Figure 6.
Active FMNL2 increases fascin movement into filopodia. (A) Representative confocal images of HeLa cells coexpressing RFP-fascin (red) and FMNL2-GFP (WT and active; ΔDAD), FMNL3-GFP, or DAAM1-GFP (green). Scale bar = 5 µm. (B and C) Analysis of fascin recovery by FRAP in filopodia (one ROI per cell/filopodia) in cells coexpressing specified formins. Fascin t1/2 values (B) and relative mobility (C; normalized to mean fascin only) extracted from monoexponential single FRAP curve fitting. One-way ANOVA; *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001, data from at least three independent experiments are shown, n = 101 (fascin only), 31 (+FMNL2), 27 (+FMNL2ΔDAD Nt), 33 (+FMNL3), 13 (+FMNL3 act), 16 (+DAAM1), and 14 (+DAAM1ΔDAD) cells. (D) FLIM-FRET analysis of HeLa cells expressing RFP-fascin and FMNL2-GFP, FMNL3-GFP, or GFP-DAAM1. Intensity images of GFP fluorescence (top panel) and FLIM data in pseudocolor (bottom panel) are shown. Warm colors show high FRET (low GFP lifetime), and cool colors indicate low FRET (high lifetime) GFP served as donor-only control and was used for calculation of FRET efficiency shown in E. Scale = 5 µm. (E) Calculated FRET efficiency (relative to GFP only) for interaction between fascin and FMNL2, FMNL3, and DAAM1 (n = 26, 35, and 11 cells). One-way ANOVA; ***, P ≤ 0.001. (F) Representative example image of fascin and active FMNL2 (FMNL2ΔDAD) FRET. Scale bar = 2 µm. (G) Interaction of FMNL2 with WT fascin, fascin-S39A (phospho-dead), or fascin-S39D (phospho-mimic) measured by FLIM-FRET in HeLa cells. FRET efficiency was calculated based on FMNL2-GFP only (donor-only control). Shown are data from three independent experiments (n = 26, 29, and 16 cells, respectively). One-way ANOVA; *, P ≤ 0.05; **, P ≤ 0.01. (H) Interaction of FMNL2 and fascin before or after disassembly of F-actin with Cytochalasin D measured by FLIM-FRET. Two-way ANOVA; ***, P ≤ 0.001; ****, P ≤ 0.0001, n = 10 and 15 cells for FMNL2 only (+ and – CytD), 20 and 28 cells for FMNL2 + fascin (+ and – CytD) from three independent experiments. Scale bar, 5 μm.

Active FMNL2 increases fascin movement into filopodia. (A) Representative confocal images of HeLa cells coexpressing RFP-fascin (red) and FMNL2-GFP (WT and active; ΔDAD), FMNL3-GFP, or DAAM1-GFP (green). Scale bar = 5 µm. (B and C) Analysis of fascin recovery by FRAP in filopodia (one ROI per cell/filopodia) in cells coexpressing specified formins. Fascin t1/2 values (B) and relative mobility (C; normalized to mean fascin only) extracted from monoexponential single FRAP curve fitting. One-way ANOVA; *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001, data from at least three independent experiments are shown, n = 101 (fascin only), 31 (+FMNL2), 27 (+FMNL2ΔDAD Nt), 33 (+FMNL3), 13 (+FMNL3 act), 16 (+DAAM1), and 14 (+DAAM1ΔDAD) cells. (D) FLIM-FRET analysis of HeLa cells expressing RFP-fascin and FMNL2-GFP, FMNL3-GFP, or GFP-DAAM1. Intensity images of GFP fluorescence (top panel) and FLIM data in pseudocolor (bottom panel) are shown. Warm colors show high FRET (low GFP lifetime), and cool colors indicate low FRET (high lifetime) GFP served as donor-only control and was used for calculation of FRET efficiency shown in E. Scale = 5 µm. (E) Calculated FRET efficiency (relative to GFP only) for interaction between fascin and FMNL2, FMNL3, and DAAM1 (n = 26, 35, and 11 cells). One-way ANOVA; ***, P ≤ 0.001. (F) Representative example image of fascin and active FMNL2 (FMNL2ΔDAD) FRET. Scale bar = 2 µm. (G) Interaction of FMNL2 with WT fascin, fascin-S39A (phospho-dead), or fascin-S39D (phospho-mimic) measured by FLIM-FRET in HeLa cells. FRET efficiency was calculated based on FMNL2-GFP only (donor-only control). Shown are data from three independent experiments (n = 26, 29, and 16 cells, respectively). One-way ANOVA; *, P ≤ 0.05; **, P ≤ 0.01. (H) Interaction of FMNL2 and fascin before or after disassembly of F-actin with Cytochalasin D measured by FLIM-FRET. Two-way ANOVA; ***, P ≤ 0.001; ****, P ≤ 0.0001, n = 10 and 15 cells for FMNL2 only (+ and – CytD), 20 and 28 cells for FMNL2 + fascin (+ and – CytD) from three independent experiments. Scale bar, 5 μm.

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