Visualization of receptor activation in plants

Perception of external signals through cell surface receptors is a basic property of all organisms. Typically, such signaling pathways are activated by binding of a specific ligand to the extracellular domain of a receptor protein followed by changes in the phosphorylation status of the intracellular kinase domain of the receptor. Induced alterations in receptor phosphorylation ultimately lead to intracellular signaling, often involving changes in gene expression. Phosphorylation of receptor kinases (RKs) is the first step in downstream signaling and progress has been made identifying important components using biochemical approaches in combination with mass spectrometry. However these approaches are invasive and no spatial and dynamic properties of biological network is obtained. This project aims to develop a fluorescence-based biosensor enabling to elucidate timing, localization and dynamics of active receptors in the model plant Arabidopsis thaliana. Visualization of receptor activation will be accomplished by mapping their phosphorylation status for unravelling signaling networks upon ligand-induced activation. The innovative aspect of this approach is to monitor active receptors or receptor complexes in a complete organism unlike what has been shown in the animal field where receptor phosphorylation is observed in cultured cells only. The brassinolide-dependent activation of the Brassinisteroid-Insensitive1 (BRI1) receptor kinase will be studied. Mass spectrometry has identified the phosphorylated serine and threonine residues in the kinase domain of the BRI1 receptor and its co-receptors, but the dynamics of (co-) receptor activation and the sub-cellular localization of these processes are hardly known, mostly because of technical limitations in visualizing specifically activated receptors. In order to overcome these limitations we will combine expertise in fluorescence spectroscopy with genetic and biochemical approaches. The developed biosensors will be used to obtain a detailed picture of membrane receptor activation through phosphorylation in cells of entire plants. The spatial and semi-quantitative analysis of receptor activation in real time will significantly add to our understanding of receptor-mediated signaling events. Furthermore, this approach has the potential to become a generic tool in studying protein modifications in signaling processes.