Imaging signal transduction in single dendritic spines

描述(申请人提供)：树突棘是从树突表面发出的兴奋性突触后间隔，小(0.1-0.01毫微秒)。CA2进入脊椎激活由数百种蛋白质组成的信号网络，从而诱导不同形式的突触可塑性。Rho GTPase蛋白，特别是rac1、RhoA和CDC42，是这些信号网络的关键组成部分，它们的激活在调节树突棘的形态和功能方面发挥着重要作用。与Rho信号在脊柱形态和功能中的重要作用一致，Rho信号通路的突变与多种形式的智力低下和自闭症有关。在这项研究中，我们将开发一种技术来测量单个树突棘在脑片中进行形态和功能可塑性时的Rho信号。为此，我们将结合双光子荧光寿命成像显微镜(2pFLIM)和基于荧光共振能量转移的Rho活性传感器，为2pFLIM进行了广泛优化。我们的初步数据表明，CDC42的活性仅限于经历突触可塑性的脊髓，而rac1和RhoA的激活沿着树突扩散到105m以上，并侵入邻近的脊髓。这些结果表明，每条Rho信号通路在不同的长度尺度上发挥作用。我们将通过测量和扰动Rho信号的时空动态来研究Rho GTPase蛋白空间扩散的机制和作用。本项目的具体目标是：1)建立Rho信号在单个脊椎中的成像技术；2)阐明Rho在突触可塑性中的时空动力学机制和作用；3)确定连接钙与Rho GTP酶激活和突触可塑性的信号通路。这项研究将阐明树突棘的形态和功能可塑性的分子机制，并将为Rho信号通路突变引起的精神疾病提供见解。与公共健康相关：突触的形状和功能由Rho蛋白介导的信号调节。许多形式的智力低下和自闭症都是由异常的Rho信号引起的。该项目将开发一种新的技术来测量单个突触中Rho蛋白的活性，以阐明Rho蛋白的活性与突触的形态和功能之间的联系机制。这将有助于理解精神发育迟滞和自闭症。

DESCRIPTION (provided by applicant): Dendritic spines are small (0.1 - 0.01 femtolitter) excitatory postsynaptic compartments emanating from the dendritic surface. Ca2+ influx into spines activates signaling networks consisting of hundreds of species of proteins that induce diverse forms of synaptic plasticity. Rho GTPase proteins, particularly Rac1, RhoA and Cdc42, are critical components of these signaling networks, and their activation plays an important role in regulating the morphology and function of dendritic spines. Consistent with the important role of Rho signaling in spine morphology and function, mutations in Rho signaling pathways are associated with many forms of mental retardation and autism. In this study, we will develop a technique to measure Rho signaling in single dendritic spines while they undergo morphological and functional plasticity in brain slices. To do so, we will combine 2-photon fluorescence lifetime imaging microscopy (2pFLIM) with fluorescent resonance energy transfer-based Rho activity sensors extensively optimized for 2pFLIM. Our preliminary data demonstrates that the activity of Cdc42 is restricted to spines undergoing synaptic plasticity, while Rac1 and RhoA activation spreads along dendrites over ~10 5m and invades neighboring spines. These results suggest that each Rho signaling pathway functions on a different length scale. We will study the mechanisms and roles of the spatial spreading of Rho GTPase proteins by measuring and perturbing the spatiotemporal dynamics of Rho signaling. The specific aims of this project are to 1) establish techniques to image Rho signaling in individual spines, 2) elucidate the mechanisms and roles of spatiotemporal dynamics of Rho during synaptic plasticity, and 3) identify signaling pathways connecting calcium with Rho GTPase activation and synaptic plasticity. This study will illuminate the molecular mechanisms of morphological and functional plasticity of dendritic spines, and will provide insights into mental diseases caused by mutations in Rho signaling pathways. PUBLIC HEALTH RELEVANCE: The shape and function of synapses are regulated by signaling mediated by Rho proteins. Many forms of mental retardation and autism are caused by abnormal Rho signaling. This project will develop a novel technique to measure the activity of Rho proteins in single synapses to elucidate the mechanisms linking the activity of Rho proteins and the morphology and function of synapses. This will facilitate understanding of mental retardation and autism.