dendritic spines. Prevention of actin polymerization with latrun- culin-B or cytochalasin-D has been reported previously to eliminate dynamic changes in spine head shape [10]. We suggest that endogenous bI-spectrin in spines maintains the spine head in a large and stable state, in part by stabilizing filamentous actin, and that decreases in bI-spectrin might facilitate actin reorgani- zation and promote dynamic changes in spine size and shape. Functional consequences of expressing the ABD of bI- spectrin The turnover of F-actin in dendritic spines is necessary for postsynaptic reorganization and the translocation of proteins into and out of the synaptic plasma membrane [14,58]. This activity is driven by rapid actin polymerization and depolymerization [59]. For example, inhibition of actin polymerization by latrunculin-A or ADF/cofilin increases the relative rate of AMPAR endocytosis and partially inhibits late-phase LTP [16,22]. Conversely, the activity of glutamate receptors can affect actin turnover [60] by modulating actin polymerization or depolymerization. Indeed, another spectrin superfamily protein, a-actinin-2, regulates not only the morphology of dendritic spines [57], but also the trafficking of AMPARs [61] and the Ca2+-dependent inactivation of NMDARs [62,63]. Larger dendritic spines contain more AMPARs and generate larger responses to glutamate [2,46]. We observed an increase in the size of dendritic spine heads after expressing the ABD of bI- spectrin, as well as an increase in the area occupied by PSD-95 in the spine head. Stabilization of F-actin by the bI-spectrin ABD, which increases the size of spine heads, is likely to account for the increase in PSD size [58]. The association of PSD-95 with F-actin is required to anchor AMPARs at postsynaptic sites [64]. We observed that the amplitudes of AMPAR-mediated mEPSCs were significantly larger in cells expressing the ABD of bI-spectrin. The doubling in PSD-95 area and spine head diameter observed in cells expressing the bI-spectrin ABD was associated with only a ,40% increase in mean mEPSC amplitude, however, indicating that a strict correlation between postsynaptic density size and AMPA receptor number [e.g. 46] is not obligatory under all conditions. Nevertheless, our data represent the first evidence that the actin-binding activity of proteins of the spectrin superfamily can affect postsynaptic sensitivity to released neurotransmitter. Rac3 and the ABD of bI-spectrin How might endogenous bI-spectrin regulate synaptic function? In addition to its ABD, bI-spectrin contains binding sites for calmodulin, protein 4.1, ankyrin, and other proteins [25,65,66]. Protein 4.1 promotes the association of the ABD of spectrin with F-actin [67,68]. The neuronal splice form, 4.1N, interacts with AMPA-type GluR1 subunits and promotes their surface expression [69]. Recruitment of protein 4.1 by the bI-spectrin ABD may therefore promote the insertion of AMPARs into the postsynaptic membrane. Small GTPases such as Rho, Rac and cdc42, promote morphological plasticity by regulating dynamic changes in the actin cytoskeleton. For example, constitutively active Rac1 disorganizes spines [70], whereas dominant-negative Rac1 dis- rupts spines [47,70]. Rac1 activity increases following activation of synaptic NMDARs and promotes the subsequent enlargement of dendritic spines [49]. Karilin-7, a guanine nucleotide exchange factor specific for Rac1, is targeted to dendritic spines, where it enlarges spine heads [71] and potentiates AMPAR-mediated synaptic activity [49]. Less is known about Rac3, although it is highly homologous to Rac1 and is expressed in neurons [51], particularly in the CA1 and CA3 region of the hippocampus during development [50]. Rac3 promotes neurite extension in cultured hippocampal neurons [72] and induces effects on cell The Role of bI-Spectrin in Dendritic Spines differentiation and morphology in neuronal cell lines that are distinct from those caused by Rac1 [73]. Mice in which Rac3 is genetically deleted are viable, learn a hippocampal dependent task normally, and have normal dendritic spine densities, although this may be due to compensation by Rac1 [74,75]. We observed that Rac3 co-immunoprecipitates with spectrin, but not Rac1. Overexpression of wild-type Rac3 increased the sizes of spine heads as effectively as overexpression of the ABD of bI-spectrin. In contrast, co-expression of a dominant-negative form of Rac3 with the ABD of bI-spectrin with inhibited its ability to enhance spine head size. Finally, neurons that overexpress Rac3 had increased mEPSC amplitudes. We therefore suggest that Rac3 activation is promoted in part by the ABD of bI-spectrin and may mediate some of the effects of endogenous spectrin on postsynaptic structure and function. How might Rac3 mediate its effects? Rac3 associates with neurabin and this association is required for its GTPase activity [72]. Furthermore, neurabin binds to actin [76] and this binding is required for its localization in dendritic spines [39]. Overexpres- sion of neurabin increases spine head size [77] and AMPAR expression and triggers actin bundling [78]. It is thus possible that the presence of high levels of bI-spectrin ABD in the spine head leads to spine enlargement and synapse strengthening by recruiting Rac3 and neurabin. Alternatively, neurabin may exert its effects on dendritic spines [39] by binding to actin and, like bI- spectrin, recruiting Rac3. It should be noted that the dominant negative Rac3 construct did not reduce dendritic spine size compared to control cells. Similarly, genetic deletion of Rac3 does not affect dendritic spine density in hippocampal cell cultures [75], although spine size was not quantified. This suggest that the interaction between endogenous bI-spectrin and Rac3 is not necessary to maintain spine head size. This possibility could be tested in future studies in which expression of endogenous bI- spectrin is reduced by targeted siRNA. Our results suggest the testable hypothesis that the ABD of endogenous bI-spectrin and Rac3 can interact in a complex that promotes both activation of Rac3 and polymerization of actin. Changes in the expression of bI-spectrin might thus affect the dynamic state of the actin cytoskeleton in spines, thereby influencing AMPAR trafficking and the balance between synapse plasticity and stability. Supporting Information Figure S1 Sequence alignment of spectrin family ABDs. Although the sequences are identical to b1-spectrin at many residues (indicated by *), and very similar (indicated by :) or homologous at others (indicated by.), there are many differences. Although all share the ability to bind and bundle actin, these regions of difference are presumably responsible for the distinct activities observed in the various experiments. (TIF) Acknowledgments We thank Ms. Wendy Resneck for her expert assistance with immuno- precipitations, Drs. T. Abrams and T. Blanpied for comments on the manuscript, and Dr. T Blanpied and Ms. E. Kong for providing the cortical cell cultures. Portions of these data have been presented to the Society for Neuroscience in 2005. Author Contributions Conceived and designed the experiments: MWN RJB SMT. Performed the experiments: MWN XC. Analyzed the data: MWN. Contributed reagents/ materials/analysis tools: MRS. Wrote the paper: MWN RJB SMT. PLoS ONE | www.plosone.org 13 January 2011 | Volume 6 | Issue 1 | e16197