The Actin Binding Domain of bI-Spectrin Regulates the Morphological and Functional Dynamics of Dendritic Spines Michael W. Nestor1,2,3¤a, Xiang Cai1, Michele R. Stone1¤b, Robert J. Bloch1,2,3, Scott M. Thompson1,2,3* 1 Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, 2 Program in Neuroscience, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, 3 Training Program in Integrative Membrane Biology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America Abstract Actin microfilaments regulate the size, shape and mobility of dendritic spines and are in turn regulated by actin binding proteins and small GTPases. The bI isoform of spectrin, a protein that links the actin cytoskeleton to membrane proteins, is present in spines. To understand its function, we expressed its actin-binding domain (ABD) in CA1 pyramidal neurons in hippocampal slice cultures. The ABD of bI-spectrin bundled actin in principal dendrites and was concentrated in dendritic spines, where it significantly increased the size of the spine head. These effects were not observed after expression of homologous ABDs of utrophin, dystrophin, and a-actinin. Treatment of slice cultures with latrunculin-B significantly decreased spine head size and decreased actin-GFP fluorescence in cells expressing the ABD of a-actinin, but not the ABD of bI-spectrin, suggesting that its presence inhibits actin depolymerization. We also observed an increase in the area of GFP- tagged PSD-95 in the spine head and an increase in the amplitude of mEPSCs at spines expressing the ABD of bI-spectrin. The effects of the bI-spectrin ABD on spine size and mEPSC amplitude were mimicked by expressing wild-type Rac3, a small GTPase that co-immunoprecipitates specifically with bI-spectrin in extracts of cultured cortical neurons. Spine size was normal in cells co-expressing a dominant negative Rac3 construct with the bI-spectrin ABD. We suggest that bI-spectrin is a synaptic protein that can modulate both the morphological and functional dynamics of dendritic spines, perhaps via interaction with actin and Rac3. Citation: Nestor MW, Cai X, Stone MR, Bloch RJ, Thompson SM (2011) The Actin Binding Domain of bI-Spectrin Regulates the Morphological and Functional Dynamics of Dendritic Spines. PLoS ONE 6(1): e16197. doi:10.1371/journal.pone.0016197 Editor: Lin Mei, Medical College of Georgia, United States of America Received August 24, 2010; Accepted December 7, 2010; Published January 31, 2011 Copyright: 2011 Nestor et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Supported by stipends to MWN from T32 GM08181, to MRS from T32 NS07375, and from grants from the Muscular Dystrophy Association (RJB) and the National Institutes of Health (R01 HL75106 to RJB and R01 MH65488 to SMT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sthom003@umaryland.edu ¤a Current address: National Institute of Child Health and Development, National Institutes of Health, Bethesda, Maryland, United States of America ¤b Current address: Aeras Global TB Vaccine Foundation, Rockville, Maryland, United States of America Introduction Most excitatory synapses are positioned on dendritic spines, which display a range of sizes and shapes. Changes in dendritic spine size accompany synapse maturation [1] and are correlated with synaptic strength [2–4]. Spine morphology is altered by changes in synaptic activity [5,6] and can accompany changes in synaptic strength in some, but not all, forms of long-term potentiation [7–9]. Conversely, abnormal dendritic spines are observed in many forms of mental dysfunction. The signaling mechanisms that regulate spine structure and couple changes in synaptic structure and function are now being actively explored. Actin, both G-actin monomers and filamentous, polymerized F- actin, is highly concentrated in dendritic spines [10] and regulates spine morphology. Dynamic changes in actin polymerization underlie morphological plasticity of spines [11,12] and can affect synaptic function [10,13–17]. Actin also serves as the scaffold to which transmembrane proteins like AMPA and NMDA receptors, as well as intracellular signaling molecules, are anchored [18–20]. Treatment with actin-depolymerizing agents reduces the number of functional AMPARs at dendritic spines [21,22]. Thus, actin and its binding proteins, as well as the signaling molecules that regulate their interaction, are essential for synapse structure and function. Spectrin is a large protein that links the actin cytoskeleton to the cytoplasmic surface of plasma membranes [23,24] and regulates the dynamic state of the actin cytoskeleton [25]. Spectrin also anchors transmembrane proteins, such as NMDARs [26], to the actin cytoskeleton, generating localized microdomains that facilitate rapid signaling cascades. Spectrin is a heterodimer, consisting of a- and b-chains [27,28]. bI-spectrin is enriched in postsynaptic spine heads in the absence of a-subunits [29]. Proteins in the spectrin superfamily are composed of a series of triple helical repeats that serve as binding sites for other cytoskeletal proteins. Spectrin also harbors a central SH3 domain and binding sites for Ca2+- activated proteins, suggesting that it may play a role in signaling cascades. The actin-binding domain (ABD) is limited to the N-terminal region of bI-spectrin and is composed of two calponin homology domains. As a first step in characterizing the role of spectrin in dendritic spines, we transfected CA1 pyramidal neurons in hippocampal PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e16197