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

大规模开发用于突触中小 GTP 酶信号成像的传感器

• 批准号: 8192088
• 负责人: Ryohei Yasuda
• 金额: $ 38.76万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-18 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8192088
• 关键词: Actins; Alzheimer' s Disease; Autistic Disorder; Biochemical; Brain; Cytoskeleton; Data; Dendrites; Dendritic Spines; Development; Diffusion; Disease; Event; Excitatory Synapse; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Genetic Transcription; Glutamates; Goals; Guanosine Triphosphate Phosphohydrolases; Hela Cells; Hippocampus (Brain); Image; Individual; Invaded; Kinetics; Learning; Length; Long-Term Potentiation; Measures; Mediating; Membrane Protein Traffic; Memory; Mental Retardation; Microscopy; Molecular; Monitor; Monomeric GTP-Binding Proteins; Morphology; N-Methyl-D-Aspartate Receptors; Neck; Neuraxis; Neuronal Plasticity; Neurons; Optics; Parents; Pattern; Photons; Play; Process; Proteins; Protocols documentation; Psyche structure; Regulation; Reporting; Resolution; Role; Running; Scheme; Schizophrenia; Sensitivity and Specificity; Signal Pathway; Signal Transduction; Signaling Protein; Slice; Structure; Surface; Synapses; Synaptic plasticity; Techniques; Testing; Time; Vertebral column; base; brain tissue; calmodulin-dependent protein kinase II; design; improved; insight; interest; light scattering; member; novel; overexpression; postsynaptic; response; rho; sensor; spatiotemporal; synaptic function

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. PUBLIC HEALTH RELEVANCE: Signaling mediated by small GTPase proteins is important for synaptic plasticity and ultimately learning and memory. This project will develop a novel technique to measure the activity of small GTPase proteins in single synapses to elucidate the mechanisms by which small GTPase signaling regulates synaptic function. This will facilitate understanding the molecular mechanisms of mental diseases related to abnormal small GTPase signaling such as mental retardation, autism, schizophrenia and Alzheimer's disease.

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，树突棘是从树突表面发出的小突触后隔室。Ca2+流入棘激活了各种形式的突触可塑性所需的信号网络。特别是，约150个小GTdR蛋白家族对于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GTdR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）的传感器报告具有高灵敏度的小GTdR活性。为了在光散射脑组织中以高灵敏度和分辨率定量成像FRET信号，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GT3蛋白。使用这些传感器，我们将筛选由NMDA受体激活的小GTdR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GTdR蛋白的传感器，2）筛选小GTdR蛋白中的那些被Ca 2+通过NMDA受体激活的小GTdR蛋白，3）测量选择的小GTdR蛋白在单个树突棘中的时空动力学。这项研究将提供深入了解小GTdR蛋白的活性是如何协调的棘产生树突棘的结构和功能可塑性，并将阐明突触可塑性的分子机制，并最终学习和记忆。 公共卫生相关性：由小G蛋白介导的信号传导对突触可塑性以及最终的学习和记忆非常重要。本项目将开发一种新的技术来测量单个突触中小GT3蛋白的活性，以阐明小GT3信号调节突触功能的机制。这将有助于理解与异常小GT3信号相关的精神疾病的分子机制，如精神发育迟滞、自闭症、精神分裂症和阿尔茨海默病。