Fig. 1. Microtubules target filopodia in B16F1 melanoma cells. Fixed cells were immunostained with monoclonal anti-tubulin antibodies followed by Cy5-labeled secondary antibodies and Alexa Fluor-488-phalloidin. (A) Combined image of microtubules (green) and F-actin (red) shows a typical lamellipodium with embedded filopodia. A line connecting the bases of all filopodia (baseline) defines the peripheral region of the lamellipodium. (B) Enlarged image of upper region (boxed in A) of lamellipodium with three peripheral microtubules. One microtubule is targeted to a filopodium (+) and two are not targeted (-). (C) Enlarged image of lower region with two peripheral microtubules targeted to filopodia (+). (D) Lamellipodia were divided into five equal zones as indicated in the inset image. Zone 1 is the lamellipodium center and zones 3 are the lamellipodium wings. Zone 2 is the transition between the lamellipodium center and wings. The relative number of targeting events, peripheral microtubules and filopodia for each zone is plotted. Bar, 2 µm (A-C). Error bars indicate standard error (^*P<0.01, unpaired t-test, n=28 cells).