Combined bimolecular fluorescence complementation and Forster resonance energy transfer reveals ternary SNARE complex formation in living plant cells.
Various fluorophore-based microscopic methods, comprising Förster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC), are suitable to study pairwise interactions of proteins in living cells. The analysis of interactions between more than two protein partners using these methods, however, remains difficult. In this study, we report the successful application of combined BiFC-FRET-fluorescence lifetime imaging microscopy and BiFC-FRET-acceptor photobleaching measurements to visualize the formation of ternary soluble N-ethylmaleimide-sensitive factor attachment receptor complexes in leaf epidermal cells. This method expands the repertoire of techniques to study protein-protein interactions in living plant cells by a procedure capable of visualizing simultaneously interactions between three fluorophore-tagged polypeptide partners.
Kwaaitaal
Mark
M
Max-Planck Institute for Plant Breeding Research, D-50829 Cologne, Germany.
Keinath
Nana F
NF
Pajonk
Simone
S
Biskup
Christoph
C
Panstruga
Ralph
R
D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D019441:Plant Epidermis / Q000166:cytology / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism; D025941:Protein Interaction Mapping; D050600:SNARE Proteins / Q000378:metabolism*
D008164:Luminescent Proteins; D010940:Plant Proteins; D050600:SNARE Proteins