Parallel Visualization of Multiple Protein Complexes in Individual Cells in Tumor Tissue
Parallel Visualization of Multiple Protein Complexes in Individual Cells in Tumor Tissue
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DOI：
10.1074/mcp.o112.023374
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发表时间：
2013-06-01
影响因子：
7
通讯作者：
Soderberg, Ola
中科院分区：
文献类型：
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作者：
Leuchowius, Karl-Johan;Clausson, Carl-Magnus;Soderberg, Ola
细胞功能由复杂的蛋白质相互作用网络调节和执行。因此，理解蛋白质之间的相互作用在确定信号级联的活性状态中是至关重要的。因此，需要新的方法来提供异质细胞群（如组织切片）中单细胞水平上不同蛋白质相互作用事件的信息。在这里，我们描述了一种多重邻近连接试验，同时可视化多个蛋白质复合物原位。该测定是原始邻位连接测定的增强，并且其基于使用用独特标签序列标记的邻位探针，所述独特标签序列可用于从探针池中读出哪些探针已结合某种蛋白质复合物。例如，使用这种方法，可以获得关于不同蛋白质复合物的成分、复合物的亚细胞位置以及不同复合物成分之间的平衡如何在正常和恶性细胞之间变化的信息。作为概念证明，我们使用该测定法在乳腺癌组织切片中在单细胞水平上同时可视化涉及EGFR、HER2和HER3同源和异源二聚体的多个蛋白质复合物。以单细胞分辨率同时研究几种蛋白质复合物形成的能力可能对系统理解具有巨大潜力，为改进疾病诊断和药物开发的可能性铺平了道路。
Cellular functions are regulated and executed by complex protein interaction networks. Accordingly, it is essential to understand the interplay between proteins in determining the activity status of signaling cascades. New methods are therefore required to provide information on different protein interaction events at the single cell level in heterogeneous cell populations such as in tissue sections. Here, we describe a multiplex proximity ligation assay for simultaneous visualization of multiple protein complexes in situ. The assay is an enhancement of the original proximity ligation assay, and it is based on using proximity probes labeled with unique tag sequences that can be used to read out which probes, from a pool of probes, have bound a certain protein complex. Using this approach, it is possible to gain information on the constituents of different protein complexes, the subcellular location of the complexes, and how the balance between different complex constituents can change between normal and malignant cells, for example. As a proof of concept, we used the assay to simultaneously visualize multiple protein complexes involving EGFR, HER2, and HER3 homo- and heterodimers on a single-cell level in breast cancer tissue sections. The ability to study several protein complex formations concurrently at single cell resolution could be of great potential for a systems understanding, paving the way for improved disease diagnostics and possibilities for drug development.