Figure 3C ) Analysis of discordance and concordance of transcriptomic responses ( DISCO ) between untreated vs . SARS - CoV - 2 infected and untreated vs IFN - γ treated organoids . The 50 most regulated genes are highlighted ; grey : discordant and black : concordant . D ) Relative expression of selected enterocyte - specific genes upon SARS - CoV - 2 infection , normalized to GAPDH . Differences in gene expression were assessed using the linear model ' lmFit ' and ' makeContrasts ' in limma . E ) Immunofluorescence for KRT20 ( green ) in human colon organoids cultured in basic medium and either non - infected ( left ) or 48 h after SARS - CoV - 2 infection ( right ) , indicating a strong increase in KRT20 expression after viral infection . Scale bar : 50 µm . Quantification of fluorescence intensity below ( n = 8 ) . Data are presented as mean + / - SD , * * * * : p ≤ 0 . 0001 , as determined by Student ' s t - testF ) qPCR data displaying the increase of INF - γ target genes ( CXCL11 , IFIT3 ) ( upper ) and enterocyte marker genes ( KRT20 , ACE2 ) ( lower ) after SARS - CoV - 2 infection , validating the results from the microarray analysis ( n = 3 ) . Data are presented as mean + / - SD , * * * * : p ≤ 0 . 0001 , as determined by Student ' s t -

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3C) Analysis of discordance and concordance of transcriptomic responses (DISCO) between untreated vs. SARS-CoV-2 infected and untreated vs IFN-γ treated organoids. The 50 most regulated genes are highlighted; grey: discordant and black: concordant.D) Relative expression of selected enterocyte-specific genes upon SARS-CoV-2 infection, normalized to GAPDH. Differences in gene expression were assessed using the linear model 'lmFit' and 'makeContrasts' in limma.E) Immunofluorescence for KRT20 (green) in human colon organoids cultured in basic medium and either non-infected (left) or 48 h after SARS-CoV-2 infection (right), indicating a strong increase in KRT20 expression after viral infection. Scale bar: 50 µm. Quantification of fluorescence intensity below (n=8). Data are presented as mean +/- SD ,****: p≤0.0001, as determined by Student's t-testF) qPCR data displaying the increase of INF-γ target genes (CXCL11, IFIT3) (upper) and enterocyte marker genes (KRT20, ACE2) (lower) after SARS-CoV-2 infection, validating the results from the microarray analysis (n=3). Data are presented as mean +/- SD ,****: p≤0.0001, as determined by Student's t-test


Figure 1 ( A ) Wild - type primary B cells were treated with 5 μg / mL of anti - kappa antibody ( a - k chain , top ) or 1 µM LatrunculinA ( LatA , bottom ) . Intracellular calcium flux was measured by flow cytometry in the presence of Src - inhibitor ( PP2 ) , Syk - inhibitor ( BAY 613606 ) or vehicle control . ( B ) Wild - type primary B cells were treated with vehicle control ( - ) , 5 μg / mL anti - kappa antibody or 1 µM LatA for the indicated time . Cells were lysed and analysed by SDS - PAGE followed by immunoblotting with phospho - Lyn ( Y507 ) and actin as loading control . The intensity of phosphorylated proteins , normalized to actin , was referred to the respective unstimulated sample , set as 1 . ( C and D ) Wild - type primary B cells were treated with vehicle control ( - ) , 5 µg / mL anti - kappa antibody or 1 µM LatA for the indicated time . Cells were lysed and analysed by SDS - PAGE followed by immunoblotting with phospho - Syk ( pSyk ) , phospho - CD19 ( pCD19 ) , phospho - Akt ( pAkt ) , phospho - ERK ( pERK ) and total ERK as loading control . The intensity of phosphorylated proteins , normalized to ERK , was referred to the respective unstimulated sample , set as 1 . ( E and F ) Wild - type primary B cells were treated with vehicle control ( - ) , 5 µg / mL anti - kappa antibody or 1 µM LatA for the indicated time . Cells were lysed and analysed by SDS - PAGE followed by immunoblotting with phospho - PLCγ2 ( pPLCγ2 ) and total ERK as loading control . The intensity of phosphorylated proteins , normalized to ERK , was referred to the respective unstimulated sample , set as 1 . ( G and H ) Wild - type primary B cells were treated with vehicle control ( - ) , 5 µg / mL anti - kappa antibody or 1 µM LatA for the indicated time . Cells were lysed and analysed by SDS - PAGE followed by immunoblotting with phospho - CD22 ( pCD22 ) , phospho - SHP1 ( pSHP1 ) , and CD22 or total ERK as loading controls . The intensity of phosphorylated proteins , normalized to CD22 or ERK , was referred to the respective unstimulated sample , set as 1 . ( I ) Wild - type and Cd22 - / - primary B cells were treated with 5 µg / mL anti - kappa antibody or 1 µM LatA and intracellular calcium flux was measured by flow cytometry . ( J ) Wild - type and Cd22Y2 , 5 , 6F primary B cells were treated with 5 µg / mL anti - kappa antibody or 1 µM LatA and intracellular calcium flux was measured by flow cytometry . ( K and L ) Wild - type , Cd22 - / - and Cd22Y2 , 5 , 6F primary B cells were treated with vehicle control ( - ) or 1 µM LatA for the indicated time . Cells were lysed and analysed by SDS - PAGE followed by immunoblotting with phospho - CD19 ( pCD19 ) , phospho - Akt ( pAkt ) , phospho - ERK ( pERK ) and actin as loading control . The intensity of phosphorylated proteins , normalized to actin , was referred to the unstimulated sample of the wild type cells , set as 1 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Wild-type primary B cells were treated with 5 μg/mL of anti-kappa antibody (a-k chain, top) or 1 µM LatrunculinA (LatA, bottom). Intracellular calcium flux was measured by flow cytometry in the presence of Src-inhibitor (PP2), Syk-inhibitor (BAY 613606) or vehicle control.(B) Wild-type primary B cells were treated with vehicle control (-), 5 μg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-Lyn (Y507) and actin as loading control. The intensity of phosphorylated proteins, normalized to actin, was referred to the respective unstimulated sample, set as 1.(C and D) Wild-type primary B cells were treated with vehicle control (-), 5 µg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-Syk (pSyk), phospho-CD19 (pCD19), phospho-Akt (pAkt), phospho-ERK (pERK) and total ERK as loading control. The intensity of phosphorylated proteins, normalized to ERK, was referred to the respective unstimulated sample, set as 1.(E and F) Wild-type primary B cells were treated with vehicle control (-), 5 µg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-PLCγ2 (pPLCγ2) and total ERK as loading control. The intensity of phosphorylated proteins, normalized to ERK, was referred to the respective unstimulated sample, set as 1.(G and H) Wild-type primary B cells were treated with vehicle control (-), 5 µg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-CD22 (pCD22), phospho-SHP1 (pSHP1), and CD22 or total ERK as loading controls. The intensity of phosphorylated proteins, normalized to CD22 or ERK, was referred to the respective unstimulated sample, set as 1.(I) Wild-type and Cd22-/- primary B cells were treated with 5 µg/mL anti-kappa antibody or 1 µM LatA and intracellular calcium flux was measured by flow cytometry.(J) Wild-type and Cd22Y2,5,6F primary B cells were treated with 5 µg/mL anti-kappa antibody or 1 µM LatA and intracellular calcium flux was measured by flow cytometry.(K and L) Wild-type, Cd22-/- and Cd22Y2,5,6F primary B cells were treated with vehicle control (-) or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-CD19 (pCD19), phospho-Akt (pAkt), phospho-ERK (pERK) and actin as loading control. The intensity of phosphorylated proteins, normalized to actin, was referred to the unstimulated sample of the wild type cells, set as 1.


