Identification of the sites of actin filament polymerization in spine heads. (A–C) The free actin filament barbed ends in mouse hippocampal neurons (DIV 12) were visualized with fluorescently labeled actin monomers (middle panels, barbed ends are indicated with arrowheads). F-actin was stained with fluorescently labeled phalloidin (left panels). Right panels show merged pictures. Barbed ends localized either as dots (A and C) or a “line” (B) at the spine head surface or to the root of the neck (C). (D) The number of sites (percentage of total spines analyzed) of barbed ends in different locations (tip of spine head [head tip] or tip of spine head + root of the neck [h. tip + n. root]) were counted from 128 spines from four independent experiments (DIV 12–16). The graph represents mean ± SEM. (E) Mouse hippocampal neurons were transfected with GFP-actin at DIV 20, and the FRAP assay was performed at DIV 21. GFP-actin was bleached from the spine head, and recovery of the GFP-actin fluorescence was followed by time-lapse imaging. The fluorescence of GFP-actin recovers mainly from the spine head tip. First sites of recovery are indicated with arrowheads. Bars, 1 µm.