A complex of Shc and Ran-GTPase localises to the cell nucleus.

The three isoforms of the adaptor protein Shc play diverse roles in cell signalling. For example, the observation of p46 Shc in the nuclei of hepatocellular carcinoma cells suggests a function quite distinct from the better characterised cytoplasmic role. Ligands responsible for the transport of various Shc isoforms into organelles such as the nucleus have yet to be reported. To identify such ligands a far western approach was used to determine the p52 Shc interactome. The Ran-GTPase nuclear transport protein was identified and found to bind to p52 Shc in vitro with low micromolar affinity. Co-immunoprecipitation, pull down and fluorescence lifetime imaging microscopy experiments in stable cells confirmed cellular interaction and nuclear localisation. The nuclear transport factor protein NTF2, which functions in cohort with Ran, was shown to form a complex with both RAN and Shc, suggesting a mechanism for Shc entry into the nucleus as part of a tertiary complex.

Fluorescence quenching by metal ions in lipid bilayers.

Fluorescence quenching of perylene by Co2+ and Ni2+ ions has been investigated both below and above the phase transition temperature in small unilamellar DPPC vesicles. Classic Förster type energy transfer was observed for perylene quenched by Co2+ ions below the phase transition when the effects of donor-donor energy transfer are taken into account. In the liquid crystalline phase a simple diffusion theory incorporating energy transfer was found to model the system well. For quenching by Ni2+ ions both above and below the phase transition temperature in lipid bilayers and in glycerol the data did not follow classic Förster type energy transfer but indicated that an additional quenching mechanism was present. A mechanism other than Förster behaviour was also observed for the quenching by Cr3+ ions in glycerol.

Biosynthesis of luminescent quantum dots in an earthworm.

The synthesis of designer solid-state materials by living organisms is an emerging field in bio-nanotechnology. Key examples include the use of engineered viruses as templates for cobalt oxide (Co(3)O(4)) particles, superparamagnetic cobalt-platinum alloy nanowires and gold-cobalt oxide nanowires for photovoltaic and battery-related applications. Here, we show that the earthworm's metal detoxification pathway can be exploited to produce luminescent, water-soluble semiconductor cadmium telluride (CdTe) quantum dots that emit in the green region of the visible spectrum when excited in the ultraviolet region. Standard wild-type Lumbricus rubellus earthworms were exposed to soil spiked with CdCl(2) and Na(2)TeO(3) salts for 11 days. Luminescent quantum dots were isolated from chloragogenous tissues surrounding the gut of the worm, and were successfully used in live-cell imaging. The addition of polyethylene glycol on the surface of the quantum dots allowed for non-targeted, fluid-phase uptake by macrophage cells.

Luminescence lifetimes of neutral nitrogen-vacancy centres in synthetic diamond containing nitrogen.

The decay time of luminescence from neutral nitrogen-vacancy (NV(0)) centres in synthetic diamond is reported. The intrinsic luminescence lifetime of NV (0) is measured as τ(r) = 19 ± 2 ns. Neutral substitutional nitrogen atoms (N(S)(0)) are shown to quench luminescence from NV(0) by dipole-dipole resonant energy transfer at a rate such that the transfer time would equal τ(r) if one (N(S)(0)) atom was ~3 nm from the NV(0). In chemical-vapour-deposited diamonds grown with a small nitrogen content, that are brown as a result of vacancy-cluster defects, the decay time of NV(0) equals τ(r) in the as-grown material. However, after annealing at ≥1700 °C to remove the brown colour, luminescence from the NV(0) centres is severely quenched. This effect is suggested to be a result of the destruction of NV(0) centres and the creation of new NV(0) centres localized in vacancy-rich regions of the crystals.

Imaging fluorescence lifetime heterogeneity applied to GFP-tagged MHC protein at an immunological synapse.

Fluorescence imaging of green fluorescent protein (GFP) may be used to locate proteins in live cells and fluorescence lifetime imaging (FLIM) may be employed to probe the local microenvironment of proteins. Here we apply FLIM to GFP-tagged proteins at the cell surface and at an inhibitory natural killer (NK) cell immunological synapse (IS). We present a novel quantitative analysis of fluorescence lifetime images that we believe is useful to determine whether apparent FLIM heterogeneity is statistically significant. We observe that, although the variation of observed fluorescence lifetime of GFP-tagged proteins at the cell surface is close to the expected statistical range, the lifetime of GFP-tagged proteins in cells is shorter than recombinant GFP in solution. Furthermore the lifetime of GFP-tagged major histocompatibility complex class I protein is shortened at the inhibitory NK cell IS compared with the unconjugated membrane. Following our previous work demonstrating the ability of FLIM to report the local refractive index of GFP in solution, we speculate that these lifetime variations may indicate local refractive index changes. This application of our method for detecting small but significant differences in fluorescence lifetimes shows how FLIM could be broadly useful in imaging discrete membrane environments for a given protein.

Multiplexed time-correlated single-photon counting.

We review the technique of multiplexed time-correlated single-photon counting whereby multiple fluorescence decay curves are recorded in parallel by statistically time-sharing a single time-to-amplitude converter. Application of the multiplexing technique to measuring the fluorescence lifetime topography of a self-absorbing sample is demonstrated. Further possibilities are discussed for multiplexed optical fiber sensor networks with built-in intelligence for detecting and discriminating between different metal ions in solution.