Visualization of BRI1 and SERK3/BAK1 Nanoclusters in <i>Arabidopsis</i> Roots

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.油菜素类固醇（Brassinosteroids，BRs）是一类植物激素，其在质膜（plasma membrane，PM）上可被配体结合受体油菜素类固醇不敏感1（BRASSINOSTEROID-INSENSITIVE1，BRI1）以及共受体体细胞胚胎发生类受体激酶3/BRI1关联激酶1（SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASE 3/BRI1 ASSOCIATED KINASE 1，SERK3/BAK1）识别。为在质膜平面可视化BRI1-GFP与SERK3/BAK1-mCherry的定位，本研究采用了可对薄层表面进行选择性照明的变角度落射荧光显微镜（variable-angle epifluorescence microscopy，VAEM）。VAEM结果显示，BRI1-GFP与SERK3/BAK1-mCherry在质膜上呈现不均匀分布，我们将其归因于特异性纳米簇的存在。内源性配体耗竭或外源性配体处理均不会影响BRI1纳米簇与SERK3/BAK1纳米簇的密度。为揭示受体复合物的相互作用群体，本研究采用了用于检测福斯特共振能量转移（Förster resonance energy transfer，FRET）的选择性表面观测-荧光寿命成像显微镜（selective-surface observation—fluorescence lifetime imaging microscopy，SSO-FLIM）。通过该方法，我们观察到纳米簇内BRI1与SERK3发生异源寡聚化，且该现象在内源性配体耗竭或信号激活状态下均未发生改变。然而，施加外源性配体后，BRI1-SERK3/BAK1异源寡聚体的数量出现减少，这可能源于质膜上处于激活状态的BRI1-SERK3/BAK1信号单元发生了内吞作用。本研究提出，植物质膜上纳米簇的形成受到生物物理约束，而这些纳米簇内部受体的化学计量比具有可变性，且该特性对信号转导至关重要。