Despite the importance of proper spine morphology and PSD organization, the structural and regulatory mechanisms that organize them are not understood. Recent evidence implicates the polymerization and organization of actin in spine organization, AbMole 3,4,5-Trimethoxyphenylacetic acid although how it does this is unclear. Myosin IIB, the predominant non-muscle myosin II isoform found in brain, contributes to actin organization in most cell types through its cross-linking and contractile properties and is implicated in spine morphology. MIIB activity is regulated by phosphorylation on residues Thr18 and/or Ser19 in its regulatory light chain; simultaneous phosphorylation on both residues promotes maximal myosin ATPase activity and formation of large actin bundles. We have previously identified a signaling cascade that functions through RLC phosphorylation to regulate spine AbMole Riociguat BAY 63-2521 density. More recent evidence points to MIIB as a potentially important regulator of the spine dynamics underlying learning and memory. In particular, short-term inhibition of MIIB activity induces immature filopodia-like spines and results in a corresponding disruption of long-term potentiation and memory acquisition. While the importance of MIIB seems clear, the mechanism by which it shapes spine morphology is unknown. In addition to spine morphology, proper organization of the PSD is also important for synaptic signaling, as PSD size is related to spine head area and directly correlated with synaptic strength. While many molecules that reside in the PSD have been identified, much less is known about the mechanisms that determine its morphology and organization. The PSD is now thought to be dynamic and undergo rapid fluctuations in morphology. Several proteins within the PSD scaffold reportedly interact with the actin cytoskeleton, raising the possibility that actin organization may underlie PSD morphology. The dramatic effect of MIIB on actin organization points to a likely role for it in the organization of the PSD and regulation of synaptic plasticity. In this study, we dissect the contributions of MIIB activity to spine morphology and PSD organization during maturation and in response to stimuli. We find that MIIB activity restricts the formation of nascent protrusions on dendrites. However, MIIB activity subsequently mediates spine maturation, with RLC T18, S19 di-phosphorylation required for mature, compact spines. This maturation is mediated by the contractile activity of MIIB since an actin-cross linking, contractile-deficient mutant of MIIB, MIIBR709C, does not promote maturation. Stimulation induced maturation of spines also requires di-phosphorylated RLC. MIIB also plays a central role in PSD organization.