Biochemical visualization of cell surface molecular clustering in living cells
Biochemical visualization of cell surface molecular clustering in living cells
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DOI：
10.1073/pnas.0710346105
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发表时间：
2008-05-27
影响因子：
11.1
通讯作者：
Honke, Koichi
中科院分区：
文献类型：
--
作者：
Kotani, Norihiro;Gu, Jianguo;Honke, Koichi
许多质膜驻留分子与其他分子聚集在一起，在多种生物事件中协同作用。在此我们报道一种灵敏且简单的方法来鉴定活细胞中细胞表面分子簇的成分。该方法包括一种近期建立的反应，称为酶介导的自由基源激活（EMARS），用于标记距离所探测的连接有辣根过氧化物酶（HRP）的分子有限距离（约200 - 300 nm）内的分子。由于这个活性区域的大小接近所报道的膜簇的大小，这表明被标记的分子与所探测的分子在同一膜区域内聚集。EMARS反应和抗体阵列分析相结合表明，在HeLa S3细胞中，多种受体酪氨酸激酶（RTKs）与β1整合素形成簇。用三种针对生长因子受体的辣根过氧化物酶标记的抗体进行EMARS反应后，类似的抗体阵列分析显示，生物素化的RTKs的模式彼此显著不同。这些结果表明，利用EMARS反应可以区分不同类型的细胞表面分子簇。因此，目前这种“生化可视化”方法有望成为在各种情况下阐明活细胞细胞表面分子聚集的有力工具。
Many plasma membrane-resident molecules cluster with other molecules to collaborate in a variety of biological events. We herein report a sensitive and simple method to identify components of cell surface molecular clusters in living cells. This method includes a recently established reaction, called the enzyme-mediated activation of radical source (EMARS), to label molecules within a limited distance (approximate to 200-300 nm) from the probed molecule on which HRP is set. Because the size of this active area is close to that of the reported membrane clusters, it is suggested that the labeled molecules cluster with the probed molecule in the same membrane domain. A combination of the EMARS reaction and antibody array analysis demonstrated that many kinds of receptor tyrosine kinases (RTKs) formed clusters with beta 1 integrin in HeLa S3 cells. A similar antibody array analysis after the EMARS reaction with three HRP-labeled antibodies against growth factor receptors showed the patterns of biotinylated RTKs to be substantially different from each other. These results suggest that different types of cell surface molecular clusters can thus be distinguished using the EMARS reaction. Therefore, the present "biochemical visualization" method is expected to be a powerful tool to elucidate molecular clustering on the cell surface of living cells in various contexts.