One Pattern Analysis (OPA) for the quantitative determination of protein interactions in plant cells

Abstract

Background: A commonly used approach to study the interaction of two proteins of interest (POIs) in vivo is measuring Förster Resonance Energy Transfer (FRET). This requires the expression of the two POIs fused to two fluorescent proteins that function as a FRET pair. A precise way to record FRET is Fluorescence Lifetime IMaging (FLIM) which generates quantitative data that, in principle, can be used to resolve both complex structure and protein affinities. However, this potential resolution is often lost in many experimental approaches. Here we introduce a novel tool for FLIM data analysis of multiexponential decaying donor fluorophores, One Pattern Analysis (OPA), which allows to obtain information about protein affinity and complex arrangement by extracting the relative amplitude of the FRET component and the FRET transfer efficiency from other FRET parameters. Results: As a proof of concept for OPA, we used FLIM-FRET, or FLIM-FRET in combination with BiFC to reassess the dimerization and tetramerization properties of known interacting MADS-domain transcription factors in Nicotiana benthamiana leaf cells and Arabidopsis thaliana flowers. Using the OPA tool and by extracting protein BINDING efficiencies from FRET parameters to dissect MADS-domain protein interactions in vivo in transient N. benthamiana experiments, we could show that MADS-domain proteins display similar proximities within dimeric or tetrameric complexes but bind with variable affinities. By combining FLIM with BiFC, we were able to identify SEPALLATA3 as a mediator for tetramerization between the other MADS-domain factors. OPA also revealed that in vivo expression from native promoters at low levels in Arabidopsis flower meristems, in situ complex formation of MADS-domain proteins cannot be detected. Conclusions We conclude that MADS-domain protein interactions are transient in situ and may involve additional, so far unknown interaction mediators. We conclude that OPA can be used to separate protein binding from information about proximity and orientation of the interacting proteins in their complexes. Visualization of individual protein interactions within the underlying interaction networks in the native environment is still restrained if expression levels are low and will require continuous improvements in fluorophore labelling, instrumentation set-ups and analysis tools.

Stem cell homeostasis in the root ofArabidopsisinvolves cell type specific complex formation of key transcription factors

“…A, B). The use of a novel analysis method allowed us to distinguish between these two scenarios, providing deeper insights into protein affinities, hereafter referred to as ‘binding’, between BRAVO, PLT3 and WOX5 (Maika et al ., 2023).…”

“…A reduction of fluorescence lifetime as a consequence of FRET can either be a result of a highly efficient energy transfer indicating close proximity or B) a high affinity of the two proteins. Figure created with BioRender.com and modified from (Maika et al ., 2023). C) Upper panel: Representative images of fluorescence lifetime imaging microscopy (FLIM) measurements of nuclei in N. benthamiana epidermal leaf cells after pixel-wise mono- or biexponential fitting.…”

“…3 C). A binding of below 10 % is interpreted as no interaction (Maika et al ., 2023), cohering with the reference and negative control samples. Upon co-expression of BRAVO-mV with PLT3-mCh, the binding increases to 28.0 ± 11.7 % (Fig.…”

“…To address this, we used a combination of bimolecular fluorescence complementation (BiFC) and FRET (Fig. 4 A, B) (Kwaaitaal et al ., 2010; Maika et al ., 2023). Here, the donor fluorophore is split into two fragments: the N-terminal part of mVenus (mV(N)) and the C-terminal part (mV(C)).…”

“…Together with previously described, independent one-on-one interactions, these findings support the hypothesis of three parallel pathways that control CSC differentiation and QC divisions in parallel. Furthermore, the observed variations of stability and probability of occurrence as indicated by a special analysis tool (Orthaus et al, 2009;Maika et al, 2023), could indicate a specific mechanism that facilitates the interaction of two POIs in a highly dynamic microenvironment, where the number of proteins is generally high, such as in the QC. (Fig.…”