Jacques Coppey

Homo-FRET Microscopy in Living Cells to Measure Monomer-Dimer Transition of GFP-Tagged Proteins

Fluorescence anisotropy decay microscopy was used to determine, in individual living cells, the spatial monomer-dimer distribution of proteins, as exemplified by herpes simplex virus thymidine kinase (TK) fused to green fluorescent protein (GFP). Accordingly, the fluorescence anisotropy dynamics of two fusion proteins (TK27GFP and TK366GFP) was recorded in the confocal mode by ultra-sensitive time-correlated single-photon counting. This provided a measurement of the rotational time of these proteins, which, by comparing with GFP, allowed the determination of their oligomeric state in both the cytoplasm and the nucleus. It also revealed energy homo-transfer within aggregates that TK366GFP progressively formed. Using a symmetric dimer model, structural parameters were estimated; the mutual orientation of the transition dipoles of the two GFP chromophores, calculated from the residual anisotropy, was 44.6 +/- 1.6 degrees, and the upper intermolecular limit between the two fluorescent tags, calculated from the energy transfer rate, was 70 A. Acquisition of the fluorescence steady-state intensity, lifetime, and anisotropy decay in the same cells, at different times after transfection, indicated that TK366GFP was initially in a monomeric state and then formed dimers that grew into aggregates. Picosecond time-resolved fluorescence anisotropy microscopy opens a promising avenue for obtaining structural information on proteins in individual living cells, even when expression levels are very low.

Picosecond-Hetero-FRET Microscopy to Probe Protein-Protein Interactions in Live Cells

By using a novel time- and space-correlated single-photon counting detector, we show that fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to herpes simplex virus thymidine kinase (TK) monomers can be used to reveal homodimerization of TK in the nucleus and cytoplasm of live cells. However, the quantification of energy transfer was limited by the intrinsic biexponential fluorescence decay of the donor CFP (lifetimes of 1.3 +/- 0.2 ns and 3.8 +/- 0.4 ns) and by the possibility of homodimer formation between two TK-CFP. In contrast, the heterodimerization of the transcriptional factor NF-E2 in the nucleus of live cells was quantified from the analysis of the fluorescence decays of GFP in terms of 1) FRET efficiency between GFP and DsRed chromophores fused to p45 and MafG, respectively, the two subunits of NF-E2 (which corresponds to an interchromophoric distance of 39 +/- 1 A); and 2) fractions of GFP-p45 bound to DsRed-MafG (constant in the nucleus, varying in the range of 20% to 70% from cell to cell). The picosecond resolution of the fluorescence kinetics allowed us to discriminate between very short lifetimes of immature green species of DsRed-MafG and that of GFP-p45 involved in FRET with DsRed-MafG.

Homo-FRET versus hetero-FRET to probe homodimers in living cells.

Publisher Summary The purpose of this chapter is to provide information on the homo-fluorescence resonance energy transfer (FRET) versus hetero-FRET to probe homodimers in living cells. FRET is a nonradiative phenomenon in which energy is transferred from a donor fluorophore to an acceptor chromophore with an efficiency that depends on the distance between the two chromophores, the extent of overlap between the donor emission and acceptor excitation spectra, the quantum yield of the donor, and the relative orientation of the donor and acceptor. For homo-FRET, because the photophysical properties of the two donor molecules are the same, the excitation energy is reversibly transferred between the fluorescent tags. Time-resolved fluorescence anisotropy monitors any process that changes the polarization of the emitted fluorescence during the excited state. Consequently, the fluorescence anisotropy decay depends on (1) rotational movements of the fluorescent molecules and (2) energy transfer taking place within the fluorescence time scale. In addition, according to the type of interaction, hetero-or homodimer, the methodology, hetero- or homo-FRET, must be judiciously chosen to obtain the best information about structural data within the macromolecular complex.

Restrained torsional dynamics of nuclear DNA in living proliferative mammalian cells.

Physical parameters, describing the state of chromatinized DNA in living mammalian cells, were revealed by in situ fluorescence dynamic properties of ethidium in its free and intercalated states. The lifetimes and anisotropy decays of this cationic chromophore were measured within the nuclear domain, by using the ultra-sensitive time-correlated single-photon counting technique, confocal microscopy, and ultra-low probe concentrations. We found that, in living cells: 1) free ethidium molecules equilibrate between extracellular milieu and nucleus, demonstrating that the cation is naturally transported into the nucleus; 2) the intercalation of ethidium into chromatinized DNA is strongly inhibited, with relaxation of the inhibition after mild (digitonin) cell treatment; 3) intercalation sites are likely to be located in chromatin DNA; and 4) the fluorescence anisotropy relaxation of intercalated molecules is very slow. The combination of fluorescence kinetic and fluorescence anisotropy dynamics indicates that the torsional dynamics of nuclear DNA is highly restrained in living cells.

Incorporation of green fluorescent protein into the essential envelope glycoprotein B of herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is a major virion component, essential for various steps of virus replication in cells, such as entry and maturation, and cell fusion. In addition, gB is a strong inducer of the immune response in humans and has been involved in neuropathogenesis. To analyze gB during infection, a recombinant HSV-1 was generated containing gB fused to the green fluorescent protein (GFP). The GFP-gB fusion protein was incorporated into fully infectious viral particles. In cells infected with the recombinant KGFP-gB, the spontaneous fluorescence emitted by the fusion protein was observed as early as 5 h post infection, and its transport through cell compartments was followed during an entire viral replication cycle. The results show that GFP can be inserted into an essential viral envelope component of HSV-1 such as gB while preserving the infectivity of the resulting recombinant. This virus allows the investigation of several events of the viral life cycle involving gB, and provides the basis for the development of new diagnostic assays.

