The cortical actin mesh opens up in NK cells expressing IFN-γ. (A) Left panels: Super-resolution images of F-actin and IFN-γ in pNK cells stimulated for 90 minutes on coverslips coated with ICAM-1 (2.5 μg/mL) or MICA-Fc (2.5 μg/mL) with ICAM-1 (2.5 μg/mL). Bars represent 5 μm. Center panels: Holes in the actin mesh as a heat map. Middle right panels: The domains in the actin mesh, which have opened up to be penetrable by a spherical particle of diameter 200-800 nm, and the regions indicated by the white square are enlarged in the far right panels. (B) Quantification of the mean areas of holes in the actin mesh for pNK cells stimulated for 90 minutes on surfaces coated as in panel A. (C) The proportion of the synapse where the actin mesh has opened up to produce domains penetrable by a particle of diameter 200-800 nm in pNK cells stimulated on surfaces coated as in panel A. Graphs represent mean ± SEM from 3 experiments; n = 30. ***P < .0001 (t test). (D) Bright-field and 2-color SI images of perforin (red) and IFN-γ (green) in pNK cells stimulated for 90 minutes on surfaces coated with MICA-Fc and ICAM-1. Bars represent 10 μm. (E) Quantification of the number of lytic granules, stained with α-perforin mAb, in the whole volume of each pNK cell after stimulation for 6 (white) or 90 minutes (black) on slides coated with poly-L-lysine or MICA-Fc (2.5 μg/mL) and ICAM-1 (2.5 μg/mL). At 90 minutes, when IFN-γ could be detected, IFN-γ⁺ and IFN-γ⁻ cells are plotted separately. Bars represent the median from 4 experiments; n > 30. ***P < .0001 (analyzed by 1-way ANOVA). (F) Comparison of the number of granules per NK cell for 4 independent donors whose cells were stimulated for 6 minutes on slides coated with MICA-Fc and ICAM-1. Each data point represents a single cell. Bars represent the median for each dataset.