B04: Dynamics of ligand induced plasmamembrane receptor complex assembly and intermembrane transport in plants

The development of plant meristems, the cell groups that harbour stem cell populations, is controlled by a set of plasmamembrane (PM) localised receptor like kinases (RLKs) that perceive extracellular peptides secreted from neighbouring cells. Peptides derived from stem cells or differentiated cells trigger intracellular signal transduction cascades via specific RLK complexes. The outcome of the signalling event often results in the cell acquiring a new fate, which is brought about by activation of target transcription factors. Genetic and molecular studies in Arabidopsis thaliana have indicated that the same peptide can signal through different RLKs, and that individual RLKs can perceive diverse peptide ligands. This raises the question of how specificity is being generated. To gain a comprehensive picture on the signalling pathways that control stem cell populations, we will investigate how two closely related and functionally overlapping, secreted peptides, CLE40 and CLV3, signal through a set of diverse RLKs and receptor-like proteins (RLPs). These RLKs and RLPs assemble into multimeric complexes and regulate similar biological processes in different plant tissues. We aim to understand the assembly of these receptor complexes in the endoplasmatic reticulum (ER) and at the PM, their composition and stoichiometry, the specificity of different receptor assemblies for the generation of distinct signalling outputs, the structure of receptor clusters in membrane microdomains, and the turnover of complex components. Multiparameter Fluorescence Imaging Spectroscopy (MFIS) of fluorescently labelled receptors in planta will allow us to monitor preformed and induced receptor complex assembly. Statistical analysis of fluorescence depolarization and fluorescence lifetime will permit to extract information on homo- and heteromerization, and brightness analysis will address the stoichiometry of complexes. Engineered versions of the RLKs will identify the amino acid motifs required for complex formation, and in vivo complementation studies with mutant receptors will indicate the relevance of receptor complex formation for the generation of signalling outputs. Temporal studies will allow to identify the different steps of RLK interaction and activation. Receptor mobility and turnover from the plasmamembrane will be studied using fluorescent protein fusions, FRAP (fluorescence recover after photobleaching) and vaTIRFM (variable angle total internal reflection fluorescence microscopy). Receptor clusters at membrane microdomains will be further resolved using STED (stimulated emission depletion) superresolution microscopy.

These studies will generate a deeper understanding how distinct assemblies of receptors and peptide ligands mediate the communication of plant stem cells with each other and their differentiating descendents.

Project leaders:
Prof. Dr. Rüdiger Simon, undefined email, undefinedInstitute of Developmental Genetics,
                           Prof. Dr. Claus Seidel, undefined email, undefinedInstitute of Molecular Physical Chemistry

Researcher: Dr. Grégoire Denay, undefined email

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