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

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...

Full description

Saved in:
Bibliographic Details
Published in:Frontiers in plant science 2018-05, Vol.9, p.639-639
Main Authors: Long, Yuchen, Stahl, Yvonne, Weidtkamp-Peters, Stefanie, Smet, Wouter, Du, Yujuan, Gadella, Jr, Theodorus W J, Goedhart, Joachim, Scheres, Ben, Blilou, Ikram
Format: Article
Language:English
Subjects:
Fluorescent proteins
In vivo FRET-FLIM
Life Sciences
Plant Science
Protein complexes
Protein-protein interaction
SCARECROW
SHORT-ROOT
Vegetal Biology
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:1664-462X
1664-462X
DOI:10.3389/fpls.2018.00639