( e ) Kymograph of Halo - TMRT7 RNAP constraining translocation of salt - resistant Scc1GFP - TEV - cohesin .

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(e) Kymograph of Halo-TMRT7 RNAP constraining translocation of salt-resistant Scc1GFP-TEV-cohesin.


( a ) Representative field of view showing Halo - TMRCTCF bound to pPlat - 4xCTCF following 75 mM NaCl , 75 mM KCl buffer wash . Arrows denote CTCF bound at predicted site of 4xCTCF array .

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(a) Representative field of view showing Halo-TMRCTCF bound to pPlat-4xCTCF following 75 mM NaCl, 75 mM KCl buffer wash. Arrows denote CTCF bound at predicted site of 4xCTCF array.


( b ) Kymograph of salt - resistant Scc1GFP - TEV - cohesin failing to bypass DNA - bound Halo - TMRCTCF . DNA was post - stained with Sytox Green .

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

(b) Kymograph of salt-resistant Scc1GFP-TEV-cohesin failing to bypass DNA-bound Halo-TMRCTCF. DNA was post-stained with Sytox Green.


C : Western blot analysis of proteins pulled down using the peptides input fraction corresponds to HeLa cell total protein extract . Bound proteins were analyzed using anti - VAP - A , anti VAP - B and anti - MOSPD2 antibodies . Actin was used as a loading control .

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C: Western blot analysis of proteins pulled down using the peptides input fraction corresponds to HeLa cell total protein extract. Bound proteins were analyzed using anti-VAP-A, anti VAP-B and anti-MOSPD2 antibodies. Actin was used as a loading control.


D : Western blot analysis of proteins pulled down using peptides comprising WT and mutant ( D478S and D478pS ) ORP1 FFAT sequence peptides Bound proteins were analyzed using anti VAP - A , anti - VAP - B and anti MOSPD2 antibodies . Actin was used as a loading control . Data information : Acidic ( D or phosphorylated residues ( pS alcoholic ( S residues are in red , green and blue , respectively ; the other residues are in black .

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

D: Western blot analysis of proteins pulled down using peptides comprising WT and mutant (D478S and D478pS) ORP1 FFAT sequence peptides Bound proteins were analyzed using anti VAP-A, anti-VAP-B and anti MOSPD2 antibodies. Actin was used as a loading control. Data information: Acidic (D or phosphorylated residues (pS alcoholic (S residues are in red, green and blue, respectively; the other residues are in black.


B : Coomassie blue staining of the recombinant wild - type MSP domains of VAP - A and VAP - B , and of the KD / MD mutant of VAP - A , after SDS - PAGE .

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B: Coomassie blue staining of the recombinant wild-type MSP domains of VAP-A and VAP-B, and of the KD/MD mutant of VAP-A, after SDS-PAGE.


C : Recombinant MSP domains pulled down with peptides corresponding to the Phospho - FFAT motif of STARD3 ( unphosphorylated ; mono - phosphorylated on S209 or S213 ; bi - phosphorylated on S209 and S213 ) , and with the control peptides were revealed by Coomassie blue staining . The recombinant proteins subjected to the assay are showed in the input fraction .

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

C: Recombinant MSP domains pulled down with peptides corresponding to the Phospho-FFAT motif of STARD3 (unphosphorylated; mono-phosphorylated on S209 or S213; bi-phosphorylated on S209 and S213), and with the control peptides were revealed by Coomassie blue staining. The recombinant proteins subjected to the assay are showed in the input fraction.


A - D : Surface Plasmon Resonance analysis of the MSP domain of VAP - A binding onto immobilized unphosphorylated ( A ) , monophosphorylated pS209 ( B ) and pS213 ( C ) , and bi - phosphorylated pS209 + pS213 ( D ) STARD3 FFAT peptides . Representative sensorgrams resulting from the interaction between the MSP domain of VAP - A injected at different concentrations and the different FFAT peptides are shown in ( A ) , ( B left ) , ( C ) , and ( D left ) . Binding curves display the SPR signal ( RU ) as a function of time . Some samples were measured twice ( concentrations printed in bold ) . Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted , as well as the bulk effect recorded with buffer only . Steady - state analysis of the interaction between pS209 ( B right ) and pS209 + pS213 ( D right ) STARD3 FFAT peptide , and the MSP domain of VAP - A . Equilibrium responses ( Req ) extracted from the left panel in association phase were plotted as a function of the dimeric MSP domain of VAP - A concentration , and fitted with a Langmuir binding model .

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

A-D: Surface Plasmon Resonance analysis of the MSP domain of VAP-A binding onto immobilized unphosphorylated (A), monophosphorylated pS209 (B) and pS213 (C), and bi-phosphorylated pS209+pS213 (D) STARD3 FFAT peptides. Representative sensorgrams resulting from the interaction between the MSP domain of VAP-A injected at different concentrations and the different FFAT peptides are shown in (A), (B left), (C), and (D left). Binding curves display the SPR signal (RU) as a function of time. Some samples were measured twice (concentrations printed in bold). Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted, as well as the bulk effect recorded with buffer only. Steady-state analysis of the interaction between pS209 (B right) and pS209+pS213 (D right) STARD3 FFAT peptide, and the MSP domain of VAP-A. Equilibrium responses (Req) extracted from the left panel in association phase were plotted as a function of the dimeric MSP domain of VAP-A concentration, and fitted with a Langmuir binding model.


E : Dissociation constants between peptides corresponding to different kind of FFAT motifs and the MSP domains of VAP - A and VAP - B . The kinetic experiments were performed at 25 ° C in Tris - HCl 50 mM , pH 7 . 5 , NaCl 75 mM buffer supplemented with 0 . 005 % ( v / v ) surfactant polysorbate 20 ( P20 , GE Healthcare ) . Mean of n independent experiments : n = 2 for pS209 and pS209 + pS213 ; 3 for pS213 ; and 4 for the non - phosphorylated FFAT . Uncertainties are obtained from the standard deviation considering a t - distribution coefficient for a risk factor of 32 % .

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

E: Dissociation constants between peptides corresponding to different kind of FFAT motifs and the MSP domains of VAP-A and VAP-B. The kinetic experiments were performed at 25 °C in Tris-HCl 50 mM, pH 7.5, NaCl 75 mM buffer supplemented with 0.005% (v/v) surfactant polysorbate 20 (P20, GE Healthcare). Mean of n independent experiments: n=2 for pS209 and pS209+pS213; 3 for pS213; and 4 for the non-phosphorylated FFAT. Uncertainties are obtained from the standard deviation considering a t-distribution coefficient for a risk factor of 32%.


A , B : Binding assays . Immunoprecipitation ( GFP - Trap ) experiments between GFP - tagged VAP - A ( WT and KD / MD mutant ; A ) , VAP - B ( B ) and Flag - tagged STARD3 ( WT and S209A , S209D and S209D / P210A mutants ) . Please note that the mutations modify the FFAT and Phospho - FFAT scores of this region of STARD3 : compared to WT STARD3 ( Phospho - FFAT : 2 . 5 ; conventional FFAT : 6 . 5 ) , the STARD3 S209A , mutant has both low Phospho - FFAT and FFAT scores ( 6 . 5 ) , the S209D mutant has a high conventional FFAT score ( 2 . 5 ) but a low Phospho - FFAT score ( 6 . 5 ) , and the double mutant has a conventional FFAT score of 1 . 5 and a Phospho - FFAT score of 5 . 5 . Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag , anti - GFP and anti - Actin antibodies . Immunoprecipitated material was analyzed using anti - Flag and anti - GFP antibodies .

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

A,B: Binding assays. Immunoprecipitation (GFP-Trap) experiments between GFP-tagged VAP-A (WT and KD/MD mutant; A), VAP-B (B) and Flag-tagged STARD3 (WT and S209A, S209D and S209D/P210A mutants). Please note that the mutations modify the FFAT and Phospho-FFAT scores of this region of STARD3: compared to WT STARD3 (Phospho-FFAT: 2.5; conventional FFAT: 6.5), the STARD3 S209A, mutant has both low Phospho-FFAT and FFAT scores (6.5), the S209D mutant has a high conventional FFAT score (2.5) but a low Phospho-FFAT score (6.5), and the double mutant has a conventional FFAT score of 1.5 and a Phospho-FFAT score of 5.5. Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag, anti-GFP and anti-Actin antibodies. Immunoprecipitated material was analyzed using anti-Flag and anti-GFP antibodies.


Recruitment assays . GFP - VAP - A ( C ; green ) were left untransfected ( C ) and labelled using anti - Flag ( magenta ) antibodies . The subpanels on the right are higher magnification ( 3 . 5 × ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Scale bars : 10 µm . Inset scale bars : 2 µm .

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

Recruitment assays. GFP-VAP-A (C ; green) were left untransfected (C) and labelled using anti-Flag (magenta) antibodies. The subpanels on the right are higher magnification (3.5×) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Scale bars: 10 µm. Inset scale bars: 2 µm.


: Recruitment assays . GFP - VAP - A D , E , G ; green ) and GFP - VAP - A KD / MD - expressing cells ( F ; green ) were transfected with Flag - STARD3 ( D , F ) , Flag - STARD3 S209A ( E ) , and Flag - STARD3 S209D / P210A ( G ) , and labelled using anti - Flag ( magenta ) antibodies . The subpanels on the right are higher magnification ( 3 . 5 × ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Scale bars : 10 µm . Inset scale bars : 2 µm .

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

: Recruitment assays. GFP-VAP-A D, E, G; green) and GFP-VAP-A KD/MD-expressing cells (F; green) were transfected with Flag-STARD3 (D, F), Flag-STARD3 S209A (E), and Flag-STARD3 S209D/P210A (G), and labelled using anti-Flag (magenta) antibodies. The subpanels on the right are higher magnification (3.5×) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Scale bars: 10 µm. Inset scale bars: 2 µm.


H : Pearson ' s correlation coefficients between VAP - A ( WT or KD / MD mutant ) and STARD3 ( WT or S209A , S209D , S209D / P210A , FA / YA mutants ) staining are shown . Each dot represents a single cell ( number of cells : VAP - A - STARD3 : 22 ; VAP - A - STARD3 S209A : 25 ; VAP - A - STARD3 S209D : 27 ; VAP - A - STARD3 S209D / P210A : 26 ; VAP - A - STARD3 FA / YA : 20 ; VAP - A KD / MD - STARD3 : 20 , from at least three independent experiments ) . Means and error bars ( SD ) are shown . Kruskal - Wallis with Dunn ' s multiple comparison test ( * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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

H: Pearson's correlation coefficients between VAP-A (WT or KD/MD mutant) and STARD3 (WT or S209A, S209D, S209D/P210A, FA/YA mutants) staining are shown. Each dot represents a single cell (number of cells: VAP-A-STARD3: 22; VAP-A-STARD3 S209A: 25; VAP-A-STARD3 S209D: 27; VAP-A-STARD3 S209D/P210A: 26; VAP-A-STARD3 FA/YA: 20; VAP-A KD/MD-STARD3: 20, from at least three independent experiments). Means and error bars (SD) are shown. Kruskal-Wallis with Dunn's multiple comparison test (**P < 0.01; ***P < 0.001).


B : SDS - PAGE gel and Western blot analysis of purified cSTD3 and pS209 cSTD3 proteins . Top : the gel was stained with Sypro Orange to visualize proteins and molecular weight markers . Bottom : two similar gels were blotted onto nitrocellulose and analysed for the presence of total and STARD3 - pS209 using specific antibodies .

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B: SDS-PAGE gel and Western blot analysis of purified cSTD3 and pS209 cSTD3 proteins. Top: the gel was stained with Sypro Orange to visualize proteins and molecular weight markers. Bottom: two similar gels were blotted onto nitrocellulose and analysed for the presence of total and STARD3-pS209 using specific antibodies.


C : Aggregation assays . LA liposomes ( 50 µM total lipids ) were incubated for 5 minutes with cSTD3 or pS209 cSTD3 ( 380 nM ) . Then , LB liposomes ( 50 µM total lipids ) and VAP - AHIS6 or VAP - A KD / MDHIS6 ( 700 nM ) were successively added . Left panels : mean radius ( black dots ) and polydispersity ( shaded area ) over time . Right panels : size distribution before ( grey bars ) and after the reaction ( black bars ) .

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

C: Aggregation assays. LA liposomes (50 µM total lipids) were incubated for 5 minutes with cSTD3 or pS209 cSTD3 (380 nM). Then, LB liposomes (50 µM total lipids) and VAP-AHIS6 or VAP-A KD/MDHIS6 (700 nM) were successively added. Left panels: mean radius (black dots) and polydispersity (shaded area) over time. Right panels: size distribution before (grey bars) and after the reaction (black bars).


D : DHE transport assay . DOPC ( 1 . 2 - dioleoyl - sn - glycero - 3 - phosphocholine ) liposomes ( 62 . 5 µM total lipids , LA ) containing 3 mol % MPB - PE were mixed with cSTD3 or pS209 cSTD3 ( 475 nM ) . After 5 minutes , liposomes ( DOPC / DOGS - NTA - Ni2 + / DNS - PE / DHE liposomes 77 . 5 / 10 / 2 . 5 / 10 mol / mol , 62 . 5 µM total lipids , LB ) , covered or not with 1 µM of VAPHIS6 or VAP - A KD / MDHIS6 , were added . FRET between DHE and DNS - PE in the LB liposomes diminishes as DHE is transported towards LA liposomes . The signal was converted into amount of DHE present in LB liposomes ( in mol % ) .

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

D: DHE transport assay. DOPC (1.2-dioleoyl-sn-glycero-3-phosphocholine) liposomes (62.5 µM total lipids, LA) containing 3 mol% MPB-PE were mixed with cSTD3 or pS209 cSTD3 (475 nM). After 5 minutes, liposomes (DOPC/DOGS-NTA-Ni2+/DNS-PE/DHE liposomes 77.5/10/2.5/10 mol/mol, 62.5 µM total lipids, LB), covered or not with 1 µM of VAPHIS6 or VAP-A KD/MDHIS6, were added. FRET between DHE and DNS-PE in the LB liposomes diminishes as DHE is transported towards LA liposomes. The signal was converted into amount of DHE present in LB liposomes (in mol%).


E : Initial DHE transport rate measured with cSTD3 or pS209 cSTD3 in the presence or absence of VAP - A HIS6 or VAP - A KD / MDHIS6 . Data are represented as mean ± S . E . M . ( n = 3 for cSTD3 - VAP and n = 4 for all other data ) . Mann - Whitney test ( * P < 0 . 05 ) .

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

E: Initial DHE transport rate measured with cSTD3 or pS209 cSTD3 in the presence or absence of VAP-A HIS6 or VAP-A KD/MDHIS6. Data are represented as mean ± S.E.M. (n = 3 for cSTD3-VAP and n=4 for all other data). Mann-Whitney test (*P < 0.05).


A - E : HeLa / Ctrl ( A ) , HeLa / STARD3 ( B ) , HeLa / STARD3 S209A ( C ) , HeLa / STARD3 S209D / P210A ( D ) and HeLa / STARD3 Conv - FFAT ( E ) cells were labeled with anti - STARD3 antibodies ( magenta ) , anti - Lamp1 antibodies ( green ) and with the fluorescent cholesterol probe filipin ( Cyan Hot ) . Nuclei are shown in gray ( TO - PRO - 3 ) . Higher magnifications images ( 2x ) of the area outlined in white are shown on the right . The filipin , STARD3 and Lamp1 merged image is labeled Overlay . Scale bars : 10 µm . Inset scale bars : 5 µm .

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

A-E: HeLa/Ctrl (A), HeLa/STARD3 (B), HeLa/STARD3 S209A (C), HeLa/STARD3 S209D/P210A (D) and HeLa/STARD3 Conv-FFAT (E) cells were labeled with anti-STARD3 antibodies (magenta), anti-Lamp1 antibodies (green) and with the fluorescent cholesterol probe filipin (Cyan Hot). Nuclei are shown in gray (TO-PRO-3). Higher magnifications images (2x) of the area outlined in white are shown on the right. The filipin, STARD3 and Lamp1 merged image is labeled Overlay. Scale bars: 10 µm. Inset scale bars: 5 µm.


F : Pearson ' s correlation coefficients between STARD3 and Lamp1 staining in HeLa / STARD3 cells . Each dot represents a single cell ( 34 cells from 4 independent experiments ) . Mean and error bars ( SD ) are shown . Note that STARD3 and Lamp1 signals are highly correlated .

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F: Pearson's correlation coefficients between STARD3 and Lamp1 staining in HeLa/STARD3 cells. Each dot represents a single cell (34 cells from 4 independent experiments). Mean and error bars (SD) are shown. Note that STARD3 and Lamp1 signals are highly correlated.


G : Relative fluorescence intensity of intracellular filipin signals in endosomes of HeLa / Ctrl , HeLa / STARD3 , HeLa / STARD3 S209A , HeLa / STARD3 S209D / P210A and HeLa / STARD3 Conv - FFAT cells . Data are displayed as a Superplot ( Lord et al , 2020 ) showing the relative filipin fluorescence intensity in endosomes of individual cells ( small circles ) from 6 independent experiments ( mean of each experiment shown as a large circle ) . Independent experiments are color - coded . Means and error bars ( SD ) are shown as black bars . Kruskal - Wallis with Dunn ' s multiple comparison test ( * P < 0 . 05 ; * * P < 0 . 01 ; n = 6 independent experiments ) .

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G: Relative fluorescence intensity of intracellular filipin signals in endosomes of HeLa/Ctrl, HeLa/STARD3, HeLa/STARD3 S209A, HeLa/STARD3 S209D/P210A and HeLa/STARD3 Conv-FFAT cells. Data are displayed as a Superplot (Lord et al, 2020) showing the relative filipin fluorescence intensity in endosomes of individual cells (small circles) from 6 independent experiments (mean of each experiment shown as a large circle). Independent experiments are color-coded. Means and error bars (SD) are shown as black bars. Kruskal-Wallis with Dunn's multiple comparison test (*P < 0.05; **P < 0.01; n=6 independent experiments).


