F-actin bundles/filopodia harbor PI3K signaling. (A) Montage of PI3K signaling (pseudocolored) and fascin-1 localization (inverted grayscale) during protrusion over actin bundles in an NIH 3T3 cell (see also Video 4), representative of 31 cells viewed. Bar, 10 µm. (B) NIH 3T3 cells coexpressing mCherry-AktPH and TFP as a volume marker were used for ratiometric analysis. Mean enrichment ratios (means ± 95% confidence interval) of PI3K signaling in filopodia (left) and the associated histograms (right) for untreated cells (blue, n = 724 filopodia) versus cells treated with 1 µM PI3K-α inhibitor IV (red, n = 119 filopodia) are shown. (C, left) Montage of mCherry-AktPH/TFP ratio (pseudocolored) showing PI3K signaling enrichment preceding lamellipodial protrusion over an actin bundle. Bar, 5 µm. The percentage of instances in which PI3K signaling first increased in filopodia was determined for each cell (right; n = 27 cells, 194 protrusions total over actin bundles). The red dot indicates the mean ± 95% confidence interval across cells.