Formins regulate fascin movement into and out of filopodia in 2D. (A) Representative Western blots showing specific silencing of FMNL2, FMNL3, or DAAM1 using siRNA (left) and morphology of control and knockdown cells (right). Scale bar = 10 µm. (B) Analysis of mEOS2-fascin movement out of filopodia in formin silenced cells. Fascin t_1/2 values and mobile versus immobile fractions from monoexponential single-curve fitting (n = 53, 12, 8, or 11 cells, one ROI per cell and one to two filopodia per ROI). One- and two-way ANOVA; *, P ≤ 0.05; ****, P ≤ 0.0001. (C) FRAP of filopodia to analyze GFP-fascin movement into filopodia in formin-silenced cells. Fascin t_1/2 values and mobile versus immobile fractions extracted from monoexponential single-curve FRAP fitting (n = 34, 30, 26, or 24 cells). One- and two-way ANOVA; *, P ≤ 0.05; **, P ≤ 0.01. (D) Small regions within single filopodia were bleached in control and formin knockdown cells and displayed as pseudocolored kymographs (space vs. time). Arrowhead depicts the base or the tip of the filopodium. Scale bar = 1 µm. (E) Analysis of kymographs shown in D. Relative base to tip (left) and tip to base (right) speeds are shown from three independent experiments (n = 25, 23, or 23), one-way ANOVA; **, P ≤ 0.01; ns, not significant. (F) Morphology of control, FMNL2, and FMNL3 knockdown cells in soft 3D collagen matrix imaged using LLSM. Scale bar = 10 µm. (G) mEOS2-fascin photoconversion and monitoring over time in cells in 3D matrices. Data from monoexponential (fascin t_1/2 and mobility) analysis of photoconversion experiments. n = 23 cells (siCTR), 22 cells (siFMNL2), and 25 cells (siFMNL3), 5–10 filopodia per cell, one- and two-way ANOVA.