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The epidermal growth factor receptor (EGFR) is a member of the erbB tyrosine kinase family of receptors. For many years it has been believed that receptor activation occurs via a monomer-dimer transition that is associated with a conformational change to activate the kinase. However, little is known about the quaternary state of the receptor at normal(More)
Graphical representation of fluorescence lifetime imaging microscopy data demonstrates that a mixture of two components with single exponential decays can be resolved by single frequency measurements. We derive a method based on linear fitting that allows the calculation of the fluorescence lifetimes of the two components. We show that introduction of(More)
Characterization of the association states of the unligated epidermal growth factor receptor (EGFR) is important in understanding the mechanism of EGFR tyrosine kinase activation in a tumor cell environment. We analyzed, in detail, the association states of unligated, immunotagged EGFR on the surface of intact epidermoid carcinoma A431 cells, using(More)
We describe a novel variant of fluorescence lifetime imaging microscopy (FLIM), denoted anisotropy-FLIM or rFLIM, which enables the wide-field measurement of the anisotropy decay of fluorophores on a pixel-by-pixel basis. We adapted existing frequency-domain FLIM technology for rFLIM by introducing linear polarizers in the excitation and emission paths. The(More)
We report the implementation and exploitation of fluorescence polarization measurements, in the form of anisotropy fluorescence lifetime imaging microscopy (rFLIM) and energy migration Förster resonance energy transfer (emFRET) modalities, for wide-field, confocal laser-scanning microscopy and flow cytometry of cells. These methods permit the assessment of(More)
The epidermal growth factor receptor (EGFR) kinase is generally considered to be activated by either ligand-induced dimerisation or a ligand-induced conformational change within pre-formed dimers. We report the relationship between ligand-induced higher-order EGFR oligomerization and EGFR phosphorylation on the surface of intact cells. We have combined(More)
Although fluorescein is a widely used fluorescent probe in the biosciences, the effect of solvent environment on its spectral properties is poorly understood. In this paper we explore the use of fluorescein as a probe of the state of hydrogen bonding in its local environment. This application is based on the observation, originally made by Martin (Chem.(More)
Fluorescence resonance energy transfer (FRET) from a donor-labelled molecule to an acceptor-labelled molecule is a useful, proximity-based fluorescence tool to discriminate molecular states on the surface and in the interior of cells. Most microscope-based determinations of FRET yield only a single value, the interpretation of which is necessarily(More)
The serotonin₁(A) receptor is a representative member of the GPCR superfamily and serves as an important drug target. The possible role of GPCR oligomerization in receptor function is an active area of research. We monitored the oligomerization state of serotonin₁(A) receptors using homo-FRET and fluorescence lifetime measurements. Homo-FRET is estimated by(More)
The epidermal growth factor receptor (EGFR) is a member of the erbB tyrosine kinase family of receptors. Structural studies have revealed two distinct conformations of the ectodomain of the EGFR: a compact, tethered, conformation and an untethered extended conformation. In the context of a monomer-dimer transition model, ligand binding is thought to(More)