Mutation of the conserved serine residue of hfascin affects filopodia dynamics and impairs its actin-bundling in vitro. (A) Similar mutations were generated in mRFP-tagged human fascin (hfascin; purple) and expressed in MDA-MB-231 cells that were depleted of endogenous fascin. Double-headed arrows highlight filopodia examined by line scan analysis in B. (B) High magnification of filopodia highlighted in A and line scan analysis show different distribution of hfascin transgenes within filopodia compared with actin (LifeAct-GFP). (A and B) Bars, 1 µm. (C) Quantification of mean filopodia length and number during transgene expression. *, P < 0.05; **, P < 0.01. See also Video 6. Error bars show means ± SD. (D) Time course of filopodia growth over 5 min (from the kymograph in Fig. S1 F) reveals that phosphomutations on residue S274 alter filopodial dynamics. (E and F) Actin-bundling (E) and actin-binding (F) cosedimentation assays show that S274A/D mutations decreased actin-bundling/binding properties of fascin in vitro. In the bundling assay, note the reduction of actin in the pellet of fascin S39D and S289A/D (red squares). In the actin-binding assay, note the reduction of these fascin mutants in the pellet (red squares). SN, supernatant; P, pellet. (G) Transmission electron micrographs reveal that S274 mutations prevent the formation of organized bundles. mag., magnification; WT, wild type.