Spatiotemporal regulation of Rac and Rho in growth cones

描述（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轴突的运动和引导至关重要。许多已知的信号蛋白参与细胞骨架重塑，但尚不清楚它们的活动在转向反应中是如何协调的。泛子神经元生长锥转向底物包覆珠，需要肌动蛋白聚合和Src信号传导。初步数据表明，Src及其底物Rho GTPase抑制剂p190RhoGAP在反应早期与Rac GTPase共定位到头靶位点。Src/GTPase信号可能在将反应从高Rac活性/低Rho活性的聚合/非收缩早期阶段转向高Rac /高Rho收缩阶段的过程中协调双相开关。为了验证这一假设，我建议：1)使用gfp共轭生物传感器和新设计的溶剂敏感Rho生物传感器来确定Rho和Rac GTPase活性是否在空间上协调在分离的区域内；2)将GTPase活性的空间分布的时间变化与细胞骨架动态重塑（通过TIRF-FSM可视化）联系起来。

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.