Spatiotemporal regulation of Rac and Rho in growth cones

描述（由申请人提供）：轴突的引导迁移是神经元发育的重要保守特征。细胞骨架动力学的精确调节对于正确的轴突运动和引导是至关重要的。许多已知的信号蛋白有助于细胞骨架重塑，但目前还不清楚它们的活动是如何协调在转向反应。失智症神经元生长锥转向基板包被珠，需要肌动蛋白聚合和信号的Src。初步数据表明，Src及其底物，Rho GTP酶抑制剂p190 RhoGAP在应答早期与Rac GTP酶一起共定位于珠靶位点沿着。潜在地，Src/GTf 3信号传导协调在从以高Rac活性/低Rho活性为特征的聚合/非收缩早期阶段转向高Rac /高Rho收缩阶段的响应期间的双相开关。为了验证这一假设，我建议1）建立Rho和Rac GTdR活动是否空间协调到隔离域使用GFP缀合的生物传感器和新设计的溶剂敏感的Rho生物传感器和2）相关的时间变化的GTdR活动的空间分布与细胞骨架动态重塑与TIRF-FSM可视化。

DESCRIPTION (provided by applicant): Guided migration of axons is an important conserved feature of neuronal development. Precise regulation of cytoskeletal dynamics is critical for proper axonal motility and guidance. Many known signaling proteins contribute to cytoskeletal remodeling, but it is not yet understood how their activities are coordinated during turning responses. Aplysia neuronal growth cones turn towards substrate-coated beads, requiring actin polymerization and signaling by Src. Preliminary data indicates that Src and its substrate, the Rho GTPase inhibitor p190RhoGAP colocalize to the bead target site along with Rac GTPase early in the response. Potentially, Src/GTPase signaling coordinates a biphasic switch during turning responses from a polymerizing/noncontractile early phase characterized by high Rac activity / low Rho activity, to a high Rac / high Rho contractile phase. To test this hypothesis, I propose to 1) establish whether Rho and Rac GTPase activities are spatially coordinated into segregated domains using GFP-conjugated biosensors and a newly designed solvent-sensitive Rho biosensor and 2) correlate temporal changes in the spatial distributions of GTPase activities with cytoskeletal dynamic remodeling as visualized with TIRF-FSM.