Figure S3.
Disturbing the organization of F-actin alters filopodia and fascin movement. (A) Filopodia length in micrometers (left) and filopodia lifetime in seconds (right) of fascin knockdown HeLa cells expressing GFP-fascin. n = 22 cells, three independent experiments. (B) Filopodia length (normalized to DMSO control) and coverage (relative filopodia number/perimeter) was evaluated after long-time (left, n = 22, 28, or 25 cells) or short-time (right, n = 51, 28, or 22 cells) treatment with SMIFH2 and CK666. One-way ANOVA; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, not significant. (C) Percentage of fast-moving fascin was extracted from filopodia photoconversion data from HeLa cells treated with DMSO, SMIFH2, or CK666. Shown are data from three independent experiments (n = 16, 18, or 11 filopodia respectively), one-way ANOVA. (D) Morphology of GFP-fascin–expressing HeLa cells embedded in soft 3D ECM after inhibition of formins (SMIFH2) and Arp2/3 complex (CK666). Images were captured using LLSM. Scale bar = 10 µm. (E) Inhibition of formins decreases pulling forces on ECM. Velocity magnitude of cell-induced bead displacement in 3D collagen matrix without (n = 220 bead velocity vectors) and with inhibitor treatment. Analysis was performed as described in Fig. S2, G and H (SMIFH2, n = 140; CK666, n = 120; Jasplakinolide, n = 137; beads only, n = 60). **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (F) Evaluation of fascin-S39A speed in comparison to WT fascin by FRAP. Filopodia were bleached in RFP-fascin WT (black) and RFP-fascin-S39A (green) expressing fascin knockdown HeLa cells, and FRAP intensities were determined over time. Representative of three independent experiments. n = 17 filopodia, two-way ANOVA with Bonferroni posttest; **, P ≤ 0.01. (G) Photoconversion of mEOS2-fascin in HeLa cells on 2D was performed to compare fascin recovery in lamellipodia and filopodia. n = 3 ROI measurements per cell area from 18 cells from three experiments. (H) Retention of fascin in lamellipodia is dependent on F-actin density. Small lamellipodial regions of DMSO control (black) and CK666-treated (magenta) GFP-fascin–expressing HeLa cells were photobleached, and FRAP was analyzed over time; n = 10 from one of three experiments. (I–K) Characterization of the CS-fascin. Filopodia length (I) and number (filopodia/perimeter; J) were measured for GFP-fascin–expressing cells and CS-fascin expressing cells. CS-fascin (tagged with both GFP and mScarlet) or GFP-fascin and mScarlet-fascin (K) were coexpressed in fascin knockdown HeLa cells; cells were fixed, and lifetime was measured by FLIM-FRET. Shown are data from two independent experiments (n = 15, 16, or 12 cells). One-way ANOVA with Bonferroni posttest; ***, P ≤ 0.001; ****, P ≤ 0.0001. (L) CS-fascin–expressing cells were treated with the Arp2/3 inhibitor CK666 and imaged on a confocal microscope to measure ratiometric FRET. Bar graph shows FRET/donor (in %) for control (n = 40 cells) and CK666 treatment (n = 15 cells). One-way ANOVA with uncorrected Fisher posttest. (M) FRET signal of HeLa cells expressing CS-fascin from time binned datasets. Binning of time-lapse images shows increased FRET signal of CS-fascin both in filopodia and at the base of filopodia after combining 50 time-lapse frames. High FRET is shown in yellow/green, and low FRET is shown in dark blue/black. Imaging was performed by SIM; representative example is shown; scale bar = 5 µm. (N) Representative image of the GFP-fascin donor-only control for 3D FRET experiment shows the lack of filopodia and no FRET signal. Scale bar = 2 µm.

Disturbing the organization of F-actin alters filopodia and fascin movement. (A) Filopodia length in micrometers (left) and filopodia lifetime in seconds (right) of fascin knockdown HeLa cells expressing GFP-fascin. n = 22 cells, three independent experiments. (B) Filopodia length (normalized to DMSO control) and coverage (relative filopodia number/perimeter) was evaluated after long-time (left, n = 22, 28, or 25 cells) or short-time (right, n = 51, 28, or 22 cells) treatment with SMIFH2 and CK666. One-way ANOVA; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, not significant. (C) Percentage of fast-moving fascin was extracted from filopodia photoconversion data from HeLa cells treated with DMSO, SMIFH2, or CK666. Shown are data from three independent experiments (n = 16, 18, or 11 filopodia respectively), one-way ANOVA. (D) Morphology of GFP-fascin–expressing HeLa cells embedded in soft 3D ECM after inhibition of formins (SMIFH2) and Arp2/3 complex (CK666). Images were captured using LLSM. Scale bar = 10 µm. (E) Inhibition of formins decreases pulling forces on ECM. Velocity magnitude of cell-induced bead displacement in 3D collagen matrix without (n = 220 bead velocity vectors) and with inhibitor treatment. Analysis was performed as described in Fig. S2, G and H (SMIFH2, n = 140; CK666, n = 120; Jasplakinolide, n = 137; beads only, n = 60). **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (F) Evaluation of fascin-S39A speed in comparison to WT fascin by FRAP. Filopodia were bleached in RFP-fascin WT (black) and RFP-fascin-S39A (green) expressing fascin knockdown HeLa cells, and FRAP intensities were determined over time. Representative of three independent experiments. n = 17 filopodia, two-way ANOVA with Bonferroni posttest; **, P ≤ 0.01. (G) Photoconversion of mEOS2-fascin in HeLa cells on 2D was performed to compare fascin recovery in lamellipodia and filopodia. n = 3 ROI measurements per cell area from 18 cells from three experiments. (H) Retention of fascin in lamellipodia is dependent on F-actin density. Small lamellipodial regions of DMSO control (black) and CK666-treated (magenta) GFP-fascin–expressing HeLa cells were photobleached, and FRAP was analyzed over time; n = 10 from one of three experiments. (I–K) Characterization of the CS-fascin. Filopodia length (I) and number (filopodia/perimeter; J) were measured for GFP-fascin–expressing cells and CS-fascin expressing cells. CS-fascin (tagged with both GFP and mScarlet) or GFP-fascin and mScarlet-fascin (K) were coexpressed in fascin knockdown HeLa cells; cells were fixed, and lifetime was measured by FLIM-FRET. Shown are data from two independent experiments (n = 15, 16, or 12 cells). One-way ANOVA with Bonferroni posttest; ***, P ≤ 0.001; ****, P ≤ 0.0001. (L) CS-fascin–expressing cells were treated with the Arp2/3 inhibitor CK666 and imaged on a confocal microscope to measure ratiometric FRET. Bar graph shows FRET/donor (in %) for control (n = 40 cells) and CK666 treatment (n = 15 cells). One-way ANOVA with uncorrected Fisher posttest. (M) FRET signal of HeLa cells expressing CS-fascin from time binned datasets. Binning of time-lapse images shows increased FRET signal of CS-fascin both in filopodia and at the base of filopodia after combining 50 time-lapse frames. High FRET is shown in yellow/green, and low FRET is shown in dark blue/black. Imaging was performed by SIM; representative example is shown; scale bar = 5 µm. (N) Representative image of the GFP-fascin donor-only control for 3D FRET experiment shows the lack of filopodia and no FRET signal. Scale bar = 2 µm.

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