Cardiff University | Prifysgol Caerdydd ORCA
Online Research @ Cardiff 
WelshClear Cookie - decide language by browser settings

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

Long, Yuchen, Stahl, Yvonne, Weidtkamp-Peters, Stefanie, Smet, Wouter, Du, Yujuan, Gadella, Theodorus W. J., Goedhart, Joachim, Scheres, Ben and Blilou, Ikram 2018. Optimizing FRET-FLIM labeling conditions to detect nuclear protein interactions at native expression levels in living arabidopsis roots. Frontiers in Plant Science 9 , 639. 10.3389/fpls.2018.00639

[img]
Preview
PDF - Published Version
Available under License Creative Commons Attribution.

Download (1MB) | Preview

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 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.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Publisher: Frontiers Media
ISSN: 1664-462X
Date of First Compliant Deposit: 10 December 2018
Date of Acceptance: 25 April 2018
Last Modified: 11 Dec 2018 12:00
URI: http://orca.cf.ac.uk/id/eprint/117552

Citation Data

Cited 3 times in Scopus. View in Scopus. Powered By Scopus® Data

Actions (repository staff only)

Edit Item Edit Item

Downloads

Downloads per month over past year

View more statistics