Picosecond time-resolved microspectrofluorometry in live cells exemplified by complex fluorescence dynamics of popular probes ethidium and cyan fluorescent protein.

Time-resolved microspectrofluorometry in live cells, based on time- and space-correlated single-photon counting, is a novel method to acquire spectrally resolved fluorescence decays, simultaneously in 256 wavelength channels. The system is calibrated with a full width at half maximum (FWHM) of 90 ps for the temporal resolution, a signal-to-noise ratio of 10(6), and a spectral resolution of 30 (Deltalambda/Lambda). As an example, complex fluorescence dynamics of ethidium and cyan fluorescent protein (CFP) in live cells are presented. Free and DNA intercalated forms of ethidium are simultaneously distinguishable by their relative lifetime (1.7 ns and 21.6 ns) and intensity spectra (shift of 7 nm). By analysing the complicated spectrally resolved fluorescence decay of CFP, we propose a fluorescence kinetics model for its excitation/desexcitation process. Such detailed studies under the microscope and in live cells are very promising for fluorescence signal quantification.

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.

Close but distinct regions of human herpesvirus 8 latency-associated nuclear antigen 1 are responsible for nuclear targeting and binding to human mitotic chromosomes.

Human herpesvirus 8 is associated with all forms of Kaposi's sarcoma, AIDS-associated body cavity-based lymphomas, and some forms of multicentric Castleman's disease. Herpesvirus 8, like other gammaherpesviruses, can establish a latent infection in which viral genomes are stably maintained as multiple episomes. The latent nuclear antigen (LANA or LNAI) may play an essential role in the stable maintenance of latent episomes, notably by interacting concomitantly with the viral genomes and the metaphase chromosomes, thus ensuring an efficient transmission of the neoduplicated episomes to the daughter cells. To identify the regions responsible for its nuclear and subnuclear localization in interphase and mitotic cells, LNAI and various truncated forms were fused to a variant of green fluorescent protein. This enabled their localization and chromosome binding activity to be studied by low-light-level fluorescence microscopy in living HeLa cells. The results demonstrate that nuclear localization of LNAI is due to a unique signal, which maps between amino acids 24 and 30. Interestingly, this nuclear localization signal closely resembles those identified in EBNA1 from Epstein-Barr virus and herpesvirus papio. A region encompassing amino acids 5 to 22 was further proved to mediate the specific interaction of LNA1 with chromatin during interphase and the chromosomes during mitosis. The presence of putative phosphorylation sites in the chromosome binding sites of LNA1 and EBNA1 suggests that their activity may be regulated by specific cellular kinases.

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.

Diet-induced thermogenesis in HIV infection.

OBJECTIVE: To assess whether postprandial dietary thermogenesis contributes to weight loss during HIV infection. METHODS: The thermogenic response to a test meal (15 kcal/kg) was evaluated with indirect calorimetry in 16 HIV-infected patients in a stable condition and compared with a control group. Patients were compared according to AIDS (n = 8) or non-AIDS (n = 8) status and to body weight loss (WL; n = 9) or no loss (NL; n = 7). Indirect calorimetry was performed after fasting 6 h and during 5 h after the test meal. RESULTS: Maximum value of energy expenditure was reached later in the WL group than in the control and NL group (200 versus 30 min, respectively). Energy expenditure returned to the initial value 300 min after the test meal (last measurement) in the control group but remained elevated in the patient group. Energy expenditure after food intake was more elevated in HIV-infected patients than in controls, especially in patients with detectable clinical change in their nutritional status (0.96 versus 0.72 kcal/kg body weight). CONCLUSION: Both kinetics and quantitative aspect of dietary thermogenesis are modified during HIV infection and the different variations are dependent on the extent of body weight loss.