Spectroscopic approach for monitoring two-photon excited fluorescence resonance energy transfer from homodimers at the subcellular level.
We have employed a spectroscopic approach for monitoring fluorescence resonance energy transfer (FRET) in living cells. This method provides excellent spectral separation of green fluorescent protein (GFP) mutant signals within a subcellular imaging volume using two-photon excited fluorescence imaging and spectroscopy (TPIS-FRET). In contrast to current FRET-based methodologies, TPIS-FRET does not rely on the selection of optical filters, ratiometric image analysis, or bleedthrough correction algorithms. Utilizing the intrinsic optical sectioning capabilities of TPIS-FRET, we have identified protein-protein interactions within discrete subcellular domains. To illustrate the applicability of this technique to the detection of homodimer formation, we demonstrated the in vivo association of promyleocyte (PML) homodimers within their corresponding nuclear body.
LaMorte
Vickie J
VJ
University of California, Beckman Laser Institute, Laser Microbeam and Medical Program, Irvine, California 92612, USA. lamorte@bli.uci.edu
Zoumi
Aikaterini
A
Tromberg
Bruce J
BJ
D045744:Cell Line, Tumor; D019281:Dimerization; D004848:Epithelium / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D007822:Laryngeal Neoplasms / Q000378:metabolism*; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D009363:Neoplasm Proteins / Q000378:metabolism*; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D025521:Tumor Suppressor Proteins
D008164:Luminescent Proteins; D046911:Macromolecular Substances; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C070576:PML protein, human; D049452:Green Fluorescent Proteins