Tracking of F-actin dynamics in T cells spreading on stimulatory coverglasses. Human T lymphoblasts were transduced with Lifeact-GFP (gray) and allowed to interact with coverglasses coated with anti-CD3 and ICAM-1 (both unlabeled). Confocal 0.5-µm-thick Z stacks were acquired by confocal microscopy using a spinning disk confocal microscope (Ultraview Perkin Elmer on an Axiovert 200 stand Carl Zeiss) every 2 s for 2 min and are shown as extended projections. Left panel shows a representative movie sequence. Right panel shows a kymograph generated along the blue line on the left. The place in the time sequence is indicated by the blue line moving along the kymograph. Red lines highlight traceable F-actin features as they undergo retrograde transport to the IS center. Actin flow rates in Figs. 3, 6, and S5 were calculated from the slopes of such lines.
