Genetically encoded probes for visualizing neuronal structure and function

用于可视化神经元结构和功能的基因编码探针

• 批准号: 7431329
• 负责人: DONALD B ARNOLD
• 金额: $ 30.16万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7431329
• 关键词: Address; Affinity; Alzheimer' s Disease; Antibodies; Architecture; Binding; Biological Assay; CAST1 Protein; Camping; Cell membrane; Cell physiology; Cell surface; Cells; Chromosome Pairing; Complex; Cyclophosphamide/Fluorouracil/Prednisone; Cytoskeletal Proteins; DLG4 gene; Development; Disease; Disruption; Disulfides; Drug Addiction; Epitopes; Fibronectins; Figs - dietary; GABA transporter; GTP-Binding Proteins; Gene Expression; Goals; Golgi Apparatus; Grant; Green Fluorescent Proteins; In Vitro; Individual; Integral Membrane Protein; Ion Channel; Label; Light; Link; Localized; Location; Long-Term Depression; Long-Term Potentiation; Maps; Measures; Membrane Proteins; Messenger RNA; Methods; Molecular; Molecular Probes; Neurons; Neurosciences; PHluorin; Parkinson Disease; Pattern; Peptide aptamers; Peptides; Personal Satisfaction; Physiological; Play; Protein Binding; Proteins; Purpose; RNA; Reporter; Reporting; Role; Score; Signal Transduction; Site; Staining method; Stains; Stereotyping; Structure; Surface; Synapses; Synaptic plasticity; System; Technology; Tertiary Protein Structure; Testing; Time; Tissues; Transgenic Mice; Visual; Work; aptamer; axon guidance; base; cell type; density; design; gephyrin; imaging probe; in vivo; interest; intracellular protein transport; optical sensor; postsynaptic; presynaptic; presynaptic density protein 95; protein localization location; protein transport; response; sensor; tool; trafficking

DESCRIPTION (provided by applicant): Studying the localization of proteins with conventional antibodies has greatly contributed to our understanding of the structure and function of neurons. However, conventional antibodies have several limitations that drastically limit their utility. Tissue must be fixed and permeabilized prior to staining and often the overlapping expression patterns of adjacent neurons are difficult to interpret because of the lack of contextual information. For these reasons, the precise subcellular localization patterns in vivo of the majority of neuronal proteins have not been well characterized. The purpose of the studies proposed in this grant is to develop genetically encoded probes that will allow the subcellular localization of neuronal proteins to be mapped in vivo and in real time with high fidelity. These probes consist of genetically encoded aptamers (intrabodies) that bind to endogenous neuronal proteins and are generated using the mRNA display system. Three different types of intrabodies will be generated: 1. Binders to individual cytoskeletal proteins that mark neuronal structures such as pre- and postsynaptic sites. 2. Binders to transmembrane proteins. These intrabodies will be modified to enable them to label either total protein or only protein that is present on the plasma membrane of the cell. 3. Binders to activated G-proteins. Intrabodies will be used to attach three types of molecules to endogenous target proteins: 1. Fluorescent molecules that can be used to report the localization of the protein. 2. proteins for measuring Ca++ concentration in the region around the protein. 3. proteins that are activated by light to produce depolarizing currents. Subcellular trafficking of proteins is crucial to virtually all neuronal functions, including establishment of synaptic connections, axon guidance and synaptic plasticity. Disruption of protein trafficking has been linked to such diseases as Alzheimer's disease and Parkinson's disease. Protein trafficking also plays a critical role in drug addiction. Intrabodies generated through RNA display will provide tools to map the subcellular localization of endogenous proteins with high fidelity, in vivo and in real time, which is not possible with current technology.

描述（由申请人提供）：研究具有常规抗体的蛋白质的定位极大地有助于我们理解神经元的结构和功能。但是，常规抗体具有多种局限性，可以极大地限制其效用。必须在染色之前固定并透化组织，并且由于缺乏上下文信息，很难解释相邻神经元的重叠表达模式。由于这些原因，大多数神经元蛋白的体内的精确亚细胞定位模式尚未得到很好的特征。该赠款提出的研究的目的是开发遗传编码的探针，以使神经元蛋白的亚细胞定位在体内和实时绘制高富度性。这些探针由与内源性神经元蛋白结合的遗传编码的适体（内植物）组成，并使用mRNA显示系统产生。将产生三种不同类型的内置遗物：1。对单个细胞骨架蛋白的粘合剂，标记神经元结构（例如突触前和突触后部位）。 2。跨膜蛋白的粘合剂。这些内遗嘱将被修改，以使它们能够标记总蛋白质或仅存在于细胞质膜上的蛋白质。 3。活化的G蛋白的粘合剂。内遗嘱将用于将三种类型的分子连接到内源性靶蛋白上：1。可用于报告蛋白质定位的荧光分子。 2。用于测量蛋白质周围区域中Ca ++浓度的蛋白质。 3。被光激活以产生去极化电流的蛋白质。蛋白质的亚细胞运输对于几乎所有神经元功能，包括建立突触连接，轴突引导和突触可塑性至关重要。蛋白质运输的破坏与诸如阿尔茨海默氏病和帕金森氏病这样的疾病有关。蛋白质运输在药物成瘾中也起着至关重要的作用。通过RNA显示生成的内构将提供工具来绘制具有高富达，体内和实时实时的内源性蛋白质的亚细胞定位，而当前技术是不可能的。