In Vivo Imaging of Brassinosteroid Endocytosis in Arabidopsis

  • Niloufer G. Irani
  • Simone Di Rubbo
  • Eugenia RussinovaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1209)


Increasing evidence shows the involvement of endocytosis in specific signaling outputs in plants. To better understand the interplay between endocytosis and signaling in plant systems, more ligand–receptor pairs need to be identified and characterized. Crucial for the advancement of this research is also the development of imaging techniques that allow the visualization of endosome-associated signaling events at a high spatiotemporal resolution. This requires the establishment of tools to track ligands and their receptors by fluorescence microscopy in living cells. The brassinosteroid (BR) signaling pathway has been among the first systems to be characterized with respect to its connection with endocytic trafficking, owning to the fact that a fluorescent version of BR, Alexa Fluor 647-castasterone (AFCS) has been generated. AFCS and the fluorescently tagged BR receptor, BR INSENSITIVE1 (BRI1) have been used for the specific detection of BRI1-AFCS endocytosis and for the delineation of their endocytic route as being clathrin-mediated. AFCS was successfully applied in functional studies in which pharmacological rerouting of the BRI1-BR complex was shown to have an impact on signaling. Here we provide a method for the visualization of endocytosis of plant receptors in living cells. The method was used to track endocytosis of BRI1-BR complexes in Arabidopsis epidermal root meristem cells by using fluorescent BRs. Pulse-chase experiments combined with quantitative confocal microscopy were used to determine the internalization rates of BRs. This method is well suited to measure the internalization of other plant receptors if fluorescent ligands are available.

Key words

Brassinosteroids Endocytosis Receptor-mediated endocytosis Confocal microscopy Live cell fluorescence microscopy Plant endomembrane systems 



We thank A. Bleys for help with manuscript preparation. This work is supported by the Odysseus program of the Research Foundation-Flanders and the BRAVISSIMO Marie-Curie Initial Training Network (predoctoral fellowships to S.D.R).


  1. 1.
    Chen X, Irani NG, Friml J (2011) Clathrin-mediated endocytosis: the gateway into plant cells. Curr Opin Plant Biol 14:674–682PubMedCrossRefGoogle Scholar
  2. 2.
    Vida TA, Emr SD (1995) A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 128:779–792PubMedCrossRefGoogle Scholar
  3. 3.
    Jelínková A, Malínská K, Simon S et al (2010) Probing plant membranes with FM dyes: tracking, dragging or blocking? Plant J 61:883–892PubMedCrossRefGoogle Scholar
  4. 4.
    Irani NG, Di Rubbo S, Mylle E et al (2012) Fluorescent castasterone reveals BRI1 signaling from the plasma membrane. Nat Chem Biol 8:583–589PubMedGoogle Scholar
  5. 5.
    Wang Z-Y, Bai M-Y, Oh E et al (2012) Brassinosteroid signaling network and regulation of photomorphogenesis. Annu Rev Genet 46:701–724PubMedCrossRefGoogle Scholar
  6. 6.
    Russinova E, Borst J-W, Kwaaitaal M et al (2004) Heterodimerization and endocytosis of Arabidopsis brassinosteroid receptors BRI1 and AtSERK3 (BAK1). Plant Cell 16:3216–3229PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Geldner N, Hyman DL, Wang X et al (2007) Endosomal signaling of plant steroid receptor kinase BRI1. Genes Dev 21:1598–1602PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Viotti C, Bubeck J, Stierhof Y-D et al (2010) Endocytic and secretory traffic in Arabidopsis merge in the trans-Golgi network/early endosome, an independent and highly dynamic organelle. Plant Cell 22:1344–1357PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Caño-Delgado A, Yin Y, Yu C et al (2004) BRL1 and BRL3 are novel brassinosteroid receptors that function in vascular differentiation in Arabidopsis. Development 131:5341–5351PubMedCrossRefGoogle Scholar
  10. 10.
    Kinoshita T, Caño-Delgado AC, Seto H et al (2005) Binding of brassinosteroids to the extracellular domain of plant receptor kinase BRI1. Nature 433:167–171PubMedCrossRefGoogle Scholar
  11. 11.
    Hothorn M, Belkhadir Y, Dreux M et al (2011) Structural basis of steroid hormone perception by the receptor kinase BRI1. Nature 474:467–471PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    She J, Han Z, Kim T-W et al (2011) Structural insight into brassinosteroid perception by BRI1. Nature 474:472–476PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Li J, Chory J (1997) A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90:929–938PubMedCrossRefGoogle Scholar
  14. 14.
    Asami T, Min YK, Nagata N et al (2000) Characterization of brassinazole, a triazole-type brassinosteroid biosynthesis inhibitor. Plant Physiol 123:93–100PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Irani NG, Russinova E (2009) Receptor endocytosis and signaling in plants. Curr Opin Plant Biol 12:653–659PubMedCrossRefGoogle Scholar
  16. 16.
    Banbury AN, Oakley JD, Sessions RB et al (2003) Tyrphostin A23 inhibits internalization of the transferrin receptor by perturbing the interaction between tyrosine motifs and the medium chain subunit of the AP-2 adaptor complex. J Biol Chem 278:12022–12028PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Niloufer G. Irani
    • 1
    • 2
    • 3
  • Simone Di Rubbo
    • 1
    • 4
  • Eugenia Russinova
    • 1
    • 2
    Email author
  1. 1.Department of Plant Systems BiologyVIB, Ghent UniversityGhentBelgium
  2. 2.Department of Plant Biotechnology and BioinformaticsGhent UniversityGhentBelgium
  3. 3.Department of Plant SciencesUniversity of OxfordOxfordUK
  4. 4.Department of BiologyUniversity of Washington-HHMI, University of WashingtonWAUSA

Personalised recommendations