mTOR direct interactions with Rheb-GTPase and raptor: sub-cellular localization using fluorescence lifetime imaging.

Background: The mammalian target of rapamycin (mTOR) signalling pathway has a key role in cellular regulation and several diseases. While it is thought that Rheb GTPase regulates mTOR, acting immediately upstream, while raptor is immediately downstream of mTOR, direct interactions have yet to be verified in living cells, furthermore the localisation of Rheb has been reported to have only a cytoplasmic cellular localization.

Results: In this study a cytoplasmic as well as a significant sub-cellular nuclear mTOR localization was shown , utilizing green and red fluorescent protein (GFP and DsRed) fusion and highly sensitive single photon counting fluorescence lifetime imaging microscopy (FLIM) of live cells.

Two-photon excitation with pico-second fluorescence lifetime imaging to detect nuclear association of flavanols.

Two-photon excitation enabled for the first time the observation and measurement of excited state fluorescence lifetimes from three flavanols in solution, which were ~1.0 ns for catechin and epicatechin, but <45 ps for epigallocatechin gallate (EGCG). The shorter lifetime for EGCG is in line with a lower fluorescence quantum yield of 0.

ULTRA: A Unique Instrument for Time-Resolved Spectroscopy.

We report the development of a high-sensitivity time-resolved infrared and Raman spectrometer with exceptional experimental flexibility based on a 10-kHz synchronized dual-arm femtosecond and picosecond laser system. Ultrafast high-average-power titanium sapphire lasers and optical parametric amplifiers provide wavelength tuning from the ultraviolet (UV) to the mid-infrared region. Customized silicon, indium gallium arsenide, and mercury cadmium telluride linear array detectors are provided to monitor the probe laser intensity in the UV to mid-infrared wavelength range capable of measuring changes in sample absorbance of ΔOD ~ 10(-5) in 1 second.

Interaction of poxvirus intracellular mature virion proteins with the TPR domain of kinesin light chain in live infected cells revealed by two-photon-induced fluorescence resonance energy transfer fluorescence lifetime imaging microscopy.

Using two-photon-induced fluorescence lifetime imaging microscopy, we corroborate an interaction (previously demonstrated by yeast two-hybrid domain analysis) of full-length vaccinia virus (VACV; an orthopoxvirus) A36 protein with the cellular microtubule motor protein kinesin. Quenching of enhanced green fluorescent protein (EGFP), fused to the C terminus of VACV A36, by monomeric red fluorescent protein (mDsRed), fused to the tetratricopeptide repeat (TPR) domain of kinesin, was observed in live chicken embryo fibroblasts infected with either modified vaccinia virus Ankara (MVA) or wild-type fowlpox virus (FWPV; an avipoxvirus), and the excited-state fluorescence lifetime of EGFP was reduced from 2.5 ± 0.

Directed single molecule diffusion triggered by surface energy gradients.

We demonstrate the diffusion of single poly(ethylene glycol) molecules on surfaces which change from hydrophilic to hydrophobic over a few micrometers. These gradients in surface energy are shown to drive the molecular diffusion in the direction of the hydrophilic component. The polymer diffusion coefficients on these surfaces are measured by fluorescence correlation spectroscopy and are shown to be elevated by more than an order of magnitude compared to surfaces without the surface energy gradient.

Specific fluorescent tracers. Imaging and applications for photodynamic therapy.

Our main objective is to enlarge the fluorescence use in biosciences, with especially the photodynamic therapy (PDT) used for cancer treatment as one of the target applications. Meta-tetra(hydroxyphenyl)chlorin (m-THPC) is a second-generation photosensitiser, applied in photodynamic therapy. The localisation of this sensitiser as well as its induced cell death mechanisms in human breast cancer cells (MCF-7 and its resistant subline MCF-7DXR, DXR: doxorubicin) were evaluated using fluorescence microscopy.

The size of lipid rafts: an atomic force microscopy study of ganglioside GM1 domains in sphingomyelin/DOPC/cholesterol membranes.

Atomic force microscopy has been used to study the distribution of ganglioside GM1 in model membranes composed of ternary lipid mixtures that mimic the composition of lipid rafts. The results demonstrate that addition of 1% GM1 to 1:1:1 sphingomyelin/dioleoylphosphatidylcholine/cholesterol monolayers leads to the formation of small ganglioside-rich microdomains (40-100 nm in size) that are localized preferentially in the more ordered sphingomyelin/cholesterol-rich phase. With 5% GM1 some GM1 microdomains are also detected in the dioleoylphosphatidylcholine-rich phase.