Distribution and dynamics of the T cell actomyosin network. (A) mKate2–NMHC II-A was transiently expressed in Jurkat T cells stably expressing GFP-actin. The cells were dropped on OKT3-coated coverglasses and imaged at a single focal plane every 3 s (also see Video 1). An image from a sequence is shown and is representative of four independent experiments. Bar, 5 µm. (B) The intensity profile of F-actin and myosin IIA was acquired along the dashed line in A. (C) A composite kymograph of actomyosin dynamics was acquired along the dashed line in A. Bar, 5 µm. (D) Jurkat T cells stably expressing GFP–SLP-76 (green) were stimulated as in A, fixed after 5 min, and stained for F-actin with phalloidin (red). Z stacks of whole cells were collected with a 0.25-µm step size and deconvolved using a calculated Point Spread Function in Volocity. A representative cell is shown in XZ and XY planes. Brackets represent 5 µm × 5 µm. (E) Kymographic analysis of F-actin features was compiled into a single graph to show the distribution of F-actin velocity across the IS radius. Mean ± SD is shown for each point (n = 13 cells). Similar results were obtained from three independent experiments.