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- Title
Optimizing FRET-FLIM Labeling Conditions to Detect Nuclear Protein Interactions at Native Expression Levels in Living Arabidopsis Roots.
- Authors
Yuchen Long; Stahl, Yvonne; Weidtkamp-Peters, Stefanie; Smet, Wouter; Yujuan Du; Gadella Jr, Theodorus W. J.; Goedhart, Joachim; Scheres, Ben; Blilou, Ikram
- Abstract
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 highlymodulated 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.
- Subjects
NUCLEAR proteins; ARABIDOPSIS
- Publication
Frontiers in Plant Science, 2018, p1
- ISSN
1664-462X
- Publication type
Article
- DOI
10.3389/fpls.2018.00639