The Role of bI-Spectrin in Dendritic Spines Figure 8. Rac3 is immunoprecipitated with the ABD of bI-spectrin and might mediate its effects on dendritic spines. A, Rac3 is specifically enriched in immunoprecipitates of detergent extracts of cortical cell cultures generated with antibodies to bI-spectrin. The input sample (lane 1) contained easily detected amounts of bI-spectrin and Rac1 but relatively little Rac3. Immunoprecipitation with an anti-bI-spectrin antibody (lane 2) specifically concentrated bI-spectrin and Rac3, but did not precipitate Rac1. Neither GTPase was detected after immunoprecipitation with a non-immune rabbit IgG (lane 3). B, GFP images of CA1 pyramidal cell dendrites co-expressing GFP and the ABD of bI-spectrin either with or without dominant negative (DN) Rac3, and co-expressing GFP with wild type Rac3. Scale bar = 10 mm. C, Pooled data illustrate the effects on the mean spine head diameter, calculated from GFP images (n = 100–300 spines/neuron, 2–5 neurons/group, p,0.001, ANOVA with Sheffe’s test). Spines in cells expressing the ABD of bI-spectrin had larger heads than cells expressing GFP alone, but not when co-expressing dominant negative Rac3. Expression of wild-type Rac3 resulted in spines with large heads, thus mimicking the effects of the ABD of bI-spectrin. doi:10.1371/journal.pone.0016197.g008 elongated dendritic spines and that this required its ABD. These results suggests that different actin-binding proteins from the spectrin superfamily can exert differential structural effects on dendritic spines, but only the ABD of bI-spectrin can enlarge postsynaptic spine heads. The ABD of bI-spectrin also affected the stability of actin filaments within the spine. In control cells, both acute (minutes) and chronic (5 hr) application of latrunculin-B, an inhibitor of actin polymerization, reduced the dimensions of spine heads and caused a loss of GFP-actin from spines, as reported previously [40]. In cells expressing the ABD of bI-spectrin, in contrast, both the latrunculin-induced loss of actin-GFP and deflation of the spine head were inhibited. Thus, the ABD of bI-spectrin reduces the rate at which actin filaments are depolymerized, even when the addition of new actin monomers has been blocked by latrunculin- B. This effect was also specific, as the ABD of a-actinin-2 did not stabilize actin-GFP in spines. These data suggest that some of the effects of expressing these constructs result not from their ability to bind to actin, but perhaps because they recruit other signaling molecules differentially. Consistent with its ability to stabilize actin filaments, the ABD of bI-spectrin significantly inhibited the constitutive morphing of PLoS ONE | www.plosone.org 12 January 2011 | Volume 6 | Issue 1 | e16197