Figure 2 ( A ) Determining the number and density of CD22 , CD19 , IgM and IgD on the surface of wild - type primary B cells . Cells were stained with PE - conjugated antibody against CD22 ( blue profile ) , CD19 ( red profile ) , IgM ( red profile ) or IgD ( red profile ) and their fluorescence profiles compared to those of standard beads ( grey profiles ) , which each possess a well - defined number of binding sites ( st0 = blank , st1 - st4 = increasing number of binding sites ) against the primary antibody . For IgM , where the range of expression is large , the low and high 25 percentiles were calculated as in Mattila et al . 2013 and shown in brackets . Data are representative of three independent experiments . ( B - E ) Dual - colour SIM analysis of CD22 , IgM , IgD and CD19 . Wild - type primary B cells were fixed , stained with Atto633 or 488 - conjugated antibody against CD22 , IgM or IgD and Alexa647 - conjugated antibody against CD19 and settled onto nonstimulatory coverslips . Cells were then embedded in agarose , imaged with SIM and analysed . ( B ) SIM images of CD22 , IgM , IgD and CD19 . ( C ) Cross - correlation function . ( D ) Pearson correlation coefficient . ( E ) Cluster size calculated from autocorrelation analysis .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Determining the number and density of CD22, CD19, IgM and IgD on the surface of wild-type primary B cells. Cells were stained with PE-conjugated antibody against CD22 (blue profile), CD19 (red profile), IgM (red profile) or IgD (red profile) and their fluorescence profiles compared to those of standard beads (grey profiles), which each possess a well-defined number of binding sites (st0=blank, st1-st4=increasing number of binding sites) against the primary antibody. For IgM, where the range of expression is large, the low and high 25 percentiles were calculated as in Mattila et al. 2013 and shown in brackets.Data are representative of three independent experiments.(B-E) Dual-colour SIM analysis of CD22, IgM, IgD and CD19. Wild-type primary B cells were fixed, stained with Atto633 or 488-conjugated antibody against CD22, IgM or IgD and Alexa647-conjugated antibody against CD19 and settled onto nonstimulatory coverslips. Cells were then embedded in agarose, imaged with SIM and analysed.(B) SIM images of CD22, IgM, IgD and CD19.(C) Cross-correlation function.(D) Pearson correlation coefficient.(E) Cluster size calculated from autocorrelation analysis.


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

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


Figure 5 ( A - C ) Single - particle - tracking of CD22 , CD19 , IgM and IgD . Wild - type primary B cells were stained with Atto633 - conjugated Fab fragments against CD22 , CD19 , IgM and IgD , settled onto nonstimulatory coverslips and imaged . Single particle trajectories were then analysed . ( A ) Trajectories of CD22 showing diffusion of single particles over 6 s . ( B ) Magnified region from ( A ) traces one particle ( current position indicated with white dashed circle , trajectory in red ) over 6 s . ( C ) Diffusion coefficients of 300 representative CD22 , CD19 , IgM and IgD particles . Bars indicate the median . Data are pooled from at least three experiments per receptor . * P < 0 . 0001 ( Student ' s t - test ) . ( D - F ) Single - particle - tracking of CD22 in primary LifeAct - GFP - expressing B cells . Cells were labelled with Atto633 - conjugated Fab fragments against CD22 , settled onto nonstimulatory coverslips and imaged . Single particle trajectories were then analysed . ( D ) Trajectories of CD22 ( left ) showing diffusion of single particles over 6 s and overlaid with a still image of F - actin intensity ( middle ) . Areas of high F - actin density ( grey line boundaries ) with trajectories of CD22 classified as being inside ( red ) and outside ( blue ) of F - actin rich areas . ( E ) Diffusion coefficients of CD22 particles from ( D ) whose trajectories were classed as inside ( in ) or outside ( out ) of actin rich areas . The overall diffusion is also shown ( all ) . Bars and numbers are the median . Data are pooled from two different experiments . * P < 0 . 0001 ( Student ' s t - test ) . ( F ) Correlation analysis . For each position in each CD22 track , the actin intensity was measured and compared to the same number of random positions on the same cell . Values from different cells were normalized so that the average intensity in F - actin rich regions corresponds to a value of one and the average for F - actin low regions to zero . ( G ) Wild - type primary B cells were labelled with Atto633 - conjugated Fab fragments against CD22 , settled onto nonstimulatory coverslips and treated with vehicle control ( DMSO ) or 1µM LatA . Single particle trajectories were then analysed . Bars and numbers indicate the median . Data are pooled from two experiments . * P < 0 . 0001 ( Student ' s t - test ) . ( H - J ) Wild - type primary B cells were stained with Alexa647 - conjugated antibody against CD22 and settled onto nonstimulatory coverslips . Cells were then fixed , imaged with dSTORM and analysed . ( H ) 2D ( top ) and pseudo 3D ( bottom ) dSTORM images were reconstructed from single molecule localizations . The white dashed square is shown in pseudo 3D ( bottom ) . ( I - J ) Quantification of the distribution of CD22 Hopkins index and with H function . Error bars ( Hopkins index ) and thin lines ( H function ) denote mean ± SEM . Data are pooled from two different experiments . ( K ) Flow cytometric analysis of F - actin by intracellular phalloidin staining of unstimulated or LatA treated wild - type B cells for the indicated length of time . ( L ) Flow cytometric analysis of CD22 surface expression in resting or stimulated B cells with 10µg / ml anti - µ chain ( Jackson ImmunoResearch ) , 1µM or 10µM LatA for 5 and 15 minutes . Error bars denote mean ± SD .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A-C) Single-particle-tracking of CD22, CD19, IgM and IgD. Wild-type primary B cells were stained with Atto633-conjugated Fab fragments against CD22, CD19, IgM and IgD, settled onto nonstimulatory coverslips and imaged. Single particle trajectories were then analysed.(A) Trajectories of CD22 showing diffusion of single particles over 6 s.(B) Magnified region from (A) traces one particle (current position indicated with white dashed circle, trajectory in red) over 6 s.(C) Diffusion coefficients of 300 representative CD22, CD19, IgM and IgD particles. Bars indicate the median. Data are pooled from at least three experiments per receptor. *P < 0.0001 (Student's t-test).(D-F) Single-particle-tracking of CD22 in primary LifeAct-GFP-expressing B cells. Cells were labelled with Atto633-conjugated Fab fragments against CD22, settled onto nonstimulatory coverslips and imaged. Single particle trajectories were then analysed.(D) Trajectories of CD22 (left) showing diffusion of single particles over 6 s and overlaid with a still image of F-actin intensity (middle). Areas of high F-actin density (grey line boundaries) with trajectories of CD22 classified as being inside (red) and outside (blue) of F-actin rich areas.(E) Diffusion coefficients of CD22 particles from (D) whose trajectories were classed as inside (in) or outside (out) of actin rich areas. The overall diffusion is also shown (all). Bars and numbers are the median. Data are pooled from two different experiments. *P < 0.0001 (Student's t-test).(F) Correlation analysis. For each position in each CD22 track, the actin intensity was measured and compared to the same number of random positions on the same cell. Values from different cells were normalized so that the average intensity in F-actin rich regions corresponds to a value of one and the average for F-actin low regions to zero.(G) Wild-type primary B cells were labelled with Atto633-conjugated Fab fragments against CD22, settled onto nonstimulatory coverslips and treated with vehicle control (DMSO) or 1µM LatA. Single particle trajectories were then analysed. Bars and numbers indicate the median. Data are pooled from two experiments.*P < 0.0001 (Student's t-test).(H-J) Wild-type primary B cells were stained with Alexa647-conjugated antibody against CD22 and settled onto nonstimulatory coverslips. Cells were then fixed, imaged with dSTORM and analysed.(H) 2D (top) and pseudo 3D (bottom) dSTORM images were reconstructed from single molecule localizations. The white dashed square is shown in pseudo 3D (bottom).(I-J) Quantification of the distribution of CD22 Hopkins index and with H function. Error bars (Hopkins index) and thin lines (H function) denote mean ± SEM.Data are pooled from two different experiments.(K) Flow cytometric analysis of F-actin by intracellular phalloidin staining of unstimulated or LatA treated wild-type B cells for the indicated length of time.(L) Flow cytometric analysis of CD22 surface expression in resting or stimulated B cells with 10µg/ml anti-µ chain (Jackson ImmunoResearch), 1µM or 10µM LatA for 5 and 15 minutes. Error bars denote mean ± SD.


