Fig 4.

Openings in the cortical actin meshwork at the immunologic synapse are not permissive for enlarged granules in LYST-deficient NK cells. A and B, EV (Fig 4, A) or LYST CRISPR NK92mi (Fig 4, B) cells forming contacts on cover slips coated with ligands for lymphocyte function–associated antigen 1 (LFA-1) and NKG2D, stained with anti-perforin antibody (red) and phalloidin (green), and visualized with STED microscopy. The images show F-actin meshwork and lytic granules proximal to the plasma membrane (contact site). Scale bars = 5 μm. Openings among actin filaments at the synapse are shown as heat maps. Close-up images show the area of the synapse containing lytic granules (white rectangles in the leftmost images). Scale bars = 2 μm. Y-Z plane projections show the position of lytic granules relative to the actin filaments. The relationship between F-actin and lytic granules is illustrated by optical sections (Z1-Z4) acquired sequentially every 220 nm and progressively distal to the contact site. C, Quantification of the average size of actin meshwork openings at the synapse center for cells stimulated as in Fig 4, A and B. D, Quantification of the proportion of the actin meshwork at the synapse area predicted to be penetrable by 200- to 800-nm-diameter vesicles, for the same cells as Fig 4, C. Graphs represent means, and error bars indicate SDs (n = 18 cells per condition from 3 experiments). There was no significant difference in actin opening sizes or actin meshwork permeability between EV and LYST CRIRPS cells. MIP, Maximum intensity projection.