F-actin governs MC dynamics but has different velocity distribution across the IS. (A) Kymographs were generated along the diameter of cells expressing GFP-actin and GFP–SLP-76 and sharpened in Photoshop as in Fig. S1 B. The arrowhead along the time axis indicates the addition of 1 µM JAS. Arrows in the kymograph of F-actin dynamics point to a feature that became immobilized upon addition of JAS. Asterisks in the kymograph of SLP-76 dynamics indicate an MC that was immobilized by addition of JAS. (B) Jurkat T cells expressing GFP–SLP-76 were pretreated with either vehicle control or Y-27632 and allowed to spread on OKT3-coated coverglasses. Velocities of SLP-76 MCs were analyzed by kymography (mean ± SD, n = 40–150 MCs from 10–19 cells). (C) Maximum-over-time (MOT) images of GFP–SLP-76 Jurkat T cells were compiled from the images acquired before or after the addition of JAS. Images are representative of three independent experiments. Bar, 10 µm. (D) Comparative analysis of SLP-76 MC centripetal velocity from a single cell overlaid with F-actin dynamics from Fig. 1 E. Mean ± SEM is shown.