Figure 6 ( A - B ) Wild - type and CD45 - deficient primary B cells were stained with Alexa647conjugated antibody against CD22 and settled onto nonstimulatory coverslips . Cells were then fixed , imaged with dSTORM and analysed . ( A ) 2D ( top ) and pseudo 3D ( bottom ) dSTORM images were reconstructed from single molecule localizations . The white dashed square is shown in pseudo 3D ( bottom ) . ( B ) Error bars ( Hopkins index ) and thin lines ( H function ) denote mean ± SEM . Data are pooled from three experiments . ( C - E ) Wild - type and CD45 - deficient primary B cells were labelled with Atto633 - conjugated Fab fragments against CD22 or IgM , settled onto nonstimulatory coverslips and imaged . Single particle trajectories were then analysed . ( C ) Trajectories of CD22 in wild - type ( left ) and CD45 - deficient cells ( right ) showing diffusion of single particles over 6 s . ( D ) Diffusion coefficients of 300 representative CD22 and IgM particles . Bars and numbers indicate the median . Data are pooled from three experiments . ( E ) Confinement analysis of CD22 and IgM . Bars and numbers indicate the median . ( F ) Two - population analysis of CD22 diffusion . Diffusion coefficient were plotted on a logarithmic scale and fitted to two Gaussian - shaped curves to account for slower ( light grey ) and faster ( dark grey ) diffusing populations . Wild - type ( blue line and circles , left ) and CD45 - deficient ( blue line and squares , left middle ) primary B cells were compared in an overlay ( right middle ) and their proportions plotted ( right ) . ( G and H ) CD45 - deficient primary B cells were labelled with Atto633 - conjugated Fab fragments against CD22 or IgM , settled onto nonstimulatory coverslips , treated with vehicle control ( DMSO ) or 1µM LatA and imaged . Single particle trajectories were then analysed and diffusion coefficients calculated . ( G ) Diffusion coefficients of 300 representative CD22 and IgM particles . Bars and numbers indicate the median . ( H ) Confinement analysis . Bars and numbers indicate the median . ( I and J ) CD45 - deficient primary B cells were stained with Alexa 647 - conjugated antibody against CD22 , settled onto nonstimulatory coverslips and treated with vehicle control or sialidase . Cells were then fixed , imaged with dSTORM and analysed . ( I ) 2D ( top ) and pseudo 3D ( bottom ) dSTORM images were reconstructed from single molecule localizations . The white dashed square is shown in pseudo 3D ( bottom ) . ( J ) Quantification of the distribution of CD22 with Hopkins index and H function . Error bars ( Hopkins index ) and thin lines ( H function ) denote mean ± SEM . Data are pooled from two experiments . * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 0001 ( Student ' s t - test ) . ( K and L ) Wild - type and CD45 - deficient primary B cells were treated with vehicle control ( - ) or 1 µM LatA for the indicated times . Cells were lysed and analysed by SDS - PAGE followed by immunoblotting with phospho - CD22 ( p - CD22 ) and total CD22 as loading control . The intensity of p - CD22 , normalized to CD22 , was referred to the unstimulated sample of the wild - type cells , set as 1 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A-B) Wild-type and CD45-deficient primary B cells were stained with Alexa647conjugated antibody against CD22 and settled onto nonstimulatory coverslips. Cells were then fixed, imaged with dSTORM and analysed.(A) 2D (top) and pseudo 3D (bottom) dSTORM images were reconstructed from single molecule localizations. The white dashed square is shown in pseudo 3D (bottom).(B) Error bars (Hopkins index) and thin lines (H function) denote mean ± SEM. Data are pooled from three experiments.(C-E) Wild-type and CD45-deficient primary B cells were labelled with Atto633-conjugated Fab fragments against CD22 or IgM, settled onto nonstimulatory coverslips and imaged. Single particle trajectories were then analysed.(C) Trajectories of CD22 in wild-type (left) and CD45-deficient cells (right) showing diffusion of single particles over 6 s.(D) Diffusion coefficients of 300 representative CD22 and IgM particles. Bars and numbers indicate the median. Data are pooled from three experiments.(E) Confinement analysis of CD22 and IgM. Bars and numbers indicate the median.(F) Two-population analysis of CD22 diffusion. Diffusion coefficient were plotted on a logarithmic scale and fitted to two Gaussian-shaped curves to account for slower (light grey) and faster (dark grey) diffusing populations. Wild-type (blue line and circles, left) and CD45-deficient (blue line and squares, left middle) primary B cells were compared in an overlay (right middle) and their proportions plotted (right).(G and H) CD45-deficient primary B cells were labelled with Atto633-conjugated Fab fragments against CD22 or IgM, settled onto nonstimulatory coverslips, treated with vehicle control (DMSO) or 1µM LatA and imaged. Single particle trajectories were then analysed and diffusion coefficients calculated.(G) Diffusion coefficients of 300 representative CD22 and IgM particles. Bars and numbers indicate the median.(H) Confinement analysis. Bars and numbers indicate the median.(I and J) CD45-deficient primary B cells were stained with Alexa 647-conjugated antibody against CD22, settled onto nonstimulatory coverslips and treated with vehicle control or sialidase. Cells were then fixed, imaged with dSTORM and analysed.(I) 2D (top) and pseudo 3D (bottom) dSTORM images were reconstructed from single molecule localizations. The white dashed square is shown in pseudo 3D (bottom).(J) Quantification of the distribution of CD22 with Hopkins index and H function. Error bars (Hopkins index) and thin lines (H function) denote mean ± SEM.Data are pooled from two experiments.*P<0.05, **P<0.001, ***P<0.0001 (Student's t-test).(K and L) Wild-type and CD45-deficient primary B cells were treated with vehicle control (-) or 1 µM LatA for the indicated times. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-CD22 (p-CD22) and total CD22 as loading control. The intensity of p-CD22, normalized to CD22, was referred to the unstimulated sample of the wild-type cells, set as 1.


