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