Three-fluorophore FRET-FLIM enables the study of trimeric protein interactions and complex formation with nanoscale resolution in living plant cells
Three-fluorophore FRET-FLIM enables the study of trimeric protein interactions and complex formation with nanoscale resolution in living plant cells
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DOI：
10.1101/722124
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发表时间：
2019-08
期刊：
影响因子：
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通讯作者：
Nina Gloeckner;S. Z. Oven-Krockhaus;F. Wackenhut;Moritz Burmeister;Friederike Wanke;E. Holzwart;
中科院分区：
文献类型：
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作者：
Nina Gloeckner;S. Z. Oven-Krockhaus;F. Wackenhut;Moritz Burmeister;Friederike Wanke;E. Holzwart;
细胞水平上的信号整合对于生物体的生存至关重要。蛋白质-蛋白质相互作用的研究为这些信号事件提供了有价值的见解。植物中最好理解的信号传导途径之一是油菜素类固醇（BR）激素信号传导途径，其由受体油菜素类固醇不敏感1（BRI 1）及其共受体BRI 1相关激酶（BAK 1）介导。BRI 1和BAK 1均显示与受体样蛋白44（RLP 44）相互作用，其通过BL信号传导的调节参与细胞壁完整性感知。在这里，我们提供的证据，通过定量在体内的三个荧光团FRET FLIM测量，RLP 44，BRI 1和BAK 1形成一个三聚体复合物在质膜的N。benthamiana叶细胞，它们之间的估计距离低于15 nm。免疫受体鞭毛蛋白传感2（FLS 2），也是一种受体样激酶，如BRI 1，不与RLP 44和BAK 1整合在类似的复合物中。我们的研究支持BRI 1和FLS 2位于PM中不同的纳米结构域中。由于供体的荧光寿命被监测，我们的方法规避了基于强度的FRET相互作用测定所需的大量计算，从而为研究具有纳米级分辨率的活植物细胞质膜中的亚区室化提供了可行的基础。
Integration of signalling on the cellular level is essential for the survival of organisms. Protein-protein interaction studies provide valuable insights in these signalling events. One of the best understood signalling pathways in plants is the brassinosteroid (BR) hormone signalling pathway, which is mediated by the receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) with its co-receptor BRI1-ASSOCIATED KINASE (BAK1). Both BRI1 and BAK1 have been shown to interact with RECEPTOR LIKE PROTEIN 44 (RLP44), which was implicated in cell wall integrity sensing by modulation of BL signalling. Here we provide evidence by quantitative in vivo three-fluorophore FRET-FLIM measurements, that RLP44, BRI1 and BAK1 form a trimeric complex in the plasma membrane of N. benthamiana leaf cells, with an estimated distance between them below 15 nm. The immune receptor FLAGELLIN SENSING 2 (FLS2), which is also a receptor-like kinase like BRI1, is not integrated in a similar complex with RLP44 and BAK1. Our study supports that BRI1 and FLS2 are localized in distinct nanodomains in the PM. As the fluorescence lifetime of the donor is monitored, our method circumvents the extensive calculations necessitated by intensity-based FRET interaction assays and thus provides a feasible base for studying the sub-compartmentalization in the plasma membrane of living plant cells with a nanoscale resolution.