A bioinformatics approach to pannexin localization and interaction in the brain

描述（由申请人提供）：最近对人类基因组的计算机模拟搜索产生了三种相关蛋白质-泛连接蛋白（Panx1、Panx2和Panx3），最初推测其作为间隙连接通道起作用，因为它们具有连接蛋白样蛋白质结构域组织.虽然连接蛋白是研究最广泛的细胞间通讯通道，但非连接蛋白间隙连接通道已在无脊椎动物中被实验发现（连接蛋白），并且pannexins比连接蛋白更类似于连接蛋白。本研究中提出的组织结构分析将使用大规模荧光显微镜蒙太奇成像来研究啮齿动物脑中的Panxl和Panx2表达，以确定具有高水平表达的区域，然后使用数据共享、挖掘和神经信息学工具来查看泛连接蛋白是否与作为相互作用伴侣的其他蛋白质共定位或指示它们是更大细胞复合物的一部分。该项目旨在将实验成像与计算分析相结合，以获得有关泛连接蛋白表达/功能的定量和跨学科信息。该提案利用数据密集型马赛克成像技术，结合神经信息学，数据挖掘和共享，对小鼠大脑进行大规模映射，以帮助整合和合成数据，从而对复杂的哺乳动物大脑建模产生更广泛的影响。具体来说，我的研究目标是：（1）应用我们最近开发的抗体工具包以高光学显微镜分辨率对Panx 1和Panx 2都高度表达的小鼠全脑进行成像，（2）将脑图谱存款并注释在公共数据库中以用于数据共享，（3）结合神经信息学和数据挖掘计算工具，使用脑图来确定是否存在"热点"。对于Panx1和Panx2在小鼠脑中的定位与其他蛋白质如连接蛋白或嘌呤能受体的高表达相关，（4）将内源性Panx1和Panx2在组织中的膜定位与细胞内定位相关联，以补充细胞培养物中外源表达的泛连接蛋白的离体研究。相关性：这是具有Panxl和Panx2的分子、细胞和器官水平成像方向的多尺度协调方法的一部分。这种多层次的方法解决了神经系统研究的特殊挑战，以弥合从组织到分子的维度范围，包括细胞网络，树突和轴突架构，突触连接，神经胶质相互作用和大分子成分。这些结构和其中的蛋白质代表了神经系统中信息处理的核心;这些蛋白质表达数据的整合，合成和共享对于我们理解大脑功能至关重要。

DESCRIPTION (provided by applicant): Recent in silico searching of the human genome yielded three related proteins - the pannexins (Panxl, Panx2 and Panx3), originally speculated to function as gap junction channels because of the their connexin- like protein domain organization. Although connexins are the most widely studied intercellular communication channels, non-connexin gap junction channels have been discovered experimentally in invertebrates (innexins) and pannexins are more similar to innexins than connexins. The tissue structure analyses proposed in this research will investigate, using large-scale fluorescence light microscopic montage imaging, Panxl and Panx2 expression in the rodent brain to determine areas with high levels of expression and then use data sharing, mining and neuroinformatic tools to see whether pannexins co- localize with other proteins as interaction partners or indicate that they are part of a larger cellular complex. This project aims to integrate experimental imaging with computational analyses in order to obtain quantitative and interdisciplinary information about pannexin expression/function. This proposal utilizes a data-intensive mosaic imaging technique for large-scale mapping of the mouse brain combined with neuroinformatics, data mining, and sharing to help integrate and synthesize data for the broader impact of modeling the complex mammalian brain. Specifically, the goals of my research are: (1) To apply our recently developed antibody tool kit to image whole mouse brain at high light microscopic resolution where both Panxl and Panx2 are highly expressed, (2) To deposit and annotate the brain maps in public databases for data sharing, (3) To use the brain maps in conjugation with neuroinformatics and data mining computational tools to determine if there are "hot spots" for Panxl and Panx2 localization in the mouse brain correlated with high expression of other proteins such as connexins or purinergic receptors, (4) To correlate membrane versus intracellular localizations of endogenous Panxl and Panx2 in tissue to complement ex vivo studies of exogenously expressed pannexins in cell culture. Relevance: This is part of a multi-scale coordinated approach with directions in molecular, cellular and organ level imaging of Panxl and Panx2. This multilevel approach addresses the particular challenge for nervous system research to bridge the dimensional range from tissues to molecules, a range encompassing cellular networks, dendritic and axonal architectures, synaptic connectivity, glial interactions and macromolecular constituents. These structures, and the proteins within them, represent the heart of information processing in the nervous system; the integration, synthesis, and sharing of this protein expression data is central to our understanding of brain function.