Abstract
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.
Highlights
Various fluorophore-based microscopic methods, comprising Forster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC), are suitable to study pairwise interactions of proteins in living cells
Before assessing ternary sensitive factor attachment receptor (SNARE) complex formation by BiFC and FRET, we aimed to demonstrate HvROR2-containing ternary complexes in barley
In analogy to experiments performed by Kwon et al (2008), we detected the presence of HvROR2-containing SDS-resistant, heat-sensitive ternary SNARE complexes by biochemical means in 7-d-old barley leaves (Fig. 1)
Summary
Various fluorophore-based microscopic methods, comprising Forster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC), are suitable to study pairwise interactions of proteins in living cells. 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 fluorophoretagged polypeptide partners. FRET measurements require more or less sophisticated microscopic equipment
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