Visualization of BRI1 and SERK3/BAK1 nanoclusters in Arabidopsis 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 northeSERK3/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 Forster 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

VAEM reveals a heterogeneous distribution of BRI1-GFP and SERK3-mCherry in the PM.

<p>Typical VAEM images of live root epidermal cells of 6 day old <i>A</i>. <i>thaliana</i> seedlings showing PM distribution of (<b>A</b>) BRI-GFP line1, (<b>B</b>) BRI1-GFP line 2 and (<b>C</b>) SERK3-mCherry, (<b>D</b>) BRI1-GFP in a <i>serk1serk3</i> mutant plant. Images taken are of epidermal cells in the early elongation zone. Scale bars represent 10 μm.</p

Mobile fractions and diffusion coefficients of BRI1 and SERK3/BAK1.

<p>Mobile fractions and diffusion coefficients of BRI1 and SERK3/BAK1.</p

FRAP analysis of BRI1-GFP and KNOLLE-GFP in epidermal cells of <i>Arabidopsis</i> roots.

<p>(<b>A</b>) Typical images of FRAP experiment prior and post bleach pulse of KNOLLE-GFP and BRI1-GFP. (<b>B</b>) Recovery-curves of KNOLLE-GFP (blue line) and BRI1-GFP (black line) in epidermal cells in the root meristem. Around 200 s after bleaching, fluorescence intensity of KNOLLE-GFP at the PM is fully restored. No such recovery is observed for BRI1-GFP. For KNOLLE-GFP n = 7, for BRI1-GFP n = 15, measured in independent replicas, error bars indicate standard error of means (SEM).</p

BRI1-GFP nanoclusters imaged using VAEM and SSO-confocal imaging.

<p>BRI1-GFP line 2 expressed in root epidermal cells imaged using VAEM (A) or SSO-confocal imaging (B). Both imaging modalities show similar nanocluster distributions. Scale bars represent 10 μm.</p

SSO-FRET-FLIM of BRI1-GFP and BRI1-GFP/SERK3/BAK1-mCherry in absence and presence of BL.

<p>(<b>A</b>) SSO fluorescence intensity image of BRI1-GFP expressed in root epidermal cells pretreated with 50 μM BRZ (<b>B</b>) SSO fluorescence lifetime image of BRI1-GFP expressed in root epidermal cells pretreated with 50 μM BRZ (<b>C</b>) SSO fluorescence lifetime image of BRI1-GFP/SERK3/BAK1-mCherry expressed in root epidermal cells pretreated with 50 μM BRZ. (<b>D</b>) SSO fluorescence lifetime image of BRI1-GFP/SERK3/BAK1-mCherry expressed in root epidermal cells pretreated with 50 μM BRZ and subsequent incubation with 1 μM BL for 1 hour. The color bar represents the false color code for fluorescence lifetime (<i>τ</i>) distribution. Scale bar represents 10 μm.</p

Quantitative analysis of SSO FRET-FLIM measurements performed in planta on roots of 5 day old <i>Arabidopsis</i> roots expressing BRI1-GFP or BRI1-GFP -SERK3/BAK1-mCherry.

<p>Quantitative analysis of SSO FRET-FLIM measurements performed in planta on roots of 5 day old <i>Arabidopsis</i> roots expressing BRI1-GFP or BRI1-GFP -SERK3/BAK1-mCherry.</p

Fluorescence lifetime distribution of high intensity nanoclusters of BRI1-GFP and BRI1-GFP- SERK3/BAK1-mCherry in the absence and presence of BL.

<p>Fluorescence lifetime distribution plots of nanoclusters with fluorescence intensities above 2000 photons per pixel. (<b>A</b>) Fluorescence lifetime distribution of BRI1-GFP nanoclusters pretreated with 50 μM BRZ (n = 91). (<b>B</b>) Fluorescence lifetime distribution of BRI1-GFP/SERK3-mCherry nanoclusters after treatment with 50 μM BRZ (n = 126). (<b>C</b>) Fluorescence lifetime distribution of BRI1-GFP-SERK3/BAK1-mCherry nanoclusters pretreated with 50 μM BRZ followed by application of 1 μM BL for 1 hour (n = 95). The data were obtained from 3 independent series of experiments.</p

Quantification of the number of receptors per nanocluster.

<p>Quantification of the number of receptors per nanocluster.</p

Quantification of clusters/ μm² and cluster size of BRI1 and SERK3.

<p>Quantification of clusters/ μm² and cluster size of BRI1 and SERK3.</p