Ligation of NKp46 by mAb requires coligation of LFA-1 to induce reorganization of cortical actin. (A) Bright-field, IRM, and fluorescence images of pNK cells stimulated on surfaces coated with mAb against NKp46, with or without ICAM-1. Symmetric rings of F-actin at the synapse periphery (indicated by white arrows) were frequently observed on slides coated with both αNKp46 and ICAM-1 (bottom right panel). Bar represents 20 μm. (B) The proportion of pNK cells that form a F-actin ring at the synapse periphery when stimulated on poly-L-lysine–coated surfaces (−) with isotype–matched mAb (IgG₁) or activating mAbs (Isotype IgG₁) against αNKG2D, αCD16, αCD2, or αNKp46, each with or without ICAM-1. Graph represents mean ± SEM, from 3 independent experiments counting 45-65 cells per condition. (C) Representative images obtained by SI microscopy of F-actin at the interface between pNK cells and surfaces coated with αNKp46 (left) or αNKp46 and ICAM-1 (right). The center of the synapse is enlarged in the panels below. Bar represents 1 μm. Third-row panels: The holes between actin filaments in the central region of the synapse as heat maps, with the smallest holes shown in blue (0.01 μm²) and largest (> 3.0 μm²) in red. Bottom panels: Regions are shown within the actin network through which a particle (such as a lytic granule) of diameter 200 nm (blue) to 800 nm (red) could fit. Bars represent 5 μm. (D) Quantification of the average size of holes in the actin mesh at the pNK synapse center for cells stimulated as for panel B. Graph represents mean ± SEM; n = 21-65 cells per condition. (E) Quantification of the proportion of the synapse penetrable by a granule of 250 nm diameter for cells stimulated as in panel B; graph represents mean ± SEM; n = 21-65 cells per condition. In all panels, data are from 3 experiments. ***P < .0001. NS indicates not significant, data compared by 1-way ANOVA.