Modulation of fascin-1 expression tunes cell morphology. (A) TIRF montage (inverted grayscale) of a branching event in a NIH 3T3 cell expressing EGFP-fascin to mark F-actin bundles (left; see also Video 3), representative of 31 cells in 13 independent experiments (see Fig. S2 A for another example). The corresponding protrusion/retraction and lamellipod/filopod overlap maps are shown at right. (B) Lamellipod/filopod overlap analysis as shown in Fig 1 B; here, both putative filopodia and submembranous bundles labeled by FP-fascin were included in the analysis. (C) Representative TIRF images of NIH 3T3 cells expressing EGFP-AktPH or coexpressing mCherry AktPH and EGFP-fascin are shown. The bar graph shows the mean numbers of morphological extensions for the control (blue, n = 152) and fascin-overexpressing (red, n = 113) cells; error bars show 95% confidence intervals. The dashed line indicates that two extensions is the de facto minimum. The histogram shows the corresponding distributions. OE, overexpression. (D) NIH 3T3 cells were depleted of fascin-1 by two targeting shRNAs, expressed separately or in combination. The immunoblot confirms shRNA-mediated loss of fascin-1 expression levels relative to a nontargeting control shRNA, with GAPDH as a loading control. Representative TIRF images of these cells as well as of cells in which fascin-1 expression was rescued are shown. The bar graph shows the mean number of extensions quantified for each population of cells (n ≥ 140 for each condition); error bars show 95% confidence intervals. The dashed line indicates that two extensions is the de facto minimum. Bars: (A) 10 µm; (C and D) 20 µm.