Abstract
Forster resonance energy transfer (FRET) due to its sensitivity of distance has been widely used to investigate the structure and interaction of biomolecules. Multiparameter fluorescence image spectroscopy (MFIS)1 provides particular advantages to FRET imaging because all the parameters are monitored simultaneously with picosecond accuracy that allows for a comprehensive analysis on the biological system2.However, the decreased donor's fluorescence lifetime in FRET can have two reasons (1) the conformational change or (2) change in fraction of interacting proteins. Here we present a comprehensive analysis method to extract all the information from low number of photons typical for fluorescence imaging. Using a 3-species model, we analyzed the sub-ensemble data and found the decay pattern of each species, and then the decay patterns were applied to obtain pixel-wise species fractions for the entire image.The method was applied to study the different protein interactions in planta. The studied protein interactions are listed below classified by the signaling pathway they belong to:1. CLAVATA pathway: CLV1, CLV2 and CORYNE (CRN).2. Flagellin pathway: brassinosteroid-insensitive1-associated kinase1 (BAK1) and flagellin-sensitive 2 (FLS2).We were able to monitor the formation of FLS2-BAK1 receptor complexes over time after infiltrating the ligand flg22. Utilizing this method, we also show that the complex of CLV1, CLV2 and CRN is already assembled before ligand-binding, but that CLV3-binding to CLV1 could induce changes in the receptor complex formation and conformation in certain region of the cell membrane.[1] Weidtkamp-Peters, S., Felekyan, S., Bleckmann, A., Simon, R., Becker, W., Kuhnemuth, R., & Seidel, C. A (2009). Photochemical & Photobiological Sciences, 8(4), 470. doi:10.1039/b903245m.[2] Stahl, Y., Grabowski, S., Bleckmann, A., Kuhnemuth, R., Weidtkamp-Peters, S., Pinto, K., et al (2013). Receptor Kinase Complexes. Current Biology, 23(5), 362-371. doi:10.1016/j.cub.2013.01.045.
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