Dendritic spines house the postsynaptic components of excitatory synapses in the brain. Synapses are inherently plastic, and dendritic spines represent an important locus for maintenance and modification of synaptic strength which is thought to underlie behavioral learning and developmental plasticity. Increasing evidence exists that spines contain within them interlinked sets of functional subdomains. Most notable of these is the postsynaptic density, the molecular machine which positions and regulates the number of postsynaptic neurotransmitter receptors. In addition, spines contain an endocytic zone positioned in the spine membrane substantially away from the synapse. The synaptic dysfunction underlying many neurological disorders is associated with impairment of processes occurring at these functional subdomains. Importantly, a number of these processes require ongoing actin polymerization. Dynamic regulation of the actin cytoskeleton within spines is necessary for spine morphological plasticity as well as maintenance of synaptic composition and function, and underlies the insertion, stabilization, and endocytosis of neurotransmitter receptors at the synapse. Changes in actin polymerization and stability within spines accompany and are required for the induction of long term potentiation and other forms of synaptic plasticity. However, little is known about the sites within spines at which actin rearrangement is specifically required for alterations in synaptic strength to be initiated and maintained. I propose that ongoing and independent regulation of actin polymerization occurs at spine subdomains such as the postsynaptic density and endocytic zone. My proposed experiments will test this hypothesis by measuring actin polymerization and movement within individual, live dendritic spines using confocal, super-resolution, and single-particle techniques, and utilizing fluorescent protein-tagged molecules to mark the postsynaptic density (PSD) and endocytic zones. Using these assays, I will then test a widely proposed mechanism that the PSD interacts with the actin cytoskeleton via the protein cortactin, and examine how the organization of actin is altered within spines during induction of LTP. These results will provide fundamental new information about cytoskeletal organization and regulation within individual dendritic spines, critical for understanding the involvement of spine actin dysregulation in disease.
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