Fig. 2. Tropomyosin is absent from regions of the leading edge that contain a dense F-actin network. Cells were either unstimulated (A-D,M), or stimulated with EGF for 50 seconds (E-H) or 3 minutes (I-L,O). Phalloidin-stained F-actin is shown in panels A, E, and I and tropomyosin (antibody LC24) in panels B, F, and J. Double-labeling overlays are shown (C,G,K; green, F-actin; red, tropomyosin), as well as phase contrast images (D,H,L). Panel M shows double-labeling of unstimulated cells and panel O shows double labeling of cells stimulated with EGF for 3 minutes. In panels M and O, the white square indicates an area that is shown at higher magnification in the panels below (panel N and P, from left to right: double labeling, F-actin, tropomyosin). The size of the square is 6 µm by 6 µm. The scale bar in panel L indicates 10 µm and applies to panels A-L. The scale bar in panel M also indicates 10 µm and applies to panels M and O. Arrows indicate regions at the leading edge that show F-actin staining but no tropomyosin. Arrowheads indicate labeling of actin stress fibers by both rhodamine-phalloidin and LC24 (tropomyosin). The diffuse, non-stress fiber actin network in the base of the lamellipodium is indicated by an asterisk and contains both F-actin and tropomyosin.