Fluorescence resonance energy transfer analysis of protein-protein interactions in single living cells by multifocal multiphoton microscopy. J Biotechnol

Department of Neurobiology, Max-Planck-Institute of Biophysical Chemistry, Göttingen, Germany.
Journal of Biotechnology (Impact Factor: 2.87). 02/2002; 82(3):267-77. DOI: 10.1016/S1389-0352(01)00042-3
Source: PubMed

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.

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Available from: Rainer Duden, May 09, 2014
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    • "The lifetime of a fluorophore can be affected by the biochemical and biophysical properties of its microenvironment, where FRET leads to a decrease in the fluorescence lifetime of the donor molecule that can accurately be measured [13]. Contrary to other spectral methods of measuring FRET, such as sensitized emission FRET (SE-FRET) [14], the ability to measure fluorescence lifetime of fluorescent proteins expressed in live cells is less dependent of relative probe concentrations and intensities, photo-bleaching as well as spectral bleed through [13], [15]. The accuracy of FRET measurement is further enhanced by this genetically-encoded triple fusion that insures a relative donor-acceptor fluorophore concentration of 1∶1. "
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    ABSTRACT: Transglutaminase type 2 (TG2) is a ubiquitously expressed member of the transglutaminase family, capable of mediating a transamidation reaction between a variety of protein substrates. TG2 also has a unique role as a G-protein with GTPase activity. In response to GDP/GTP binding and increases in intracellular calcium levels, TG2 can undergo a large conformational change that reciprocally modulates the enzymatic activities of TG2. We have generated a TG2 biosensor that allows for quantitative assessment of TG2 conformational changes in live cells using Förster resonance energy transfer (FRET), as measured by fluorescence lifetime imaging microscopy (FLIM). Quantifying FRET efficiency with this biosensor provides a robust assay to quickly measure the effects of cell stress, changes in calcium levels, point mutations and chemical inhibitors on the conformation and localization of TG2 in living cells. The TG2 FRET biosensor was validated using established TG2 conformational point mutants, as well as cell stress events known to elevate intracellular calcium levels. We demonstrate in live cells that inhibitors of TG2 transamidation activity can differentially influence the conformation of the enzyme. The irreversible inhibitor of TG2, NC9, forces the enzyme into an open conformation, whereas the reversible inhibitor CP4d traps TG2 in the closed conformation. Thus, this biosensor provides new mechanistic insights into the action of two TG2 inhibitors and defines two new classes based on ability to alter TG2 conformation in addition to inhibiting transamidation activity. Future applications of this biosensor could be to discover small molecules that specifically alter TG2 conformation to affect GDP/GTP or calcium binding.
    PLoS ONE 08/2012; 7(8):e44159. DOI:10.1371/journal.pone.0044159 · 3.23 Impact Factor
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    • "FRET is a technique used to measure the distance between two fluorescence molecules (donor and acceptor) with the overlapping emission wavelength from the donor and excitation wavelength of the acceptor. The technology has been extended to measure the protein–protein interactions when the proteins are tagged with fluorescence donor and acceptor (Majoul et al. 2002). In this study, the emission wavelength from PGIS–CFP of 480 nm (±15 nm) is overlapped with the excitation wavelength of 520 nm (±15 nm) of COX- RFP. "
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    ABSTRACT: Our aim is to understand the molecular mechanisms of the selective nonsteroidal anti-inflammatory drugs (NSAID), cyclooxygenase-2 (COX-2) inhibitors', higher "priority" to reduce synthesis of the vascular protector, prostacyclin (PGI2), compared to that of nonselective NSAIDs. COX-1 or COX-2 was co-expressed with PGI2 synthase (PGIS) in COS-7 cells. The Km and initial velocity (½t Vmax) of the coupling reaction between COX-1 and COX-2 to PGIS were established. The experiment was further confirmed by a kinetics study using hybrid enzymes linking COX-1 or COX-2 to PGIS. Finally, COX-1 or COX-2 and PGIS were respectively fused to red (RFP) and cyanic (CFP) fluorescence proteins, and co-expressed in cells. The distances between COXs and PGIS were compared by FRET. The Km for converting arachidonic acid (AA) to PGI2 by COX-2 coupled to PGIS is ~2.0μM; however, it was 3-fold more (~6.0μM) for COX-1 coupled to PGIS. The Km and ½t Vmax for COX-2 linked to PGIS were ~2.0μM and 20s, respectively, which were 2-5 folds faster than that of COX-1 linked to PGIS. The FRET study found that the distance between COX-2-RFP and PGIS-CFP is shorter than that between COX-1-RFP and PGIS-CFP. The study provided strong evidence suggesting that the low Km, faster ½t Vmax, and closer distance are the basis for COX-2 dominance over COX-1 (coupled to PGIS) in PGI2 synthesis, and further demonstrated the mechanisms of selective COX-2 inhibitors with higher potential to reduce synthesis of the vascular protector, PGI2.
    Life sciences 10/2010; 88(1-2):24-30. DOI:10.1016/j.lfs.2010.10.017 · 2.70 Impact Factor
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    • "energy transfer will occur only when the donor and acceptor are very close in space and in a particular relative orientation , making FRET a highly sensitive method (Clegg, 1996; Stryer, 1978). Green fluorescent protein (GFP)-based FRET has been applied in many experimental systems to study protein interactions as well as to measure local calcium concentrations, phosphorylation kinetics, protein cleavage kinetics, and other processes (Damelin and Silver, 2000; Day, 1998; Heim and Tsien, 1996; Mahajan et al., 1998; Miyawaki et al., 1997; Mochizuki et al., 2001; Jiang and Sorkin, 2002; Majoul et al., 2001, 2002; Warren et al., 2002; Sato et al., 2002; Immink et al., 2002; Ting et al., 2001; Weiss et al., 2001; Wilson et al., 2002; Ruiz-Velasco and Ikeda, 2001; Truong et al., 2001). For the ECFP-EYFP pair, the interfluorophore distance corresponding to 50% FRET efficiency, termed the Forster radius R o , is 49 –52 Å (Tsien, 1998). "
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    ABSTRACT: Macromolecular transport between the nucleus and cytoplasm occurs through the nuclear pore complexes (NPCs). The NPC in the budding yeast Saccharomyces cerevisiae is a 60-MDa structure embedded in the nuclear envelope and composed of ~30 proteins, termed nucleoporins or nups. Here we present a large-scale analysis of spatial relationships between nucleoporins using fluorescence resonance energy transfer (FRET) in living yeast cells. Energy transfer was measured in a panel of strains, each of which coexpresses the enhanced cyan and yellow fluorescent proteins as fusions to distinct nucleoporins. With this approach, we have determined 13 nucleoporin pairs yielding FRET signals. Independent experiments are consistent with the FRET results: Nup120 localization is perturbed in the nic96-1 mutant, as is Nup82 localization in the nup116Delta mutant. To better understand the spatial relationship represented by an in vivo FRET signal, we have investigated the requirements of these signals. We demonstrate that in one case FRET signal is lost upon insertion of a short spacer between the nucleoporin and its enhanced yellow fluorescent protein label. We also show that the Nup120 FRET signals depend on whether the fluorescent moiety is fused to the N- or C-terminus of Nup120. Combined with existing data on NPC structure, the FRET pairs identified in this study allow us to propose a refined molecular model of the NPC. We suggest that the approach may serve as a prototype for the in situ study of other large macromolecular complexes.
    Biophysical Journal 01/2003; 83(6):3626-36. DOI:10.1016/S0006-3495(02)75363-0 · 3.97 Impact Factor
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