Figure 2.

MyH9 mediates LFA-1 de-adhesion at the uropod. (A) Human T lymphocytes were transfected with MyH9-GFP or β-actin–GFP, and cell migration on ICAM-1 and CXCL-12 was analyzed by time-lapse fluorescence microscopy at 37°C (see also Video S5 for MyH9-GFP and Video S6 for β-actin–GFP). Arrows mark the direction of migration. Two-dimensional images and three-dimensional histograms of fluorescence intensity and cell surface distribution are shown in a pseudo-color scale (from low [black] to high [red]). Bar, 20 μm. (B and C) Human T lymphocytes were pretreated with DMSO or 50 μM blebbistatin and allowed to migrate on ICAM-1– and CXCL-12–coated cover glass for 20 min at 37°C (B). The polarization index of cells was calculated as the ratio of x to y, where x is the longest distance across cells (from head to tail) and y is the greatest width perpendicular to x (C). Bar, 25 μm (B). (D) T lymphocytes were pretreated with DMSO or 50 μM blebbistatin for 2 h and stimulated with CXCL-12 for 20 min. LFA-1 immunoprecipitates (TS2/4 antibody) were then obtained and subjected to silver staining. (E and F) Human T lymphocytes were pretreated with DMSO or 50 μM blebbistatin for 1 h at 37°C, and cell migration on ICAM-1/CXCL-12–coated cover glasses was tracked over a 30-min period. Each line represents one cell. Experiments were repeated on T lymphocyte preparations from three independent donors.