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. 2018 Feb 19;28(4):489–502.e9. doi: 10.1016/j.cub.2017.12.044

Figure 1.

Figure 1

Mature NK Cell Lytic Synapse Is Defined by a Pervasive F-Actin Network

(A) Representative frames from NK92.LifeAct-mEmerald stained with LysoTracker red and seeded on the indicated antibody-coated glass surface and imaged by live confocal microscopy. Scale bars, 5 μm.

(B and C) Spreading speed measured from initial cell contact (B) until the cell footprint reaches its plateau value reported (C). N = 37, 21, and 35 cells, respectively, per condition from 3, 4, and 4 independent experiments. The p value was calculated by one-way ANOVA Kruskal-Wallis test (Dunn’s) in (B) and ordinary one-way ANOVA with Tukey’s post hoc comparison in (C). n.s., not significant.

(D) Overlay of cell outlines throughout 30 min for each substrate condition (top row); the representative cells are from (A). Each outline was used to define a 2-μm-thick inner rim where the fluorescence intensity (F.I.) of the LifeAct-mEmerald probe was reported in the kymographs below. The small black arrowheads highlight the presence of the actin-rich lamellipodium. Scale bar, 10 μm.

(E) Z projection of a 3D volume from live confocal microscopy of HeLa target cells and NK92.LifeAct-mEmerald effectors (see Movie S1). The IS at the interface between target and effector cells is highlighted in yellow. Two representative ISs (red and green insets) are shown en face after a 90° rotation along the x axis (i.e., y axis projection). Representative image of 3 independent repeats. Scale bar, 10 μm.

(F) Single frames from live TIRF-SIM microscopy showing the continuous presence of actin fibers throughout the formation and maturation of the IS (from 2 to 5 min following initial contact with the glass). The color scheme of the images within the white square has been inverted, filtered and magnified below to allow the visualization of the dimmest structure while retaining the linearity of the fluorescence signal (see Movie S2). Representative images of 43 cells from 6 experiments are shown. Scale bar, 5 μm (top) and 1 μm (bottom).

(G) Frequency of NK92.LAMP1-pHluorin cells undergoing degranulation detected by TIRF microscopy at the times indicated. N = 60, 67, and 64 cells, respectively, per condition from 3 independent experiments.