The N-terminal actin binding domain is followed by four tandem spectrin repeats and a calmodulin-like domain, that determines each isoform’s calcium sensitivity, at its Cterminus. Although three of the four a-actinin isoforms, aactinin-1, -2, and -4, have been identified in rat PSD fractions by mass spectrometry and RT-PCR of cultured hippocampal neurons, immunofluorescence and electron microscopy studies have shown specific enrichment of a-actinin-2 in the PSD of excitatory synapses in pyramidal neurons of the cortex and hippocampus. In addition to cross-linking actin filaments, a-actinin interacts with several membrane-associated proteins, including integrins, acatenin, and the L-type Ca2+ channel Cav1.2, and through these interactions a-actinin is thought to couple these molecules to actin filaments. In vitro binding assays suggest a-actinin-2 interacts directly with the NR1 and NR2B subunits of the NMDA receptor. In vitro studies also suggest a-actinin-2 binds to densin-180 to form a ternary complex with CaMKIIa and NR2B. These observations are supported by studies in HEK293 cells, in which a-actinin-2 targets CaMKIIa to F-actin and enhances the interaction between CaMKIIa and NR2B. These putative interactions suggest a-actinin-2 could interpret signals and mediate interactions between PSD components and the actin cytoskeleton, and thus play a pivotal role in post-synaptic organization. a-Actinin regulation and function in spines is poorly understood and relies largely on in vitro binding interactions or studies in nonneuronal cells. PtdInsP2, PIP2, binds to the actin-binding domain of a-actinin-2 and tethers it to the plasma membrane, a function thought to maintain the open state of the NMDA receptor. Neurons expressing an a-actinin-2 mutant unable to interact with PIP2 display significantly reduced peak and steadystate NMDA current compared to neurons expressing wild-type aactinin-2. In one study, overexpression of a-actinin-2 increased the length and density of dendritic protrusions in cultured hippocampal neurons, suggesting a role in determining spine morphology. To ascertain a biological function for a-actinin-2 in spines, we knocked down a-actinin-2 in hippocampal neurons via short interfering RNA. We find that loss of a-actinin-2 increases spine density and the presence of filopodia-like spines that lack a PSD. These immature spines do not form synapses and therefore do not mature in response to chemical stimulation. We further show the Ca2+-insensitive EF-hand motif in a-actinin-2 is critical for its role in spine morphogenesis and PSD organization. Expression of either a-actinin-4 or a Ca2+-sensitive a-actinin-2 mutant does not rescue spine morphology and PSD assembly in neurons lacking endogenous a-actinin-2. However, expression of a Ca2+-insensitive a-actinin-4 mutant does rescue PSD organization. These studies suggest a-actinin-2 BEZ235 re-organizes the actin cytoskeleton in filopodialike dendritic protrusions to promote assembly of the PSD and mediate its transition to a mature, mushroom-shaped morphology.