Figure 7 ( A - E ) Wild - type and Cd22R130E primary B cells were labelled with Atto 633 - conjugated Fab fragments against CD22 or IgM , settled onto nonstimulatory coverslips and imaged . Single particle trajectories were then analysed . ( A ) Trajectories of CD22 in Wild - type ( left ) and Cd22R130E cells ( right ) showing diffusion of single particles over 6 s . B ) Diffusion coefficients of 300 representative CD22 and IgM particles . Bars and numbers indicate the median . ( C ) Confinement analysis . Bars and numbers indicate the median . ( D and E ) Two - population analysis of CD22 diffusion . Analysis of CD22 diffusion plotted on a logarithmic scale and fitted to two Gaussian - shaped curves to account for slower and faster diffusing populations . ( D ) Wild - type ( blue line and circles ) and Cd22R130E ( blue dotted line and squares middle ) primary B cells were compared in an overlay ( left ) and the proportions of the faster diffusing populations plotted ( right ) . ( E ) Proportions of faster - diffusing populations . ( F and G ) Wild - type and Cd22R130E primary B cells were stained with Alexa 647 - conjugated antibody against CD22 and settled onto nonstimulatory coverslips . Cells were then fixed , imaged with dSTORM and analysed . ( F ) 2D ( top ) and pseudo 3D ( bottom ) dSTORM images were reconstructed from single molecule localizations . The white dashed square is shown in pseudo 3D ( bottom ) . ( G ) Quantification of the distribution of CD22 with Hopkins index and H function . Error bars ( Hopkins index ) and thin lines ( H function ) denote mean ± SEM . Data are pooled from three experiments . ( H ) Wild - type and Cd22R130E primary B cells were treated with 1 µM LatA and intracellular calcium flux was measured by flow cytometry . ( I - K ) Wild - type and Cd22R130E primary B cells were treated with vehicle control ( - ) or 1 µM LatA for the indicated time . Cells were lysed and analysed by SDS - PAGE followed by immunoblotting with phospho - CD19 ( p - CD19 ) , phospho - Akt ( pAkt ) , phospho - ERK ( pERK ) and actin and total ERK as loading controls . The intensity of phosphorylated proteins , normalized to actin or ERK , was referred to the unstimulated sample of the wild type cells , set as 1 . Data are representative of at least two experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A-E) Wild-type and Cd22R130E primary B cells were labelled with Atto 633-conjugated Fab fragments against CD22 or IgM, settled onto nonstimulatory coverslips and imaged. Single particle trajectories were then analysed.(A) Trajectories of CD22 in Wild-type (left) and Cd22R130E cells (right) showing diffusion of single particles over 6 s.B) Diffusion coefficients of 300 representative CD22 and IgM particles. Bars and numbers indicate the median.(C) Confinement analysis. Bars and numbers indicate the median.(D and E) Two-population analysis of CD22 diffusion. Analysis of CD22 diffusion plotted on a logarithmic scale and fitted to two Gaussian-shaped curves to account for slower and faster diffusing populations.(D) Wild-type (blue line and circles) and Cd22R130E (blue dotted line and squares middle) primary B cells were compared in an overlay (left) and the proportions of the faster diffusing populations plotted (right).(E) Proportions of faster-diffusing populations.(F and G) Wild-type and Cd22R130E primary B cells were stained with Alexa 647-conjugated antibody against CD22 and settled onto nonstimulatory coverslips. Cells were then fixed, imaged with dSTORM and analysed.(F) 2D (top) and pseudo 3D (bottom) dSTORM images were reconstructed from single molecule localizations. The white dashed square is shown in pseudo 3D (bottom).(G) Quantification of the distribution of CD22 with Hopkins index and H function. Error bars (Hopkins index) and thin lines (H function) denote mean ± SEM. Data are pooled from three experiments.(H) Wild-type and Cd22R130E primary B cells were treated with 1 µM LatA and intracellular calcium flux was measured by flow cytometry.(I-K) Wild-type and Cd22R130E primary B cells were treated with vehicle control (-) or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-CD19 (p-CD19), phospho-Akt (pAkt), phospho-ERK (pERK) and actin and total ERK as loading controls. The intensity of phosphorylated proteins, normalized to actin or ERK, was referred to the unstimulated sample of the wild type cells, set as 1.Data are representative of at least two experiments.


Figure 1A ) z - stacks were acquired from FM4 - 64 labeled vacuoles in wildtype cells ( BJ3505 ) , using a spinning disc microscope ( step size 200 nm ) . 3D reconstructions from these stacks reveal a peripherally located fusion pore . Scale bar 2 µm . B ) Wildtype ( BJ3505 ) and isogenic pmc1 ∆ / vcx1Δ mutant cells were labeled with FM4 - 64 . The number of vacuoles per cell was determined in 3 independent experiments evaluating 200 cells each . Scale bar : 2 µm . C ) Frequency of visible fusion pores . Cells were grown as in ( B ) . The percentage of cells showing visible fusion pore was determined from z - stacks . Means s . d . are shown for 100 stained vacuoles from 3 independent experiments . E ) Distribution of Vph1 - GFP and FM4 - 64 in vacuole - vacuole contact sites . Vph1 - GFP expressing cells , labeled with FM4 - 64 , were analyzed under a spinning disk confocal microscope equipped with two cameras for the simultaneous acquisition of GFP and FM4 - 64 fluorescence signals . Separate channels are shown on the right . The entire contact surface , but not the pore , is accessible to both probes . Scale bar : 2 µm . F ) Expansion of a vacuolar fusion pore in real time . pmc1 ∆ / vcx1Δ cells were labeled with FM4 - 64 and immobilized in a 50 µL flow chamber ( Ibidi ) . The osmotic value of the medium was changed by perfusion with water , using a pump with a flow of 30 µL / s . A frame was acquired every 2 s for a total period of the 30 s , using the laser at minimal intensity .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A) z-stacks were acquired from FM4-64 labeled vacuoles in wildtype cells (BJ3505), using a spinning disc microscope (step size 200 nm). 3D reconstructions from these stacks reveal a peripherally located fusion pore. Scale bar 2 µm.B) Wildtype (BJ3505) and isogenic pmc1∆/vcx1Δ mutant cells were labeled with FM4-64. The number of vacuoles per cell was determined in 3 independent experiments evaluating 200 cells each. Scale bar: 2 µm. C) Frequency of visible fusion pores. Cells were grown as in (B). The percentage of cells showing visible fusion pore was determined from z-stacks. Means s.d. are shown for 100 stained vacuoles from 3 independent experiments. E) Distribution of Vph1-GFP and FM4-64 in vacuole-vacuole contact sites. Vph1-GFP expressing cells, labeled with FM4-64, were analyzed under a spinning disk confocal microscope equipped with two cameras for the simultaneous acquisition of GFP and FM4-64 fluorescence signals. Separate channels are shown on the right. The entire contact surface, but not the pore, is accessible to both probes. Scale bar: 2 µm.F) Expansion of a vacuolar fusion pore in real time. pmc1∆/vcx1Δ cells were labeled with FM4-64 and immobilized in a 50 µL flow chamber (Ibidi). The osmotic value of the medium was changed by perfusion with water, using a pump with a flow of 30 µL/s. A frame was acquired every 2 s for a total period of the 30 s, using the laser at minimal intensity.


Figure 2A ) Vacuolar membranes of wildtype ( BY4741 ) , nyv1 ∆ vam3 ∆ , ypt7 ∆ or vma16F190Y cells were labeled with the vital dye FM4 - 64 . A vacuole that was in a sufficiently peripheral location to be selectively photobleached was exposed to a laser pulse ( white arrows indicate the bleaching area ) . Recovery of fluorescence in this area was assayed 10 seconds later by spinning disc confocal microscopy . Scale bar : 2 µm . B ) Kinetics of FM4 - 64 recovery in ( A ) . Means and s . d . are shown for 20 vacuole clusters from 3 independent experiments . C ) In vivo lipid and content mixing . Vacuoles in living yeast were labeled with the indicated lumenal ( CDCFDA ) and membrane ( FM4 - 64 or PX - GFP ) probes . Note that CRY1 cells lack the ADE2 gene and hence naturally accumulate 5 - amino - 1 - ( 5 - phospho - D - ribosyl ) imidazole as fluorescent fluid phase marker in the vacuolar lumen . FRAP - experiments were performed ( arrows indicate the bleaching area ) . 1 . Non - bleached lumenal area . 2 . Bleached lumenal area . 3 . Bleached membrane area . Scale bar : 2 µm . D and E ) Kinetics of fluorescence recovery after photobleaching of the probes in ( C ) . Numbers denote the areas shown in ( C ) : 1 . Non - bleached lumenal area . 2 . Bleached lumenal area . 3 . Bleached membrane area . Means and s . d . are shown for 20 vacuole clusters from 3 independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A) Vacuolar membranes of wildtype (BY4741), nyv1∆ vam3∆, ypt7∆ or vma16F190Y cells were labeled with the vital dye FM4-64. A vacuole that was in a sufficiently peripheral location to be selectively photobleached was exposed to a laser pulse (white arrows indicate the bleaching area). Recovery of fluorescence in this area was assayed 10 seconds later by spinning disc confocal microscopy. Scale bar: 2 µm. B) Kinetics of FM4-64 recovery in (A). Means and s.d. are shown for 20 vacuole clusters from 3 independent experiments. C) In vivo lipid and content mixing. Vacuoles in living yeast were labeled with the indicated lumenal (CDCFDA) and membrane (FM4-64 or PX-GFP) probes. Note that CRY1 cells lack the ADE2 gene and hence naturally accumulate 5-amino-1-(5-phospho-D-ribosyl)imidazole as fluorescent fluid phase marker in the vacuolar lumen. FRAP-experiments were performed (arrows indicate the bleaching area). 1. Non-bleached lumenal area. 2. Bleached lumenal area. 3. Bleached membrane area. Scale bar: 2 µm. D and E) Kinetics of fluorescence recovery after photobleaching of the probes in (C). Numbers denote the areas shown in (C): 1. Non-bleached lumenal area. 2. Bleached lumenal area. 3. Bleached membrane area. Means and s.d. are shown for 20 vacuole clusters from 3 independent experiments.


