Fig. 5. Quantification of the relative locations of tropomyosin, F-actin, and the Arp2/3 complex at the leading edge. Cells analyzed were double-labeled for tropomyosin (LC24) and F-actin (rhodamine-phalloidin, A,B,C,J,K), tropomyosin (f9d) and barbed ends (biotin-labeled G-actin, D,E,F), and tropomyosin (LC24) and the Arp2/3 complex (anti-p34, G,H,I) as in Figs 2,3,4. For panels A-I, cells were either unstimulated (A,D,G), or stimulated with EGF for 50 seconds (B,E,H) or 3 minutes (C,F,I). The fluorescence intensity (arbitrary units) is the mean of the cell perimeter in regions of the lamellipodium for 0.22 µm wide steps (see Materials and Methods). Quantification is shown within 1 µm of the cell membrane. After EGF stimulation, the F-actin, barbed ends and the Arp2/3 complex distribution peak within 0.5 µm of the membrane, while tropomyosin increases deeper in the lamellipodium. In panel J, quantification of F-actin is shown up to 4 µm beyond the cell membrane without EGF stimulation, 50 seconds and 3 minutes after EGF stimulation. Regions corresponding to the leading edge and the base of the lamellipodium are indicated. In panel K, quantification of tropomyosin (LC24) is shown up to 4 µm beyond the cell membrane without EGF stimulation, 50 seconds and 3 minutes after EGF stimulation. Error bars indicate standard error, with n=15 (A,B,C,J,K) and n=25 (D,E,F,G,H,I).

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