Two-hybrid fluorescence cross-correlation spectroscopy detects protein-protein interactions in vivo.
Fluorescence cross-correlation spectroscopy (FCCS) uses the correlated motion of two distinct fluorophores to detect their interaction. Whereas FCCS has been used with chemically or genetically labeled interaction partners in vitro, FCCS has never been demonstrated in vivo between two autofluorescent proteins. At least one reaction partner was always chemically labeled. Fos and Jun, two components of the AP-1 transcription factor, are known to exert their function as a dimer and can therefore serve as a reference for dimer formation. Expressing fusion proteins between Fos and the enhanced green fluorescent protein (EGFP), as well as Jun and the monomeric red fluorescent protein 1 (mRFP1) in HeLa cells, we show here, for the first time, in vivo FCCS detection of protein-protein interactions. The mobility of the dimerized species is slow, indicating that DNA-binding might stabilize dimerization. The technique has rich potential applications for the rapid screening of protein-protein interactions in vivo, which are able to clarify events during the whole life of cells.
Baudendistel
Nina
N
Division Biophysics of Macromolecules (B040), German Cancer Research Center, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany.
Müller
Gabriele
G
Waldeck
Waldemar
W
Angel
Peter
P
Langowski
Jörg
J
D001703:Biophysics / Q000379:methods*; D004247:DNA / Q000737:chemistry; D019281:Dimerization; D005456:Fluorescent Dyes / Q000737:chemistry; D017353:Gene Deletion; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D015233:Models, Statistical; D010957:Plasmids / Q000378:metabolism; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D016760:Proto-Oncogene Proteins c-fos / Q000737:chemistry; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry; D020798:Two-Hybrid System Techniques
D005456:Fluorescent Dyes; D011506:Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D004247:DNA