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

描述(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，即从树突表面发出的小突触后间隔。Ca~(2+)进入脊椎后，激活了多种形式突触可塑性所需的信号网络。特别是，~150个小GTP酶蛋白家族对突触可塑性的许多方面都是重要的，包括对肌动蛋白细胞骨架、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脑片中单个树突棘中60多个小GTP酶蛋白的活性。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GTP酶活性。为了对光散射脑组织中高灵敏度和高分辨率的FRET信号进行定量成像，我们将使用双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GTP酶蛋白。使用这些传感器，我们将筛选由NMDA受体激活的小GTP酶蛋白，并在经历结构和功能可塑性的单个树突棘中成像它们的活性。我们的具体目标是1)开发和测试小GTP酶蛋白的传感器，2)筛选那些被钙离子通过NMDA受体激活的小GTP酶蛋白，3)测量单个树突棘中选定的小GTP酶蛋白的时空动态。这项研究将提供关于小GTPase蛋白的活性如何在棘突中协调以产生树突棘的结构和功能可塑性的见解，并将阐明突触可塑性以及最终学习和记忆的分子机制。

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.