Mst1 regulates Myosin IIa localization and is required for partitioning of low and higher affinity LFA-1 molecules.

<p><b>A</b>) Wt and <i>Mst1^h/h</i> CD4 T cells expressing Myosin IIa-GFP were seeded into slide chambers pre-coated with 1 μg/mL ICAM-1-Fc and stimulated with CCL19 prior to fixation and staining of F-actin with Rhodamine-phalloidin. Three-dimensional image reconstructions from z-stacks of confocal micrographs are displayed. <b>B</b>) Wt and <i>Mst1^h/h</i> CD4 T cells expressing Myosin IIa-GFP were visualized by live TIRF microscopy. Arrows indicate bipolar morphology. Data are representative of 2 individual experiments with 150 cells per genotype. <b>C, D</b>) Wt and <i>Mst1^h/h</i> CD4 T cells were stimulated as above and stained with 2D7 (anti-low affinity CD11a/LFA-1, green) and M17/4 (anti-CD11a/LFA-1, red). <b>E</b>) Wt CD4 T cells stimulated as above with or without Blebbistatin treatment were stained with 2D7 and visualized by immunofluorescence. <b>F</b>) Quantification of cells with 2D7 localization at the trailing edge of untreated or Blebbistatin-treated Wt and Mst1^h/h CD4 T cells (data are representative of 2–3 individual experiments, n = 13–23).