Figure 1.
Fascin–actin interactions are highly dynamic and spatially regulated. (A) Photoconverted mEOS2-fascin in fascin knockdown cells imaged by confocal microscopy. Top panel shows surface plot visualization (intensity in x,y). Cell border in preconverted frame is highlighted in gray, and photoconversion region, in purple (conversion ROI contained multiple filopodia per cell). Bottom panel shows the respective cell and the intensity of the photoconverted channel (red). Inset picture shows the unconverted cell (green channel). One representative time lapse is shown, scale bar = 10 µm. Arrowheads depict photoconverted fascin in filopodia outside of photoconversion ROI. (B) Representative relative intensities of photoconverted and unconverted regions over time (black and purple, respectively; 4-s frame rate). Peak intensity values (overall maximum intensity) are depicted with arrows. (C) Fascin half-life (t1/2 in seconds, left) showing the time needed for 50% of fascin molecules to exit filopodia after photoconversion and fascin mobile and immobile fractions (right) are shown. Values were extracted from monoexponential curves of photoconverted areas. n = 9 cells (ROIs with multiple filopodia). (D) Percentage of fast moving fascin and t1/2 of fast- and slow-moving fascin. Values were extracted from biexponential curves of photoconverted areas. n = 9 cells (ROIs with multiple filopodia). (E) Small area photobleaching (FRAP) along single filopodium of GFP-fascin expressing cells. Bottom panel shows representative pseudocolored kymograph of fascin recovery. Scale bar, 2 μm. (F) Single molecules of mEOS2-fascin along filopodia (top panel, filopodia borders are outlined as dashed lines, 15 ms frame rate, scale bar = 0.5 µm). Bottom graph: Multiple single-molecule tracks of fascin along the filopodial shaft. The base is located at xy = 0. (G) Unbound fascin fraction estimated by single-molecule tracking analysis as described in Supplemental Methods; average single-molecule tracks of n = 15 filopodia. (H) Interaction of GFP-fascin and RFP-actin measured with multiconfocal FLIM-FRET microscope. Left, intensity image; right, FLIM-FRET data. High interactions are red (low lifetime), whereas low interactions are blue (high lifetime). Scale bar = 2 µm. (I) Fascin–actin interaction measured over time in single filopodium of living cell in 2D with live FLIM-FRET at a 3-s frame rate. Lifetime values are plotted over time (circles) with linear regression (solid line) and 95% confidence interval (dotted line). (J) Fluctuation range of fascin–actin interaction measurement by live FLIM-FRET in different cellular compartments. Lowess residuals were calculated as positive or negative residual value of lifetime data at a certain time point in respect to the fitted Lowess curve of lifetime data. Lowess residuals are plotted as minimum to maximum floating bar graphs for cytoplasm, lamellipodia, and filopodia measurements. The line represents the median. n = 28 cells. (K) FRET efficiency data of GFP-fascin interaction with RFP-actin obtained for the base and the tip of filopodia. n = 29 filopodia, Student’s t test; **, P ≤ 0.01. (L) Fascin–actin interaction at the base versus the tip in growing, shrinking, or stable filopodia measured by live FLIM-FRET. *, P ≤ 0.05. n = 16, 15, or 17 filopodia from two independent experiments. (M) Comparison of live FLIM-FRET measurements of fascin–actin interaction at the base and the tip of actively growing, shrinking, or stable filopodia. Fold change over time (e.g., fold FRET change between the beginning and the end of the growth phase) is shown. n = 16, 14, or 16 filopodia from one of two independent experiments.

Fascin–actin interactions are highly dynamic and spatially regulated. (A) Photoconverted mEOS2-fascin in fascin knockdown cells imaged by confocal microscopy. Top panel shows surface plot visualization (intensity in x,y). Cell border in preconverted frame is highlighted in gray, and photoconversion region, in purple (conversion ROI contained multiple filopodia per cell). Bottom panel shows the respective cell and the intensity of the photoconverted channel (red). Inset picture shows the unconverted cell (green channel). One representative time lapse is shown, scale bar = 10 µm. Arrowheads depict photoconverted fascin in filopodia outside of photoconversion ROI. (B) Representative relative intensities of photoconverted and unconverted regions over time (black and purple, respectively; 4-s frame rate). Peak intensity values (overall maximum intensity) are depicted with arrows. (C) Fascin half-life (t1/2 in seconds, left) showing the time needed for 50% of fascin molecules to exit filopodia after photoconversion and fascin mobile and immobile fractions (right) are shown. Values were extracted from monoexponential curves of photoconverted areas. n = 9 cells (ROIs with multiple filopodia). (D) Percentage of fast moving fascin and t1/2 of fast- and slow-moving fascin. Values were extracted from biexponential curves of photoconverted areas. n = 9 cells (ROIs with multiple filopodia). (E) Small area photobleaching (FRAP) along single filopodium of GFP-fascin expressing cells. Bottom panel shows representative pseudocolored kymograph of fascin recovery. Scale bar, 2 μm. (F) Single molecules of mEOS2-fascin along filopodia (top panel, filopodia borders are outlined as dashed lines, 15 ms frame rate, scale bar = 0.5 µm). Bottom graph: Multiple single-molecule tracks of fascin along the filopodial shaft. The base is located at xy = 0. (G) Unbound fascin fraction estimated by single-molecule tracking analysis as described in Supplemental Methods; average single-molecule tracks of n = 15 filopodia. (H) Interaction of GFP-fascin and RFP-actin measured with multiconfocal FLIM-FRET microscope. Left, intensity image; right, FLIM-FRET data. High interactions are red (low lifetime), whereas low interactions are blue (high lifetime). Scale bar = 2 µm. (I) Fascin–actin interaction measured over time in single filopodium of living cell in 2D with live FLIM-FRET at a 3-s frame rate. Lifetime values are plotted over time (circles) with linear regression (solid line) and 95% confidence interval (dotted line). (J) Fluctuation range of fascin–actin interaction measurement by live FLIM-FRET in different cellular compartments. Lowess residuals were calculated as positive or negative residual value of lifetime data at a certain time point in respect to the fitted Lowess curve of lifetime data. Lowess residuals are plotted as minimum to maximum floating bar graphs for cytoplasm, lamellipodia, and filopodia measurements. The line represents the median. n = 28 cells. (K) FRET efficiency data of GFP-fascin interaction with RFP-actin obtained for the base and the tip of filopodia. n = 29 filopodia, Student’s t test; **, P ≤ 0.01. (L) Fascin–actin interaction at the base versus the tip in growing, shrinking, or stable filopodia measured by live FLIM-FRET. *, P ≤ 0.05. n = 16, 15, or 17 filopodia from two independent experiments. (M) Comparison of live FLIM-FRET measurements of fascin–actin interaction at the base and the tip of actively growing, shrinking, or stable filopodia. Fold change over time (e.g., fold FRET change between the beginning and the end of the growth phase) is shown. n = 16, 14, or 16 filopodia from one of two independent experiments.

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