The Role of bI-Spectrin in Dendritic Spines Figure 1. Actin binding domains from various spectrin superfamily proteins differentially bundle actin and localize to spines in CA1 pyramidal neurons. Two-dimensional projections of confocal images of neurons expressing GFP (A) or the ABDs of different proteins fused to DsRed (B–F). (B) Neurons expressing the ABD of bI-spectrin show actin bundling in the cell soma and in dendrites. The ABD of utrophin (C) also bundles actin in the soma. Neurons expressing the ABD of actinin (D) did not display actin bundling in the soma, dendrites, or spines (Scale right = 25 mm, Scale left = 100 mm). E, Merged image of a dendrite from a CA1 pyramidal neuron co-expressing actin-GFP and the ABD of bI-spectrin fused to DsRed. The arrow indicates a bundled actin filament in the dendrite. Images at the right show the co-localization of actin and the ABD of bI- spectrin in spine heads (scale bar = 1 mM). F, Co-expression of the ABDs of a-actinin, utrophin, dystrophin, or filamin, all fused to DsRed, and GFP in CA1 pyramidal cells demonstrate their differential targeting to dendritic spines (E, F Scale bar = 10 mm). doi:10.1371/journal.pone.0016197.g001 but significant, decrease in average spine density as compared to GFP transfected neurons (n = 5 neurons/group p,0.01, ANOVA and Tukey’s test)(Fig. 3C). Only the ABD of bI-spectrin affected dendritic spines. Dendritic spine heads in cells expressing GFP plus the ABDs of a-actinin-2, dystrophin, utrophin and filamin were not significantly larger than spines in cells expressing GFP alone (n = 5 neurons/group p.0.05, ANOVA and Tukey’s test)(Fig. 2C). Because all of these constructs bind actin, some other action of the bI-spectrin ABD must be responsible for the enlargement of the dendritic spine head. PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e16197