Fig. 2: NK cell cytoskeleton visualization by expanding α–tubulin and actin in NK cells alone and co–cultured with A. fumigatus.

Conventional CLSM images of NK cells settled on PDL–treated coverslips prior to expansion (a α–tubulin, h actin) and respective ExM images (b, c α–tubulin, i actin) are shown. Note the difference in NK cell size post ~4x expansion. ExM images of NK/A. fumigatus co–cultures are shown in d–g (alpha–tubulin) and j–l (actin) post 3 h (e), 3.5 h (d) and 5.5 h (f, g, j–l) of co–incubation. NK–cell cytoskeleton structures show a prominent re–organization towards A. fumigatus hyphae. Interacting NK cells show MTOC polarization towards A. fumigatus hyphae and accumulation of actin at the site of interaction. In contrast, cells that are not yet interacting with the fungus showed equal distribution of the actin signal on the cell surface. All images represent maximal intensity z–projections of the whole image stack, with the exception of panels g and l, that represent maximal intensity z–projections of only 3 (g slices 19–21) or 4 (l slices 19–22) slices of the samples shown in panels f and k, where interaction of the NK cell with the fungus was most pronounced. Dotted lines indicate borders of NK cells (grey) and fungal hyphae (yellow). Representative images out of 5 (a–h) and 2 (i–l) biological replicates. Scale bars, 10 µm (a–l).