Binary 2in1 Vectors Improve in Planta (Co)localization and Dynamic Protein Interaction Studies

Abstract

Fluorescence-based protein-protein interaction techniques are vital tools for understanding in vivo cellular functions on a mechanistic level. However, only under the condition of highly efficient (co)transformation and accumulation can techniques such as Förster resonance energy transfer (FRET) realize their potential for providing highly accurate and quantitative interaction data. FRET as a fluorescence-based method unifies several advantages, such as measuring in an in vivo environment, real-time context, and the ability to include transient interactions as well as detecting the mere proximity of proteins. Here, we introduce a novel vector set that incorporates the benefit of the recombination-based 2in1 cloning system with the latest state-of-the-art fluorescent proteins for optimal coaccumulation and FRET output studies. We demonstrate its utility across a range of methods. Merging the 2in1 cloning system with new-generation FRET fluorophore pairs allows for enhanced detection, speeds up the preparation of clones, and enables colocalization studies and the identification of meaningful protein-protein interactions in vivo.Two technological advances allowed the field of modern cell biology to emerge: the development of high-resolution microscopes and the groundbreaking work in the development of fluorescent proteins (FPs) that were originally isolated from jellyfish (for review, see Day and Davidson, 2009;Zimmer, 2009). Genetically encoded FPs can be fused directly to proteins of choice, offering insights into their functional properties by allowing researchers to monitor subcellular localization, chemical environment, interaction, movement, and/or turnover rate.Research on the FPs themselves has exploded in the past two decades, and a range of technological improvements to and variations of the FPs were generated. These improvements include novel spectral properties from all areas of the color palette and from a diverse range of organisms. They also extend to a multitude of parameters, such as brightness, folding efficiency, chromophore oxidation rate, quantum yield, pH, and photostability (Day and Davidson, 2009). Taken together, these optimizations further the possibilities of available methods or prompt the development of new techniques.In 1946, the German scientist Theodor Förster laid the theoretical foundation for resonance energy transfer, now known as Förster resonance energy transfer (FRET;Forster, 1946). According to this theory, a donor chromophore in an excited state can transfer the excitation energy to a nearby acceptor chromophore via dipole-dipole coupling without the emission of a photon (for review, see Clegg, 2009;Ishikawa-Ankerhold et al., 2012). FRET depends on the spectral overlap between the emission spectrum of the donor and the absorbance spectrum of the acceptor chromophores. It also depends on the fluorescence quantum yield and the excited state lifetime of the donor in the absence of an acceptor as well as the relative orientation of the two chromophores. Most importantly, FRET ef...

Techniques for the analysis of protein-protein interactions in vivo

“…A nucleus expressing two interacting proteins attached to a donor and an acceptor fluorophore. After bleaching (right), the acceptor fluorescence is almost completely lost, but an increase in donor fluorescence can be detected (image from Hecker et al, 2015, modified for visualization purposes only). D, Exemplary fluorescence lifetime decay curve of a putative donor molecule when FRET is occurring (red solid curve; t 1 ) or in the absence of FRET (cyan solid line; t 2 ).…”

“…This method is straightforward, but it must be carefully executed, as unequal loading of the fluorescent proteins will give dramatically different results. Figure 5B shows the absorption (dashed lines) and emission spectra (solid lines) of mTurquoise2 and mVenus (modified from Hecker et al, 2015). The l 4 weighted spectral overlap, which is depicted in dark gray, is one of three important factors for efficient FRET.…”

“…For example, mixing of two individual Agrobacterium tumefaciens strains only yields approximately 80% cotransfection, in addition to high expression variability, which is most likely a consequence of unequal gene dosage (see Fig. 6A below; Hecker et al, 2015).…”

“…The dark gray surface depicts the l 4 weighted overlap integral. Acceptor and donor spectral bleed through are shown in magenta and green, respectively (see "FRET: Lifetime Versus Intensity" for details; image modified from Hecker et al, 2015). C, Example of a FRET acceptor photobleaching experiment.…”

“…Conversely, when the abundance of the acceptor exceeds that of the donor and is excited by the same light source, more emission in the acceptor would be taken as an indication of FRET when none exists. One solution in these circumstances is to adjust the relative amount of fluorescent proteins by controlling gene dosage (Hecker et al, 2015).…”