Early apoptosis-related changes triggered by HSV-1 in individual neuronlike cells

Early events of apoptosis following HSV-1 infection were investigated at the single-cell level using intensified fluorescence digital-imaging microscopy. The results provide evidence that infection of differentiated ND7 neuronlike cells by HSV-1 triggers detectable alterations indicative of physiological changes associated with the early stages of apoptosis. Less than 1 h after infection with HSV-1 (KOS strain) or K26GFP (GFP being fused to HSV-1 capsid protein VP26) we observed (i) moderate decrease in mitochondrial membrane potential (about 20%), (ii) exposure of phosphatidyl serine, (iii) morphological change in the mitochondria that became spherical instead of filamentous, and (iv) activation of caspase-8. Within 3 h changes reverted to normal, which indicated that apoptosis was counteracted very early following HSV-1 infection. Similar results were obtained with KOS-TK27GFP, lacking TK and UL24 proteins, suggesting that TK and UL24 play no role in apoptosis. In Vero cells mitochondrial changes characteristic of the apoptotic process were not observed following HSV-1 infection. The UV-inactivated K26GFP had the capacity to induce apoptosis in neuronlike cells. This real-time multiparametric analysis, in combination with relevant viral mutants, could be a useful approach for dissecting the roles of various viral genes in modulating apoptotic pathways during infection.

Modulation of clonogenicity, growth, and radiosensitivity of three human epidermoid tumor cell lines by a fibroblastic environment

PURPOSE To develop a model vitro system to examine the influence of fibroblasts on the growth and survival of human tumor cells after exposure to ionizing radiation. METHODS AND MATERIALS The cell system of three epidermoid carcinoma cell lines derived from head and neck tumors having differing growth potentials and intrinsic radiosensitivities, as well as a low passage skin fibroblast strain from a normal human donor. The tumor cells were seeded for five days prior to exposure to radiation: (a) in the presence of different numbers of fibroblasts, (b) in conditioned medium from stationary fibroblast cultures, and (c) on an extracted fibroblastic matrix. RESULTS When grown with fibroblasts, all three tumor cell lines showed increased clonogenicity and increased radioresistance. The radioprotective effect was maximal at a density of approximately 10(5) fibroblasts/100 mm Petri dish, and was greatest in the intrinsically radiosensitive tumor cell line. On the other hand, the effects of incubation with conditioned medium or on a fibroblastic matrix varied among the tumor cell lines. Thus, the protective effect afforded by coculture with fibroblasts must involve several cellular factors related to the fibroblast itself. CONCLUSIONS These observations emphasize the importance of cultural conditions on the apparent radiosensitivity of human tumor cell lines, and suggest that the fibroblastic connective tissue enveloping the malignant cells should be considered when the aim is to establish a radiopredictive assay from surgical tumor fragments.

Adriamycin uptake and metabolism in organotypic culture of A 549 human adenocarcinoma cells according to the exposure time

SummaryIn organotypic cultures (nodules) of A 549 human lung adenocarcinoma cells, the long-term cytotoxicity of Adriamycin is strongly improved by shortening the exposure time to the drug. In order to gain insight into the mechanisms of Adriamycin toxicity in this system, we have examined the drug uptake, retention and metabolism by fluorescence microscopy and HPLC analysis. A 549 nodules efficiently metabolize Adriamycin, two major metabolites, adriamycinol and an aglycone derivative, as yet chemically unidentified, are formed and efficiently excreted. Kinetic data show that a long exposure to Adriamycin triggers its efflux from both the nucleus and the cytoplasm while stimulating its metabolism. Therefore, a long exposure time to the drug appears to trigger a process of cellular detoxification by favouring its excretion from the cells via increased metabolism.

Fluorescence lifetime imaging of cells on the picosecond timescale

Novel MCP detectors for time- and space-correlated single photon counting (TSCSPC) spectroscopy, featuring delay-line (DL) or quadrant anode (QA), are employed in microscopic fluorescence lifetime imaging on the picosecond time scale. The linear DL-MCP-PMT is characterized by a spatial instrument response function (IRF) of 100 micrometer FWHM, resulting in 200 space channels, whereas the QA-MCP-PMT is a 2D imager with 400 by 400 pixel at 40 micrometer resolution. The detectors have a temporal IRF of 75 ps (DL) and 120 ps (QA) FWHM, sufficient for 10 ps time resolution. First results on TOTO-fixed cell systems are presented, demonstrating high-quality kinetics at subcellular resolution, with up to 6 lifetime species at higher dye concentrations, characteristically distributed among individual cell compartments. A comparison with TOTO/DNA- suspensions is made that serve as reference system.

A moderate but not total decrease of mitochondrial membrane potential triggers apoptosis in neuron-like cells

The effects of various degrees of perturbation of the mitochondrial membrane potential (mtΔψ) on apoptosis was investigated by intensified fluorescence digital-imaging microscopy on neuron-like cells, ND7. MtΔψ was either decreased by 40% by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP 100 nM, 15 min) or completely collapsed (FCCP 10 μM, 60 min). A moderate decrease of mtΔψ induced a reduction of mitochondrial NADH, followed by exposure of phosphatidyl serine and then by chromatin condensation, 36% of nuclei being condensed 60 min after FCCP treatment. During these stages, mitochondrion morphology was fully preserved. In contrast, no chromatin condensation was observed after a rapid and total dissipation of mtΔψ. These results suggest that a partial decrease of mtΔψ would allow mitochondrial functions required to trigger apoptosis to be sustained.