The C-type lectin dendritic cell (DC)–specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood. By high resolution electron microscopy, we demonstrate a direct relation between DC-SIGN function as viral receptor and its microlocalization on the plasma membrane. During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm. Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts. Finally, we show that the organization of DC-SIGN in microdomains on the plasma membrane is important for binding and internalization of virus particles, suggesting that these multimolecular assemblies of DC-SIGN act as a docking site for pathogens like HIV-1 to invade the host.

We exploit the strong excitonic coupling in a superradiant trimer molecule to distinguish between longlived collective dark states and photobleaching events. The population and depopulation kinetics of the dark states in a single molecule follow power-law statistics over 5 orders of magnitude in time. This result is consistent with the formation of a radical unit via electron tunneling to a time-varying distribution of trapping sites in the surrounding polymer matrix. We furthermore demonstrate that this radicalization process forms the dominant pathway for molecular photobleaching.

The blinking behavior of perylene diïmide molecules is investigated at the single-molecule level. We observe long-time scale blinking of individual multi-chromophoric complexes embedded in a poly(methylmethacrylate) matrix, as well as for the monomeric dye absorbed on a glass substrate at ambient conditions. In both these different systems, the blinking of single molecules is found to obey analogous power-law statistics for both the on and off periods. The observed range for single-molecular power-law blinking extends over the full experimental time window, covering four orders of magnitude in time and six orders of magnitude in probability density. From molecule to molecule, we observe a large spread in off-time power-law exponents. The distributions of off-exponents in both systems are markedly different whereas both on-exponent distributions appear similar. Our results are consistent with models that ascribe the power-law behavior to charge separation and (environment-dependent) recombination by electron tunneling to a dynamic distribution of charge acceptors. As a consequence of power-law statistics, single molecule properties like the total number of emitted photons display non-ergodicity.

Recent studies on the newly cloned red fluorescence protein DsRed from the Discosoma genus have shown its tremendous advantages: bright red fluorescence and high resistance against photobleaching. However, it has also become clear that the protein forms closely packed tetramers, and there is indication for incomplete protein maturation with unknown proportion of immature green species. We have applied single-molecule methodology to elucidate the nature of the fluorescence emission in the DsRed. Real-time fluorescence trajectories have been acquired with polarization sensitive detection. Our results indicate that energy transfer between identical monomers occurs efficiently with red emission arising equally likely from any of the chromophoric units. Photodissociation of one of the chromophores weakly quenches the emission of adjacent ones. Dual color excitation (at 488 and 568 nm) single-molecule microscopy has been performed to reveal the number and distribution of red vs. green species within each tetramer. We find that 86% of the DsRed contain at least one green species with a red-to-green ratio of 1.2-1.5. On the basis of our findings, oligomer suppression would not only be advantageous for protein fusion but will also increase the fluorescence emission of individual monomers.T he cloning of the red fluorescent protein (drFP583, commercially available as DsRed) from the Indo pacific reef coral Discosoma sp (1) has triggered intense biological interest as a potential expression marker and fusion partner that would be complementary to the Aequorea victoria green fluorescent protein (avGFP). The main advantage of these naturally fluorescent proteins is that they provide strong visible fluorescence and can be genetically fused to other proteins. Since the cloning of the avGFP (2, 3), different mutants have been produced to extend the palette of available colors. However, no GFP mutant has been produced with emission maxima longer than 529 nm (3). The newly cloned DsRed has bright red fluorescence with emission maxima at 583 nm, one of the longest wavelength emissions reported so far in a wild-type species [Fradkov et al. (4) have reported a wild type highly homologous to the drFP583 with 593-nm emission and a hybrid with 616-nm emission]. Potentially, DsRed is an ideal partner of GFP in fluorescence resonance energy transfer (FRET) experiments, particularly in cell biological applications in which cellular autofluorescence poses a problem (5).In the last few months, extensive research has been done in the biochemical and photophysical investigation of DsRed. The absorption spectrum of the mature protein exhibits a strong band with a peak at 558 nm and two minor shoulders at 526 and 490 nm (6). An extinction coefficient of 75,000 mol Ϫ1 ͞cm Ϫ1 and a fluorescence quantum yield of 0.7 at 558-nm excitation wavelength have been reported (7), much higher than initially published by Matz et al. (1). The protein proves to be stable under harsh pH conditions and is extremely resistant to photobleaching (7, 8). However, t...