Skip to Main content Skip to Navigation
Journal articles

Optimizing FRET-FLIM labeling conditions to detect nuclear protein interactions at native expression levels in living Arabidopsis roots

Abstract : Protein complex formation has been extensively studied using Forster 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.
Document type :
Journal articles
Complete list of metadata

Cited literature [77 references]  Display  Hide  Download

https://hal.inrae.fr/hal-02621264
Contributor : Migration Prodinra Connect in order to contact the contributor
Submitted on : Tuesday, May 26, 2020 - 1:38:31 AM
Last modification on : Wednesday, November 3, 2021 - 5:42:51 AM

File

2018_Long_Front Plant Sci_1.pd...
Publisher files allowed on an open archive

Licence


Distributed under a Creative Commons Attribution 4.0 International License

Identifiers

Collections

Citation

Yuchen Long, Yvonne Stahl, Stefanie Weidtkamp-Peters, Wouter Smet, Yujuan Du, et al.. Optimizing FRET-FLIM labeling conditions to detect nuclear protein interactions at native expression levels in living Arabidopsis roots. Frontiers in Plant Science, Frontiers, 2018, 9, ⟨10.3389/fpls.2018.00639⟩. ⟨hal-02621264⟩

Share

Metrics

Record views

74

Files downloads

141