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Visualization of BRI1 and SERK3/BAK1 Nanoclusters in Arabidopsis Roots
Stefan J Hutten1, Danny S Hamers1, Marije Aan den Toorn1
1Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, WE Wageningen, The Netherlands.
Plos One
|January 24, 2017
Summary
Plant hormone receptors brassinosteroid-insensitive 1 (BRI1) and SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASE 3/BRI1 ASSOCIATED KINASE 1 (SERK3/BAK1) form nanoclusters at the plasma membrane. Ligand binding reduces BRI1-SERK3/BAK1 hetero-oligomers, suggesting endocytosis regulates signaling.
Area of Science:
- Plant molecular biology
- Cell signaling
- Biophysics
Background:
- Brassinosteroids (BRs) are crucial plant hormones regulating growth and development.
- BR signaling initiates at the plasma membrane (PM) via the receptor BRASSINOSTEROID-INSENSITIVE1 (BRI1) and its co-receptor SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASE 3/BRI1 ASSOCIATED KINASE 1 (SERK3/BAK1).
- Understanding receptor complex dynamics at the PM is key to deciphering BR signal transduction.
Purpose of the Study:
- To visualize and characterize the distribution and interaction of BRI1 and SERK3/BAK1 at the plant PM.
- To investigate the role of ligand binding in the formation and stoichiometry of BRI1-SERK3/BAK1 complexes within nanoclusters.
- To elucidate the biophysical mechanisms governing receptor complex organization and signaling.
Main Methods:
- Variable-angle epifluorescence microscopy (VAEM) for visualizing BRI1-GFP and SERK3/BAK1-mCherry distribution in the PM.
- Selective-surface observation-fluorescence lifetime imaging microscopy (SSO-FLIM) coupled with Förster resonance energy transfer (FRET) to detect receptor hetero-oligomerization.
- Manipulation of endogenous and exogenous BR ligands to assess their impact on receptor complex dynamics.
Main Results:
- VAEM revealed inhomogeneous distribution of BRI1 and SERK3/BAK1, forming distinct nanoclusters at the PM.
- Neither BRI1 nor SERK3/BAK1 nanocluster density was affected by ligand depletion or application.
- SSO-FLIM-FRET demonstrated hetero-oligomerization of BRI1 and SERK3/BAK1 within nanoclusters, which was unaffected by ligand absence or signal activation but reduced upon ligand application, potentially due to endocytosis.
Conclusions:
- Plant PM nanocluster formation is governed by biophysical constraints.
- The stoichiometry of BRI1-SERK3/BAK1 receptors within nanoclusters is variable and crucial for signal transduction.
- Ligand-induced reduction in BRI1-SERK3/BAK1 hetero-oligomers suggests endocytosis of active signaling units regulates BR perception.
