Thus, myosin IIB serves as a critical regulator of post-synaptic plasticity, consistent with the observation that myosin IIB is necessary for memory formation. Our observations and previous literature lead to a model for the role of MIIB in spine formation and maturation. Spines form in regions of inactive MIIB and can extend into long filopodia-like structures in the absence of high MIIB activity. The most likely mechanism for this formation and extension is due to localized activation of Rac. The GIT1/PIX/PAK complex, which contains the Rac-activator PIX and Rac-effector PAK, is one mechanism by which Rac activation is localized to generate spines. These filopodia-like spines are highly dynamic and protrude and retract frequently; since MIIB is not required for this activity, it is likely that this arises largely from actin polymerization and depolymerization. In contrast, the maturation into a compact, mushroom-shaped structure requires MIIB contractile activity; however, Arp2/3-driven actin polymerization may contribute as well to drive spine head expansion, in analogy with the broad protrusions it mediates in migrating fibroblasts. Finally, MIIB may also serve to localize signals that affect spine morphology and function, such as GEFs that mediate Rac activity, e.g., ß-PIX and Kalirin-7, or other mechanoresponsive molecules that regulate signaling in other cell types. Our holistic view of the effect of myosin II on the component processes of postsynaptic development provides the framework for the identification of critical therapeutic targets, such as ROCK, for the treatment of learning and memory disorders. Postsynaptic density-95 monoclonal antibody was purchased from Santa Cruz Biotechnology and used at ratio of 1:100 for immunostaining. Non-muscle myosin heavy chain II-B polyclonal antibody was obtained from Covance and used at a ratio of 1:1000. A polyclonal antibody against phosphorylated RLC-T18, S19 was purchased from Cell Signaling Technologies and used at a ratio of 1:100-1:200. Secondary anti-mouse and anti-rabbit antibodies conjugated to Alexa488, 568 and 647 were from Invitrogen. Blebbistatin, Calyculin A, and Y-27632 were purchased from Calbiochem and used at the concentrations indicated in the figures. Tetrodotoxin and strychnine were purchased from Sigma and reconstituted in dH2O. The shRNA knockdown vector for MIIB has been described elsewhere. GFP-MIIB was a gift from Robert S. Adelstein. RNAi-insensitive GFP-MIIB and GFP-MIIB-R709C mutants have been described previously. The 39-UTR encompassing 1500nt’s was cut out of both GFP-MIIB and GFP-MIIB-R709C vectors using XmaI restriction enzyme. The 1.5 kb DNA piece was ligated into the 9 kb vector backbone and sequenced to verify correct orientation of the insert. PSD-95-GFP was a gift from David Bredt. RLC-GFP constructs were kindly provided by Kathleen Kelly, and RLC-AD-GFP was generated as previously described. Low-density hippocampal cultures were prepared from E19 rat embryos as described previously. All experiments were carried out in compliance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the University of Virginia Animal Care and Use Committee. Neurons were plated on glass coverslips coated with 1 mg/ml poly-L-lysine at an AZD152 Abmole SIX3, a tumor suppressor, inhibits astrocytoma tumorigenesis by transcriptional repression of AURKA/B approximate density of 70 cells/mm2 and were transfected using a modified calcium phosphate precipitation method as described previously. Cortical neurons were nucleofected with DsRed2 as described by, and plated on poly-L-lysine coated imaging dishes. DIV 5-12 cortical neurons were micropipetted with 100 mM-1 mM blebbistatin for 10 msec-1 sec with 5psi pressure using an IM 300 Microinjector from Narishige International USA, Inc.. For the chemical stimulation experiments involving knockdown or inhibition of MIIB, DIV14-17 neurons were removed from the glia-feeder layer and placed in 1X Mg2+ -free extracellular solution containing 15 mM NaCl, 0.5 mM KCl, 0.2 mM CaCl2, 3 mM glucose, 1 mM Hepes, 0.5 mM tetrodotoxin, and 1 mM strychnine, pH7.4. Stimulated neurons are treated with 200 mM glycine and incubated at 35uC, 5% CO2 for 3 min. The solution is removed and replaced with 1X Mg2+ -free extracellular solution with tetrodotoxin and strychnine and incubated at 35uC, 5% CO2 for 20 minutes before fixation.