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搜索发现了三种相关蛋白- pannexins (Panxl， Panx2和Panx3)，最初由于其连接蛋白样蛋白结构域组织而推测其功能为间隙连接通道。虽然连接蛋白是研究最广泛的细胞间通讯通道，但在无脊椎动物中也发现了非连接蛋白的间隙连接通道（连接蛋白），而泛连接蛋白更类似于连接蛋白而不是连接蛋白。本研究提出的组织结构分析将利用大规模荧光显微镜蒙太奇成像，研究Panxl和Panx2在啮齿动物大脑中的表达，以确定高水平表达的区域，然后使用数据共享、挖掘和神经信息学工具来观察pannexins是否与其他蛋白质作为相互作用伙伴共定位，或者表明它们是更大细胞复合体的一部分。本项目旨在将实验成像与计算分析相结合，以获得关于pannexin表达/功能的定量和跨学科信息。该建议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测绘，以帮助整合和合成数据，为复杂哺乳动物大脑建模提供更广泛的影响。具体来说，我的研究目标是：(1)利用我们最新开发的抗体试剂盒对Panxl和Panx2高表达的小鼠全脑进行高光显微成像；(2)将大脑图谱存入公共数据库并注释，以便数据共享；(3)结合神经信息学和数据挖掘计算工具，使用脑图谱来确定小鼠大脑中是否存在与连接蛋白或嘌呤能受体等其他蛋白高表达相关的Panxl和Panx2定位的“热点”；(4)将组织中内源性Panxl和Panx2的膜定位与细胞内定位相关联，以补充细胞培养中外源性pannexins的体外研究。相关性：这是Panxl和Panx2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，从组织到分子的维度范围，包括细胞网络，树突和轴突结构，突触连通性，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信息处理的核心；整合、合成和共享这些蛋白质表达数据是我们理解大脑功能的核心。

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.