Accumulation of F-actin at the NK cell–macrophage IS. (A) NK cells and autologous macrophages were coincubated, fixed, and then stained with anti–MHC class II mAb (red) and with Alexa Fluor 488–conjugated phalloidin (green) to mark the location of F-actin. Analysis of the fluorescence intensity around the cell membrane demonstrates a 3-fold increase in the amount of F-actin, but not MHC class II protein, at the synapse. (B) The organization of F-actin at the IS was often ring-shaped as shown. Bar chart shows the frequency of a clearly ring-shaped distribution of F-actin at the IS between NK cells and unstimulated macrophages or macrophages^{high LPS}. (C) The percent of NK cell–macrophage conjugates where F-actin clearly accumulated at the IS was scored and then compared when each cell type was pretreated with cytochalasin D or sodium azide. (D) The percent of NK cell–macrophages^{high LPS} conjugates where F-actin clearly accumulated at the IS was scored and then compared when each cell type was pretreated with cytochalasin D or sodium azide. The means ± SD from 3 independent experiments are shown with the total number of cells indicated above each bar. Scale bars, 10 μm.