Picosecond-Hetero-FRET Microscopy to Probe Protein-Protein Interactions in Live Cells

Abstract

By using a novel time- and space-correlated single-photon counting detector, we show that fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to herpes simplex virus thymidine kinase (TK) monomers can be used to reveal homodimerization of TK in the nucleus and cytoplasm of live cells. However, the quantification of energy transfer was limited by the intrinsic biexponential fluorescence decay of the donor CFP (lifetimes of 1.3 +/- 0.2 ns and 3.8 +/- 0.4 ns) and by the possibility of homodimer formation between two TK-CFP. In contrast, the heterodimerization of the transcriptional factor NF-E2 in the nucleus of live cells was quantified from the analysis of the fluorescence decays of GFP in terms of 1) FRET efficiency between GFP and DsRed chromophores fused to p45 and MafG, respectively, the two subunits of NF-E2 (which corresponds to an interchromophoric distance of 39 +/- 1 A); and 2) fractions of GFP-p45 bound to DsRed-MafG (constant in the nucleus, varying in the range of 20% to 70% from cell to cell). The picosecond resolution of the fluorescence kinetics allowed us to discriminate between very short lifetimes of immature green species of DsRed-MafG and that of GFP-p45 involved in FRET with DsRed-MafG.

Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins

“…One shorter lifetime of 1.1 ns contributing for 33% and one longer lifetime of 3.7 ns contributing for 67% could be recovered. The departure of single-exponential fluorescence decay is also observed by others [18][19][20]. The presence of multiple fluorescent states is most probably due to two different conformations of ECFP in the crystal structure in which two amino acids, Tyr145 and His148, are positioned differently with respect to the fluorophore [21].…”

High-precision FLIM–FRET in fixed and living cells reveals heterogeneity in a simple CFP–YFP fusion protein

“…Previous time-resolved studies have also found that the fluorescence lifetime of CFP in mammalian cells is biexponential. Tramier et al [23] …”

“…In other photon-counting FLIM studies of FRET between CFP and YFP in cells, the extent of FRET has been described by biexponential kinetics, with two lifetimes that are different to the donor-only decays [23][24][25]. More complex kinetics were not observed.…”

Dynamique d’assemblage des machineries nucléolaires après la mitose, en temps réel et en cellules vivantes

“…Nous avons comparé les concentrations des protéi-nes de maturation précoces ou tardives dans les PNB. Enfin nous avons utilisé une méthode de FRET (fluorescence resonance energy transfer) fondé sur la décroissance de la durée de vie de la GFP (donneur) en présence de DsRed (accepteur) [7]. Cette analyse non invasive, est réalisée pendant l'assemblage du nucléole et mesure la distance entre les deux étiquettes fluorescentes.…”