A : Coomassie blue staining of the recombinant MSP domain of MOSPD2 after SDS - PAGE .

A: Coomassie blue staining of the recombinant MSP domain of MOSPD2 after SDS-PAGE.


B : Recombinant MSP domains pulled down with STARD3 FFAT peptides ( unphosphorylated , mono - phosphorylated on S209 or S213 , bi - phosphorylated on S209 and S213 , or tri - phosphorylated on S203 , S209 and S213 . and control peptides were revealed by Coomassie blue staining . The recombinant proteins subjected to the assay are shown in the input fraction .

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B: Recombinant MSP domains pulled down with STARD3 FFAT peptides (unphosphorylated, mono-phosphorylated on S209 or S213, bi-phosphorylated on S209 and S213, or tri-phosphorylated on S203, S209 and S213.and control peptides were revealed by Coomassie blue staining. The recombinant proteins subjected to the assay are shown in the input fraction.


SPR analysis of the MSP domain of MOSPD2 binding onto immobilized unphosphorylated ( C ) STARD3 FFAT . Representative sensorgrams resulting from the interaction between the MSP domain of MOSPD2 injected at different concentrations and the different FFAT peptides are shown in ( C ) Binding curves display the SPR signal ( RU ) as a function of time . Concentrations printed in bold indicate samples measured twice . Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted , as well as the bulk effect recorded with buffer only .

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SPR analysis of the MSP domain of MOSPD2 binding onto immobilized unphosphorylated (C) STARD3 FFAT. Representative sensorgrams resulting from the interaction between the MSP domain of MOSPD2 injected at different concentrations and the different FFAT peptides are shown in (C) Binding curves display the SPR signal (RU) as a function of time. Concentrations printed in bold indicate samples measured twice. Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted, as well as the bulk effect recorded with buffer only.


SPR analysis of the MSP domain of MOSPD2 binding onto immobilized pS209 ( D ) STARD3 FFAT . Representative sensorgrams resulting from the interaction between the MSP domain of MOSPD2 injected at different concentrations and the different FFAT peptides are shown in ( D left ) . Binding curves display the SPR signal ( RU ) as a function of time . Concentrations printed in bold indicate samples measured twice . Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted , as well as the bulk effect recorded with buffer only . ( D right ) steady - state analysis of the interaction between pS209 STARD3 FFAT peptide and the MSP domain of MOSPD2 . Equilibrium responses ( Req ) extracted from the left panel were plotted as a function of the monomeric concentration of the MSP domain of MOSPD2 , and fitted with a 1 : 1 binding model . The experiments were performed at 25 ° C in Tris - HCl 50 mM , pH 7 . 5 , NaCl 75 mM buffer supplemented with 0 . 005 % ( v / v ) surfactant polysorbate 20 ( P20 , GE Healthcare ) . Mean of 2 independent experiments . Uncertainties are obtained from the standard deviation considering a t - distribution coefficient for a risk factor of 32 % .

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SPR analysis of the MSP domain of MOSPD2 binding onto immobilized pS209 (D) STARD3 FFAT. Representative sensorgrams resulting from the interaction between the MSP domain of MOSPD2 injected at different concentrations and the different FFAT peptides are shown in (D left). Binding curves display the SPR signal (RU) as a function of time. Concentrations printed in bold indicate samples measured twice. Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted, as well as the bulk effect recorded with buffer only. (D right) steady-state analysis of the interaction between pS209 STARD3 FFAT peptide and the MSP domain of MOSPD2. Equilibrium responses (Req) extracted from the left panel were plotted as a function of the monomeric concentration of the MSP domain of MOSPD2, and fitted with a 1:1 binding model. The experiments were performed at 25 °C in Tris-HCl 50 mM, pH 7.5, NaCl 75 mM buffer supplemented with 0.005% (v/v) surfactant polysorbate 20 (P20, GE Healthcare). Mean of 2 independent experiments. Uncertainties are obtained from the standard deviation considering a t-distribution coefficient for a risk factor of 32%.


E : Interaction dissociation constants between the different STARD3 FFAT peptides and the MSP domains of MOSPD2 .

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E: Interaction dissociation constants between the different STARD3 FFAT peptides and the MSP domains of MOSPD2.


F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - tagged MOSPD2 and Flag - tagged STARD3 ( WT and S209A , S209D and S209D / P210A mutants ) . Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag , anti - GFP and anti - Actin antibodies . Immunoprecipitated material was analyzed using anti - Flag and anti - GFP antibodies .

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F: Immunoprecipitation (GFP-Trap) experiments between GFP-tagged MOSPD2 and Flag-tagged STARD3 (WT and S209A, S209D and S209D/P210A mutants). Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag, anti-GFP and anti-Actin antibodies. Immunoprecipitated material was analyzed using anti-Flag and anti-GFP antibodies.


G - I : GFP - MOSPD2 - expressing cells ( green ) were left untransfected ( G ) or transfected with Flag - STARD3 ( H ) and Flag - STARD3 S209A ( I ) , and labelled using anti - Flag ( magenta ) antibodies . The subpanels on the right are higher magnification ( 3 . 5 × ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Scale bars : 10 µm .

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G-I: GFP-MOSPD2-expressing cells (green) were left untransfected (G) or transfected with Flag-STARD3 (H) and Flag-STARD3 S209A (I), and labelled using anti-Flag (magenta) antibodies. The subpanels on the right are higher magnification (3.5×) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Scale bars: 10 µm.


J : Pearson ' s correlation coefficients between MOSPD2 ( WT or RD / LD ) and STARD3 ( WT , S209A or FA / YA ) staining are shown . Each dot represents a single cell ( number of cells : MOSPD2 - STARD3 : 22 ; MOSPD2 - STARD3 S209A : 21 ; MOSPD2 - STARD3 FA / YA : 20 ; MOSPD2 RD / LD - STARD3 : 17 , from at least three independent experiments ) . Means and error bars ( SD ) are shown . Kruskal - Wallis with Dunn ' s multiple comparison test ( * * * P < 0 . 001 ) .

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J: Pearson's correlation coefficients between MOSPD2 (WT or RD/LD) and STARD3 (WT, S209A or FA/YA) staining are shown. Each dot represents a single cell (number of cells: MOSPD2-STARD3: 22; MOSPD2- STARD3 S209A: 21; MOSPD2-STARD3 FA/YA: 20; MOSPD2 RD/LD-STARD3: 17, from at least three independent experiments). Means and error bars (SD) are shown. Kruskal-Wallis with Dunn's multiple comparison test (***P < 0.001).


An S . typhimurium strain devoid of its SPI - 1 TTSS effector proteins is able to induce programmed cell death in macrophages . BMDPM from wild - type ( A ) or caspase - 1 − / − ( B ) mice ( Kuida et al . , 1995 ) were infected with wild - type S . typhimurium or mutant derivative either lacking all TTSS effector proteins ( effectorless ) or defective for TTSS secretion by virtue a having a mutation on an essential component of this system ( invG ) . 6 h after infection , cytotoxicity was evaluated by lactate dehydrogenase release or by terminal deoxynucleatide transferase ( TUNEL ) staining . Values for the lactate dehydrogenase release represent the average of at least three independent measurements . Equivalent results were obtained in several repetitions of these experiments .

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An S. typhimurium strain devoid of its SPI-1 TTSS effector proteins is able to induce programmed cell death in macrophages. BMDPM from wild-type (A) or caspase-1−/− (B) mice (Kuida et al., 1995) were infected with wild-type S. typhimurium or mutant derivative either lacking all TTSS effector proteins (effectorless) or defective for TTSS secretion by virtue a having a mutation on an essential component of this system (invG). 6 h after infection, cytotoxicity was evaluated by lactate dehydrogenase release or by terminal deoxynucleatide transferase (TUNEL) staining. Values for the lactate dehydrogenase release represent the average of at least three independent measurements. Equivalent results were obtained in several repetitions of these experiments.


Transient expression of SipB in BMDPM from caspase - 1 − / − mice results in cell death . Plasmids expressing either SipB , SipC , or SipD fused to YFP were introduced into BMDPM from caspase - 1 − / − mice as indicated in the Materials and methods section . Macrophages were then fixed , stained with DAPI ( to visualize chromatin ) , and observed under a fluorescence microscope . Cells expressing SipC or SipD do not show any signs of cytotoxicity . In contrast , cells expressing SipB show clear signs of cytotoxicity and chromatin condensation . Arrows indicate the nucleus of transfected cells .

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Transient expression of SipB in BMDPM from caspase-1−/−mice results in cell death. Plasmids expressing either SipB, SipC, or SipD fused to YFP were introduced into BMDPM from caspase-1−/− mice as indicated in the Materials and methods section. Macrophages were then fixed, stained with DAPI (to visualize chromatin), and observed under a fluorescence microscope. Cells expressing SipC or SipD do not show any signs of cytotoxicity. In contrast, cells expressing SipB show clear signs of cytotoxicity and chromatin condensation. Arrows indicate the nucleus of transfected cells.


Transient expression of SipB in COS - 2 cells . ( A ) A plasmid expressing YFP - tagged SipB was transfected into COS - 2 cells , and its localization was examined under a confocal microscope .

