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Plant Hormones pp 135-146 | Cite as

FRET-FLIM for Visualizing and Quantifying Protein Interactions in Live Plant Cells

  • Alejandra Freire Rios
  • Tatyana Radoeva
  • Bert De Rybel
  • Dolf Weijers
  • Jan Willem BorstEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1497)

Abstract

Proteins are the workhorses that control most biological processes in living cells. Although proteins can accomplish their functions independently, the vast majority of functions require proteins to interact with other proteins or biomacromolecules. Protein interactions can be investigated through biochemical assays such as co-immunoprecipitation (co-IP) or Western blot analysis, but such assays lack spatial information. Here we describe a well-developed imaging method, Förster resonance energy transfer (FRET) analyzed by fluorescence lifetime imaging microscopy (FLIM), that can be used to visualize protein interactions with both spatial and temporal resolution in live cells. We demonstrate its use in plant developmental research by visualizing in vivo dimerization of AUXIN RESPONSE FACTOR (ARF) proteins, mediating auxin responses.

Key words

FRET FLIM Protein interactions Fluorescent proteins 

Notes

Acknowledgements

This study was supported by the Netherlands Organization for Scientific Research (NWO; NWO-NSFC grant number 846.11.001 and ECHO grant 711.011.002 to D.W.). B.D.R. was funded by the Netherlands Organization for Scientific Research (NWO; VIDI 864.13.001) and by The Research Foundation—Flanders (FWO; Odysseus II G0D0515N and Post-doc grant 12D1815N). The authors would like to thank Dr. S. Lindhoud for providing us with Fig. 1. FRET-FLIM experiments were performed on a multimode confocal microscope supported by an NWO Middelgroot Investment Grant (721.011.004; J.W.B.).

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Alejandra Freire Rios
    • 1
  • Tatyana Radoeva
    • 1
  • Bert De Rybel
    • 1
    • 2
    • 3
  • Dolf Weijers
    • 1
  • Jan Willem Borst
    • 1
    • 4
    Email author
  1. 1.Laboratory of BiochemistryWageningen UniversityWageningenThe Netherlands
  2. 2.Department of Plant Systems Biology, Flemish Institute of BiotechnologyVIB, Ghent UniversityGhentBelgium
  3. 3.Department of Plant Biotechnology and BioinformaticsGhent UniversityGhentBelgium
  4. 4.Microspectroscopy CenterWageningen UniversityWageningenThe Netherlands

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