Three-fluorophore FRET-FLIM enables the study of trimeric protein interactions and complex formation with nanoscale resolution in living plant cells

Gloeckner, N.; zur Oven-Krockhaus, S.; Wackenhut, F.; Burmeister, M.; Wanke, F.; Holzwart, E.; Meixner, A. J.; Wolf, S.; Harter, K.

doi:10.1101/722124

bioRxiv · 10.1101/722124

Three-fluorophore FRET-FLIM enables the study of trimeric protein interactions and complex formation with nanoscale resolution in living plant cells

Gloeckner, N.·zur Oven-Krockhaus, S.·Wackenhut, F.·Burmeister, M.·Wanke, F.·Holzwart, E.·Meixner, A. J.·Wolf, S.·Harter, K.

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Abstract

Protein-protein interaction studies provide valuable insights into cellular signaling. The well-studied brassinosteroid (BR) signaling is initiated by the hormone-binding receptor Brassinosteroid Insensitive 1 (BRI1) and its co-receptor BRI1 Associated Kinase 1 (BAK1). BRI1 and BAK1 were shown to interact independently with the Receptor-Like Protein 44 (RLP44), which is implicated in BRI1/BAK1-dependent cell wall integrity perception. To demonstrate the proposed complex formation of BRI1, BAK1 and RLP44, we established three-fluorophore intensity-based spectral Forster resonance energy transfer (FRET) and FRET-fluorescence lifetime imaging microscopy (FLIM) for living plant cells. Our evidence indicates that RLP44, BRI1 and BAK1 form a ternary complex in a distinct plasma membrane nanodomain. In contrast, although the immune receptor Flagellin Sensing 2 (FLS2) also forms a heteromer with BAK1, the FLS2/BAK1 complexes are localized in other nanodomains. In general, our three-fluorophore FRET approaches provide a feasible basis for studying the interaction and sub-compartmentalization of proteins in great detail.

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Gloeckner, N., zur Oven-Krockhaus, S., Wackenhut, F., Burmeister, M., Wanke, F., Holzwart, E., Meixner, A. J., Wolf, S., Harter, K.. 2019-08-01. Three-fluorophore FRET-FLIM enables the study of trimeric protein interactions and complex formation with nanoscale resolution in living plant cells. https://doi.org/10.1101/722124

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