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Transient expression of SipB in COS-2 cells. (A) A plasmid expressing YFP-tagged SipB was transfected into COS-2 cells, and its localization was examined under a confocal microscope.


( B ) SipB colocalized with mitochondrial markers in transfected cells . COS - 2 cells were transfected with a plasmid encoding either SipB - CFP ( top ) or SipB - YFP ( bottom ) . Transfected cells were stained with MitoTracker ® Red ( Molecular Probes , Inc . ) to visualize mitochondria and were examined by confocal microscopy . The SipB - induced structures were readily stained with mitochondrial markers ( top ) . Mitochondria were also seen contained within the SipB - induced structures ( bottom ) .

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(B) SipB colocalized with mitochondrial markers in transfected cells. COS-2 cells were transfected with a plasmid encoding either SipB-CFP (top) or SipB-YFP (bottom). Transfected cells were stained with MitoTracker® Red (Molecular Probes, Inc.) to visualize mitochondria and were examined by confocal microscopy. The SipB-induced structures were readily stained with mitochondrial markers (top). Mitochondria were also seen contained within the SipB-induced structures (bottom).


( C ) SipB - induced structures were labeled with the ER marker Sec - 61 . COS - 2 cells were cotransfected with plasmids encoding SipB - YFP and Sec61 - CFP , and transfected cells were examined by confocal microscopy .

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(C) SipB-induced structures were labeled with the ER marker Sec-61. COS-2 cells were cotransfected with plasmids encoding SipB-YFP and Sec61-CFP, and transfected cells were examined by confocal microscopy.


Immuno - EM of SipB - transfected COS - 2 cells . COS - 2 cells were transfected with a plasmid expressing SipB - YFP , and 24 h after transfection , cells were fixed and prepared for immuno - EM using an antibody specific for GFP and a gold - labeled anti - rabbit antibody . SipB was seen localized to structures made up of closely apposed membranes that were often in close proximity to mitochondria ( A ) . Mitochondria were often seen apparently fusing to the SipB - induced membranous structure ( B , see arrows in inset on top ; C , inset to the right ) . Mitochondria were often seen contained with the SipB - induced membrane structures ( D ) . Bars , 250 nm .

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Immuno-EM of SipB-transfected COS-2 cells. COS-2 cells were transfected with a plasmid expressing SipB-YFP, and 24 h after transfection, cells were fixed and prepared for immuno-EM using an antibody specific for GFP and a gold-labeled anti-rabbit antibody. SipB was seen localized to structures made up of closely apposed membranes that were often in close proximity to mitochondria (A). Mitochondria were often seen apparently fusing to the SipB-induced membranous structure (B, see arrows in inset on top; C, inset to the right). Mitochondria were often seen contained with the SipB-induced membrane structures (D). Bars, 250 nm.


The nonfusogenic SipB428 - 593 mutant localizes to the mitochondria , but does not form multivesicular structures and interferes with the activity of the wild - type protein . ( A ) COS - 2 cells were transfected with a plasmid expressing YFP - tagged SipB428 - 593 , stained with an antibody to the mitochondrial protein CoxIV , subunit VIc ( Molecular Probes , Inc . ) , and examined by confocal microscopy . SipB428 - 593 - YFP localized to mitochondria and did not induce the formation of multivesicular structures seen after expression of the wild - type protein .

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The nonfusogenic SipB428-593 mutant localizes to the mitochondria, but does not form multivesicular structures and interferes with the activity of the wild-type protein. (A) COS-2 cells were transfected with a plasmid expressing YFP-tagged SipB428-593, stained with an antibody to the mitochondrial protein CoxIV, subunit VIc (Molecular Probes, Inc.), and examined by confocal microscopy. SipB428-593-YFP localized to mitochondria and did not induce the formation of multivesicular structures seen after expression of the wild-type protein.


( B ) COS - 2 cells were cotransfected with plasmids expressing either SipB428 - 593 - YFP or SipB - CFP , and transfected cells were examined under a confocal microscope . Expression of SipB428 - 593 - YFP prevented wild - type SipB - CFP from forming multivesicular structures ( notice that vesicular structures are only apparent in the absence , but not in the presence , of SipB428 - 593 - YFP ; right ) .

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(B) COS-2 cells were cotransfected with plasmids expressing either SipB428-593-YFP or SipB-CFP, and transfected cells were examined under a confocal microscope. Expression of SipB428-593-YFP prevented wild-type SipB-CFP from forming multivesicular structures (notice that vesicular structures are only apparent in the absence, but not in the presence, of SipB428-593-YFP; right).


The S . typhimurium SPI - 1 TTSS - secreted protein SipB localizes to the mitochondria of infected macrophages . ( A ) Bone marrow - derived macrophages were infected with either wild - type S . typhimurium or the TTSS translocation - defective sipD mutant strain with a multiplicity of infection of 50 . 3 h after infection , macrophages were collected and processed for immuno - EM using gold - labeled antibodies . SipB could be readily detected in macrophages infected with wild - type ( A and B ) , but not in macrophages infected with the sipD mutant strain ( C ) . Bars , 100 nm .

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The S. typhimurium SPI-1 TTSS-secreted protein SipB localizes to the mitochondria of infected macrophages. (A) Bone marrow-derived macrophages were infected with either wild-type S. typhimurium or the TTSS translocation-defective sipD mutant strain with a multiplicity of infection of 50. 3 h after infection, macrophages were collected and processed for immuno-EM using gold-labeled antibodies. SipB could be readily detected in macrophages infected with wild-type (A and B), but not in macrophages infected with the sipD mutant strain (C). Bars, 100 nm.


( B ) Alternatively , mitochondria were isolated from the infected macrophages and the presence of SipB or the resident mitochondrial protein CoxIV , subunit IV , and were examined by Western immunoblot analysis using specific antibodies .

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(B) Alternatively, mitochondria were isolated from the infected macrophages and the presence of SipB or the resident mitochondrial protein CoxIV, subunit IV, and were examined by Western immunoblot analysis using specific antibodies.


S . typhimurium - infected macrophages exhibited signs of autophagocytosis . Caspase - 1 − / − BMDPM were infected with wild - type S . typhimurium or it isogenic translocation - defective sipD mutant . 6 h after infection , macrophages were either stained with MDC to label autophagosomes ( I ) or processed for EM ( II ) as described in the legend to Fig . 3 . Macrophages infected with wild - type S . typhimurium ( but not those infected with the sipD mutant ) exhibited a large number of vesicular structures that stained with MDC ( I )

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

S. typhimurium-infected macrophages exhibited signs of autophagocytosis. Caspase-1−/− BMDPM were infected with wild-type S. typhimurium or it isogenic translocation-defective sipD mutant. 6 h after infection, macrophages were either stained with MDC to label autophagosomes (I) or processed for EM (II) as described in the legend to Fig. 3. Macrophages infected with wild-type S. typhimurium (but not those infected with the sipD mutant) exhibited a large number of vesicular structures that stained with MDC (I)


Under the electron microscope ( II ) , macrophages infected with wild - type Salmonella exhibited multivesicular structures resembling autophagosomes ( A - D ) Often , electron - dense material , presumably damaged organelles , were readily observed within the multivesicular structures ( B - D ) . In contrast , macrophages infected with type III secretion - defective sipD mutant did not exhibit the large number of multivesicular structures observed in wild - type - infected cells . Bars ( A and E ) 500 nm ; ( B , C , D and F ) 250 nm .

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

Under the electron microscope (II), macrophages infected with wild-type Salmonella exhibited multivesicular structures resembling autophagosomes (A-D) Often, electron-dense material, presumably damaged organelles, were readily observed within the multivesicular structures (B-D). In contrast, macrophages infected with type III secretion-defective sipD mutant did not exhibit the large number of multivesicular structures observed in wild-type-infected cells. Bars (A and E) 500 nm; (B, C, D and F) 250 nm.


The S . typhimurium - induced macrophage death does not require caspase activity . BMDPM were infected with wild - type S . typhimurium or the TTSS - defective invG mutant in the presence or absence of 50 μM of the pan - caspase inhibitor Z - VAD , and cell death was measured as indicated in the legend to Fig . 1 .

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

The S. typhimurium-induced macrophage death does not require caspase activity. BMDPM were infected with wild-type S. typhimurium or the TTSS-defective invG mutant in the presence or absence of 50 μM of the pan-caspase inhibitor Z-VAD, and cell death was measured as indicated in the legend to Fig. 1.


B Volcano plot showing proteins enrichment ( fold change in log10 scale ) after affinity purification in Jurkat T cells expressing ARHGAP45OST molecules compared to affinity purification in control Jurkat T cells expressing similar levels of WT ( untagged ) ARHGAP45 proteins prior to ( unstimulated ) and at 120 s after ( stimulated ) TCR stimulation . ARHGAP45 interacting proteins with a greater than 2 - fold enrichment and a p - value < 0 . 01 were selected as specific ARHGAP45 interactors ( Dataset EV1 ) and some of them are specified in red . Red lines represent the thresholds set on P - value and enrichment to identify specific ARHGAP45 interactors .