Figure 3A ) FM4 - 64 and GFP excitation / emission spectra . C ) Assay by microscopy . Half of two cultures of cells expressing Vph1 - EGFP or Nyv1 - EGFP were labeled with FM4 - 64 , washed and mixed with the other , non - labeled half of the same culture . Cells were analyzed by spinning disc confocal microscopy . The intensities of GFP fluorescence were tracked along the dashed lines and compared between neighboring FM4 - 64 - labeled and unlabeled cells . D ) FACS analysis . The strains were labeled washed and analyzes by FACS . Shown are side ( SSC - A ) and forward scatter ( FSC - A ) , as well as the fluorescence intensity distributions for FM4 - 64 and for GFP in the absence or presence of FM4 - 64 . E ) Comparison of the GFP mean fluorescence from FM4 - 64 - stained cells and unstained cells ( 20 ' 000 each ) .

O B-PANEL_START O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A) FM4-64 and GFP excitation/emission spectra.C) Assay by microscopy. Half of two cultures of cells expressing Vph1-EGFP or Nyv1-EGFP were labeled with FM4-64, washed and mixed with the other, non-labeled half of the same culture. Cells were analyzed by spinning disc confocal microscopy. The intensities of GFP fluorescence were tracked along the dashed lines and compared between neighboring FM4-64-labeled and unlabeled cells.D) FACS analysis. The strains were labeled washed and analyzes by FACS. Shown are side (SSC-A) and forward scatter (FSC-A), as well as the fluorescence intensity distributions for FM4-64 and for GFP in the absence or presence of FM4-64. E) Comparison of the GFP mean fluorescence from FM4-64-stained cells and unstained cells (20'000 each).


Figure 4C , D ) FRAP assays for fusion pores . The indicated cells expressing ( C ) yeGFP - LT or ( D ) LT - yeGFP were labeled with FM4 - 64 and subjected to FRAP experiments Cells were imaged before and 0 ( bleached ) or 30 seconds after photobleaching ( recovery ) . The bleached areas are indicated by an arrow . E , F ) FRAP assays for fusion pores were performed as in ( C , D ) , but with nyv1 ∆ cells . Scale bar : 2 µm . G ) The histogram reports the fraction of cells showing FRAP . Means and s . d . are shown from 3 independent experiments , with 100 cells analyzed in each .

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Figure 4C,D) FRAP assays for fusion pores. The indicated cells expressing (C) yeGFP-LT or (D) LT-yeGFP were labeled with FM4-64 and subjected to FRAP experiments Cells were imaged before and 0 (bleached) or 30 seconds after photobleaching (recovery). The bleached areas are indicated by an arrow. E,F) FRAP assays for fusion pores were performed as in (C, D), but with nyv1∆ cells. Scale bar: 2 µm. G) The histogram reports the fraction of cells showing FRAP. Means and s.d. are shown from 3 independent experiments, with 100 cells analyzed in each.


Figure 5A - F ) The indicated yeast strains were transformed with expression plasmids for FYVE2 - GFP and their vacuolar membranes were labeled with FM4 - 64 . FRAP was performed using a spinning disk microscope equipped with an emission beam splitter and two synchronized cameras , allowing to simultaneously analyze FM4 - 64 and GFP fluorescence . The histograms show the kinetics of signal recovery for ( A ) wildtype ( WT ) , ( B ) vam3tsf , ( C ) ypt7T22N , ( D ) vma16F190Y , ( E ) vps33P184L , and ( F ) nyv1 ∆ cells . Scale bar : 2 µm . Arrows indicate the bleaching area . Means and s . d . are shown for 20 stained vacuoles from 3 independent experiments .

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Figure 5A-F) The indicated yeast strains were transformed with expression plasmids for FYVE2-GFP and their vacuolar membranes were labeled with FM4-64. FRAP was performed using a spinning disk microscope equipped with an emission beam splitter and two synchronized cameras, allowing to simultaneously analyze FM4-64 and GFP fluorescence. The histograms show the kinetics of signal recovery for (A) wildtype (WT), (B) vam3tsf, (C) ypt7T22N, (D) vma16F190Y, (E) vps33P184L, and (F) nyv1∆ cells. Scale bar: 2 µm. Arrows indicate the bleaching area. Means and s.d. are shown for 20 stained vacuoles from 3 independent experiments.


Figure 6A - D ) Wild - type ( A , B ) or nyv1 ∆ cells ( C , D ) , which lack the vacuolar R - SNARE Nyv1 , were labeled with FM4 - 64 . Cells were immobilized on a chambered slide and subjected to FRAP experiments ( C ) Vacuole fission induced by hypertonic shift . FRAP experiments were performed before ( 0 ' ) or 15 min after addition of NaCl to 0 . 5 mol / l . The fluorescence images show the vacuoles inside a single cell . ( B , D ) Cells in S - phase were selected , which grow a daughter cell and therefore fission their vacuoles into a chain of tubulo - vesicular structures , part of which are transported into the daughter cell . FRAP experiment on vesicles in the inheritance structure were performed . Arrows indicate the bleaching area , dashed orange lines the cell cortex . Scale bar : 2 µm .

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Figure 6A-D) Wild-type (A, B) or nyv1∆ cells (C,D), which lack the vacuolar R-SNARE Nyv1, were labeled with FM4-64. Cells were immobilized on a chambered slide and subjected to FRAP experiments (C) Vacuole fission induced by hypertonic shift. FRAP experiments were performed before (0') or 15 min after addition of NaCl to 0.5 mol/l. The fluorescence images show the vacuoles inside a single cell. (B, D) Cells in S-phase were selected, which grow a daughter cell and therefore fission their vacuoles into a chain of tubulo-vesicular structures, part of which are transported into the daughter cell. FRAP experiment on vesicles in the inheritance structure were performed. Arrows indicate the bleaching area, dashed orange lines the cell cortex. Scale bar: 2 µm.


Figure 7A ) Wildtype ( BY4741 ) cells labeled with FM4 - 64 show extensive contact zones between vacuoles . Scale bar : 2 µm . C ) FRAP analysis of FM4 - 64 - labeled wildtype cells was performed by acquiring pictures in continuous acquisition mode after bleaching , for a total period of 5 s . Three representative frames after photobleaching are shown . The bars in the magnified parts of frames 2 and 3 illustrate the positions at which the recovery of FM4 - 64 fluorescence was quantified . Scale bar : 2 µm . D ) Quantification of recovery experiments in ( C ) . A delay of > 50 ms between the appearance of the signal on both sides of the photobleached vacuole was considered as asymmetric recovery . The histogram shows means and s . d . from 3 independent experiments evaluating 50 stained vacuoles each . A Schematic view of the various possible positions of a fusion pore and its consequences for our ability to detect asymmetric FM4 - 64 recovery . The contact zone between the two vacuoles is tilted by 90 ° in the middle panel , which gives four examples for the positioning of the pore ( shown as a black dot ) . Asymmetric recovery could only be detected if the pore is sufficiently far from the center of the contact zone and close to the optical plane that is imaged ( the cases marked in red ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A) Wildtype (BY4741) cells labeled with FM4-64 show extensive contact zones between vacuoles. Scale bar: 2 µm.C) FRAP analysis of FM4-64-labeled wildtype cells was performed by acquiring pictures in continuous acquisition mode after bleaching, for a total period of 5 s. Three representative frames after photobleaching are shown. The bars in the magnified parts of frames 2 and 3 illustrate the positions at which the recovery of FM4-64 fluorescence was quantified. Scale bar: 2 µm. D) Quantification of recovery experiments in (C). A delay of >50 ms between the appearance of the signal on both sides of the photobleached vacuole was considered as asymmetric recovery. The histogram shows means and s.d. from 3 independent experiments evaluating 50 stained vacuoles each. A Schematic view of the various possible positions of a fusion pore and its consequences for our ability to detect asymmetric FM4-64 recovery. The contact zone between the two vacuoles is tilted by 90° in the middle panel, which gives four examples for the positioning of the pore (shown as a black dot). Asymmetric recovery could only be detected if the pore is sufficiently far from the center of the contact zone and close to the optical plane that is imaged (the cases marked in red).


