experimenter blinded to the condition. Spine head width was defined as the maximum dimension of the spine head, whereas spine length was defined as the distance from the base of the spine neck to the tip of the spine head. Spine density was calculated by manually counting the number of spines expressed over a 30 mm segment of dendrite. For all measurements of spine density or dimensions, at least 2 well isolated apical dendrites were used per neuron. In experiments involving dynamic imaging, these same analyses were performed on images taken from living cells in unfixed cultures collected using an epifluorescence wide-field microscope (Nikon; 60X/0.8 NA, water immersion objective) and a CCD camera (Hamamatsu Orca ER; effective pixel si- 2 ze = 0.012 mm ). Photodamage was minimized by adding Trolox (0.2 mM) to the saline. For post-hoc analysis of time-lapse image sequences, spine heads were traced manually and the length and width were computed. This method underestimates the morphological dynamics of spines because the measurements are only taken in x and y planes [36]. Images were captured every second for 3 min and binned into 10 s intervals. Morphological dynamics were measured by calculating the coefficient of variation of the spine head diameter within each 10 s interval. Out-of-focus images were removed from the analyses. Fluorescence intensity was calculated after subtract- ing the average background intensity of two regions of interest surrounding the dendritic spine. To measure PSD-95 area, slice cultures were co-transfected biolistically with GFP fused to PSD-95 and DsRed fused to the bI- spectrin ABD. PSD-95 - GFP was a gift from Dr. T. A. Blanpied, and was constructed by subcloning PSD-95 at HindIII-EcoR1 into N1 versions of the fusion protein expression constructs. After 24– 48 h, slices were placed in a standard recording chamber filled with control saline and wide-field images were taken, as above. After background subtraction, each cluster of PSD-95-GFP was traced manually and an area was calculated using SimplePCI software. Analyses of dendritic spine dimensions and PSD-95-GFP area were performed by comparing the cumulative probability distributions with the Kolmogorov-Smirnov test. Otherwise, unpaired t-tests were used to establish significance. When comparing more than two groups, ANOVA was used with a Tukey’s or Scheffe’s post-hoc analysis to establish significance unless otherwise noted. All values are presented as mean values 6 standard error of the mean. Immunoprecipitation Cultures of cortical neurons were washed with buffered saline with complete protease inhibitors (Roche Applied Science, Indianapolis, IN), placed in 0.32 M sucrose in the same solution, and scraped. After brief centrifugation, the cellular pellet was mixed with an equal volume of two-fold concentrated RIPA buffer to obtain the following final concentrations of detergents: 1% NP- 40, 0.5% deoxycholate, 0.1% SDS. Extracts were triturated in the cold, briefly, and stored on ice. Aliquots (70 ml) of Dynabeads (Invitrogen, Carlsbad, CA), pre-coated with sheep anti-rabbit IgG, in buffered saline were bound with 6 mg affinity-purified rabbit antibodies to bI-spectrin or with purified non-immune rabbit IgG and washed. The bead pellet was mixed with 1 mg of protein of the cellular extract and mixed gently overnight. The immunopre- cipitates were collected, washed briefly in buffered saline containing 0.5% Tween, solubilized with SDS-PAGE sample buffer, and subjected to SDS-PAGE, followed by electro-transfer to nitrocellulose. Blots were incubated with antibodies to bI- spectrin, Rac1, or Rac3. Bound antibody was detected with peroxidase-conjugated secondary antibodies (Roche Diagnostics, The Role of bI-Spectrin in Dendritic Spines Indianapolis, IN; Amersham Biosciences, England) and the SuperSignal West Femto Kit (Thermo Scientific, Waltham, MA). Samples of the cell extract were run in parallel, to mark the positions of the proteins of interest. Affinity-purified rabbit antibodies to bI-spectrin have been described [37]. Antibodies to Rac1 were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to Rac3 were the generous gift of Dr. I. De Curtis (San Raffaele Scientific Inst., Milan, Italy). Results The ABD of bI-spectrin induces actin bundling in CA1 pyramidal neurons To investigate the interaction between spectrin family proteins and the actin cytoskeleton, we created constructs of the highly homologous ABDs of bI-spectrin, a-actinin-2, utrophin, dystro- phin, and filamin (supporting information Fig. S1), all fused to the fluorophore DsRed, and introduced them biolistically into CA1 pyramidal cells in hippocampal slice cultures. We co-transfected with a GFP plasmid in most experiments to fill the cells and allow visualization of dendrites and dendritic spines (Fig. 1A), and quantified spine shape and size from images of GFP distribution. The DsRed-tagged bI-spectrin ABD labeled filamentous structures in the cell body and apical and basal dendrites (n = 12)(Fig. 1B). We observed a distribution of the ABD of bI- spectrin that was similar to what has been reported for endogenous spectrin in hippocampal pyramidal neurons [29]. Co-transfection of GFP-tagged actin revealed that these structures were bundles of filamentous actin, which were not seen with expression of actin- GFP alone, suggesting that the ABD of bI-spectrin associates with actin filaments, promoting their formation and inducing bundling. The ABD of bI-spectrin, either alone or together with actin-GFP, also labeled spine heads in apical and basal dendrites (Fig. 1E). Expression of the ABD of other members of spectrin superfamily tagged with DsRed gave different results. Expression of the ABD of utrophin resulted in actin bundling in the neuronal cell bodies, as well as apical and basal dendrites (n = 12)(Fig. 1C), but minimal labeling of dendritic spines (Fig. 1F). Expression of the ABDs of actinin-2 (n = 6) and filamin (n = 7), in contrast, produced diffuse labeling of the soma and principal dendrites (Fig. 1D), without actin bundling, as well as prominent labeling of dendritic spines (Fig. 1F). The ABD of dystrophin (n = 12) produced no actin bundling in the cell bodies or dendrites and did not label spines (Fig. 1F). Co-transfection of GFP with the utrophin and dystrophin constructs revealed that spines were present, but unlabeled by their ABDs. We observed that the expression level for all of the ABD constructs were similar based on the distribution and fluorescence intensity of each ABD. These results demonstrate that the ABDs of spectrin superfamily members caused distinct effects on actin filaments and were targeted differentially to dendritic spines. Although these ABDs are highly homologous, their effects are therefore likely to be mediated by more than mere binding to actin. The ABD of bI-spectrin enlarges dendritic spine heads Endogenous bI-spectrin is expressed at high levels in the heads of postsynaptic spine [29]. Analysis of confocal images of GFP- filled dendritic spines revealed that the heads of dendritic spines co-expressing the ABD of bI-spectrin were more than twice as large as spines expressing GFP alone (n = 500 spines/5 neurons/ group; p,0.05, K-S test)(Fig. 2). Spine length was not significantly different between the two groups, however (n = 500 spines/5 neurons/group, p.0.05, K-S test)(Fig. 2B). Neurons co-expressing the ABD of bI-spectrin and GFP also displayed a small (,20%), PLoS ONE | www.plosone.org 3 January 2011 | Volume 6 | Issue 1 | e16197