Single molecules of myosin IIA associate with NK-cell lytic granules. Lytic granules isolated from resting YTS cells were evaluated by platinum rotary shadowing electron microscopy (A). Whole resting YTS cells or lytic granules isolated from resting YTS cells were evaluated by STED microscopy (B-E). (A) YTS cell lytic granule with emanating structures resembling myosin IIA, which are highlighted in color in the image at right. Scale bar indicates 200 nm. (B) YTS cell isolated lytic granule visualized with an antibody against the myosin IIA tailpiece (green, left). (C) Lytic granule within fixed YTS cell visualized with anti-perforin (red, confocal) and anti-myosin (green, STED) antibodies. The distance between myosin IIA antibody molecules was measured using a line drawn around the periphery of the lytic granules (B-C, white, right). Scale bar indicates 500 nm. (D-E) Normalized intensity plots along the line drawn at the periphery of the lytic granules shown in panels B-C. Values are normalized such that the maximum intensity in the plot is equal to 1. Numbers at the top indicate the distance between adjacent antibody molecules. Results are representative of > 10 (A), 3 (B,D), or > 30 (C,E) images. (F) Proposed model of myosin IIA interaction with NK-cell lytic granules. Unphosphorylated myosin IIA tailpiece obscures the binding site for lytic granules (highlighted in turquoise). Myosin IIA tailpiece phosphorylated at S1943 bends backward onto the rod, revealing the putative granule binding site. 1933x myosin IIA is missing a large portion of the granule-binding site, and therefore cannot sustain an interaction with granules under force. S1943A myosin IIA has an intact tailpiece, but cannot be phosphorylated and therefore cannot reveal its putative granule-binding site. Myosin molecules containing one 1933x and one WT myosin are able to bind to granules with a weaker interaction, whereas molecules containing 1 S1943A and 1 wild-type myosin are sterically hindered from binding.