Disruption of ERM function impairs B cell spreading and antigen aggregation. (A) Schematic of GFP-tagged ezrin constructs. (B) Localization of GFP-tagged WT and mutated versions of ezrin were visualized by confocal microscopy in unstimulated DT40 B cells. (C) Scanning electron microscopy images of DT40 B cells expressing WT and mutated versions of ezrin after settling onto planar lipid bilayers containing anti-IgM as antigen. (D–F) Individual cells were quantified for the number of filopodia (D; *, P = 0.047), length of filopodia (E; *, P = 0.0369; ***, P < 0.0001), and frequency of cells with ruffles (F). (E) Red bars indicate mean. (G–I) DT40 B cells expressing WT Ezrin-GFP (G), Ezrin-CA–GFP (H), and Ezrin-DN–GFP (I) were settled onto lipid bilayers containing anti-IgM. Contact area was visualized by IRM (top), and fluorescently labeled antigen (Ag; middle) and GFP-tagged ezrin (bottom) were visualized by confocal microscopy at the indicated times. Antigen and GFP are mapped to a pseudocolor scale to aid visualization. (J and K) Contact area (J) and total antigen intensity within the contact area (K) were quantified over time. Data are representative of at least two independent experiments. (D, F, J, and K) Error bars indicate SEM. Bars, 2 µm.