Abstract
II Modulation of the actin cytoskeleton dictates the morphological changes associated with dendritic spine dynamics, which serve as the structural basis underlying learning and memory. These micron-sized protrusions mature from a filopodia-like morphology into a mushroom-shape with an enlarged post-synaptic density (PSD). The PSD contains an assembly of synaptic adhesion molecules, glutamate receptors, and signaling scaffolds; many of which respond to glutamate receptor activation and relay signals to the underlying cytoskeleton to induce structural changes in spine and PSD morphology. Non-muscle myosin IIB (MIIB) and -actinin-2 (ACTN2) directly effect actin organization and both proteins localize to dendritic spines. Both molecules cross-link actin filaments and MIIB also mediates contraction through its ATPase activity. Knockdown of either ACTN2 or MIIB creates an immature spine morphology that fails to mature into a mushroom-shaped spine during development and in response to chemical stimulation. Additionally, loss of ACTN2 increases spine density. Expression of an actin cross-linking, non-contractile mutant, MIIB R709C, showed that spine maturation requires contractile activity. Additionally, di-phosphorylation of the myosin regulatory light chain (RLC) by Rho kinase is required for spine maturation. Inhibition of MIIB activity via blebbistatin treatment, knockdown, or expression of a mono-phosphomimetic mutant of RLC similarly abrogated spine maturation. MIIB and ACTN2 also determine PSD size, morphology, and placement in the spine. Loss of ACTN2 prevents the recruitment and stabilization of a PSD and NMDA- type glutamate receptors in the spine, resulting in defective synaptic formation. III Conversely, a PSD is still seen in neurons with MIIB knocked down, but its loss creates an elongated PSD morphology that is no longer restricted to the spine tip, resulting in a less-clustered distribution of NMDA receptors. In contrast, increased MIIB activity, through either over-expression of wild type MIIB or a RLC di-phosphomimetic mutant, enlarges the PSD area and creates an increased density of mature spines. These observations support a model whereby ACTN2 nucleates PSD formation and recruits the NMDA-type glutamate receptor to the spine, which leads to a functioning synapse. Subsequent NMDA receptor activation increases RLC di-phosphorylation to stimulate MIIB contractility, resulting in a mushroom-shaped spine with an enlarged PSD.
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