FIGURE 4.

Fascin‐K471 acetylation regulates filopodia dynamics by modulating its bundling to F‐actin. A, The distribution of actin and the mean filopodial length and density (filopodium numbers per μm of cell perimeter; at least 50 cells were measured) were analyzed in KYSE150‐shNC and KYSE150‐shFSCN1 cells expressing LifeAct‐RFP. B, Fascin and F‐actin distribution and the mean filopodial length and density in KYSE150‐shFSCN1‐#1 cells that co‐expressed different types of Fascin‐GFP (WT, K471R, or K471Q) along with LifeAct‐RFP (at least 50 cells were measured). GFP and RFP fluorescence signals are shown in green and magenta, respectively. C and D, Representative confocal images of cell filopodia (arrowheads) during their lifetime (right; C). The lifetime of filopodia during one extension and retraction cycle was quantified (D; n ≥ 100 filopodia in at least 20 cells). E and F, FRAP assays were used to analyze the diffusion of different types of Fascin‐GFP (WT, K471R, or K471Q) and LifeAct‐RFP at the filopodia base of cells. GFP (green) and RFP (magenta) fluorescence signals of cell filopodia were obtained for an overview (left) with time‐lapse sequence images of the boxed region in one filopodium before and after photo‐bleaching (right; E). The recovery profile is curve‐fit by a double exponential function with recovery constant K_inflow and K_outflow for intra‐filopodia exchange and filopodia‐cytoplasmic exchange, respectively (F; n = 30). G and H, F‐actin bundling activity of Fascin and its mutant forms was analyzed by Western blotting. The pellet (P) and supernatant (S) represent F‐actin and G‐actin, respectively. Bar charts show the densitometric G‐actin/F‐actin ratio. Data were obtained from three independent experiments and are presented as the mean ± standard error of the mean (SEM). Student's t‐test was used for statistical analysis (*P < 0.05; **P < 0.01). Abbreviations: GFP: Green fluorescent protein; RFP: Red fluorescent protein; sh: short hairpin; NC: negative control; FRAP: fluorescence recovery after photobleaching; P: pellet; S: supernatant; SEM: standard error of the mean