Optimizing FRET-FLIM Labeling Conditions to Detect Nuclear Protein Interactions at Native Expression Levels in Living Arabidopsis Roots
Gadella, Theodorus W. J.
KAUST DepartmentBiological and Environmental Sciences and Engineering (BESE) Division
Desert Agriculture Initiative
MetadataShow full item record
AbstractProtein 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.
CitationLong Y, Stahl Y, Weidtkamp-Peters S, Smet W, Du Y, et al. (2018) Optimizing FRET-FLIM Labeling Conditions to Detect Nuclear Protein Interactions at Native Expression Levels in Living Arabidopsis Roots. Frontiers in Plant Science 9. Available: http://dx.doi.org/10.3389/fpls.2018.00639.
SponsorsThe authors are grateful to Prof Anna Akhmanova for providing mammalian cell line and lab facilities to conduct transfections in Hela cells and to Prof Rudiguer Simon for critical reading of the manuscript.
PublisherFrontiers Media SA
JournalFrontiers in Plant Science