WASP colocalizes with the SCAR complex at the leading edge of neutrophils. Microscopy of HL-60 cells expressing TagRFP-WASP and Hem-1–YFP, a component of the SCAR regulatory complex. (A) TIRF images of live HL-60 cells. Top: WASP localization in three sequential time points, overlaid on the far right (0 s in red, 20 s in green, and 40 s in blue). Middle: same sequence of images but for Hem-1. Bottom: overlay of WASP and Hem-1 at each time point. The plot shows line scans of normalized fluorescence intensity of WASP (red) and Hem-1 (green). The location for generating the line scans is shown in the adjacent image (white line). (B) Two additional examples of live HL-60 cells in TIRF and corresponding line scans (indicated by white lines in the images). (C) Spinning-disk confocal images of two fixed HL-60 cells showing an axial slice through the middle of thick pseudopods. The slices shown were taken 3 µm above the coverslip. See also Fig. S1 (for additional images and kymographs) and Video 1. For all cells, the direction of migration is to the left.