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Mapping Transient Protein Interactions at the Nanoscale in Living Mammalian Cells
ACS Nano ( IF 15.8 ) Pub Date : 2018-09-07 00:00:00 , DOI: 10.1021/acsnano.8b01227
Herlinde De Keersmaecker , Rafael Camacho , David Manuel Rantasa , Eduard Fron , Hiroshi Uji-i 1 , Hideaki Mizuno , Susana Rocha
Affiliation  

Protein–protein interactions (PPIs) form the basis of cellular processes, regulating cell behavior and fate. PPIs can be extremely transient in nature, which hinders their detection. In addition, traditional biochemical methods provided limited information on the spatial distribution and temporal dynamics of PPIs that is crucial for their regulation in the crowded cellular environment. Given the pivotal role of membrane micro- and nanodomains in the regulation of PPIs at the plasma membrane, the development of methods to visualize PPIs with a high spatial resolution is imperative. Here, we present a super-resolution fluorescence microscopy technique that can detect and map short-lived transient protein–protein interactions on a nanometer scale in the cellular environment. This imaging method is based on single-molecule fluorescence microscopy and exploits the effect of the difference in the mobility between cytosolic and membrane-bound proteins in the recorded fluorescence signals. After the development of the proof of concept using a model system based on membrane-bound modular protein domains and fluorescently labeled peptides, we applied this imaging approach to investigate the interactions of cytosolic proteins involved in the epidermal growth factor signaling pathway (namely, Grb2, c-Raf, and PLCγ1). The detected clusters of Grb2 and c-Raf were correlated with the distribution of the receptor at the plasma membrane. Additionally, the interactions of wild type PLCγ1 were compared with those detected with truncated mutants, which provided important information regarding the role played by specific domains in the interaction with the membrane. The results presented here demonstrate the potential of this technique to unravel the role of membrane heterogeneity in the spatiotemporal regulation of cell signaling.

中文翻译:

在哺乳动物细胞的纳米尺度上绘制瞬态蛋白质相互作用。

蛋白质间相互作用(PPI)构成细胞过程,调节细胞行为和命运的基础。PPI本质上可能是非常短暂的,这会阻碍其检测。此外,传统的生化方法提供了关于PPI的空间分布和时间动态的有限信息,这对于它们在拥挤的细胞环境中的调节至关重要。鉴于膜微域和纳米域在质膜上调节PPI的关键作用,迫切需要开发出具有高空间分辨率的PPI可视化方法。在这里,我们提出了一种超高分辨率的荧光显微镜技术,该技术可以在细胞环境中以纳米尺度检测和绘制短暂的瞬时蛋白质-蛋白质相互作用。这种成像方法基于单分子荧光显微镜,并利用了所记录的荧光信号中胞质和膜结合蛋白之间迁移率差异的影响。在使用基于膜结合的模块化蛋白质结构域和荧光标记的肽的模型系统开发概念验证后,我们应用了这种成像方法来研究参与表皮生长因子信号传导途径(即Grb2, c-Raf和PLCγ1)。检测到的Grb2和c-Raf簇与质膜上受体的分布相关。此外,将野生型PLCγ1的相互作用与截短突变体检测到的相互作用进行了比较,其中提供了有关特定结构域在与膜相互作用中所起的作用的重要信息。此处介绍的结果证明了该技术有潜力揭示膜异质性在细胞信号时空调节中的作用。
更新日期:2018-09-07
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