Dynamic visualization of polyribosomes

多核糖体的动态可视化

• 批准号: 9809553
• 负责人: Young Jun Yoon
• 金额: $ 20.88万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-13 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9809553
• 关键词: Affinity; Alzheimer' s Disease; Asparagine; Binding; Biochemical; Biochemical Genetics; Biological Assay; Cells; Dendrites; Disabled Persons; Disease; Disease model; Dyes; Event; FMR1; Fluorescence; Fluorescence Resonance Energy Transfer; Fractionation; Fragile X Syndrome; Frequencies; Functional disorder; Gene Silencing; Genes; Genetic; Goals; Hereditary Disease; Image; Imagery; Imaging Techniques; Individual; Inherited; Intellectual functioning disability; Isoleucine; KH Domain; Kinetics; Knock-out; Knowledge; Label; Ligands; Link; Location; Maps; Measurement; Measures; Mental Retardation; Messenger RNA; Methods; Missense Mutation; Molecular; Monitor; Movement; Mutation; Neurons; Parkinson Disease; Patients; Permeability; Phenocopy; Photobleaching; Polyribosomes; Production; Property; Protein Biosynthesis; Protein C; Proteins; RNA; Resolution; Ribosomes; Risk; S-nitro-N-acetylpenicillamine; Synapses; Testing; Translating; Translations; Work; autism spectrum disorder; fighting; fluorescence lifetime imaging; fluorophore; genetic approach; high resolution imaging; imaging modality; improved; innovation; light microscopy; loss of function mutation; mutant; mutant mouse model; nervous system disorder; particle; protein function; protein protein interaction; response; small molecule; spatiotemporal; targeted treatment

Project Summary/Abstract Fragile X syndrome (FXS) is the leading single-gene cause of autism and the most common form of inherited intellectual disability. Although we understand the genetic mechanism of fragile X mental retardation (FMR1) gene silencing and the loss of function mutations, what is not clear is the molecular function of fragile X mental retardation protein (FMRP). There is biochemical and genetic evidence that FMRP interacts with RNA and polysomes to regulate protein synthesis. However, these approaches are unable to detect the dynamic interactions between ribosomes and FMRP. Our plan is to characterize the polysome-FMRP association using imaging techniques. Light microscopy can provide high resolution map of when and where molecular interactions take place. We will first demonstrate fluorescence resonance energy transfer between ribosome subunits (riboFRET) by fluorescence lifetime imaging microscopy (FLIM). In addition to riboFRET from fluorescent proteins, we will utilize orthogonal self-labeling tags that can be conjugated to organic dyes. The enhanced brightness and photostability of synthetic dyes will improve FLIM-FRET measurements. Next, co-expression of FMRP can show which polysomes within dendrites are bound by FMRP and we allow us to test how synaptic stimulation can alter this interaction. We will perform single-particle tracking (SPT) to monitor individual ribosomal subunits and FMRP assembly or disassembly in response to synaptic activity. Innovations in self-labeling tags, organic dyes and FLIM will be combined to provide robust and high resolution FRET and SPT results in neurons. Our strategy intends to provide a comprehensive understanding of molecular interactions between ribosomes and FMRP.

项目总结/摘要 脆性X综合征（FXS）是自闭症的主要单基因原因，也是遗传性自闭症的最常见形式。 智力残疾。虽然我们了解脆性X智力低下（FMR 1）的遗传机制， 基因沉默和功能丧失突变，目前尚不清楚的是脆性X染色体的分子功能 阻滞蛋白（FMRP）。有生物化学和遗传学证据表明FMRP与RNA相互作用， 多聚核糖体调节蛋白质合成。然而，这些方法无法检测动态交互 核糖体和FMRP之间的联系我们的计划是使用成像技术来表征多聚体-FMRP的关联 技术.光学显微镜可以提供分子相互作用发生的时间和地点的高分辨率地图 地方我们将首先展示核糖体亚基之间的荧光共振能量转移（riboFRET） 荧光寿命成像显微镜（FLIM）除了来自荧光蛋白的riboFRET，我们将 利用可与有机染料缀合的正交自标记标签。增强的亮度和 合成染料的光稳定性将改善FLIM-FRET测量。接下来，FMRP的共表达可以显示 树突内的哪些多核糖体被FMRP结合，我们允许我们测试突触刺激如何改变 这种互动。我们将进行单粒子跟踪（SPT）来监测单个核糖体亚基， FMRP的组装或拆卸对突触活动的反应。自标记标签、有机染料的创新 和FLIM将结合起来，以提供强大的和高分辨率的FRET和SPT结果的神经元。我们的战略 旨在提供一个全面的理解核糖体和FMRP之间的分子相互作用。