Figure 4.

Foci Are Present at Sites of Local Actin Rearrangement

(A and B) Representative NK92.LifeAct-mEmerald cell activated (A) (inverted color from Figure 3B) for 20 min on anti-CD18 and anti-NKp30 prior to imaging by TIRF-SIM including F-actin foci that undergo dynamic behaviors highlighted by the orange arrowheads in the time series in (B) (see Movie S6). Colored tracks indicate their displacement over 95 s. Scale bars, 5 μm (A) and 1 μm (B).

(C) Quantification of the lifetime of the actin foci. Data are pooled from 20 cell averages at 42 s ± 43.3 s. Additionally, a small proportion (3.06%) of all actin foci measured have a turnover longer than the acquisition of the time lapse (>3 min).

(D) Quantification of the number of F-actin-rich foci in NK92 cells activated for 10 min prior to administration of 50 μM CK666 for an additional 20 min. Images were acquired by time-gated STED microscopy, and the number of F-actin foci present at the synapse were measured using image segmentation. N = 10 and 13 cells, respectively, from 1 experiment representative of 4 experiments. The p value was calculated by Mann-Whitney unpaired two-tailed test.

(E) Representative live-cell imaging experiment using TIRF-SIM where NK92.LifeAct-mEmerald cells have been plated on an antibody-coated surface and imaged through the treatment and washout of 50 μM CK666. The actin foci are sensitive to inhibition of CK666 but recover shortly after washout of the compound (see Movie S7). The cell is representative of 12 cells from 4 independent repeats. Scale bar, 5 μm.