title: Visualization of BRI1 and SERK3/BAK1 Nanoclusters in Arabidopsis Roots.
abstract: Brassinosteroids (BRs) are plant hormones that are perceived at the plasma membrane (PM) by the ligand binding receptor BRASSINOSTEROID-INSENSITIVE1 (BRI1) and the co-receptor SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASE 3/BRI1 ASSOCIATED KINASE 1 (SERK3/BAK1). To visualize BRI1-GFP and SERK3/BAK1-mCherry in the plane of the PM, variable-angle epifluorescence microscopy (VAEM) was employed, which allows selective illumination of a thin surface layer. VAEM revealed an inhomogeneous distribution of BRI1-GFP and SERK3/BAK1-mCherry at the PM, which we attribute to the presence of distinct nanoclusters. Neither the BRI1 nor the SERK3/BAK1 nanocluster density is affected by depletion of endogenous ligands or application of exogenous ligands. To reveal interacting populations of receptor complexes, we utilized selective-surface observation-fluorescence lifetime imaging microscopy (SSO-FLIM) for the detection of Förster resonance energy transfer (FRET). Using this approach, we observed hetero-oligomerisation of BRI1 and SERK3 in the nanoclusters, which did not change upon depletion of endogenous ligand or signal activation. Upon ligand application, however, the number of BRI1-SERK3 /BAK1 hetero-oligomers was reduced, possibly due to endocytosis of active signalling units of BRI1-SERK3/BAK1 residing in the PM. We propose that formation of nanoclusters in the plant PM is subjected to biophysical restraints, while the stoichiometry of receptors inside these nanoclusters is variable and important for signal transduction.
authors.affiliation: Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, WE Wageningen, The Netherlands.
authors.forename: Stefan J
authors.initials: SJ
authors.lastname: Hutten
authors.affiliation: Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, WE Wageningen, The Netherlands.
authors.forename: Danny S
authors.initials: DS
authors.lastname: Hamers
authors.affiliation: Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, WE Wageningen, The Netherlands.
authors.forename: Marije
authors.initials: M
authors.lastname: Aan den Toorn
authors.affiliation: Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, WE Wageningen, The Netherlands.
authors.forename: Wilma
authors.initials: W
authors.lastname: van Esse
authors.affiliation: Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, WE Wageningen, The Netherlands.
authors.forename: Antsje
authors.initials: A
authors.lastname: Nolles
authors.affiliation: Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, WE Wageningen, The Netherlands.
authors.forename: Christoph A
authors.initials: CA
authors.lastname: Bücherl
authors.affiliation: Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, WE Wageningen, The Netherlands.
authors.forename: Sacco C
authors.initials: SC
authors.lastname: de Vries
authors.affiliation: Laboratory of Biophysics, Wageningen University & Research, Stippeneng 4, WE Wageningen, The Netherlands.|Microspectroscopy Centre, Wageningen University & Research, Stippeneng 4, WE Wageningen, The Netherlands.
authors.forename: Johannes
authors.initials: J
authors.lastname: Hohlbein
authors.affiliation: Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, WE Wageningen, The Netherlands.|Microspectroscopy Centre, Wageningen University & Research, Stippeneng 4, WE Wageningen, The Netherlands.
authors.forename: Jan Willem
authors.initials: JW
authors.lastname: Borst
mesh_terms: D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D049329:Nanostructures; D018517:Plant Roots / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D011494:Protein Kinases; C463660:BAK1 protein, Arabidopsis; C108270:BRI1 protein, Arabidopsis; D017346:Protein Serine-Threonine Kinases