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Fluorescent castasterone reveals BRI1 signaling from the plasma membrane

Nature Chemical Biology volume 8, pages 583–589 (2012)Cite this article

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Abstract

Receptor-mediated endocytosis is an integral part of signal transduction as it mediates signal attenuation and provides spatial and temporal dimensions to signaling events. One of the best-studied leucine-rich repeat receptor–like kinases in plants, BRASSINOSTEROID INSENSITIVE 1 (BRI1), perceives its ligand, the brassinosteroid (BR) hormone, at the cell surface and is constitutively endocytosed. However, the importance of endocytosis for BR signaling remains unclear. Here we developed a bioactive, fluorescent BR analog, Alexa Fluor 647–castasterone (AFCS), and visualized the endocytosis of BRI1–AFCS complexes in living Arabidopsis thaliana cells. Impairment of endocytosis dependent on clathrin and the guanine nucleotide exchange factor for ARF GTPases (ARF-GEF) GNOM enhanced BR signaling by retaining active BRI1-ligand complexes at the plasma membrane. Increasing the trans-Golgi network/early endosome pool of BRI1–BR complexes did not affect BR signaling. Our findings provide what is to our knowledge the first visualization of receptor-ligand complexes in plants and reveal clathrin- and ARF-GEF–dependent endocytic regulation of BR signaling from the plasma membrane.

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Figure 1: Characterization of AFCS.
Figure 2: AFCS follows a similar trafficking route as BRI1 via the TGN/EEs and MVBs and is sequestered into the vacuole.
Figure 3: Inhibition of BRI1 endocytosis at the plasma membrane results in enhanced BR signaling.
Figure 4: Inhibition of GNOM-dependent endocytosis at the plasma membrane but not accumulation of BRI1–AFCS in the TGN/EE results in enhanced BR signaling.

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Acknowledgements

We thank N. Geldner, Y. Yin, S. Clouse, Xiaofeng Wang, S. Huber and Xuelu Wang for sharing materials and protocols; S. Figaroli for technical assistance; M. De Cock for the help with manuscript preparation; and J. Martins for NMR and structure analysis. This work is supported by the Odysseus program of the Research Foundation-Flanders (to J.F.), the BRAVISSIMO Marie-Curie Initial Training Network (predoctoral fellowships to S.D.R. and J.S.-P.), the Research Foundation-Flanders (postdoctoral fellowship to D.V.D.), the Agency for Innovation by Science and Technology (postdoctoral fellowship to K.M.), the Research Foundation-Flanders (research grant to E.R.) and projects 1M06030 and Z4 055 0506 (to J.H., M.S. and L.K.). The 700-MHz equipment of the Interuniversitary NMR Facility used in this work was financed by Ghent University, the Free University of Brussels and the University of Antwerp via the 'Zware Apparatuur' Incentive of the Flemish Government.

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Author notes

  1. Niloufer G Irani and Simone Di Rubbo: These authors contributed equally to this work.

Affiliations

  1. Department of Plant Systems Biology, VIB, Ghent, Belgium.

    Niloufer G Irani, Simone Di Rubbo, Evelien Mylle, Joanna Schneider-Pizoń, Anna-Mária Szatmári, Daniël Van Damme, Kiril Mishev, Mirela-Corina Codreanu, Jiří Friml & Eugenia Russinova

  2. Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium

    Niloufer G Irani, Simone Di Rubbo, Evelien Mylle, Joanna Schneider-Pizoń, Anna-Mária Szatmári, Daniël Van Damme, Kiril Mishev, Mirela-Corina Codreanu, Jiří Friml & Eugenia Russinova

  3. Department of Organic Chemistry, Laboratory for Organic and Biomimetic Chemistry, Ghent University, Ghent, Belgium

    Jos Van den Begin, Dieter Buyst & Annemieke Madder

  4. Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic

    Jaroslava Hniliková & Ladislav Kohout

  5. Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Olomouc, Czech Republic

    Miroslav Šíša & Miroslav Strnad

  6. Department of Molecular Genetics, Centre for Research in Agricultural Genomics, Barcelona, Spain

    Josep Vilarrasa-Blasi & Ana I Caño-Delgado

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  1. Niloufer G Irani
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Contributions

N.G.I., S.D.R., J.F. and E.R. conceived the study and designed the experiments. N.G.I., S.D.R., E.M., J.S.-P., A.-M.S., J.V.-B., K.M., M.-C.C. and D.V.D. performed experiments and analyzed the results. N.G.I., J.V.d.B., J.H., M.S., M.Š., L.K., D.B. and A.M. performed the chemical synthesis and analyzed the results. N.G.I., S.D.R., A.I.C.-D., D.V.D., A.M., J.F. and E.R. planned experiments and analyses. N.G.I., S.D.R., J.F. and E.R. wrote the manuscript. All authors commented on the results and the manuscript.

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Correspondence to Eugenia Russinova.

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Irani, N., Di Rubbo, S., Mylle, E. et al. Fluorescent castasterone reveals BRI1 signaling from the plasma membrane. Nat Chem Biol 8, 583–589 (2012). https://doi.org/10.1038/nchembio.958

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