KIR2DL2 signaling inhibits cell spreading and the initiation of Ca2+ flux. (A and B) NKL cells expressing KIR2DL2 were stained with PKH26 and imaged using TIRF microscopy on lipid bilayers containing the indicated proteins. (A) Representative time-lapse montages (∼90-s intervals) under both activating (top) and inhibitory (bottom) conditions. (B) Bar graph representing the distribution of cell behavior on surfaces containing the indicated ligands. Only cells visible in the imaging field for ≥5 min were analyzed. Cells were described as spread if they formed a stationary footprint at least 10 µm in diameter (yellow arrow in A), collapsed if they engaged in minimal dynamic interactions with the membrane (magenta arrow in A), or motile if they exhibited directional migration (cyan arrow in A). Occasionally, cells would display two phenotypes during the imaging period. (C) NKL cells expressing KIR2DL2 (KIR-WT) or KIR2DL2(mut) (KIR-Mut) were loaded with Fura-2AM and imaged on lipid bilayers containing ULBP3, ICAM, and the indicated HLA-Cw3 proteins. (right) Representative time-lapse montages (4-min intervals) showing a pseudocolored Fura-2AM ratio (warmer colors indicate higher intracellular Ca2+ concentrations). (left) Background-corrected mean Fura-2AM ratios for all imaging fields are plotted versus time for each condition. Error bars show SEM. All data are representative of at least two independent experiments. Bars, 10 µm.
