NKG2D stimulation induces the formation of activating receptor microclusters. (A) NKL cells expressing DAP10-mCherry were imaged using TIRF microscopy on bilayers containing ULBP3 and ICAM. (left) A kymograph showing centripetally mobile and stationary DAP10 clusters, indicated by the cyan arrow and arrowhead, respectively. The line used to generate the kymograph is shown on the right. (B–D) NKL cells expressing DAP10-GFP were fixed and stained with antibodies against phosphotyrosine (pY) on bilayers containing ULBP3 and ICAM. (B) Representative images showing DAP10 fluorescence (left), phosphotyrosine fluorescence (right), and the overlay (center). (C) Linescans depicting DAP10 and phosphotyrosine fluorescence (yellow and blue, respectively) in specific microclusters within the contact region. The lines used for each linescan are shown in the central image in B. (D, left) Schematic showing how images were divided into central and peripheral zones for quantification. (right) Before and after graph showing the ratio of normalized phosphotyrosine fluorescence intensity (F_pY) to normalized DAP10-GFP fluorescence intensity (F_DAP10) for peripheral and central regions. Ratios calculated from the same cell are connected by lines. All data are representative of at least two independent experiments. arb., arbitrary. Bars, 5 µm.