EAT-2 enhances granule polarization and exocytosis. (A) YT-S cells and CellTrace violet–loaded K562 cells were stained with phalloidin (actin), anti-perforin, and anti-tubulin. Conjugates were then analyzed by confocal microscopy. Individual stainings for a representative conjugate are shown. Scale bars are shown in B. Shown is a representative of 4 experiments. (B) Stained conjugates were categorized into different stages to describe progression of cytotoxicity, as detailed in the Methods section. Conjugates representative of each stage are shown. Bars, 5 µm. (C) YT-S cells expressing GFP alone or in combination with WT EAT-2 or tyrosine 127-to-phenylalanine 127 (Y127F) EAT-2 were incubated for 10 min with K562 cells expressing CD48. They were then stained as described for A and percentages of YT-S cells (NK cells) at each stage of cytotoxicity were determined. At least 100 conjugates were analyzed for each cell type. A bar graph representation of one experiment is shown on the left. A correlation coefficient (r) analysis between YT-S GFP cells and YT-S cells expressing WT or Y127F EAT-2 for 2 independent experiments is shown on the right. Mean values with standard deviations are shown. Shown is a representative of 2 experiments. (D) Data from C were corrected after assuming that the reduced conjugate formation in cells expressing EAT-2 observed in Fig. 9 B occurred only for stage 0 and 1 conjugates. Mean values with error bars and standard deviations of four independent experiments are shown. *, P < 0.05. (E) This experiment was performed as detailed for C, except that cells were preincubated with BAPTA-AM (BAPTA), PD98059, or both. Cells exposed to DMSO alone were used as control. Shown is a representative of 2 independent experiments. (F) Splenocytes from poly I:C–primed mice were incubated in the presence of RMA-S and tested by flow cytometry for their ability to up-regulate CD107a expression at the surface. Unstimulated cells (Unst) or cells stimulated with phorbol myristate acetate and ionomycin (P/I) were used as controls. NK cells were identified by gating on NK1.1⁺ TCR-β⁻ cells. Representative dot plots are shown on the left, whereas a statistical analysis of three experiments is depicted on the right. On the left, percentages of CD107a⁺ cells are shown. On the right, values for unstimulated cells are subtracted from those of stimulated cells. Mean values with standard deviations are shown. *, P ≤ 0.02. Shown is a representative of 3 independent experiments. EAT-2 KO, EAT-2–deficient mice; EAT-2 KI, mice expressing Y120,127F EAT-2.