Manko Hanna, Normant Vincent, Perraud Quentin, Steffan Tania, Gasser Véronique, Boutant Emmanuel, Réal Éléonore, Schalk Isabelle J, Mély Yves, Godet Julien
Université de Strasbourg, Laboratoire de Bioimagerie et Pathologies, UMR CNRS 7021.
Université de Strasbourg, UMR 7242, ESBS; CNRS, UMR 7242, ESBS.
J Vis Exp. 2020 Aug 25(162). doi: 10.3791/61602.
Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with Förster resonance energy transfer (FRET) provide accurate information about PPIs in live cells. FLIM-FRET relies on measuring the fluorescence lifetime decay of a FRET donor at each pixel of the FLIM image, providing quantitative and accurate information about PPIs and their spatial cellular organizations. We propose here a detailed protocol for FLIM-FRET measurements that we applied to monitor PPIs in live Pseudomonas aeruginosa in the particular case of two interacting proteins expressed with highly different copy numbers to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. This protocol describes in detail all the necessary steps for PPI characterization - starting from bacterial mutant constructions up to the final analysis using recently developed tools providing advanced visualization possibilities for a straightforward interpretation of complex FLIM-FRET data.
蛋白质-蛋白质相互作用（PPIs）控制着细胞内的各种关键过程。荧光寿命成像显微镜（FLIM）与Förster共振能量转移（FRET）相结合，可提供活细胞中PPIs的准确信息。FLIM-FRET依赖于在FLIM图像的每个像素处测量FRET供体的荧光寿命衰减，从而提供有关PPIs及其空间细胞组织的定量和准确信息。我们在此提出一种用于FLIM-FRET测量的详细方案，该方案应用于监测活的铜绿假单胞菌中的PPIs，具体是针对以高度不同的拷贝数表达的两种相互作用蛋白的情况，以证明该技术在揭示PPIs关键特征方面的质量和稳健性。该方案详细描述了PPI表征所需的所有必要步骤——从细菌突变体构建到最终分析，使用最近开发的工具，为复杂的FLIM-FRET数据提供直接解释的高级可视化可能性。
