Fig. 3

Roles of wild-type or phosphomutant fascin 2b proteins in the formation of long filopodia.

Schematics of proteins expressed in COS-7 cells and corresponding confocal micrographs are displayed: GFP-WT fascin 2b (A,E,I), GFP-espin (B,F,J), GFP (C,G,K), GFP-WT fascin 2b and espin (D,H,L), GFP-S38A fascin 2b (Q,T,W), GFP-S38E fascin 2b (R,U,X), or GFP-S38D fascin 2b (S,V,Y). Confocal images are of representative live cells. Filopodia are observable in cells expressing GFP-WT fascin 2b (E,I), GFP-espin (F,J), or GFP-S38A fascin 2b (T,W). None are apparent in cells in which only GFP was introduced, as exhibited by this representative cell (G,K). Cells expressing GFP-S38E fascin 2b (U,X) or GFP-S38D fascin 2b (V,Y) have no filopodia visible by confocal microscopy. Cells that express both GFP-WT fascin 2b and espin have longer cytoplasmic protrusions (H,L,Z) than cells that express GFP-WT fascin 2b or GFP-espin separately (E,F). Scanning electron micrographs of cells expressing GFP-WT fascin 2b (M), GFP-espin (N), or GFP-WT fascin 2b and espin (P) show results similar to those of cells observed using confocal microscopy, except that diminutive filopodia are seen on the surfaces of cells that express the GFP control, as shown in image (O). The mean lengths ± SEM of the cellular protrusions are shown for cells expressing GFP-espin (N_filopodia = 400), GFP-WT fascin 2b (N_filopodia = 130), or GFP-WT fascin 2b together with espin (N_filopodia = 330), and they indicate synergism between fascin 2b and espin (Z). Asterisk indicates P<0.0001 for Student′s t test. Scale bars of confocal images and electron micrographs are 5 μm and 1 μm, respectively.