(A, B) Two Jurkat T cells expressing F-tractin-P-tdTom and PAGFP-actin were stimulated on anti-CD3 coated coverslips in 0.5 mM Ca2+, and PAGFP-actin was photoactivated in the ADZ regions indicated in the F-tractin-P-tdTom TIRF images (left, yellow circles) 2 min after perfusion of 0 Ca2+o (A) or 2 mM Ca2+o (B). Incorporation of fluorescent PAGFP-actin is shown as a function of time after photoactivation. The lamella/lamellipod border in 2/0.5 mM Ca2+o and cell edge are indicated by pink dashed lines. Images are from Video 11. Time after photoactivation is in min:sec; scale bar, 5 µm; color scale indicates fluorescence intensity (0–1 a.u.). (C, D) Normalized PAGFP-actin fluorescence intensity (see Materials and methods) along the line indicated (top right) as a function of radial position. The fluorescence profile before photoactivation is shown in black; the color scale applies to subsequent profiles acquired every 1.5 s after photoactivation. The cell edge (red arrowhead), the lamellipod/lamella border (blue arrowhead) and the edge of the ADZ (green arrowhead) are indicated. Data are representative of 12–13 cells. (E) Representative TIRF images of Jurkat cells stimulated on anti-CD3 in 0.5 mM Ca2+then transferred to 0.5 Ca2+ (left) or 0 Ca2+ (right) for 2.5 min, labeled with Alexa-594 phalloidin (top) and anti-WAVE2 (bottom) (see Materials and methods). Ca2+ promotes localization of WAVE2 to the edge of the lamellipod. (F) Average anti-WAVE2 fluorescence in a 1-µm band around the perimeter of the synapse (Fperimeter) relative to the average fluorescence across the whole synapse (Fcell) in 0 and 0.5 mM Ca2+ (n = 63 cells each). Error bars indicate SEM; p-values from Student’s two-tailed t-test. (G) TIRF images of a Jurkat cell expressing F-tractin-P-tdTom (top) and EGFP-Abi1 (bottom) stimulated on anti-CD3 in 0.5 mM Ca2+o (left), 1.5 min after Ca2+o removal (center), and 1.5 min after readdition of 2 mM Ca2+o (right). Ca2+ promotes Abi1 localization to the edge of the lamellipod. Images are taken from Video 12. Scale bar, 5 µm; color scale indicates fluorescence intensity (0–1 a.u.). (H) The average fluorescence of EGFP-Abi1 in a 1-µm band around the perimeter of the synapse (Fperimeter) relative to the average fluorescence across the entire synapse (Fcell) versus time (n = 8 cells). (I) The fluorescence intensity (a.u.) of EGFP-Abi1 along the line indicated (top right, pink) in 0.5 mM Ca2+o (blue) and 1.5 min after Ca2+o removal (red).