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

B Volcano plot showing proteins enrichment (fold change in log10 scale) after affinity purification in Jurkat T cells expressing ARHGAP45OST molecules compared to affinity purification in control Jurkat T cells expressing similar levels of WT (untagged) ARHGAP45 proteins prior to (unstimulated) and at 120 s after (stimulated) TCR stimulation. ARHGAP45 interacting proteins with a greater than 2-fold enrichment and a p-value < 0.01 were selected as specific ARHGAP45 interactors (Dataset EV1) and some of them are specified in red. Red lines represent the thresholds set on P-value and enrichment to identify specific ARHGAP45 interactors.


B The ratio of CTV / CMTPX - labeled cells present in each organ ( Ro ) was determined and normalized by dividing it with Ri , the ratio of CTV / CMTPX - labeled cells present in the cell mixtures prior to injection . Ro / Ri ratio corresponding to each of the specified mice are shown . Data shown for Arhgap45 - / - mice correspond to 4 independent experiments ( E1 to E4 ) involving a total of 15 individual mice whereas data shown for WT mice correspond to 2 independent experiments ( E1 and E2 ) involving a total of 5 individual mice . The Arhgap45 - / - mice analyzed on the same day as the control mice corresponded to the E1 and E2 experiments . Mean and SD are shown . ns , non - significant , * * * * P ≤ 0 . 0001 ; multiple unpaired T tests .

O O O O B-SMALL_MOLECULE 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 B-SMALL_MOLECULE 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 B-ORGANISM O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM O O B-GENEPROD O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O 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 The ratio of CTV/CMTPX-labeled cells present in each organ (Ro) was determined and normalized by dividing it with Ri, the ratio of CTV/CMTPX-labeled cells present in the cell mixtures prior to injection. Ro/Ri ratio corresponding to each of the specified mice are shown. Data shown for Arhgap45-/-mice correspond to 4 independent experiments (E1 to E4) involving a total of 15 individual mice whereas data shown for WT mice correspond to 2 independent experiments (E1 and E2) involving a total of 5 individual mice. The Arhgap45-/- mice analyzed on the same day as the control mice corresponded to the E1 and E2 experiments. Mean and SD are shown. ns, non-significant, **** P ≤ 0.0001; multiple unpaired T tests.


C Expression of CCR7 , and LFA - 1 on naïve CD4 + and CD8 + T cells found in LNs of WT and Arhgap45 - / - mice , analyzed by flow cytometry . Gray shaded curves , isotype control staining . D Quantification of data shown in ( C ) . The MFI corresponding to each analyzed mouse is shown together with mean and SD . Each dot represents an individual mouse and CD4 + T cells from at least 4 mice have been analyzed for each condition . Mice analyzed on the same day are represented by similar dots ( see key in bottom right corner ) . ns , non - significant , * * P ≤ 0 . 002 ; multiple unpaired T tests .

O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O B-GENEPROD O O O B-ORGANISM O O O B-EXP_ASSAY I-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 B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O 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 Expression of CCR7, and LFA-1 on naïve CD4+ and CD8+ T cells found in LNs of WT and Arhgap45-/- mice, analyzed by flow cytometry. Gray shaded curves, isotype control staining. D Quantification of data shown in (C). The MFI corresponding to each analyzed mouse is shown together with mean and SD. Each dot represents an individual mouse and CD4+ T cells from at least 4 mice have been analyzed for each condition. Mice analyzed on the same day are represented by similar dots (see key in bottom right corner). ns, non-significant, ** P ≤ 0.002; multiple unpaired T tests.


The cells specified below were loaded into the upper chamber of a Transwell migration device without ( no ) or with the specified chemokines in the lower chamber . The percentage of cells that migrated to the bottom chamber after 2 h ( T cells ) or 4 h ( B cells ) is shown . A Chemotaxis of naïve CD4 + T cells from WT and Arghap45 − / − mice in response to CCL19 , CCL21 and CXCL12 . B Chemotaxis of naïve CD8 + T cells from WT and Arghap45 − / − mice in response to CCL19 , CCL21 and CXCL12 . C Chemotaxis of naïve B cells from WT and Arghap45 − / − mice in response to CCL19

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

The cells specified below were loaded into the upper chamber of a Transwell migration device without (no) or with the specified chemokines in the lower chamber. The percentage of cells that migrated to the bottom chamber after 2 h (T cells) or 4 h (B cells) is shown. A Chemotaxis of naïve CD4+ T cells from WT and Arghap45−/− mice in response to CCL19, CCL21 and CXCL12. B Chemotaxis of naïve CD8+ T cells from WT and Arghap45−/− mice in response to CCL19, CCL21 and CXCL12. C Chemotaxis of naïve B cells from WT and Arghap45−/− mice in response to CCL19


D Adding CCL19 in both the upper and lower chambers ( CCL19 UL ) prevented migration of WT and Arghap45 − / − naïve T cells whereas the use of Transwell membrane inserts precoated with ICAM - 1 ( CCL19 + ICAM - 1 ) did not change the pattern of chemotaxis observed using ' bare ' membrane inserts ( CCL19 ) . E Chemotaxis of naive CD4 + T cells and B cells from WT , Arghap45 − / − , and Arhgap45 ∆ T / ∆ T mice in response to CCL19 . F Chemotaxis of activated CD4 + T cells from WT and Arghap45 − / − mice in response to CCL19 . Data information : In ( A ) to ( F ) each dot represents an individual mouse and as shown CD4 + T cells from 3 to 9 mice have been analyzed for each condition . Mice analyzed on the same day are represented by similar dots ( see key in bottom right corner ) . Mean and SD are shown . * P ≤ 0 . 033 , * * P ≤ 0 . 002 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ; A , B , C , D , F multiple unpaired T test ; E two - way ANOVA test .

O O B-GENEPROD 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 B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-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 B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-ORGANISM O O O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Adding CCL19 in both the upper and lower chambers (CCL19 UL) prevented migration of WT and Arghap45−/− naïve T cells whereas the use of Transwell membrane inserts precoated with ICAM-1 (CCL19 + ICAM-1) did not change the pattern of chemotaxis observed using 'bare' membrane inserts (CCL19). E Chemotaxis of naive CD4+ T cells and B cells from WT, Arghap45−/−, and Arhgap45∆T/∆T mice in response to CCL19. F Chemotaxis of activated CD4+ T cells from WT and Arghap45−/− mice in response to CCL19. Data information: In (A) to (F) each dot represents an individual mouse and as shown CD4+ T cells from 3 to 9 mice have been analyzed for each condition. Mice analyzed on the same day are represented by similar dots (see key in bottom right corner). Mean and SD are shown. *P ≤ 0.033, ** P ≤ 0.002, *** P ≤ 0.001, **** P ≤ 0.0001; A, B, C, D, F multiple unpaired T test; E two-way ANOVA test.


Analysis of migration patterns of WT and Arghap45 − / − activated T cells on 2D surface coated with ICAM - 1 . Each track represents the migratory path of individual WT and Arghap45 − / − activated T cells recorded over 466 s in a single field of view , Trajectories were plotted to a common starting point , and > 200 T cells were recorded per plot using time - lapse microscopy at 10 x magnification . Analysis of migration patterns of WT and Arghap45 − / − naive T cells on 2D surface coated with ICAM - 1 and CCL21 . Same conditions as in ( A ) .

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

Analysis of migration patterns of WT and Arghap45−/− activated T cells on 2D surface coated with ICAM-1. Each track represents the migratory path of individual WT and Arghap45−/− activated T cells recorded over 466 s in a single field of view, Trajectories were plotted to a common starting point, and > 200 T cells were recorded per plot using time-lapse microscopy at 10 x magnification. Analysis of migration patterns of WT and Arghap45−/− naive T cells on 2D surface coated with ICAM-1 and CCL21. Same conditions as in (A).


A , B Representative kymographs of WT ( A ) and Arghap45 − / − ( B ) naive CD4 + T cells on 2D surface coated with ICAM - 1 and CCL21 .

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

A, B Representative kymographs of WT (A) and Arghap45−/− (B) naive CD4+ T cells on 2D surface coated with ICAM-1 and CCL21.


A Irradiated H - 2 Kb - positive spleen cells isolated from T cell - deficient mice were pulsed for 2 hours with the N4 agonist OVA peptide and cultured with CD8 + T cells purified from WT OT - I ( WT ) or Arhgap45 − / − OT - I ( Arhgap45 − / − ) mice . Cell proliferation was measured by luminescence after 48 h . Data are representative of 2 independent experiments with 3 mice per genotype ( mean and SEM of triplicate are shown ) .

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

A Irradiated H-2 Kb- positive spleen cells isolated from T cell-deficient mice were pulsed for 2 hours with the N4 agonist OVA peptide and cultured with CD8+ T cells purified from WT OT-I (WT) or Arhgap45−/− OT-I (Arhgap45−/−) mice. Cell proliferation was measured by luminescence after 48 h. Data are representative of 2 independent experiments with 3 mice per genotype (mean and SEM of triplicate are shown).


( A ) NIH3T3 fibroblasts co - expressing DsRed - CA7 and EGFP - CA2 ( n = 4 independent replicates ) .

