Genetically encoded probes for visualizing neuronal structure and function

描述（由申请人提供）：研究蛋白质与常规抗体的定位，极大地促进了我们对神经元结构和功能的理解。然而，传统抗体有一些限制，这极大地限制了它们的效用。在染色之前，组织必须被固定和渗透，并且由于缺乏上下文信息，相邻神经元的重叠表达模式通常难以解释。由于这些原因，大多数神经元蛋白在体内的精确亚细胞定位模式尚未得到很好的表征。这项研究的目的是开发基因编码探针，使神经元蛋白的亚细胞定位能够在体内实时高保真地绘制出来。这些探针由基因编码的适体（体内）组成，这些适体与内源性神经元蛋白结合，并通过mRNA显示系统产生。将产生三种不同类型的体内体：单个细胞骨架蛋白的结合物，标记神经元结构，如突触前和突触后位点。2. 跨膜蛋白的结合物。这些小体将被修饰，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g蛋白的结合物。体内将用于将三种类型的分子附着到内源性靶蛋白上：荧光分子可以用来报告蛋白质的定位。2. 用于测量蛋白质周围区域的钙离子浓度。3. 被光激活产生去极化电流的蛋白质。亚细胞蛋白质的运输对几乎所有的神经元功能都至关重要，包括突触连接的建立、轴突的引导和突触的可塑性。蛋白质运输的中断与阿尔茨海默病和帕金森病等疾病有关。蛋白质贩运在吸毒成瘾中也起着关键作用。通过RNA展示产生的体内将提供工具，以高保真度，在体内和实时绘制内源性蛋白质的亚细胞定位，这是目前技术无法实现的。

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.