Sites of actin filament polymerization in dendritic filopodia. (A and B) The free actin filament barbed ends in mouse hippocampal neurons were visualized with fluorescently labeled actin monomers (middle panels). F-actin was stained with fluorescently labeled phalloidin (left panels). Right panels show merged images. The cell in A was fixed at DIV 16 and the cell in B was fixed at DIV 12. (C) The number of sites (percentage of total amount) of barbed ends in different locations (tip, root, or tip + root) was counted from 76 filopodia from four independent experiments (DIV 12–16). Data are presented as mean ± SEM. (D and E) Mouse cortical neurons were transfected with a construct expressing GFP–β-actin at DIV 8, and the FRAP assay was performed at DIV 9. GFP-actin was bleached from total filopodium (red), and the appearance of nonbleached actin (green) was followed by time-lapse imaging. In D, GFP-actin fluorescence recovers (actin filaments polymerize) from the filopodium tip; in E, recovery occurs mainly from the root. Bars, 1 µm.