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

(A) NIH3T3 fibroblasts co-expressing DsRed-CA7 and EGFP-CA2 (n = 4 independent replicates).


( B - E ) Co - localization of EGFP and the two CA isoforms with filamentous actin studied in fibroblasts expressing ( B ) EGFP , ( C ) EGFP - CA2 , or ( D , E ) EGFP - CA7 and stained with phalloidin - 594 to visualize F - actin ( n = 4 , 10 and 8 independent replicates , respectively ) . A magnification of the area marked with the yellow rectangle in ( C ) and ( D ) shows the localization of EGFP - CA2 and EGFP - CA7 compared to phalloidin - 594 . ( E ) EGFP - CA7 caused a prominent overexpression phenotype with thick and curvy cytosolic actin bundles ( arrow ) and plasmalemmal protrusions ( arrowhead ) .

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

(B-E) Co-localization of EGFP and the two CA isoforms with filamentous actin studied in fibroblasts expressing (B) EGFP, (C) EGFP-CA2, or (D, E) EGFP-CA7 and stained with phalloidin-594 to visualize F-actin (n = 4, 10 and 8 independent replicates, respectively). A magnification of the area marked with the yellow rectangle in (C) and (D) shows the localization of EGFP-CA2 and EGFP-CA7 compared to phalloidin-594. (E) EGFP-CA7 caused a prominent overexpression phenotype with thick and curvy cytosolic actin bundles (arrow) and plasmalemmal protrusions (arrowhead).


( F - H ) The normalized fluorescence emission intensity profiles for F - actin ( red line ) and ( F ) EGFP , ( G ) EGFP - CA2 , or ( H ) EGFP - CA7 ( black line ) .

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

(F-H) The normalized fluorescence emission intensity profiles for F-actin (red line) and (F) EGFP, (G) EGFP-CA2, or (H) EGFP-CA7 (black line).


( A ) Actin co - sedimentation assay shows that CA7 binds to F - actin . The binding is enhanced at more acidic pH ( 6 . 5 vs . 7 . 4 ) n = 3 independent replicates at each actin concentration , two - way ANOVA , P < 0 . 001 .

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

(A) Actin co-sedimentation assay shows that CA7 binds to F-actin. The binding is enhanced at more acidic pH (6.5 vs. 7.4) n = 3 independent replicates at each actin concentration, two-way ANOVA, P < 0.001.


( B ) Fluorescence time ‐ lapse images of F - actin bundling in an in vitro bundling assay . A mixture of unlabeled and Rhodamine labelled non - muscle actin was polymerized in the absence ( PBS control , upper panel ) or presence of mCA7 ( lower panel ) . Numbers in images indicate the time after the onset of the experiment ( 0 , 5 and 23 min ) . Intensity based Fire - coloring ( Fiji ) was used to visualize intensity changes . Scale bar 10 µm . ( C ) Quantification of the mean increase in filament length ( n = 10 filaments at each time point ) and the mean relative fluorescence intensity values of cross - sections for individual filaments / bundles ( n = 30 - 31 ) in the absence and presence of mCA7 ( 1 . 12 µM ) . The data were analyzed using a general mixed model with time as a within - unit factor and the presence of CA7 as a between - unit factor . n = 3 independent repetitions , experiment repeats were included as a covariate and were non - significant .

O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O B-TISSUE 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 B-SUBCELLULAR O O O O O O O O O O O O O O O O O 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 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) Fluorescence time‐lapse images of F-actin bundling in an in vitro bundling assay. A mixture of unlabeled and Rhodamine labelled non-muscle actin was polymerized in the absence (PBS control, upper panel) or presence of mCA7 (lower panel). Numbers in images indicate the time after the onset of the experiment (0, 5 and 23 min). Intensity based Fire-coloring (Fiji) was used to visualize intensity changes. Scale bar 10 µm. (C) Quantification of the mean increase in filament length (n = 10 filaments at each time point) and the mean relative fluorescence intensity values of cross-sections for individual filaments /bundles (n = 30 - 31) in the absence and presence of mCA7 (1.12 µM). The data were analyzed using a general mixed model with time as a within-unit factor and the presence of CA7 as a between-unit factor. n = 3 independent repetitions, experiment repeats were included as a covariate and were non-significant.


( D ) Kymographs showing different time points in the line of interest ( line width 1 µm ) from the experiments analyzed in ( C ) . Kymographs were generated with Fiji Multi Kymograph function . Total time is 159 frames = 26 . 5 minutes . Scale bar 5 μm ( full height is 20 μm ) . F - actin bundles can be detected as clear lines in kymographs .

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

(D) Kymographs showing different time points in the line of interest (line width 1 µm) from the experiments analyzed in (C). Kymographs were generated with Fiji Multi Kymograph function. Total time is 159 frames = 26.5 minutes. Scale bar 5 μm (full height is 20 μm). F-actin bundles can be detected as clear lines in kymographs.


( E ) Fluorescence time ‐ lapse images of F - actin bundling in an in vitro bundling assay . A mixture of unlabeled and Rhodamine labelled non - muscle actin was polymerized in the presence of mCA7 ( 0 . 11 µM ) . Numbers above images indicate the time after the onset of the experiment ( 22 min - 23 min 20 sec ) . Intensity based Fire - coloring ( Fiji ) was used to visualize intensity changes . White arrows highlight the bundling filaments . Scale bar 5 μm . This is a representative video from 8 similar experiments .

O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O B-TISSUE 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 B-SUBCELLULAR O O O O O O O O O O O O O O O O

(E) Fluorescence time‐lapse images of F-actin bundling in an in vitro bundling assay. A mixture of unlabeled and Rhodamine labelled non-muscle actin was polymerized in the presence of mCA7 (0.11 µM). Numbers above images indicate the time after the onset of the experiment (22 min -23 min 20 sec). Intensity based Fire-coloring (Fiji) was used to visualize intensity changes. White arrows highlight the bundling filaments. Scale bar 5 μm. This is a representative video from 8 similar experiments.


( A , B ) NIH3T3 cells transfected with DsRed ( A ) or DsRed - CA7 ( B ) were incubated in growth medium with 5 µM Latrunculin B for 0 , 2 , 5 , 10 , or 30 minutes , or in an equal amount of DMSO for 60 minutes . Analyses of experiments show that in cells transfected with DsRed - CA7 F - actin structures collapse more slowly ( 0 min : 83 % " normal " ; 2 min : 80 % , p = 0 . 44 ; 5 min : 74 % , p = 0 . 39 ; 10 min : 27 % , p = 0 . 08 ; 30 min : 16 % , p = 0 . 02 ; DMSO : 82 % , p = 0 . 99 ; tested against 0 min with two - way ANOVA , Dunnett ' s multiple comparison test ) than in the DsRed - transfected ones ( 0 min : 89 % " normal " ; 2 min : 48 % , p = 0 . 14 ; 5 min : 31 % , p = 0 . 04 ; 10 min : 2 % , p = 0 . 001 ; 30 min : 0 . 7 % , p = 0 . 001 ; DMSO : 88 % , p = 0 . 8 ; tested against 0 min with two - way ANOVA , Dunnett ' s multiple comparison test ) . For the analysis , cells were categorized to three groups as " normal " , " some shape / F - actin left " and " round " . The upper panel shows example images of the cells in all three categories for ( A ) DsRed - or ( B ) DsRed - CA7 transfected cells ( actin visualized with Phalloidin - 488 ) .

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(A,B) NIH3T3 cells transfected with DsRed (A) or DsRed-CA7 (B) were incubated in growth medium with 5 µM Latrunculin B for 0, 2, 5, 10, or 30 minutes, or in an equal amount of DMSO for 60 minutes. Analyses of experiments show that in cells transfected with DsRed-CA7 F-actin structures collapse more slowly (0 min: 83% "normal"; 2 min: 80%, p = 0.44; 5 min: 74%, p = 0.39; 10 min: 27%, p = 0.08; 30 min: 16%, p = 0.02; DMSO: 82%, p = 0.99; tested against 0 min with two-way ANOVA, Dunnett's multiple comparison test) than in the DsRed-transfected ones (0 min: 89% "normal"; 2 min: 48%, p = 0.14; 5 min: 31%, p = 0.04; 10 min: 2%, p = 0.001; 30 min: 0.7%, p = 0.001; DMSO: 88%, p = 0.8; tested against 0 min with two-way ANOVA, Dunnett's multiple comparison test) . For the analysis, cells were categorized to three groups as "normal", "some shape/F-actin left" and "round". The upper panel shows example images of the cells in all three categories for (A) DsRed- or (B) DsRed-CA7 transfected cells (actin visualized with Phalloidin-488).


( A - C ) NIH3T3 fibroblasts expressing EGFP - CA7 - mutant1 ( A ) , EGFP - CA7 - mutant2 ( B ) , and EGFP - CA7 - mutant3 ( C ) . F - actin is visualized with Phalloidin - 594 . In the right - most panel of ( A - C ) are the normalized fluorescence intensity profiles of the mutated CA7 EGFP signal ( black ) and actin ( red ) and the yellow line in left - most panels indicates the cross - section from which the pixel intensities were measured . Scale bars 20 µm .

