PIP2 dynamics at NK-cell cytolytic synapse. Primary cultured NK cells were infected with recombinant lentiviruses encoding GFP (GFP-ctr) or GFP-PH and used for time-lapse confocal microscopy. (A) Membrane distribution of GFP-PH and (B) GFP-ctr. Anti-CD16-treated GFP-PH-expressing cells were added to Fc receptor-positive targets. Images were obtained at 30-second intervals. (C-F) Representative images show dichroic phase contrast and fluorescence. The numbers indicate the time after NK cells have made contact with target cells. Bar represents 5 μm. (G) Levels of membrane GFP-PH was quantified and binned into 30-second intervals. The mean fluorescence intensity was calculated on NK cells forming synapses with target cells on randomly acquired fields in 3 independent experiments (mean ± SD; n = 100). Abscissa: time in seconds on NK/target cell contact. Ordinate: relative fluorescence. To allow comparison between experiments, fluorescence intensity of a defined area within cytolytic synapse (♦) or contact-free membrane area (■) was normalized to the maximum signal recorded for each individual area. (H) Biochemical quantification of PIP2 during the cytotoxic event. ³²P-radiolabeled primary cultured NK cells were treated with anti-MHCI (ctr) or anti-CD16 mAb and allowed to interact with P815 target cells at 37°C for the indicated times. PIP2 value of control-mAb stimulated sample was assumed as 100%. Data are the mean plus or minus SD of 3 separate experiments.