Intracellular compartments containing IFN-γ preferentially localize to regions where the actin mesh has opened up. (A) Right panel: Two-color SI images of F-actin (red) and IFN-γ (green) in a pNK cell activated on a surface coated with MICA-Fc (2.5 μg/mL) and ICAM-1 (2.5 μg/mL) for 90 minutes. Bar represents 5 μm. White box represents the region of the immune synapse that is enlarged in the center panel. Right panel: The domains within the enlarged region that have opened up in the cortical actin mesh to be 200-800 nm in diameter. Bar represents 1μm. (B) Relative frequencies of the volumes of IFN-γ stained compartments located at 0-250 nm (left) or 250-500 nm (right) up into the cell away from the contact with the surface. See main text for precise numeric quantification of this data. (C) The average volume of IFN-γ compartments located within 250 nm or 250-500 nm of the cell surface interface. Graphs represent mean ± SEM from 3 experiments; n = 250. ***P < .0001. (D) The ratio of surface area of compartments containing IFN-γ to surface area of a sphere with an equal volume is plotted against the measured volume of the compartment. Graph represents mean ± SEM from 3 experiments; n = 473. (E) Odds ratios for IFN-γ to be localized on regions within the cortical actin mesh in which holes 200-800 nm in diameter have opened are plotted as mean ± SEM at axial distances for IFN-γ located 0-1 μm above the surface of slides coated with MICA-Fc and ICAM-1. Graph represents n = 10 cells from 3 experiments.