Thus, MIIB activity determines spine formation and orchestrates the spine and PSD morphologies that underlie post-synaptic plasticity. Since MIIB inhibition creates filopodia-like protrusions and inhibits spine development into compact, mushroom-shaped structures, we hypothesized that MIIB also mediates the acute, activity-induced morphology changes that underlie spine maturation. To test this, we selectively activated synaptic NMDA receptors with the co-agonist glycine and assayed for morphological changes indicative of spine maturation, including decreased spine length and increased spine tip width. At DIV 14-17, neurons display many immature filopodialike spines, allowing us to observe an accelerated, acute maturation response to stimulation. Glycine stimulation of control neurons promotes extensive maturation, including spine shortening and spine tip enlargement, resulting in the appearance of numerous mushroom-shaped spines. In contrast, acute inhibition of MIIB with blebbistatin prevented both spine shortening and increased spine tip width; instead, spines persisted as filopodia-like projections even when stimulated with glycine. However, shRNA knockdown of MIIB did not prevent spine shortening in response to glycine, but did prevent an increase in spine tip width. Thus, shRNA knockdown of MIIB also leads to the persistence of filopodia-like protrusions. Together these results demonstrate that MIIB mediates the morphological transition from immature filopodia-like protrusions into mature mushroom-shaped spines. Non-muscle myosin II plays a major role in the organization of actin filaments and dictates the diverse morphologies and directional movement of various cell types. These include the apical constriction of epithelial cells, nuclear positioning, orientation of the AbMole Folic acid microtubule-organizing AbMole Riociguat BAY 63-2521 center, Golgi and the contractile ring of dividing cells, and polarization of migrating fibroblasts. Of the MII isoforms, MIIB is the predominant one found in hippocampal neurons, and its activity and effective affinity for actomyosin filaments is regulated by RLC. Previous studies have implicated MIIB as a target of a signaling pathway that is mutated in non-syndromic mental retardation and in spine development and memory formation. We now address the mechanisms by which MIIB acts on spines and show that differential MIIB activity determines where spines form, creates diverse post-synaptic spine morphologies, and mediates the morphology, size, and positioning of the PSD. It also mediates the changes in spine morphology in response to stimuli. Thus, MIIB emerges as a major downstream regulator of the component processes underlying post-synaptic plasticity, and implicitly, learning and memory. Spine maturation consists of three stages: emergence of protrusions along the dendritic shaft, spine elongation, and maturation into a mushroom-shape. Our results demonstrate that differential MIIB activity mediates and coordinates these diverse stages of spine development.