The myosin IIA tailpiece and S1943 phosphorylation are required for its interaction with lytic granules. (A) Confocal fluorescent micrographs of YTS cells expressing wild-type myosin IIA-GFP (MyoIIA-GFP), 1933x myosin IIA-GFP (1933x-GFP), or S1943A myosin IIA-GFP (S1943A-GFP) conjugated to 721.221 target cells (721) Differential interference contrast (DIC, left) in addition to fluorescent signal for actin (red), perforin (blue), or GFP-myosin (green) are shown, along with an overlay of fluorescent channels (right). Scale bar indicates 5 μm. (B) Percentage of total perforin fluorescent volume colocalized with GFP volume ± SD; > 20 cells per condition were analyzed. (C) Western blot of isolated lytic granules from resting YTS cells expressing wild-type myosin IIA-GFP (MIIA), 1933x myosin IIA-GFP (1933x), or S1943A myosin IIA-GFP (S1943A). GFP, myosin IIA, and granzyme B (GzmB) blots are shown. Gel was loaded for equal granule protein content to facilitate comparison of GFP-myosin quantity on lytic granules. Numbers below blots indicate the ratio of the GFP, and the myosin band intensities to the total intensity for granzyme B normalized to the ratio for MIIA. Results shown are representative of 3 independent assays.