Nanoscale organization and dynamics of the siglec CD22 coorporate with the cytoskeleton in restraining BCR signalling (2015)
Figure 6

Figure 6. CD45 has a major contribution in CD22 organization and mobility

(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.