Myosin IIA is not required for F-actin flow but is necessary for long-term maintenance of the IS. (A) GFP-actin–expressing Jurkat T cells were pretreated for 15 min with vehicle or 25 µM Y-27632 and imaged while spreading on anti-CD3–coated coverslips. Kymographs were generated along the radii of fully spread cells. The arrowhead indicates the time when photobleaching of lamellipodia was induced. Brackets represent 2 µm × 15 s. (B, top) Analysis of velocities calculated from kymographs as in A (mean ± SD, n = 15 cells). (bottom) To verify inhibitory activity of Y-27632, Jurkat T cells were pretreated with Y-27632 as in A and stimulated with OKT3 (1 µg/ml) for 5 min. Cells were lysed and analyzed by Western blot analysis with antibodies to phosphorylated MLC (pMLC) or GAPDH. (C) Jurkat T cells were transiently transfected with F-tractin tdTomato and pretreated with 50 µM blebbistatin (Bleb.) or vehicle for 30 min. Actin retrograde flow was analyzed as in B. Values are mean ± SD of 80–90 kymographs from 14–20 cells. (D) GFP-actin–expressing Jurkat T cells were transfected with oligonucleotides specific for myosin IIA heavy chain (SiM) or control oligonucleotides (SiC) and cultured for 48 h, at which time suppression was found to be optimal. (top) Retrograde flow was analyzed as in B. (bottom) Lysates were analyzed by Western blotting to assess efficiency of suppression. Values represent relative NMHC II-A levels, normalized to GAPDH. (E) Jurkat T cells were untreated or pretreated with 25 µM Y-27632 or 50 µM blebbistatin and allowed to spread for the indicated times on anti-CD3–coated coverslips before fixation and labeling with phalloidin and anti–NMHC II-A. Bar, 5 µm. (F) Morphometric analysis of cells prepared as in E. Data represent mean ± SEM (n = 67–125 cells per condition). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Similar results were obtained in two independent experiments.