Figure 1 | Representative analysis of B220 and IgM surface expression in bone marrow ( left ) and spleen cells ( right ) of mice from the indicated genotypes . Representative flow cytometric analysis of splenocytes from mice of the indicated genotypes ( pre - gated on B cells : B220 + / CD19 + ) for surface expression of IgM and the 3 - 83 idiotype ( 54 . 1 ) . Shown data are representative 11 - 35 individual mice per genotype . PCR fragments amplified with specific primers for Vκ and Jκ5 from genomic DNA of purified splenic B cells from Ptenf / f x 3 - 83ki and Ptenf / f x mb1 - cre x 3 - 83ki mice on the respective backgrounds . Genomic tail DNA from a 3 - 83ki mouse and DNA from purified splenic B cells of a control ( ctrl ) mouse were used as controls . PCR for the splicing factor Srp20 served as a loading control . Kb and Kd indicate the respective background of the mice , ( H2 - Kb : + Ag ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1| Representative analysis of B220 and IgM surface expression in bone marrow (left) and spleen cells (right) of mice from the indicated genotypes.Representative flow cytometric analysis of splenocytes from mice of the indicated genotypes (pre-gated on B cells: B220+/CD19+) for surface expression of IgM and the 3-83 idiotype (54.1). Shown data are representative 11 - 35 individual mice per genotype.PCR fragments amplified with specific primers for Vκ and Jκ5 from genomic DNA of purified splenic B cells from Ptenf/f x 3-83ki and Ptenf/f x mb1-cre x 3-83ki mice on the respective backgrounds. Genomic tail DNA from a 3-83ki mouse and DNA from purified splenic B cells of a control (ctrl) mouse were used as controls. PCR for the splicing factor Srp20 served as a loading control. Kb and Kd indicate the respective background of the mice, (H2-Kb: +Ag).


Figure 2A | Intracellular expression of IgM ( Ig - μHC ic ) in bone marrow ( left ) and spleen cells ( right ) was determined by flow cytometry and compared between the populations of B cells ( green , identified by B220 and CD19 expression ) and non - B cells ( gray ) . Numbers in the histograms indicate the percentages of the positive populations . Figures are representative of 11 - 35 individual mice per genotype . B | Intracellular Ca2 + influx was measured in CD90 . 2 / Thy1 . 2 - splenocytes derived from mice of the indicated genotypes following stimulation with 10 μg / ml α - κLC antibody . Figures are representative of at least 3 individual mice per genotype . C | Serum IgM concentrations measured in mice of the indicated genotypes . Mean ± SD , symbols represent IgM concentrations from individual mice ( n ) . Statistical significance was calculated by using the Kruskal - Wallis test ( see also Table S1 ) , n . s . = not significant ; * * * * p ≤ 0 . 0001 . D , E | Absolute numbers of B cells in bone marrow ( D ) and spleens ( E ) from mice of the respective genotypes , determined by B220 / CD19 surface expression . Mean ± SD , symbols indicate the numbers of B cells in individual mice ( n ) . Statistical significance was calculated by using the Kruskal - Wallis test ( see also Tables S2 - 3 ) , n . s . = not significant ; * p ≤ 0 . 05 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A | Intracellular expression of IgM (Ig-μHC ic) in bone marrow (left) and spleen cells (right) was determined by flow cytometry and compared between the populations of B cells (green, identified by B220 and CD19 expression) and non-B cells (gray). Numbers in the histograms indicate the percentages of the positive populations. Figures are representative of 11 - 35 individual mice per genotype.B | Intracellular Ca2+ influx was measured in CD90.2/Thy1.2- splenocytes derived from mice of the indicated genotypes following stimulation with 10 μg/ml α-κLC antibody. Figures are representative of at least 3 individual mice per genotype.C | Serum IgM concentrations measured in mice of the indicated genotypes. Mean ±SD, symbols represent IgM concentrations from individual mice (n). Statistical significance was calculated by using the Kruskal-Wallis test (see also Table S1), n. s. = not significant; **** p ≤ 0.0001.D, E | Absolute numbers of B cells in bone marrow (D) and spleens (E) from mice of the respective genotypes, determined by B220/CD19 surface expression. Mean ±SD, symbols indicate the numbers of B cells in individual mice (n). Statistical significance was calculated by using the Kruskal-Wallis test (see also Tables S2-3), n. s. = not significant; * p ≤ 0.05; *** p ≤ 0.001; **** p ≤ 0.0001.


Figure 3Splenocytes from mice of the indicated genotypes , ( A ) Ptenf / f , Ptenf / f x mb1 - cre and Ptenf / f x mb1 - cre 3 - 83ki mice on the respective backgrounds were analyzed by flow cytometry . B cells were pre - gated by B220 and CD19 expression and the IgM / IgD surface expression ( dot plots , left ) was determined . Histograms ( right ) show a comparison of intracellular IgD expression ( Ig - δHC ic ) between the splenic populations of B cells ( orange , identified by B220 and CD19 expression ) and non - B cells ( gray ) . Numbers in the histograms indicate the percentages of the positive populations . Data are representative of 11 - 35 individual mice per genotype . ( B ) Ptenf / f x MD4tg and Ptenf / f x mb1 - cre x MD4tg mice in presence and absence of antigen ( ML5tg ) , were analyzed by flow cytometry . B cells were pre - gated by B220 and CD19 expression and the IgM / IgD surface expression ( dot plots , left ) was determined . Histograms ( right ) show a comparison of intracellular IgD expression ( Ig - δHC ic ) between the splenic populations of B cells ( orange , identified by B220 and CD19 expression ) and non - B cells ( gray ) . Numbers in the histograms indicate the percentages of the positive populations . Data are representative of 11 - 35 individual mice per genotype . C | Quantification of surface IgD mean fluorescence intensity ( MFI ) in Ptenf / f x MD4 - trangenic mice in presence ( mb1 - cre + / + ) and absence ( mb1 - cre + / ki ) of Pten . Single dots represent the average MFI of 4 independent measurements per mouse ( n ) , mean ± SD . Statistical significance was calculated by using an unpaired two - tailed t - test . D | Serum anti - HEL titers . Sera from MD4tg mice of the indicated genotypes were adjusted to an IgM concentration of 500 μg / ml and applied in triplicates to HEL - coated plates in dilution steps of 1 : 3 , mean ± SEM . Statistical significance was calculated by using the Kruskal - Wallis test ( see also Table S4 ) . Statements of significance in the figure refer to the comparison between Ptenf / f x MD4tg ( filled red squares ) and Ptenf / f x MD4tg x MD5tg sera ( filled orange triangles ) , * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 . E | Immunohistochemistry of sections from spleens of Ptenf / f , Ptenf / f x mb1 - cre , Ptenf / f x mb1 - cre x 3 - 83ki and Ptenf / f x mb1 - cre x MD4tg mice for CD169 ( green ) , CD5 ( red ) and IgM ( cyan ) at 10x magnification . Pictures in the second row show enlarged areas indicated by the white squares , respectively . Double - arrows indicate the distance between T cell ( red ) and marginal zone ( green ) . Shown pictures are representative of 2 - 3 mice per

