Genetically encoded probes for visualizing neuronal structure and function

描述（由申请人提供）：用常规抗体研究蛋白质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，常规抗体具有几个限制，这极大地限制了它们的效用。在染色之前，组织必须被固定和透化，并且由于缺乏上下文信息，通常难以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的探针，使神经元蛋白质的亚细胞定位能够在体内和真实的时间内以高保真度绘制。这些探针由与内源性神经元蛋白结合的遗传编码适体（胞内抗体）组成，并使用mRNA展示系统生成。将产生三种不同类型的胞内抗体：1.与单个细胞骨架蛋白结合，标记神经元结构，如突触前和突触后部位。2.跨膜蛋白的结合剂。这些胞内抗体将被修饰以使它们能够标记总蛋白或仅标记存在于细胞质膜上的蛋白。3.与活化的G蛋白结合。胞内抗体将用于将三种类型的分子连接至内源性靶蛋白：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的Ca++浓度的蛋白质。3.被光激活产生去极化电流的蛋白质。蛋白质的亚细胞运输对几乎所有神经元功能都至关重要，包括建立突触连接、轴突导向和突触可塑性。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。蛋白质运输在药物成瘾中也起着关键作用。通过RNA展示产生的胞内抗体将提供在体内和真实的时间内以高保真度绘制内源性蛋白质的亚细胞定位的工具，这在当前技术下是不可能的。

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.