Non-muscle Myosin-IIB and A-actinin-2 Determine Dendritic Spine Morphology and Post-synaptic Organization

Hodges, Jennifer Lorraine, Department of Molecular Cell Biology, University of Virginia
Horwitz, Rick, Department of Cell Biology, University of Virginia
Casanova, James, Department of Cell Biology, University of Virginia
Schafer, Dorothy, Department of Biology, University of Virginia
Bloom, George, Department of Biology, University of Virginia

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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PHD (Doctor of Philosophy)
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