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Splenocytes from mice of the indicated genotypes, (A) Ptenf/f, Ptenf/f x mb1-cre and Ptenf/f x mb1-cre 3-83ki mice on the respective backgrounds were analyzed by flow cytometry. B cells were pre-gated by B220 and CD19 expression and the IgM/IgD surface expression (dot plots, left) was determined. Histograms (right) show a comparison of intracellular IgD expression (Ig-δHC ic) between the splenic populations of B cells (orange, identified by B220 and CD19 expression) and non-B cells (gray). Numbers in the histograms indicate the percentages of the positive populations. Data are representative of 11 - 35 individual mice per genotype.(B) Ptenf/f x MD4tg and Ptenf/f x mb1-cre x MD4tg mice in presence and absence of antigen (ML5tg), were analyzed by flow cytometry. B cells were pre-gated by B220 and CD19 expression and the IgM/IgD surface expression (dot plots, left) was determined. Histograms (right) show a comparison of intracellular IgD expression (Ig-δHC ic) between the splenic populations of B cells (orange, identified by B220 and CD19 expression) and non-B cells (gray). Numbers in the histograms indicate the percentages of the positive populations. Data are representative of 11 - 35 individual mice per genotype.C | Quantification of surface IgD mean fluorescence intensity (MFI) in Ptenf/f x MD4-trangenic mice in presence (mb1-cre +/+) and absence (mb1-cre +/ki) of Pten. Single dots represent the average MFI of 4 independent measurements per mouse (n), mean ±SD. Statistical significance was calculated by using an unpaired two-tailed t-test.D | Serum anti-HEL titers. Sera from MD4tg mice of the indicated genotypes were adjusted to an IgM concentration of 500 μg/ml and applied in triplicates to HEL-coated plates in dilution steps of 1 : 3, mean ±SEM. Statistical significance was calculated by using the Kruskal-Wallis test (see also Table S4). Statements of significance in the figure refer to the comparison between Ptenf/f x MD4tg (filled red squares) and Ptenf/f x MD4tg x MD5tg sera (filled orange triangles), ** p ≤ 0.01; *** p ≤ 0.001.E | Immunohistochemistry of sections from spleens of Ptenf/f, Ptenf/f x mb1-cre, Ptenf/f x mb1-cre x 3-83ki and Ptenf/f x mb1-cre x MD4tg mice for CD169 (green), CD5 (red) and IgM (cyan) at 10x magnification. Pictures in the second row show enlarged areas indicated by the white squares, respectively. Double-arrows indicate the distance between T cell (red) and marginal zone (green). Shown pictures are representative of 2 - 3 mice per genotype


Figure 4B | 3 days post transduction the surface expression of IgM / IgD ( dot plots , top ) , IgD and CD23 ( histograms , bottom ) was measured by flow cytometry and compared between EV - and FoxO1 - A3 - transduced cells . Fluorescence minus one ( FMO ) stainings , lacking α - IgD or α - CD23 antibodies respectively , served as controls ( ctrl ) . Bar diagrams below flow cytometry data display the percentages of IgD + cells ( n = 12 ; left ) and MFI of CD23 ( as relative units , RU ) , normalized to the MFI measured in the respective EV transduced cells ; n = 12 ; middle ) . Fcer2 transcript levels were measured by qRT - PCR ( right , n = 4 ) . Mean ± SD , symbols indicate data from individual mice . Statistical significance of IgD expression was determined by applying the Wilcoxon matched - pairs signed rank test for CD23 and by applying the one sample two - tailed t - test for Fcer2 expression . C | Cstf64 ( n = 8 ) , Ell2 ( n = 8 ) and Zfp318 ( n = 11 ) expression levels were measured at 3 days post transduction by qRT - PCR . FoxO1 - A3 - and EV - transduced splenic B cells were FACS - purified according to the gating strategy Mean ± SD , symbols indicate the expression in individual mice . Statistical significance was calculated by using the one - sample two - tailed t - test . E : IgD ( n = 6 ) , CD23 ( n = 4 ) and Fcer2 ( n = 4 ) . F : Cstf64 ( n = 4 ) , Ell2 ( n = 4 ) and Zfp318 ( n = 4 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O

Figure 4B | 3 days post transduction the surface expression of IgM/IgD (dot plots, top), IgD and CD23 (histograms, bottom) was measured by flow cytometry and compared between EV- and FoxO1-A3-transduced cells. Fluorescence minus one (FMO) stainings, lacking α-IgD or α-CD23 antibodies respectively, served as controls (ctrl). Bar diagrams below flow cytometry data display the percentages of IgD+ cells (n = 12; left) and MFI of CD23 (as relative units, RU), normalized to the MFI measured in the respective EV transduced cells; n = 12; middle). Fcer2 transcript levels were measured by qRT-PCR (right, n = 4). Mean ±SD, symbols indicate data from individual mice. Statistical significance of IgD expression was determined by applying the Wilcoxon matched-pairs signed rank test for CD23 and by applying the one sample two-tailed t-test for Fcer2 expression.C | Cstf64 (n = 8), Ell2 (n = 8) and Zfp318 (n = 11) expression levels were measured at 3 days post transduction by qRT-PCR. FoxO1-A3- and EV-transduced splenic B cells were FACS-purified according to the gating strategy Mean ±SD, symbols indicate the expression in individual mice. Statistical significance was calculated by using the one-sample two-tailed t-test.E: IgD (n = 6), CD23 (n = 4) and Fcer2 (n = 4).F: Cstf64 (n = 4), Ell2 (n = 4) and Zfp318 (n = 4).


Figure 5B | Representative flow cytometric analysis of Ramos cells reconstituted with HH10 - mu IgM , HH10 - mu IgD . Cells were stained with α - murine μHC - , δHC - and κLC - antibodies , respectively . EV - transduced HL - KO cells , expressing GFP only , were used as negative control ( gray ) . C | Representative flow cytometric analysis of HEL - binding in reconstituted Ramos cells after staining with fluorescently - labeled HEL . EV - transduced HL - KO cells , expressing GFP only , were used as negative control ( gray ) . D | Representative intracellular Ca2 + influx in HH10 - mu IgM - and HH10 - mu IgD - expressing cells upon stimulation with multivalent HEL ( complex cHEL ) , monovalent ( soluble sHEL ) ( both at a concentration of 1 μg / ml ) , or 10 μg / ml α - mouse κLC antibody , respectively . E | Representative intracellular Ca2 + influx of HH10 - mu IgM - ( top ) and HH10 - mu IgD - ( bottom ) expressing cells upon stimulation with indicated ratios of 1 μg / ml cHEL and sHEL , and a 1 : 25 mixture of cHEL with bovine serum albumin ( BSA ) , where 1 = 1 μg / ml . F | Schematic overview of reconstitution of BCR - deficient Ramos ( HL - KO ) cells with HH10 - specific human IgM ( HH10 - hu IgM ) and IgD ( HH10 - hu IgD ) BCR isotype . G - J | using HH10 - hu IgM - and HH10 - hu IgD - expressing cells instead of HH10 - mu IgM - and HH10 - mu IgD - expressing cells , respectively . Data shown in G to J are representative of minimum three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5B | Representative flow cytometric analysis of Ramos cells reconstituted with HH10-mu IgM, HH10-mu IgD. Cells were stained with α-murine μHC-, δHC- and κLC-antibodies, respectively. EV-transduced HL-KO cells, expressing GFP only, were used as negative control (gray).C | Representative flow cytometric analysis of HEL-binding in reconstituted Ramos cells after staining with fluorescently-labeled HEL. EV-transduced HL-KO cells, expressing GFP only, were used as negative control (gray).D | Representative intracellular Ca2+ influx in HH10-mu IgM- and HH10-mu IgD-expressing cells upon stimulation with multivalent HEL (complex cHEL), monovalent (soluble sHEL) (both at a concentration of 1 μg/ml), or 10 μg/ml α-mouse κLC antibody, respectively.E | Representative intracellular Ca2+ influx of HH10-mu IgM- (top) and HH10-mu IgD- (bottom) expressing cells upon stimulation with indicated ratios of 1 μg/ml cHEL and sHEL, and a 1 : 25 mixture of cHEL with bovine serum albumin (BSA), where 1 = 1 μg/ml.F | Schematic overview of reconstitution of BCR-deficient Ramos (HL-KO) cells with HH10-specific human IgM (HH10-hu IgM) and IgD (HH10-hu IgD) BCR isotype. G - J | using HH10-hu IgM- and HH10-hu IgD-expressing cells instead of HH10-mu IgM- and HH10-mu IgD-expressing cells, respectively. Data shown in G to J are representative of minimum three independent experiments.


