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.

The crossroads of receptor‐mediated signaling and endocytosis in plants

“…Although the distribution and composition of BRI1/BAK1‐containing MDs did not change upon ligand treatment, heterogeneous fluorescence lifetime distribution in the MDs was detected by means of the selective‐surface observation with Förster (or fluorescence) resonance energy transfer and fluorescence lifetime imaging (SSO‐FRET‐FLIM) (Hutten et al ). This discovery hinted at a different receptor stoichiometry within the MDs, supporting the notion that the BRI1 and BAK1 complexes are probably preassembled before reaching the PM (Bücherl et al ; Wang et al ; Hutten et al ). The difference in receptor stoichiometry within the cluster might reflect the various signaling outputs regulated by BAK1, a coreceptor shared among different signaling pathways.…”

Specifying the role of BAK1‐interacting receptor‐like kinase 3 in brassinosteroid signaling

“…These computational results open the possibility that a physical association of BRI1 and BAK1 is per se energetically possible at a site independent of the one observed for the complex in Figure C, even while BAK1 is in interaction with BIR3 (Figure ). This putative trimeric complex can provide the structural background for the experimentally observed hormone‐independent interaction of BRI1 and BAK1 in planta (Caesar et al ; Bücherl et al ; Hutten et al ). In these modeled complexes BRI1 cannot access the catalytic center of BAK1 but binds to another site of BAK1 that does not allow unspecific activation of BAK1 and BRI1.…”

“…Several groups have reported an interaction between BRI1 and BAK1 in the plasma membrane and co‐existence of BAK1 and BRI1 in membrane nanoclusters in the absence of exogenous BR or in BR‐depleted cells (Caesar et al ; Bücherl et al ; Hutten et al ). However, based on the broad and constitutive expression of BIR3 in Arabidopsis (Imkampe et al ) and our computational results, the interaction between the surface regions in the cytoplasmic domains of BIR3 and BAK1 appears to be too favorable to be interfered by the cytoplasmic domain of BRI1 in the absence of the hormonal ligand.…”

“…Cell biology and in vivo protein‐protein interaction studies showed that in the absence of BR and in BR‐depleted cells, BRI1 and BAK1 interact with each other and establish preformed hetero‐oligomers in nanoclusters located in the plasma membrane (Bücherl et al ; Hutten et al ). Remarkably, experiments indicate that the spatial distance between the BRI1 and BAK1 molecules and the hetero‐oligomerization status of BRI1 and BAK1 in the nanoclusters do not dramatically change upon BR application (Caesar et al ; Hutten et al ).…”

“…Cell biology and in vivo protein‐protein interaction studies showed that in the absence of BR and in BR‐depleted cells, BRI1 and BAK1 interact with each other and establish preformed hetero‐oligomers in nanoclusters located in the plasma membrane (Bücherl et al ; Hutten et al ). Remarkably, experiments indicate that the spatial distance between the BRI1 and BAK1 molecules and the hetero‐oligomerization status of BRI1 and BAK1 in the nanoclusters do not dramatically change upon BR application (Caesar et al ; Hutten et al ). The switch from the preformed inactive to the active BRI1‐BAK1 hetero‐oligomer is thought to be initiated by the binding of the BAK1's extracellular leucine‐repeat‐rich (LRR) domain to the BR‐bound extracellular LRR domain of BRI1 (Santiago et al ; Sun et al ; Hohmann et al ).…”