Cdc42 plays a role in early BCR signaling. (A) Purified splenic Cdc42 KO and WT B cells were settled on TIB120-coated coverslips. Representative scanning electron microscopy images (top row) and direct stochastic optical reconstruction microscopy for IgM nanoscale organization are shown (bottom row). Quantifications were performed on at least 50 cells and are shown in the right-hand columns and show the percentage of cells with more than 30 protrusions and the H function (see text). Data are representative of two independent experiments. (B) Purified splenic WT and Cdc42 KO B cells were stained intracellularly with phalloidin and analyzed by flow cytometry. Data are representative of 3 independent experiments. (C) Purified splenic WT B cells were stimulated with 10 µg.ml⁻¹ anti-IgM F(ab’)₂ for different times and lysed. Nonstimulated lysates incubated with GTPgS (positive) or GDP (negative) were used as controls. Lysates were incubated with GST-PAK beads (binding to Cdc42-GTP) and both total lysate and the bound fraction were analyzed by Western blot. Cdc42-GTP (top row) and actin (bottom row) protein levels are shown. Densitometric analysis was used to quantify the signal intensity of Cdc42-GTP normalized to actin. Data are representative of at least 3 independent experiments. (D and F) Flow cytometry analysis for levels of pSrc and pSyk (D), or pErk and pAkt (F) in purified splenic WT (blue lines) or Cdc42 KO (red lines) B cells at steady state (gray shaded histogram) or 2 min after stimulation with 10 µg ml⁻¹ anti-κ light chain (colored lines). Quantifications are shown in the right column and were calculated as the difference between the geometric mean of the fluorescence between stimulated and nonstimulated samples (ΔMFI). Data are representative of >3 independent experiments. (E) Intracellular Ca²⁺ influx in purified splenic WT (blue lines) or Cdc42 KO (red lines) B cells after stimulation with either anti-IgM F(ab’)₂ (top; 1 µg ml⁻¹ [dotted lines], 10 µg ml⁻¹ [solid lines]) or latrunculin A (bottom; 1 µmol/liter⁻¹). Data are representative of two independent experiments. (G) Western blot analysis showing protein levels of phospho-CD19 (top) and ERK (bottom) in splenic WT or Cdc42 KO B cells after stimulation with 10 µg.ml⁻¹ anti-κ light chain antibody. Densitometric analysis was used to quantify the signal intensity of pCD19 normalized to ERK, referred to the respective controls. Quantification is shown in the chart below. (H) Representative images by total internal reflection microscopy (TIRF) of splenic WT (left column) or Cdc42 KO B cells (right column) settled on planar lipid bilayers loaded with Alexa Fluor 633-streptavidin and anti-κ light chain antibody. Representative images were taken at 1 and 15 min after settling and show the spreading (top row) and contraction (bottom row) phases. Quantification chart in the right-hand column indicates maximum spreading area. Data are representative of 3 independent experiments. (I) Representative TIRF (top row) and SEM (bottom row) of WT (left column) and Cdc42 KO (right column) splenic B cells stimulated on glass coverslips coated with anti-κ light chain antibody for 10 min, before fixation and staining with phalloidin for TIRF or processing for SEM. Quantifications (right column) were performed on >100 cells and indicate circularity index calculated with Image J (top right) and percentage of fully spread cells (bottom right). Data are representative of 3 independent experiments. (J) Internalization of soluble anti-IgM antibody measured by flow cytometry in WT (blue line) or Cdc42 KO splenic B cells (red line) after 15 min of stimulation with biotinylated anti-IgM antibody. Gray shaded histograms indicate basal IgM levels. The right-hand chart shows internalization kinetics. Data are representative of >3 independent experiments. (Bars, 3 µm). Error bars are mean ± SEM. Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.0001; ****, P < 0.00001. All charts are representative of at least 3 independent experiments.