Figure 6A | Quantification of surface IgM ( top ) and IgD ( bottom ) MFI in B cells from mice of the indicated genotypes . Single dots represent the average MFI of 4 independent measurements per mouse ( n ) , mean ± SD . Statistical significance was calculated by using the Kruskal - Wallis test n . s . = not significant ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 . | Representative intracellular Ca2 + influx measured in Thy1 . 2 - splenocytes , derived from mice of the indicated genotypes , upon stimulation with cHEL , monovalent sHEL ( both at a concentration of 1 μg / ml ) or 10 μg / ml α - mouse κLC antibody , respectively . Line diagrams below the dot plots show the overlayed MFI of the Ca2 + influx kinetics displayed in the plots above . C | Quantification of intracellular Ca2 + influx The median area under the curve of the Ca2 + influx kinetics was calculated , single dots represent data from individual mice , mean ± SD . Statistical significance was calculated by applying the Mann - Whitney - U test n . s . = not significant , * * p ≤ 0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A | Quantification of surface IgM (top) and IgD (bottom) MFI in B cells from mice of the indicated genotypes. Single dots represent the average MFI of 4 independent measurements per mouse (n), mean ±SD. Statistical significance was calculated by using the Kruskal-Wallis test n. s. = not significant; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.| Representative intracellular Ca2+ influx measured in Thy1.2- splenocytes, derived from mice of the indicated genotypes, upon stimulation with cHEL, monovalent sHEL (both at a concentration of 1 μg/ml) or 10 μg/ml α-mouse κLC antibody, respectively. Line diagrams below the dot plots show the overlayed MFI of the Ca2+ influx kinetics displayed in the plots above.C | Quantification of intracellular Ca2+ influx The median area under the curve of the Ca2+ influx kinetics was calculated, single dots represent data from individual mice, mean ±SD. Statistical significance was calculated by applying the Mann-Whitney-U test n. s. = not significant, ** p ≤ 0.01.


Figure 7B | Analysis of GC B cells in splenic B220 + B cells from wild type ( WT , top ) and IgD - / - ( bottom ) animals after 4 , 7 and 10 days ( left to right ) post immunization . Percentages of follicular B cells ( Fo . B ; CD38 + / CD95 - ) and GC B cells ( CD38 - / CD95 + ) are depicted in the plots . GC B cells ( CD38 - / CD95 + ) were further analyzed for GL - 7 expression , compared to Fo . B cells and quantified . C | Quantification of Fo . B cells ( top , percentages ) and CD38 - / CD95 + / GL - 7 + GC cells ( percentages and absolute numbers , bottom panels ) in WT ( black ) and IgD - / - ( green ) animals after 4 , 7 , 10 and 14 days post immunization , mean ± SD ( numbers of animals indicated in the top plot ) . Statistical significance was analyzed by a two - tailed unpaired t - test . D | Analysis of GC in spleen sections from WT and IgD - / - animals after 4 , 7 and 10 days post immunization . Representative images of 2 x 2 mm sections stained with DAPI ( nuclei ) and peanut agglutinin ( PNA ) are shown for the indicated time points . Scale bar represents 500 μm . Dashed white or yellow lines mark distinct GC foci in the merged images . E | Representative 800 x 800 μm regions of areas marked by yellow dashed lines within the images of D are shown as enlarged single GCs . As no distinct GC foci were observed in IgD - / - samples from days 4 and 7 , an identical 800 x 800 μm region is shown in each case . F | Quantification of number of GCs per mm2 area of spleen sections from WT ( black ) and IgD - / - ( green ) animals after 4 , 7 and 10 days post immunization . Data represent mean ± SD of two sections ( from 3 animals per group and time point ) and were analyzed by two - tailed unpaired t - test . G | Total serum α - TNP IgG . Sera from PBS - or TNP - Ova - immunized WT ( left ) and IgD - / - ( middle ) mice , collected at day 7 following immunization , were adjusted to an IgG concentration of 1 μg / ml and applied in duplicates and dilution steps of 1 : 3 to TNP - coated plates , respectively , mean ± SEM . Right : Overlay of α - TNP IgG titers from TNP - Ova - immunized WT and IgD - / - mice . Statistical significance was calculated by using the Mann - Whitney - U test ( see also Table S7 ) , n . s . = not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B | Analysis of GC B cells in splenic B220+ B cells from wild type (WT, top) and IgD-/- (bottom) animals after 4, 7 and 10 days (left to right) post immunization. Percentages of follicular B cells (Fo.B; CD38+/CD95-) and GC B cells (CD38-/CD95+) are depicted in the plots. GC B cells (CD38-/CD95+) were further analyzed for GL-7 expression, compared to Fo.B cells and quantified.C | Quantification of Fo.B cells (top, percentages) and CD38-/CD95+/GL-7+ GC cells (percentages and absolute numbers, bottom panels) in WT (black) and IgD-/- (green) animals after 4, 7, 10 and 14 days post immunization, mean ±SD (numbers of animals indicated in the top plot). Statistical significance was analyzed by a two-tailed unpaired t-test.D | Analysis of GC in spleen sections from WT and IgD-/- animals after 4, 7 and 10 days post immunization. Representative images of 2 x 2 mm sections stained with DAPI (nuclei) and peanut agglutinin (PNA) are shown for the indicated time points. Scale bar represents 500 μm. Dashed white or yellow lines mark distinct GC foci in the merged images. E | Representative 800 x 800 μm regions of areas marked by yellow dashed lines within the images of D are shown as enlarged single GCs. As no distinct GC foci were observed in IgD-/- samples from days 4 and 7, an identical 800 x 800 μm region is shown in each case. F | Quantification of number of GCs per mm2 area of spleen sections from WT (black) and IgD-/- (green) animals after 4, 7 and 10 days post immunization. Data represent mean ±SD of two sections (from 3 animals per group and time point) and were analyzed by two-tailed unpaired t-test. G | Total serum α-TNP IgG. Sera from PBS- or TNP-Ova-immunized WT (left) and IgD-/- (middle) mice, collected at day 7 following immunization, were adjusted to an IgG concentration of 1 μg/ml and applied in duplicates and dilution steps of 1 : 3 to TNP-coated plates, respectively, mean ±SEM. Right: Overlay of α-TNP IgG titers from TNP-Ova-immunized WT and IgD-/- mice. Statistical significance was calculated by using the Mann-Whitney-U test (see also Table S7), n. s. = not significant, * p ≤ 0.05, ** p ≤ 0.01.


Figure 1C : 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 .

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Figure 1C: 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.


Figure 2B : 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 .

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Figure 2B: 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.


Figure 3A - 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 % .

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Figure 3A-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%.


Figure 4A , 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 . : 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A,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.: 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).


Figure 5B : 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 ) . 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 ) .

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Figure 5B: 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).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).