KIR2DL2 photostimulation blocks the formation of activating receptor microclusters. (A) NKL cells expressing DAP10-GFP and KIR2DL2-mCherry were imaged in TIRF mode and photostimulated on bilayers containing ULBP3, ICAM, and HLA-Cw3(cage). (top) A representative time-lapse montage (∼80-s intervals), with UV irradiation indicated by magenta text. (bottom) Two single-cell kymographs showing KIR2DL2-mCherry and DAP10-GFP clusters both before and after UV irradiation, which is indicated by the magenta line. Mobile clusters of DAP10 are denoted by arrows. Brackets indicate areas of the kymographs showing peripheral regions rich in KIR2DL2 microclusters but devoid of mobile DAP10 microclusters. Lines used for kymographs are shown in the first image of the time lapse. (B and D) Tracks of DAP10 microclusters in representative single cells. (B) Cells were imaged and UV irradiated on bilayers containing ULBP3, ICAM, and either HLA-Cw3(Tyr) (left) or HLA-Cw3(cage) (right). Paths traveled before UV irradiation are shown in red, and paths after UV irradiation are shown in blue. (D) Cells were treated with latrunculin (Lat) on bilayers containing ULBP3 and ICAM. Paths traveled before latrunculin addition are shown in red, and paths after latrunculin addition are shown in blue. (C) Bar graphs showing the relative amounts of mobile versus immobile DAP10 microclusters. (left and center) Cells were photostimulated on bilayers containing either HLA-Cw3(Tyr) (left) or HLA-Cw3(cage) (center). (right) Cells were treated with latrunculin on bilayers containing ULBP3 and ICAM. The total number of analyzed microclusters is indicated above each bar, and the number of analyzed cells for each experiment is shown between bars. The mean starting ratio of mobile to immobile microclusters differed from experiment to experiment. Hence, two independent experiments, each derived from cells imaged the same day, are shown for each condition. Bars, 5 µm.