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

Handle URI:
http://hdl.handle.net/10754/627904
Title:
Optimizing FRET-FLIM Labeling Conditions to Detect Nuclear Protein Interactions at Native Expression Levels in Living Arabidopsis Roots
Authors:
Long, Yuchen; Stahl, Yvonne; Weidtkamp-Peters, Stefanie; Smet, Wouter; Du, Yujuan; Gadella, Theodorus W. J.; Goedhart, Joachim; Scheres, Ben; Blilou, Ikram ( 0000-0001-8003-3782 )
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.
KAUST Department:
Biological and Environmental Sciences and Engineering (BESE) Division; Desert Agriculture Initiative
Citation:
Long 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.
Publisher:
Frontiers Media SA
Journal:
Frontiers in Plant Science
Issue Date:
15-May-2018
DOI:
10.3389/fpls.2018.00639
Type:
Article
ISSN:
1664-462X
Sponsors:
The 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.
Additional Links:
https://www.frontiersin.org/articles/10.3389/fpls.2018.00639/full
Appears in Collections:
Articles; Desert Agriculture Initiative; Biological and Environmental Sciences and Engineering (BESE) Division

Full metadata record

DC FieldValue Language
dc.contributor.authorLong, Yuchenen
dc.contributor.authorStahl, Yvonneen
dc.contributor.authorWeidtkamp-Peters, Stefanieen
dc.contributor.authorSmet, Wouteren
dc.contributor.authorDu, Yujuanen
dc.contributor.authorGadella, Theodorus W. J.en
dc.contributor.authorGoedhart, Joachimen
dc.contributor.authorScheres, Benen
dc.contributor.authorBlilou, Ikramen
dc.date.accessioned2018-05-17T06:24:05Z-
dc.date.available2018-05-17T06:24:05Z-
dc.date.issued2018-05-15en
dc.identifier.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.en
dc.identifier.issn1664-462Xen
dc.identifier.doi10.3389/fpls.2018.00639en
dc.identifier.urihttp://hdl.handle.net/10754/627904-
dc.description.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.en
dc.description.sponsorshipThe 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.en
dc.publisherFrontiers Media SAen
dc.relation.urlhttps://www.frontiersin.org/articles/10.3389/fpls.2018.00639/fullen
dc.rightsArchived with thanks to Frontiers in Plant Scienceen
dc.titleOptimizing FRET-FLIM Labeling Conditions to Detect Nuclear Protein Interactions at Native Expression Levels in Living Arabidopsis Rootsen
dc.typeArticleen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
dc.contributor.departmentDesert Agriculture Initiativeen
dc.identifier.journalFrontiers in Plant Scienceen
dc.eprint.versionPublisher's Version/PDFen
dc.contributor.institutionPlant Developmental Biology, Wageningen University and Research Centre, Wageningen, Netherlandsen
dc.contributor.institutionInstitute for Developmental Genetics, Heinrich Heine University, Düsseldorf, Germanyen
dc.contributor.institutionCenter for Advanced Imaging, Heinrich Heine University, Düsseldorf, Germanyen
dc.contributor.institutionSection of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, Netherlandsen
kaust.authorBlilou, Ikramen
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