In situ Protein-Protein Interaction Networks (PIN) of Neurons

神经元原位蛋白质-蛋白质相互作用网络 (PIN)

• 批准号: 7941008
• 负责人: AKIRA CHIBA
• 金额: $ 94.02万
• 依托单位: UNIVERSITY OF MIAMI CORAL GABLES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7941008
• 关键词: Affect; Age; Axon; Bioinformatics; Brain; Cataloging; Catalogs; Complement; Computer Analysis; Data; Databases; Dendrites; Deposition; Development; Drosophila genus; Drug Addiction; Energy Transfer; Genetic; Genomics; Goals; Green Fluorescent Proteins; Image; In Situ; Left; Libraries; Life; Literature; Maps; Microscopy; Modeling; Molecular; Neurons; Neurosciences; Proteins; Proteome; Proteomics; Reagent; Research Infrastructure; Research Personnel; Resources; Robotics; Staging; Study models; Surveys; Synapses; System; Three-Dimensional Image; Transgenic Organisms; Universities; Whole Organism; base; design; fly; genome-wide; in vivo; molecular dynamics; nervous system disorder; neuronal cell body; protein protein interaction; public health relevance; reconstruction; red fluorescent protein; repaired

DESCRIPTION (provided by applicant): A team of University of Miami investigators requests $4,970,522 to undertake a readily deployable collaborative project that will redefine the proteomics as a context-rich molecular bioinformatics. Proteomics has been hailed as 'the next big thing' after genomics. It has progressed from cataloging the whole complement of proteins, or proteome, to charting their interactions, or interactome. Yet the major predicament in proteomics today is its paucity of in situ contexts. The team proposes to launch an imaging-based survey of protein-protein interaction networks within neurons. Its ultimate goal is reconstruction of genome-wide protein- protein interaction networks within each and every subcellular compartment of neurons at progressive steps of their development. This project will be the first systematic inspection of when and where each protein-protein interaction takes place in vivo. The investigators bring their expertise in neuronal imaging, Drosophila genetics, and computational analysis. The project will isolate GFP (green fluorescent protein) protein-trap lines for endogenous proteins present in well-studied model CNS and PNS neurons, and create inducible transgenic lines for neurologically 'relevant' proteins tagged with either GFP or monomeric RFP (red fluorescent protein). Using crosses to combine the green and red tagged proteins in a single fly, the project will both determine the localization of each protein and reveal the dynamic interactions between proteins in cell body, axon, dendrite and/or synapse of intact neurons within a whole organism during development. To achieve high-content screens within two years, the project takes advantage of a robotic imaging system as well as a FLIM (fluorescent lifetime microscopy) cluster. The latter quantifies GFP-to-RFP FRET (Forster resonance energy transfer), an indicator of direct association, between each of approximately 100,000 protein protein pairs. Over one million 3D images of model neurons will be analyzed to construct proteomic maps specific for different neuronal types, developmental stages and subcellular compartments. This image library will then undergo cross-correlation analysis to arrive at a model of the dynamics of the molecular networks of wild type neurons. At its completion, the project not only delivers the first context-rich proteomics resource, but also offers a new intellectual infrastructure for determining the molecular circuitries affected by neurological disorders, aging or drug addiction and designing strategies to repair and/or protect neurons. PUBLIC HEALTH RELEVANCE: Proteomics remains virtually context-less. The proposed project will show context-specific proteomic maps of wild type neurons in the intact brain, furnishing a much-needed intellectual infrastructure to transform definitions of the molecular circuitries affected by neurological disorders as well as strategies to repair/protect neurons.

描述（由申请人提供）：迈阿密大学的一个研究小组要求4，970，522美元进行一个易于部署的合作项目，将重新定义蛋白质组学作为一个上下文丰富的分子生物信息学。蛋白质组学被誉为继基因组学之后的“下一件大事”。它已经从对蛋白质的整个补充或蛋白质组进行编目，发展到绘制它们的相互作用或相互作用组。然而，当今蛋白质组学的主要困境是缺乏原位背景。该团队建议对神经元内的蛋白质-蛋白质相互作用网络进行基于成像的调查。它的最终目标是在神经元发育的各个阶段，在神经元的每个亚细胞区室中重建全基因组蛋白质-蛋白质相互作用网络。该项目将是第一次系统检查每个蛋白质-蛋白质相互作用在体内发生的时间和地点。研究人员带来了他们在神经元成像，果蝇遗传学和计算分析方面的专业知识。该项目将分离GFP（绿色荧光蛋白）蛋白陷阱线的内源性蛋白质存在于研究模型CNS和PNS神经元，并创建可诱导的转基因线的神经'相关'蛋白标记的GFP或单体RFP（红色荧光蛋白）。使用交叉将绿色和红色标记的蛋白质结合在一只苍蝇中，该项目将确定每种蛋白质的定位，并揭示整个生物体发育过程中完整神经元的细胞体，轴突，树突和/或突触中蛋白质之间的动态相互作用。为了在两年内实现高内容的屏幕，该项目利用了机器人成像系统以及FLIM（荧光寿命显微镜）集群。后者定量GFP到RFP FRET（福斯特共振能量转移），这是大约100，000个蛋白质蛋白质对中的每一个之间的直接关联的指标。将分析超过100万张模型神经元的3D图像，以构建针对不同神经元类型、发育阶段和亚细胞区室的蛋白质组图谱。然后，该图像库将进行互相关分析，以获得野生型神经元的分子网络的动力学模型。在其完成后，该项目不仅提供了第一个上下文丰富的蛋白质组学资源，而且还提供了一个新的知识基础设施，用于确定受神经系统疾病，衰老或药物成瘾影响的分子电路，并设计修复和/或保护神经元的策略。 公共卫生关系：蛋白质组学仍然几乎没有上下文。拟议的项目将显示完整大脑中野生型神经元的上下文特异性蛋白质组学图谱，提供急需的知识基础设施，以改变受神经系统疾病影响的分子电路的定义以及修复/保护神经元的策略。