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(A-C) NIH3T3 fibroblasts expressing EGFP-CA7-mutant1 (A), EGFP-CA7-mutant2 (B), and EGFP-CA7-mutant3 (C). F-actin is visualized with Phalloidin-594. In the right-most panel of (A-C) are the normalized fluorescence intensity profiles of the mutated CA7 EGFP signal (black) and actin (red) and the yellow line in left-most panels indicates the cross-section from which the pixel intensities were measured. Scale bars 20 µm.


( D ) Analysis of the mutated CA7 and F - actin co - localization in cultured fibroblasts . Scatterplots of fluorescent intensities per pixel ( EGFP vs . Phalloidin - 594 ) along a cross section through a representative cell . Pearson ' s correlation coefficient ( r ) for the analyzed cell is given in each panel .

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(D) Analysis of the mutated CA7 and F-actin co-localization in cultured fibroblasts. Scatterplots of fluorescent intensities per pixel (EGFP vs. Phalloidin-594) along a cross section through a representative cell. Pearson's correlation coefficient (r) for the analyzed cell is given in each panel.


( E ) Pearson ' s correlation coefficient values calculated for the depicted constructs and compared to CA7 . F - actin had a strong positive correlation coefficient with EGFP - CA7 ( r = 0 . 91 ± 0 . 02 , n = 26 cells ) . Neither EGFP alone ( r = 0 . 01 ± 0 . 03 , n = 56 ) nor EGFP - CA2 ( r = 0 . 09 ± 0 . 03 , n = 53 ) co - localized with F - actin ( P < 0 . 0001 for both constructs , when compared to CA7 ) . From the five mutated CA7 constructs , EGFP - CA7 - mutant1 ( r = 0 . 51 ± 0 . 04 , P < 0 . 0001 , n = 25 ) and EGFP - CA7 - mutant3 ( r = 0 . 72 ± 0 . 03 , P = 0 . 05 , n = 21 ) co - localized less with F - actin actin when compared to CA7 . The co - localization of the other four mutated CA7 constructs , EGFP - CA7 - mutant2 ( r = 0 . 92 ± 0 . 02 , P = 0 . 99 , n = 18 ) , EGFP - CA7 - R223E ( r = 0 . 86 ± 0 . 02 , P = 0 . 99 , n = 24 ) and EGFP - CA7 - H96 / 98C ( r = 0 . 79 ± 0 . 04 , P = 0 . 14 , n = 18 ) did not differ significantly from that of CA7 . Data are shown as mean ± SEM . Data did not pass Shapiro - Wilk test for normality , statistical comparison against CA7 was done with Kruskall - Wallis test corrected for multiple comparisons .

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(E) Pearson's correlation coefficient values calculated for the depicted constructs and compared to CA7. F-actin had a strong positive correlation coefficient with EGFP-CA7 (r = 0.91 ± 0.02, n = 26 cells). Neither EGFP alone (r = 0.01 ± 0.03, n = 56) nor EGFP-CA2 (r = 0.09 ± 0.03, n = 53) co-localized with F-actin (P < 0.0001 for both constructs, when compared to CA7). From the five mutated CA7 constructs, EGFP-CA7-mutant1 (r= 0.51 ± 0.04, P < 0.0001, n = 25) and EGFP-CA7-mutant3 (r = 0.72 ± 0.03, P = 0.05, n = 21) co-localized less with F-actin actin when compared to CA7. The co-localization of the other four mutated CA7 constructs, EGFP-CA7-mutant2 (r = 0.92 ± 0.02, P = 0.99, n = 18), EGFP-CA7-R223E (r = 0.86 ± 0.02, P = 0.99, n = 24) and EGFP-CA7-H96/98C (r = 0.79 ± 0.04, P = 0.14, n = 18) did not differ significantly from that of CA7. Data are shown as mean ± SEM. Data did not pass Shapiro-Wilk test for normality, statistical comparison against CA7 was done with Kruskall-Wallis test corrected for multiple comparisons.


( A ) Isoform - specific subcellular localization shown in cultured hippocampal neurons ( DIV14 ) co - expressing DsRed - CA7 ( left ) and EGFP - CA2 ( middle ) .

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(A) Isoform-specific subcellular localization shown in cultured hippocampal neurons (DIV14) co-expressing DsRed-CA7 (left) and EGFP-CA2 (middle).


( B , C ) Representative confocal images of precocious in vivo expression of ( B ) EGFP - CA2 and ( C ) EGFP - CA7 in P40 mouse cortical layer 2 / 3 pyramidal neurons . Neurons were transfected at E14 . 5 with EGFP - CA2 or EGFP - CA7 using in utero electroporation and images were taken from fixed slices . Right panels in ( B ) and ( C ) show higher magnification of the primary apical dendrite marked with a box . ( D ) The expression of EGFP - CA7 disrupted the normal spine morphology and induced the formation of thick , filopodia - like protrusions .

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(B,C) Representative confocal images of precocious in vivo expression of (B) EGFP-CA2 and (C) EGFP-CA7 in P40 mouse cortical layer 2/3 pyramidal neurons. Neurons were transfected at E14.5 with EGFP-CA2 or EGFP-CA7 using in utero electroporation and images were taken from fixed slices. Right panels in (B) and (C) show higher magnification of the primary apical dendrite marked with a box. (D) The expression of EGFP-CA7 disrupted the normal spine morphology and induced the formation of thick, filopodia-like protrusions.


( A ) Comparison of mEPSCs in cortical layer 2 / 3 pyramidal neurons from P30 - P40 WT and CA7 KO mice . Sample traces of mEPSC recordings from WT and CA7 KO neurons , low - pas filtered at 1 kHz ( left ) . The data are summarized in the bar diagrams ( right ) . mEPSC frequency ( P = 0 . 63 ) and amplitude ( P = 0 . 90 ) were not significantly different between WT and CA7 KO and neurons ( n = 7 and 5 neurons , respectively , Student ' s independent samples t - test ) .

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(A) Comparison of mEPSCs in cortical layer 2/3 pyramidal neurons from P30 - P40 WT and CA7 KO mice. Sample traces of mEPSC recordings from WT and CA7 KO neurons, low-pas filtered at 1 kHz (left). The data are summarized in the bar diagrams (right). mEPSC frequency (P = 0.63) and amplitude (P = 0.90) were not significantly different between WT and CA7 KO and neurons (n = 7 and 5 neurons, respectively, Student's independent samples t-test).


( B ) Representative confocal images of apical dendrites from Lucifer Yellow injected cortical layer 2 / 3 pyramidal neurons from WT and CA7 KO mice . The dendritic spine density and spine head size were examined in fixed slice preparations from P34 - P37 mice . Scale bar 2 µm .

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(B) Representative confocal images of apical dendrites from Lucifer Yellow injected cortical layer 2/3 pyramidal neurons from WT and CA7 KO mice. The dendritic spine density and spine head size were examined in fixed slice preparations from P34 - P37 mice. Scale bar 2 µm.


( C ) Summary of the spine density analysis done from the Lucifer Yellow injected neurons . Spine density was a higher in CA7 KO neurons both in apical and basal dendrites ( n = 28 neurons for both ) compared to WT ( n = 29 neurons for apical and n = 30 for basal dendrite analysis ) ( P = 0 . 000002 for apical dendrites , analyzed with Mann - Whitney test , and P = 6 , 8 x 10 - 8 for basal dendrites , Student ' s t - test with Welch - correction . ) A total of 8279 spines were analyzed from four CA7 KO mice and 8730 spines from two WT control mice .

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(C) Summary of the spine density analysis done from the Lucifer Yellow injected neurons. Spine density was a higher in CA7 KO neurons both in apical and basal dendrites (n = 28 neurons for both) compared to WT (n= 29 neurons for apical and n=30 for basal dendrite analysis) (P = 0.000002 for apical dendrites, analyzed with Mann-Whitney test, and P = 6,8 x 10-8 for basal dendrites, Student's t-test with Welch-correction.) A total of 8279 spines were analyzed from four CA7 KO mice and 8730 spines from two WT control mice.


( D ) The spine head width distribution7differed significantly between the genotypes ( n = 467 spines from WT and n = 421 spines from CA7 KO animals , 15 neurons analyzed from both genotypes , Wilcoxon rank sum test with continuity correction , W = 134540 , P < 0 . 001 ) .

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(D) The spine head width distribution7differed significantly between the genotypes (n= 467 spines from WT and n = 421 spines from CA7 KO animals, 15 neurons analyzed from both genotypes, Wilcoxon rank sum test with continuity correction, W = 134540, P < 0.001).


( F - G ) Thermal shift assay traces of human Teneurin4WT ( F ) and human Teneurin4Mut ( G ) in the presence of physiological calcium concentrations ( + Ca2 + ; black ) , low calcium concentration ( + / - Ca2 + ; blue and red , respectively ) and no calcium ( - Ca2 + ; grey ) .

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(F-G) Thermal shift assay traces of human Teneurin4WT (F) and human Teneurin4Mut (G) in the presence of physiological calcium concentrations (+ Ca2+; black), low calcium concentration (+/- Ca2+; blue and red, respectively) and no calcium (- Ca2+; grey).


