title: Fluorescence resonance energy transfer analysis of protein-protein interactions in single living cells by multifocal multiphoton microscopy.
abstract: Fluorescence resonance energy transfer (FRET) resolved by multifocal multiphoton microscopy (MMM) was successfully used to measure transport phenomena in living cells. We expressed different pairs of CFP-/YFP-fusion proteins involved in retrograde Golgi-to-ER transport to analyze sorting of the occupied KDEL-receptor into retrograde transport vesicles triggered by application of the external cholera toxin mutant CTXK63. FRET observed as a sensitized emission of the acceptor was confirmed by acceptor photobleaching and the dequenching of the donor was measured. FRET-MMM data obtained from single cells were compared with bulk cell experiments employing spectrofluorimetry. The importance of controlling the degree of overexpression of CFP-/YFP-fusion proteins for FRET analysis is stressed in this article. Using MMM we showed for the first time that FRET can be measured across the Golgi membrane. Finally, FRET-MMM records performed continuously over 2 h allowed to analyze intracellular retrograde transport and sorting events and to discuss these mechanisms on a single cell level.
authors: Department of Neurobiology, Max-Planck-Institute of Biophysical Chemistry, Göttingen, Germany. imajoul@gwdg.de
authors: Irina
authors: I
authors: Majoul
authors: Martin
authors: M
authors: Straub
authors: Rainer
authors: R
authors: Duden
authors: Stefan W
authors: SW
authors: Hell
authors: Hans-Dieter
authors: HD
authors: Söling
mesh_terms: D001426:Bacterial Proteins; D001693:Biological Transport, Active; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D018000:Receptors, Peptide / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; C064459:KDEL receptor; D008164:Luminescent Proteins; D011506:Proteins; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins