Large Scale Development of Sensors for Imaging Small GTPase Signals in Synapses

描述（由申请人提供）：在中枢神经系统中，大多数兴奋性突触终止于树突棘，树突棘是树突表面发出的小突触后区室。钙离子流入脊柱激活了多种形式的突触可塑性所需的信号网络。特别是，约150个小GTPase蛋白家族对突触可塑性的许多方面都很重要，包括肌动蛋白细胞骨架、膜运输、囊泡运输和基因转录的调节。在这项研究中，我们将开发一种技术来监测大脑切片中单个树突棘中60多种小GTPase蛋白的活性。为此，我们将开发可扩展的设计和优化方案，使基于荧光共振能量转移（FRET）的传感器报告高灵敏度的小GTPase活性。为了在光散射脑组织中以高灵敏度和高分辨率定量成像FRET信号，我们将使用2光子荧光寿命成像显微镜（2pFLIM）。我们的初步数据表明，我们的设计可以应用于许多小的GTPase蛋白。利用这些传感器，我们将筛选NMDA受体激活的小GTPase蛋白，并成像它们在单个树突棘中经历结构和功能可塑性的活性。我们的具体目标是1)开发和测试小GTPase蛋白的传感器，2)筛选那些通过NMDA受体被Ca2+激活的小GTPase蛋白，3)测量单个树突棘中选定的小GTPase蛋白的时空动态。该研究将揭示小GTPase蛋白的活性如何在棘中协调产生树突棘的结构和功能可塑性，并将阐明突触可塑性和最终学习记忆的分子机制。

DESCRIPTION (provided by applicant): In the central nervous system, most excitatory synapses terminate on dendritic spines, small postsynaptic compartments emanating from the dendritic surface. Ca2+ influx into spines activates a signaling network required for diverse forms of synaptic plasticity. In particular, the family of ~150 small GTPase proteins is important for many aspects of synaptic plasticity, including regulation of the actin cytoskeleton, membrane trafficking, vesicular transport and gene transcription. In this study, we will develop a technique to monitor the activity of more than 60 small GTPase proteins in single dendritic spines in brain slices. To do so, we will develop scalable designs and optimization schemes to make fluorescence resonance energy transfer (FRET)-based sensors reporting small GTPase activity with high sensitivity. To quantitatively image FRET signal with high sensitivity and resolution in light scattering brain tissue, we will use 2-photon fluorescence lifetime imaging microscopy (2pFLIM). Our preliminary data demonstrates that our design can be applied to many small GTPase proteins. Using these sensors, we will screen small GTPase proteins activated by NMDA receptors, and image their activity in single dendritic spines undergoing structural and functional plasticity. Our specific aims are 1) to develop and test sensors for small GTPase proteins, 2) to screen small GTPase proteins for those activated by Ca2+ through NMDA receptors, 3) to measure the spatiotemporal dynamics of selected small GTPase proteins in single dendritic spines. This study will provide insights into how the activity of small GTPase proteins is coordinated in spines to produce structural and functional plasticity of dendritic spines, and will illuminate the molecular mechanisms of synaptic plasticity and ultimately learning and memory.