Nascent adhesions form underneath F-actin bundles and colocalize with maximal PI3K signaling. (A) TIRF montages of mCherry-fascin (grayscale) and EGFP-paxillin (pseudocolored) coexpressed in a NIH3T3 cell, along with the overlay of the two channels (red, fascin; green, paxillin; see also Video 5). Bar, 10 µm. The sequence is representative of nine cells viewed and shows the formation of adhesions along the fascin-containing actin bundle during protrusion. (B) TIRF montages of mCherry-AktPH and EGFP-paxillin (both pseudocolored) coexpressed in a NIH 3T3 cell, along with the overlay of the two channels (red, AktPH; green, paxillin; see also Video 6). Bar, 5 µm. The sequence is representative of 13 cells viewed and shows the emergence of PI3K signaling colocalized with nascent adhesions formed along the bundle. (C) TIRF images of FP-AktPH–expressing NIH 3T3 cells, plated on either fibronectin or poly-lysine, before and after FAK inhibition (Inhib; 10 µM FAK inhibitor II; see also Video 7). Bars, 20 µm. For each cell, the after/before ratio compares the difference between the mean intensity of the morphological extension with the highest intensity and the mean intensity of the center region of the contact area; a value significantly <1 indicates that the pattern became more uniform after FAK inhibition. Values are reported as means ± 95% confidence interval for the fibronectin (n = 19) and poly-lysine (n = 24) data.