The visible touch: in planta visualization of protein-protein interactions by fluorophore-based methods

Abstract

Non-invasive fluorophore-based protein interaction assays like fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC, also referred to as "split YFP") have been proven invaluable tools to study protein-protein interactions in living cells. Both methods are now frequently used in the plant sciences and are likely to develop into standard techniques for the identification, verification and in-depth analysis of polypeptide interactions. In this review, we address the individual strengths and weaknesses of both approaches and provide an outlook about new directions and possible future developments for both techniques.

Figure 1

Excitation and emission spectra of a commonly used FRET pair. The scheme depicts simplified absorbance and emission spectra of CFP (cyan fluorescent protein; donor; D) and YFP (yellow fluorescent protein; acceptor, A). Overlap between CFP emission and YFP absorption (shaded region) is a prerequisite for FRET. D_abs – Donor absorbance; D_em – Donor emission; A_abs – Acceptor absorbance; A_em – Acceptor emission.

Figure 2

Detection of protein-protein interactions via FRET. FRET between cyan fluorescent protein (CFP) as a donor fused to protein A and yellow fluorescent protein (YFP) fused as an acceptor to protein B. Under favorable spatial and angular conditions, interaction between A and B causes a decrease in the intensity of donor (CFP) fluorescence concomitant with an increase in acceptor (YFP) fluorescence. CFP and YFP are depicted as cyan and yellow ribbon models fused to putative interacting proteins A and B, respectively.

Figure 3

Principle and quantitative assessment of FRET via DFRAP. (a) In case of FRET between the donor CFP and the acceptor YFP due to interaction between two proteins A and B, the photochemical destruction of the acceptor abolishes FRET and leads to an increased emission from the donor, CFP. CFP and YFP are depicted as cyan and yellow ribbon models fused to putative interacting proteins A and B respectively. (b, c). Time-course analysis of fluorescence intensity before and after photobleaching in the presence or absence of a protein-protein interaction. Blue and yellow curves indicate the levels of CFP and YFP fluorescence before and after photobleaching, respectively. In case of FRET, bleaching of the acceptor molecule leads to an increase in donor fluorescence (b). In the absence of interaction between proteins A and B, CFP levels before and after the bleach do not vary considerably (c). BB – Before bleach, AB – After bleach.

Figure 4

FRET-FLIM analysis of the MLO-calmodulin interaction. Barley MLO is a plant-specific calmodulin-binding protein that functions as a modulator of defence against the common powdery mildew pathogen [90]. YFP-tagged wild-type barley MLO or mutant variants thereof (W423R and L420R W423R, bearing amino acid substitutions in the calmodulin binding domain [90]) were co-expressed with CFP-tagged calmodulin in single barley leaf epidermal cells. FRET-FLIM analysis was performed as described in [32]. Donor fluorophore lifetimes are color-coded according to the scale indicated on top of the Figure. "Warmer" colors are indicative of shorter donor fluorophore lifetimes and thus interaction between MLO and calmodulin. Size bar, 20 μm.

Figure 5

Principle of the BiFC assay. The scheme depicts the principle of the BiFC assay, exemplified by a split YFP fluorophore. Proteins A and B are fused to N- and C-terminal fragments of YFP, respectively. In the absence of an interaction between A and B, the fluorophore halves remain non-functional. Following interaction between A and B, a functional fluorophore is reconstituted which exhibits emission of fluorescence upon excitation with an appropriate wavelength.

Figure 6

Confocal images of bimolecular fluorescence complementation (BiFC) studies. The micrographs show a positive result (HSP90 dimerization; [91]) as well as a negative result (expected absence of interaction between HSP90 and importinα, a mediator of nuclear transportation) of the BiFC assay. HSP90 tagged with the N-terminal fragment of YFP (HSP90-YN) was co-expressed in Nicotiana benthamiana leaves by Agrobacterium tumefaciens transient transformation with the C-terminal fragment of YFP fused to either HSP90 (YC-HSP90; left side) or importinα (YC-IMP; right side). Yellow colour results from the functional complementation of the two halves of the YFP fluorophore and indicates interaction of corresponding fusion proteins. Size bar, 10 μm.