The Role of bI-Spectrin in Dendritic Spines Figure 7. The ABD of bI-spectrin enhances single spine AMPAR-mediated synaptic responses. A, mEPSCs from an untransfected control cell (upper trace) and a cell transfected with the ABD of bI-spectrin (lower trace) were recorded at 270 mV in the presence of TTX (1 mm). Average sized single mEPSCs recorded from the untransfected cell (black) and transfected cell (gray) are shown superimposed at right. B, Group data showing that neurons expressing the ABD of bI-spectrin had significantly greater mEPSC amplitudes, compared to untransfected controls or cells expressing GFP alone, suggesting an increase in AMPARs at these synapses (n = 7 neurons/group; p,0.05, K-S test). The increase in mEPSC amplitude produced by the spectrin ABD was mimicked in cells expressing CA Rac3, in which mEPSC amplitude was also larger than in controls or GFP expressing cells (p,0.05, K-S test). C, The frequency of mEPSCs in transfected neurons decreased significantly, suggesting fewer functional synapses (n = 7 neurons/ group;* = p,0.05, t-test). doi:10.1371/journal.pone.0016197.g007 detected in dendritic spines, but its functions there remain unknown. In this study, we expressed the actin binding domain of bI- spectrin and related spectrin superfamily proteins, an approach used successfully as a first step in revealing the function of the full- length proteins, such as a-actinin [30]. Our assumption is that the exogenous ABD construct will mimic the function of the N- terminal ABD of the full length protein. We cannot exclude, however, that overexpression of the ABD of bI-spectrin may negatively affect endogenous spectrin or induce other downstream compensatory effects [30]. Indeed, a full characterization of spectrin requires complimentary approaches, such as knock-down of endogenous spectrin and overexpression of the full length protein, that are beyond the scope of the current study. Nevertheless, spectrin is a prominent substrate of the Ca2+- activated protease calpain and activation of calpain may result in the creation of fragments not unlike those we have expressed. Interactions between bI-spectrin and actin Although all of the proteins of the spectrin superfamily and related proteins share highly homologous ABDs, they are all unique at many sites (Fig. S1). Indeed, we found a surprising heterogeneity in the way these ABDs were targeted in CA1 pyramidal cells. The ABDs of bI-spectrin and utrophin, but not dystrophin, colocalized with GFP-tagged actin and bundled actin filaments in the cell body and proximal dendrites. The ABDs of bI-spectrin, a-actinin-2 and filamin, but not utrophin and dystrophin, were concentrated with actin in the heads of dendritic spines. Our results with the ABD of a-actinin-2 are in contrast to those of Nakagawa et al. [30], who reported that the ABD of a- actinin-2 did not localize to spines, although comparable regions of the protein were used in both studies. Even more unique to the ABD of bI-spectrin was its ability to enlarge the spine head. None of the other ABDs we assayed produced this effect. Nakagawa et al. [30] observed that overexpression of full length a-actinin-2 PLoS ONE | www.plosone.org 11 January 2011 | Volume 6 | Issue 1 | e16197