72 siRNA and transfected with mCherry-EGFP-LC3 were treated with vehicle (Ctrl), Torin1 (250 nM; 3 h), bafilomycin A1 (100 nM; 6 h - BafA1) or combinations thereof as indicated. Autophagosomes (green+red) and autolysosomes (red only) were quantified per cell (Mean ± SEM from 3 independent experiments; one-way ANOVA with Fisher's LSD test: ns, not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; N (cells) = Ctrl/Ctrl: 120; Ctrl/Torin1: 101; C9orf72/Ctrl: 99; C9orf72/Torin1: 106; Ctrl/BafA1: 116; Ctrl/Torin1/BafA1: 118; C9orf72/BafA1: 109; C9orf72/Torin1/BafA1: 106). Scale bar = 20 µm. C9orf72 knockdown was confirmed by RT-qPCR (Appendix Fig S2).


C , D . HEK293 cells treated with non - targeting ( Ctrl ) or C9orf72 siRNA were incubated with BafA1 , BafA1 + Torin1 ( C ) or BafA1 + rapamycin ( D ) and levels of LC3 - I and II were determined on immunoblots . Levels of LC3 - II were normalized against α - tubulin and are shown relative to the BafA1 - treated sample ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test : ns , not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 ; N = 3 experiments ) .

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

C, D. HEK293 cells treated with non-targeting (Ctrl) or C9orf72 siRNA were incubated with BafA1, BafA1+Torin1 (C) or BafA1+rapamycin (D) and levels of LC3-I and II were determined on immunoblots. Levels of LC3-II were normalized against α-tubulin and are shown relative to the BafA1-treated sample (Mean ± SEM; one-way ANOVA with Fisher's LSD test: ns, not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; N = 3 experiments).


E , F . Primary cortical neurons ( DIV5 / 6 ) were transfected with ECFP non - targeting ( Ctrl ) or C9orf72 miRNA ( cyan ) and EGFP - LC3 ( green ) . 3 days post transfection neurons were treated with vehicle ( Ctrl ) , Torin1 ( 250 nM ; 3 h ) , BafA1 ( 100 nM ; 5 h ) or combinations thereof as indicated . Autophagosomes were quantified as the number of EGFP - LC3 positive puncta per soma from 2 independent experiments ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test : ns , not significant , * p ≤ 0 . 05 ; * * * P ≤ 0 . 001 ; * * * * P ≤ 0 . 0001 ; N ( cells ) = Ctrl miRNA / Ctrl : 77 ; Ctrl miRNA / Torin1 : 68 ; C9orf72 miRNA / Ctrl : 57 ; C9orf72 miRNA / Torin1 : 69 ; Ctrl miRNA / BafA1 : 35 ; Ctrl miRNA / Torin1 / BafA1 : 57 ; C9orf72 miRNA / BafA1 : 66 ; C9orf72 miRNA / Torin1 / BafA1 : 64 ) . Scale bar = 5 µm .

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

E, F. Primary cortical neurons (DIV5/6) were transfected with ECFP non-targeting (Ctrl) or C9orf72 miRNA (cyan) and EGFP-LC3 (green). 3 days post transfection neurons were treated with vehicle (Ctrl), Torin1 (250 nM; 3 h), BafA1 (100 nM; 5 h) or combinations thereof as indicated. Autophagosomes were quantified as the number of EGFP-LC3 positive puncta per soma from 2 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test: ns, not significant, * p ≤ 0.05; *** P ≤ 0.001; **** P ≤ 0.0001; N (cells) = Ctrl miRNA/Ctrl: 77; Ctrl miRNA/Torin1: 68; C9orf72 miRNA/Ctrl: 57; C9orf72 miRNA/Torin1: 69; Ctrl miRNA/BafA1: 35; Ctrl miRNA/Torin1/BafA1: 57; C9orf72 miRNA/BafA1: 66; C9orf72 miRNA/Torin1/BafA1: 64). Scale bar = 5 µm.


A . 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 .

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

A. 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 O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

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).


A , 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 .

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

A, 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 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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 ) .

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

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 ) .

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

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 ) .

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).


A . 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 ) .

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

A. 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 .

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

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


C . 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 .

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

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


D . HeLa 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 O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O

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


A , B . HeLa 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 .

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

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


C . SH - SY5Y neuroblastoma 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 O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

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


B . 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 .

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

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


C . 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 .

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

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


D . 35S - radiolabelled recombinant Myc - Rab1a 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 ) .

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

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


E . Increasing volumes of 35S - radiolabelled recombinant Myc - C9orf72L 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 O O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

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


A . HEK293 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 .

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

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


B . HeLa 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 .

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

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


C . HeLa 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 O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

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


A . HeLa 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 .

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

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


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

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


A , B . Two C9ALS / FTD iNeuron cultures ( 183 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


( A ) Wild - type 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 .

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

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


( B ) Wild - type 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 .

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

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


( C 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 .

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

(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 .

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

(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 .

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

(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 .

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

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


( J ) Wild - type 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 .

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

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


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

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


( A ) Determining 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 .

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

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

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


Figure 3 . CD22 is highly clustered on 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 O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-GENEPROD O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


( A - C ) Single - particle - tracking of CD22 , CD19 , IgM 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 ) .

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

(A-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 .

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

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


( G ) Wild - type 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 ) .

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

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


( H - J ) Wild - type 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 .

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

(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 .

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

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


( L ) Flow cytometric 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 O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

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


( A - B ) Wild - type 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 .

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

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