Dataset Information

Live-cell fluorescence imaging reveals high stoichiometry of Grb2 binding to the EGF receptor sustained during endocytosis.

ABSTRACT: Activation of epidermal growth factor (EGF) receptor (EGFR) leads to its interaction with Grb2, a dual-function adapter mediating both signaling through Ras and receptor endocytosis. We used time-lapse three-dimensional imaging by spinning disk confocal microscopy to analyze trafficking of EGFR and Grb2 in living HeLa cells stimulated with low, physiological concentrations of EGFR ligands. Endogenous Grb2 was replaced in these cells by Grb2 fused to yellow fluorescent protein (YFP). After transient residence in the plasma membrane, Rhodamine-conjugated EGF (EGF-Rh) and Grb2-YFP were rapidly internalized and accumulated in endosomes. Quantitative image analysis revealed that on average two Grb2-YFP molecules were colocalized with one EGF-Rh in cells stimulated with 2 ng/ml EGF-Rh, and the excess of Grb2-YFP over EGF-Rh was even higher when a receptor-saturating concentration of EGF-Rh was used. Therefore, we hypothesize that a single EGFR molecule can be simultaneously associated with functionally distinct Grb2 interaction partners during and after endocytosis. Continuous presence of Grb2-YFP in endosomes was also observed when EGFR was activated by transforming growth factor-? and amphiregulin, suggesting that endosomal EGFRs remain ligand occupied and signaling competent, despite the fact that these growth factors are thought to dissociate from the receptor at acidic pH. The prolonged localization and activity of EGFR-Grb2 complexes in endosomes correlated with the sustained activation of extracellular stimulus-regulated kinase 1/2, suggesting that endosomal EGFRs contribute significantly to this signaling pathway. We propose that endosomal EGFRs function to extend signaling in time and space to compensate for rapid downregulation of surface EGFRs in cells with low receptor expression levels.

SUBMITTER: Fortian A

PROVIDER: S-EPMC3889400 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Live-cell fluorescence imaging reveals high stoichiometry of Grb2 binding to the EGF receptor sustained during endocytosis.

Fortian Arola A Sorkin Alexander A

Journal of cell science 20131120 Pt 2

Activation of epidermal growth factor (EGF) receptor (EGFR) leads to its interaction with Grb2, a dual-function adapter mediating both signaling through Ras and receptor endocytosis. We used time-lapse three-dimensional imaging by spinning disk confocal microscopy to analyze trafficking of EGFR and Grb2 in living HeLa cells stimulated with low, physiological concentrations of EGFR ligands. Endogenous Grb2 was replaced in these cells by Grb2 fused to yellow fluorescent protein (YFP). After transi ...[more]

PMID: 24259669

Similar Datasets

Project description:Thermally activated delayed fluorescence (TADF) molecules offer nowadays a powerful tool in the development of novel organic light emitting diodes due to their capability of harvesting energy from non-emissive triplet states without using heavy-metal complexes. TADF emitters have very small energy difference between the singlet and triplet excited states, which makes thermally activated reverse intersystem crossing from the triplet states back to the singlet manifold viable. This mechanism generates a long-lived delayed fluorescence component which can be explored in the sensing of oxygen concentration, local temperature, or used in time-gated optical cell-imaging, to suppress interference from autofluorescence and scattering. Despite this strong potential, until recently the application of TADF outside lighting devices has been hindered due to the low biocompatibility, low aqueous solubility and poor performance in polar media shown by the vast majority of TADF emitters. To achieve TADF luminescence in biological media, careful selection or design of emitters is required. Unfortunately, most TADF molecules are not emissive in polar media, thus complexation with biomolecules or the formation of emissive aggregate states is required, in order to retain the delayed fluorescence that is characteristic of these compounds. Herein, we demonstrate a facile method with great generalization potential that maintains the photophysical properties of solvated dyes by combining luminescent molecules with polymeric nanoparticles. Using an established swelling procedure, two known TADF emitters are loaded onto polystyrene nanoparticles to prepare TADF emitting nanomaterials able to be used in live-cell imaging. The obtained particles were characterized by optical spectroscopy and exhibited the desired TADF emission in aqueous media, due to the polymeric matrix shielding the dye from solvent polarity effects. The prepared nanoparticles were incubated with live human cancer cells and showed very low cytotoxicity and good cellular uptake, thus making fluorescence microscopy imaging possible at low dye concentrations.

Dataset Information

Live-cell fluorescence imaging reveals high stoichiometry of Grb2 binding to the EGF receptor sustained during endocytosis.

Publications

Live-cell fluorescence imaging reveals high stoichiometry of Grb2 binding to the EGF receptor sustained during endocytosis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets