Figure 3.
Local F-actin organization regulates fascin movement into and out of filopodia. (A) Filopodia of mEOS2-fascin–expressing HeLa cells were photoconverted, and exit of photoconverted fascin was monitored over time using a confocal microscope at a 1-s frame rate. Upper panel, control cells (DMSO treated cells); middle panel, formin inhibition (SMIFH2-treated cells); bottom panel, Arp 2/3 complex inhibition (CK666-treated cells). Shown are pseudocolored ratios of photoconverted/unconverted intensities before (first column, −1 s) and after (shown are every 5 s) conversion. Scale bar = 5 µm. (B) Fascin movement out of filopodia. Photoconversion intensities plotted versus time for control and SMIFH2- and CK666-treated cells. Early and late phases were compared using two-way ANOVA with uncorrected Fisher’s posttest; shown are data (n = 15, 24, or 17 cells, one ROI per cell, and two to four filopodia per ROI) from three independent experiments; *, P ≤ 0.05; **, P ≤ 0.01; ns, not significant. (C) Fascin t1/2 values and mobility versus immobility (in %) extracted from monoexponential single-curve photoconversion fitting (n = 15, 24, or 17 cells, one ROI per cell, and two to four filopodia per ROI). One-way ANOVA; *, P ≤ 0.05; **, P ≤ 0.01. (D) Fascin movement into filopodia. Filopodia of GFP-fascin–expressing HeLa were bleached (FRAP), and recovery intensities were plotted over time for control and SMIFH2- and CK666-treated cells. Early and late phases were compared using two-way ANOVA with uncorrected Fisher’s posttest; shown are data (n = 18, 21, or 17 cells) from three independent experiments; *, P ≤ 0.05; **, P ≤ 0.01. (E) Fascin t1/2 values and mobile versus immobile fractions extracted from monoexponential single-curve FRAP fitting (n = 18, 21, or 17 cells). One-way ANOVA; *, P ≤ 0.05; ****, P ≤ 0.0001. (F) Small regions within single filopodia were bleached in control and SMIFH2- and CK666-treated cells and displayed as pseudocolored kymographs (space vs. time). Arrowheads point to the base or the tip of the filopodium. Scale bar, 1 μm. (G) Analysis of kymographs shown in F. Relative base to tip (left) and tip to base (right) speeds are shown. Data are from three independent experiments (n = 18, 21, or 17 single filopodia from ≥15 individual cells per condition), one-way ANOVA; *, P ≤ 0.05; ***, P ≤ 0.001. (H) Interaction of GFP-fascin and RFP-actin measured in live cells by multiconfocal FLIM-FRET imaging. Examples of control cells (top panel) and SMIFH2 treated cells (bottom panel) are shown. Intensity images on the left and pseudocolored FLIM image on the right. Warm colors indicate FRET (low lifetime) and cool colors indicate no FRET (high lifetime). Scale bar = 2 µm. (I) Mean FRET efficiency (left, n = 16) and lifetime Lowess residuals (right, n = 28) of fascin–actin interaction of single filopodia measured by live FLIM in cells with and without SMIFH2 treatment. Student’s t test; *, P ≤ 0.05.

Local F-actin organization regulates fascin movement into and out of filopodia. (A) Filopodia of mEOS2-fascin–expressing HeLa cells were photoconverted, and exit of photoconverted fascin was monitored over time using a confocal microscope at a 1-s frame rate. Upper panel, control cells (DMSO treated cells); middle panel, formin inhibition (SMIFH2-treated cells); bottom panel, Arp 2/3 complex inhibition (CK666-treated cells). Shown are pseudocolored ratios of photoconverted/unconverted intensities before (first column, −1 s) and after (shown are every 5 s) conversion. Scale bar = 5 µm. (B) Fascin movement out of filopodia. Photoconversion intensities plotted versus time for control and SMIFH2- and CK666-treated cells. Early and late phases were compared using two-way ANOVA with uncorrected Fisher’s posttest; shown are data (n = 15, 24, or 17 cells, one ROI per cell, and two to four filopodia per ROI) from three independent experiments; *, P ≤ 0.05; **, P ≤ 0.01; ns, not significant. (C) Fascin t1/2 values and mobility versus immobility (in %) extracted from monoexponential single-curve photoconversion fitting (n = 15, 24, or 17 cells, one ROI per cell, and two to four filopodia per ROI). One-way ANOVA; *, P ≤ 0.05; **, P ≤ 0.01. (D) Fascin movement into filopodia. Filopodia of GFP-fascin–expressing HeLa were bleached (FRAP), and recovery intensities were plotted over time for control and SMIFH2- and CK666-treated cells. Early and late phases were compared using two-way ANOVA with uncorrected Fisher’s posttest; shown are data (n = 18, 21, or 17 cells) from three independent experiments; *, P ≤ 0.05; **, P ≤ 0.01. (E) Fascin t1/2 values and mobile versus immobile fractions extracted from monoexponential single-curve FRAP fitting (n = 18, 21, or 17 cells). One-way ANOVA; *, P ≤ 0.05; ****, P ≤ 0.0001. (F) Small regions within single filopodia were bleached in control and SMIFH2- and CK666-treated cells and displayed as pseudocolored kymographs (space vs. time). Arrowheads point to the base or the tip of the filopodium. Scale bar, 1 μm. (G) Analysis of kymographs shown in F. Relative base to tip (left) and tip to base (right) speeds are shown. Data are from three independent experiments (n = 18, 21, or 17 single filopodia from ≥15 individual cells per condition), one-way ANOVA; *, P ≤ 0.05; ***, P ≤ 0.001. (H) Interaction of GFP-fascin and RFP-actin measured in live cells by multiconfocal FLIM-FRET imaging. Examples of control cells (top panel) and SMIFH2 treated cells (bottom panel) are shown. Intensity images on the left and pseudocolored FLIM image on the right. Warm colors indicate FRET (low lifetime) and cool colors indicate no FRET (high lifetime). Scale bar = 2 µm. (I) Mean FRET efficiency (left, n = 16) and lifetime Lowess residuals (right, n = 28) of fascin–actin interaction of single filopodia measured by live FLIM in cells with and without SMIFH2 treatment. Student’s t test; *, P ≤ 0.05.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal