Data_Sheet_1_Optimizing FRET-FLIM Labeling Conditions to Detect Nuclear Protein Interactions at Native Expression Levels in Living Arabidopsis Roots.PDF

Protein complex formation has been extensively studied using Förster resonance energy transfer (FRET) measured by Fluorescence Lifetime Imaging Microscopy (FLIM). However, implementing this technology to detect protein interactions in living multicellular organism at single-cell resolution and under native condition is still difficult to achieve. Here we describe the optimization of the labeling conditions to detect FRET-FLIM in living plants. This study exemplifies optimization procedure involving the identification of the optimal position for the labels either at the N or C terminal region and the selection of the bright and suitable, fluorescent proteins as donor and acceptor labels for the FRET study. With an effective optimization strategy, we were able to detect the interaction between the stem cell regulators SHORT-ROOT and SCARECROW at endogenous expression levels in the root pole of living Arabidopsis embryos and developing lateral roots by FRET-FLIM. Using this approach we show that the spatial profile of interaction between two transcription factors can be highly modulated in reoccurring and structurally resembling organs, thus providing new information on the dynamic redistribution of nuclear protein complex configurations in different developmental stages. In principle, our optimization procedure for transcription factor complexes is applicable to any biological system.

蛋白质复合物的形成已广泛通过Förster共振能量转移（FRET）及荧光寿命成像显微镜（FLIM）进行的研究。然而，将此技术在单细胞分辨率和原生条件下应用于检测活体多细胞生物中的蛋白质相互作用，仍然是一项艰巨的挑战。本研究描述了优化标记条件以检测活植物中的FRET-FLIM的过程。该研究以标记位置的优化为例，探讨了在N端或C端区域寻找最佳标记位置，以及选择明亮且适宜的荧光蛋白作为供体和受体标记以进行FRET研究的策略。通过有效的优化策略，我们能够在活体拟南芥胚胎的根极和发育中的侧根中，检测到干细胞调节因子SHORT-ROOT和SCARECROW在原生表达水平下的相互作用。采用此方法，我们揭示了两个转录因子之间的相互作用在重复出现且结构相似的器官中的空间分布可以高度调节，从而为不同发育阶段中细胞核蛋白质复合体配置的动态重新分配提供了新的见解。原则上，我们对转录因子复合体的优化程序适用于任何生物系统。