( A ) Dimensionless Kratky plots for Teneurin4WT ( left ) and Teneurin4Mut ( right ) in physiological calcium concentrations ( + Ca2 + ; black ) , low calcium concentrations ( + / - Ca2 + ; blue and red , respectively ) and no calcium ( - Ca2 + ; grey ) . I ( 0 ) and Rg were obtained from Guinier analysis ( Fig EV5A , B ) . Crosshairs indicate the maximum position for a fully folded globular protein .

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(A) Dimensionless Kratky plots for Teneurin4WT (left) and Teneurin4Mut (right) in physiological calcium concentrations (+ Ca2+; black), low calcium concentrations (+/- Ca2+; blue and red, respectively) and no calcium (- Ca2+; grey). I(0) and Rg were obtained from Guinier analysis (Fig EV5A, B). Crosshairs indicate the maximum position for a fully folded globular protein.


( B ) Pair - distance distribution plots for Teneurin4 proteins colored as in A .

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(B) Pair-distance distribution plots for Teneurin4 proteins colored as in A.


( A ) Representative images of HEK293 - T cells transfected with GFP ( top ) , GFP - Teneurin4WT ( GFP - Ten4WT ; middle ) or GFP - Teneurin4Mut ( GFP - Ten4Mut ; bottom ) for 24h and stained with Alexa 568 Phalloidin ( magenta ) and Dapi ( blue ) . Images are maximum intensity projections of 14 - 16 stacks . Scale bar , 10 µm . ( B ) Zoom shows the regions indicated in A . White arrows indicate the tip of the filopodium from the transfected cell . Scale bar , 2 µm . (

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(A) Representative images of HEK293-T cells transfected with GFP (top), GFP-Teneurin4WT (GFP-Ten4WT; middle) or GFP- Teneurin4Mut (GFP-Ten4Mut; bottom) for 24h and stained with Alexa 568 Phalloidin (magenta) and Dapi (blue). Images are maximum intensity projections of 14-16 stacks. Scale bar, 10 µm. (B) Zoom shows the regions indicated in A. White arrows indicate the tip of the filopodium from the transfected cell. Scale bar, 2 µm. (


( C ) Average number of filopodia per cell in cells transfected with GFP , GFP - Teneurin4WT ( Ten4 WT ; red ) and GFP - Teneurin4Mut ( Ten4Mut ; blue ) . ( D ) Fraction of filopodia enriched with GFP per cell in GFP - , GFP - Teneurin4WT ( Ten4WT ; blue ) - and GFP - Teneurin4Mut ( Ten4Mut ; red ) - transfected cells . (

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(C) Average number of filopodia per cell in cells transfected with GFP, GFP-Teneurin4WT (Ten4 WT; red) and GFP- Teneurin4Mut (Ten4Mut; blue). (D) Fraction of filopodia enriched with GFP per cell in GFP-, GFP-Teneurin4WT (Ten4WT; blue)- and GFP-Teneurin4Mut (Ten4Mut; red)-transfected cells. (


( A ) Representative images of non - adherent K562 cells electroporated with GFP ( left ) , GFP - Teneurin4WT ( GFP - Ten4WT ; middle ) or GFP - Teneurin4Mut ( GFP - Ten4Mut ; right ) . Zoomed images show the formation of cellular clusters in cells expressing GFP - Ten4WT and GFP - Ten4Mut , but not in GFP - expressing cells . Scale bar top panel , 50 µm . Scale bar bottom panel , 20 µm .

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(A) Representative images of non-adherent K562 cells electroporated with GFP (left), GFP-Teneurin4WT (GFP-Ten4WT; middle) or GFP-Teneurin4Mut (GFP-Ten4Mut; right). Zoomed images show the formation of cellular clusters in cells expressing GFP-Ten4WT and GFP-Ten4Mut, but not in GFP-expressing cells. Scale bar top panel, 50 µm. Scale bar bottom panel, 20 µm.


( C ) Fraction of cell clusters detected per image in GFP - ( black ) , GFP - Teneurin4WT ( blue ) - and GFP - Teneurin4Mut ( red ) - electroporated cells .

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(C) Fraction of cell clusters detected per image in GFP- (black), GFP-Teneurin4WT (blue)- and GFP-Teneurin4Mut (red)-electroporated cells.


WB analysis of smc5 - AID clones in the absence or presence of Auxin using FLAG . Tubulin is used as loading control and wt cells is used as a negative control for the FLAG tag .

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WB analysis of smc5-AID clones in the absence or presence of Auxin using FLAG. Tubulin is used as loading control and wt cells is used as a negative control for the FLAG tag.


Proliferation curve of wt and smc5 - AID cells in the presence or absence of Auxin . The data represents means ± SD of 3 independent experiments .

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Proliferation curve of wt and smc5-AID cells in the presence or absence of Auxin. The data represents means ± SD of 3 independent experiments.


Proliferation curves of wt and 2 independent smc5 clones grown at 39 . 5ºC , with the estimated doubling time on the right . Data represents means ± SD of 4 experiments . Asterisks indicate p value ≤ 0 . 05 , as derived from unpaired t - test type 3 , two - sample unequal variance , carried out to check statistical significance between smc5 clones and wt . smc5 clone 1 versus wt , p = 0 . 016 , smc5 clone 2 versus wt , p = 0 . 04 .

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Proliferation curves of wt and 2 independent smc5 clones grown at 39.5ºC, with the estimated doubling time on the right. Data represents means ± SD of 4 experiments. Asterisks indicate p value ≤ 0.05, as derived from unpaired t-test type 3, two-sample unequal variance, carried out to check statistical significance between smc5 clones and wt. smc5 clone 1 versus wt, p=0.016, smc5 clone 2 versus wt, p=0.04.


Bidimensional FACS analysis shows significant accumulation in G2 / M and decrease in the S phase in smc5 mutants . The data represents means ± SD of 3 independent experiments for smc5 clones 2 and 3 , and 4 independent experiments for wt and smc5 clone 1 . Asterisks indicate p value ≤ 0 . 05 , detailed below , using paired t - test . S - phase ( smc5 clones 1 , 2 , 3 versus wt : p = 0 . 0031 ; p = 0 . 0002 ; p = 0 . 004 , respectively ) . G2 / M ( smc5 clones 1 , 2 , 3 versus wt : p = 0 . 029 ; p = 0 . 0014 ; p = 0 . 02 , respectively ) .

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Bidimensional FACS analysis shows significant accumulation in G2/M and decrease in the S phase in smc5 mutants. The data represents means ± SD of 3 independent experiments for smc5 clones 2 and 3, and 4 independent experiments for wt and smc5 clone 1. Asterisks indicate p value ≤ 0.05, detailed below, using paired t-test. S-phase (smc5 clones 1, 2, 3 versus wt: p= 0.0031; p=0.0002; p=0.004, respectively). G2/M (smc5 clones 1, 2, 3 versus wt: p= 0.029; p=0.0014; p=0.02, respectively).


Survival curve of smc5 cells treated with different concentrations of cisplatin ( CDDP ) . Data represents means ± SD of 3 experiments .

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Survival curve of smc5 cells treated with different concentrations of cisplatin (CDDP). Data represents means ± SD of 3 experiments.


Survival curve of smc5 cells complemented or not with chicken SMC5 cDNA and treated with different concentrations of cisplatin . The experiment is carried out at 39 . 5ºC . Data represents means ± SD of at least 3 experiments .

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Survival curve of smc5 cells complemented or not with chicken SMC5 cDNA and treated with different concentrations of cisplatin. The experiment is carried out at 39.5ºC. Data represents means ± SD of at least 3 experiments.


Survival curve of smc5 ku70 cells treated with different concentrations of cisplatin . Data represents means ± SD of the 3 experiments .

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Survival curve of smc5 ku70 cells treated with different concentrations of cisplatin. Data represents means ± SD of the 3 experiments.


Survival curve of smc5 fancc cells treated with different concentrations of cisplatin . Data represents means ± SD of 3 experiments .

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Survival curve of smc5 fancc cells treated with different concentrations of cisplatin. Data represents means ± SD of 3 experiments.


WB analysis of CHK1 - P levels at steady state or after treatment with 1 μM cisplatin for 3 hours . CHK1 total protein is used as loading control . Quantitative analysis of CHK1 - P levels at steady state versus total CHK1 is shown in the bottom panel . Data represents means ± SD of 4 experiments .

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WB analysis of CHK1-P levels at steady state or after treatment with 1 μM cisplatin for 3 hours. CHK1 total protein is used as loading control. Quantitative analysis of CHK1-P levels at steady state versus total CHK1 is shown in the bottom panel. Data represents means ± SD of 4 experiments.


WB analysis of FANCD2 ubiquitination levels at steady state after treatment with 1 μM cisplatin for 3 hours . Analysis of the ratio between FANCD2 ubiquitinated / non - ubiquitinated forms is shown on the bottom panel . Data represents means ± SD of 3 experiments .

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WB analysis of FANCD2 ubiquitination levels at steady state after treatment with 1 μM cisplatin for 3 hours. Analysis of the ratio between FANCD2 ubiquitinated/non-ubiquitinated forms is shown on the bottom panel. Data represents means ± SD of 3 experiments.