Activation via CD16 triggers the assembly of a cytolytic kinapse. (A) NK cells were incubated for 5 min on slides coated with rituximab (Rtx) or MICA, both with ICAM-1, or ICAM-1 alone (−) and then fixed. Panels show representative confocal images of F-actin stained with Alexa Fluor 488–labeled phalloidin. (B) Cells were scored according to their F-actin distribution; dense symmetrical rings (gray) accumulated asymmetrically at the leading edge (yellow) or more evenly distributed across the interface (green). n = 3 independent experiments. (C) IRM live-cell imaging of the contact between NK cells and glass slides coated with rituximab or MICA, both with ICAM-1. An overlay of all 360 frames (6 min at one frame per second) is shown colored according to time. Bars, 20 µm. (D and E) Surface area (D) and circularity (E) of cells was analyzed. Circularity values approaching 1 indicate a more circular shape, whereas 0 indicates an elongated shape (n = 3 independent experiments; mean ± SEM). (F) NK cell motility on activating surfaces. NK cells were labeled with Calcein and allowed to settle on surfaces coated with ICAM-1 alone (−), rituximab, or MICA, both with ICAM-1 ±1 µM TAPI-0 (where indicated). Images were acquired every 30 s for 45 min, and cells were tracked using Cell Tracker, a MATLAB plugin. n = 20 individual cell tracks from one representative donor. Axes are ±300 µm. (G) Speed of NK cells on differently coated surfaces. Each point represents the average speed of an individual cell from a representative donor (median ± IQR). (H) Mean NK cell speed (n = 4 different donors; mean ± SEM). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 calculated by Kruskal-Wallis test (G) or one-way ANOVA (H). See also Fig. S5 and Videos 1 and 2.