Nanoscale organization and dynamics of the siglec CD22 coorporate with the cytoskeleton in restraining BCR signalling (2015)
Figure 7

Figure 7. CD22 mobility, nanoscale organization and function is dependent on its sialic-acid binding activity

(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.

(L) Computer simulation of diffusing CD22 nanoclusters with the nanocluster size from Cd22R130E primary B cells.

(M-O) Area covered by diffusing CD22 with the nanocluster parameters or the diffusion coefficient of Cd22R130E. Data are from seven independent simulation runs.

(M) Computer simulation of diffusing CD22 with the nanocluster parameters of Cd22R130E cells.

(N) Computer simulation of diffusing CD22 with the median diffusion coefficient of Cd22R130E.

(O) Computer simulation of diffusing CD22 nanoclusters with the real diffusion coefficient and nanocluster parameters for CD22 observed in wild-type and Cd22R130E cells. The graph shows the area covered.

Data are from seven independent simulation runs.

*P <0.01, **P <0.001, ***P <0.0001 (Student’s t-test).