Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells.
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.
Tramier
Marc
M
Institut Jacques Monod, UMR 7592, CNRS, Universités P6/P7, 2 place Jussieu, 75251 Paris Cedex 05, France.
Gautier
Isabelle
I
Piolot
Tristan
T
Ravalet
Sylvie
S
Kemnitz
Klaus
K
Coppey
Jacques
J
Durieux
Christiane
C
Mignotte
Vincent
V
Coppey-Moisan
Maïté
M
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D050981:Erythroid-Specific DNA-Binding Factors; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D018259:Herpesvirus 1, Human / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D046911:Macromolecular Substances; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013937:Thymidine Kinase / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D014709:Vero Cells
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; C494692:NFE2 protein, human; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C413663:fluorescent protein 583; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase