Proximity biotinylation provides insight into the molecular composition of focal adhesions at the nanometer scale
Proximity biotinylation provides insight into the molecular composition of focal adhesions at the nanometer scale
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DOI：
10.1126/scisignal.aaf3572
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发表时间：
2016-06-14
影响因子：
7.3
通讯作者：
Manser, Ed
中科院分区：
文献类型：
--
作者：
Dong, Jing-Ming;Tay, Felicia Pei-Ling;Manser, Ed
焦点黏附是通过整合素家族跨膜蛋白将后生动物细胞与细胞外基质连接起来的蛋白质复合体。整合素将许多蛋白质招募到这些复合体中，被称为“粘性蛋白”。我们在U2OS骨肉瘤细胞中使用邻近依赖的生物素化(BioID)标记细胞质焦点黏附蛋白paxlin和直接结合β整合素的kindlin-2之间15到25 nm范围内的蛋白质。利用生物素化蛋白的质谱分析，我们鉴定了27个已知的粘附性蛋白和8个先前未知的靠近巴西林的组分。然而，这些蛋白中只有7种与巴西林直接相互作用，其中一种是适配蛋白Kank2。接近b整合素的蛋白质包括15种在paxlin BioID数据集中鉴定的黏附蛋白。BioID还正确地将kindlin-2确立为细胞-细胞连接蛋白。通过关注这个较小的数据集，Kindlin-2的新合作伙伴被发现，即促进内吞作用的蛋白质liprin beta 1和EFR3A，但与之前的报告相反，不是丝胺结合蛋白Midfilin。一个基于这两个数据集的模型表明，局灶性粘连包含的成分比之前怀疑的要少，而且帕克西林远离质膜。这些数据不仅说明了使用BioID和稳定同位素标记的质谱学来定义大分子复合体的能力，而且还能够正确地识别粘附物中的治疗目标。
Focal adhesions are protein complexes that link metazoan cells to the extracellular matrix through the integrin family of transmembrane proteins. Integrins recruit many proteins to these complexes, referred to as the "adhesome." We used proximity-dependent biotinylation (BioID) in U2OS osteosarcoma cells to label proteins within 15 to 25 nm of paxillin, a cytoplasmic focal adhesion protein, and kindlin-2, which directly binds beta integrins. Using mass spectrometry analysis of the biotinylated proteins, we identified 27 known adhesome proteins and 8 previously unknown components close to paxillin. However, only seven of these proteins interacted directly with paxillin, one of which was the adaptor protein Kank2. The proteins in proximity to b integrin included 15 of the adhesion proteins identified in the paxillin BioID data set. BioID also correctly established kindlin-2 as a cell-cell junction protein. By focusing on this smaller data set, new partners for kindlin-2 were found, namely, the endocytosis-promoting proteins liprin beta 1 and EFR3A, but, contrary to previous reports, not the filamin-binding protein migfilin. A model adhesome based on both data sets suggests that focal adhesions contain fewer components than previously suspected and that paxillin lies away from the plasma membrane. These data not only illustrate the power of using BioID and stable isotope-labeled mass spectrometry to define macromolecular complexes but also enable the correct identification of therapeutic targets within the adhesome.