Activity-dependent changes in the number and shape of dendritic spines during development and synaptic plasticity are essential for the formation of neuronal circuits, in learning and memory, and in the etiology of mental retardation and mental illness. Our long-term objective is to understand the signaling mechanisms which regulate activity-dependent plasticity of dendritic spines. Rho-like small GTPases are central regulators of the actin cytoskeleton and spine morphogenesis. We previously identified the Rac1-guanine- nucleotide exchange factor (GEF) kalirin-7 as a key regulator of spine morphogenesis in neurons. However, it is not clear whether kalirin-7 and Rac1 are regulated by synaptic activity and whether they regulate activity-dependent spine structural plasticity. In the Preliminary data section, we show that: 1) kalirin-7 recruitment to synapses is regulated by tyrosine phosphorylation and potentially synaptic activity; 2) kalirin-7 tyrosine phosphorylation is regulated by synaptic activity; 3) structural plasticity can be induced in cultured neurons by an NMDA receptor-dependent mechanism; 4) NMDA-receptor-induced spine structural plasticity is mediated by the PDZ domain-containing protein AF-6, which interacted with kalirin-7 in a yeast 2-hybrid screen. Based on these observations, we hypothesize that kalirin-7 and Rac1 regulate activity-dependent synaptic structural plasticity. To test this hypothesis we propose the following specific aims: 1) to examine the synaptic activity-dependent regulation of kalirin-7 enzymatic GEF activity and GTP-binding by Rac1; 2) to examine the association of kalirin-7 with NMDA receptors and the regulation of synaptic translocation of kalirin-7 and Rac1 by synaptic activity; 3) to visualize the real-time translocation of EGFP-tagged kalirin-7 and Rac1 in neurons by time-lapse imaging and fluorescence recovery after photobleaching (FRAP); 4) to assess the role and requirement of kalirin-7 and Rac1 in activity-dependent spine morphogenesis. These studies will use primary cultures of cortical and hippocampal neurons.
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