The Role of bI-Spectrin in Dendritic Spines Figure 3. The ABD of bI-spectrin prevents actin depolymerization. A, After application of latrunculin (5 mm) for 5 hr, the diameter of the spine head in cells expressing GFP alone decreased significantly (n = 500 spines/5 neurons/group; p,0.05, K-S test), whereas spine length was not changed. B, Latrunculin treatment produced no significant decrease in spine head diameter or spine length in cells co-expressing the ABD of bI- spectrin fused to DsRed and GFP, in contrast. Scale bar = 2 mm for A and B. C, Application of latrunculin-B resulted in a significant decrease in spine density in spines expressing GFP alone (n = 5 neurons/group; p,0.01 ANOVA with Tukey’s test), but not in spines expressing the ABD of bI-spectrin. Spine density was significantly lower in cells expressing the b1 spectrin ABD, as compared to cells expressing GFP alone. Scale bar = 6 mm. doi:10.1371/journal.pone.0016197.g003 The ABD of bI-spectrin stabilizes actin filaments in dendritic spines Differences in the sizes of spine heads correlate with different states of actin polymerization [38]. We further probed the activity of the ABD of bI-spectrin on actin by testing its ability to stabilize actin filaments in spines in the presence of latrunculin-B, which depolymerizes F-actin by preventing addition of G-actin monomers [15,39]. F-actin in spines is significantly depolymerized after 1 hour of latrunculin treatment, with complete depolymerization requiring 4–8 h. This is accompanied by the collapse of the dendritic spine head, known as ‘‘deflation’’ [15,21,39]. In agreement with these results, we found that application of latrunculin-B (6 mM) for 5 h to slice cultures caused a 50% decrease in spine head diameter in cells transfected with GFP alone (GFP+latrunculin = 0.4 mm60.1 mm vs. GFP alone = 0.8 mm60.1 mm; n = 500 spines/5 neurons/group; p,0.05, K-S test)(Fig. 3A). Spine length was not affected significantly, however (1.2 mm60.06 mm control vs. 1.2 mm60.04 mm latruncu- lin; n = 500 spines/5 neurons/group; p.0.05, K-S test). In contrast, application of latrunculin to cells transfected with the ABD of bI- spectrin had no significant effect on either spine head diameter (ABD of bI-spectrin+latrunculin = 2.0 mm60.20 mm vs. ABD of bI-spec- trin = 2.1 mm60.2 mm; n = 500 spines/5 neurons/group; p.0.05, PLoS ONE | www.plosone.org 6 January 2011 | Volume 6 | Issue 1 | e16197