Fluorescence resonance energy transfer analysis of protein-protein interactions in single living cells by multifocal multiphoton microscopy.
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.
Majoul
Irina
I
Department of Neurobiology, Max-Planck-Institute of Biophysical Chemistry, Göttingen, Germany. imajoul@gwdg.de
Straub
Martin
M
Duden
Rainer
R
Hell
Stefan W
SW
Söling
Hans-Dieter
HD
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*
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