epidermal-growth-factor and cyanine-dye-3

Fluorescence resonance energy transfer (FRET) has found widespread uses in biosensing, molecular imaging, and light harvesting. Plasmonic metal nanostructures offer the possibility of engineering photonic environment of specific fluorophores to enhance the FRET efficiency. However, the potential of plasmonic nanostructures to enable tailored FRET enhancement on planar substrates remains largely unrealized, which are of considerable interest for high-performance on-surface bioassays and photovoltaics. The main challenge lies in the necessitated concurrent control over the spectral properties of plasmonic substrates to match that of fluorophores and the fluorophore-substrate spacing. Here, a self-assembled plasmonic substrate based on polydopamine (PDA)-coated plasmonic nanocrystals is developed to effectively address this challenge. The PDA coating not only drives interfacial self-assembly of the nanocrystals to form closely packed arrays with customized optical properties, but also can serve as a tailored nanoscale spacer between the fluorophores and plasmonic nanocrystals, which collectively lead to optimized fluorescence enhancement. The biocompatible plasmonic substrate that allows convenient bioconjugation imparted by PDA has afforded improved FRET efficiency in DNA microarray assay and FRET imaging of live cells. It is envisioned that the self-assembled plasmonic substrates can be readily integrated into fluorescence-based platforms for diverse biomedical and photoconversion applications.

Topics: Biocompatible Materials; Carbocyanines; Cell Line, Tumor; Epidermal Growth Factor; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Gold; Humans; Indoles; Metal Nanoparticles; Microscopy, Confocal; Oligonucleotide Array Sequence Analysis; Polymers; Silver

We have developed cell stimulative system by covalently immobilized signalling molecules on the surface of coverslips on which cells are later cultured. N-(6-maleimidocaproyloxy)sulfo-succinimide (sulfo-EMCS) cross-links the amino-terminal of epidermal growth factors (EGF) with the thiol-modified glass surface without degrading EGF's physiological activity. The glass surface was covered up to about 1.0 EGF moleculesnm(-2) with uniform density. This density can be controlled by changing concentration of the maleimide-modified EGF in the solution reacting with the thiol-modified glass coverslips. When the density of EGF was only slightly lower than that of EGF receptor dimers, cellular response was dramatically decreased. The EGF receptor molecules bound with the immobilized EGF were prevented from lateral diffusion and internalization and kept their initial position. These properties are useful for quantitative, spatial and temporal control of the input signal stimulating cells in cellular signaling system studies. In addition, the immobility of the EGF in this system makes suitable targets for stable single-molecule observation under total internal reflection fluorescence microscopy (TIR-FM) to study EGF signalling mechanism, preventing lateral diffusion and internalization of EGF receptors. Here we show results of single-molecule observations of the association and dissociation between phosphorylated EGF receptors and Cy3-labeled growth factor receptor-binding protein 2 (Grb2) proteins in A431 cells stimulated by the immobilized EGF and discuss the utility of this method for the study of intracellular signal transduction.

Topics: Biological Assay; Carbocyanines; Cell Culture Techniques; Epidermal Growth Factor; Glass; GRB2 Adaptor Protein; Humans; Microscopy, Fluorescence; Phosphorylation; Signal Transduction; Tumor Cells, Cultured

Topics: Carbocyanines; Cells, Cultured; Dimerization; Epidermal Growth Factor; ErbB Receptors; Fluorescent Dyes; Humans; Microscopy, Fluorescence; Phosphorylation; Signal Transduction; Time Factors

The early events in signal transduction from the epidermal growth factor (EGF) receptor (EGFR) are dimerization and autophosphorylation of the receptor, induced by binding of EGF. Here we observe these events in living cells by visualizing single molecules of fluorescent-dye-labelled EGF in the plasma membrane of A431 carcinoma cells. Single-molecule tracking reveals that the predominant mechanism of dimerization involves the formation of a cell-surface complex of one EGF molecule and an EGFR dimer, followed by the direct arrest of a second EGF molecule, indicating that the EGFR dimers were probably preformed before the binding of the second EGF molecule. Single-molecule fluorescence-resonance energy transfer shows that EGF-EGFR complexes indeed form dimers at the molecular level. Use of a monoclonal antibody specific to the phosphorylated (activated) EGFR reveals that the EGFR becomes phosphorylated after dimerization.

Topics: Antibodies, Monoclonal; Calcium; Carbocyanines; Carcinoma; Cell Membrane; Dimerization; Energy Transfer; Epidermal Growth Factor; ErbB Receptors; Fluorescent Dyes; Humans; Intracellular Fluid; Microscopy, Fluorescence; Phosphorylation; Rhodamines; Signal Transduction; Tumor Cells, Cultured

Evidence for a new signaling mechanism consisting of ligand-independent lateral propagation of receptor activation in the plasma membrane is presented. We visualized the phosphorylation of green fluorescent protein (GFP)-tagged ErbB1 (ErbB1-GFP) receptors in cells focally stimulated with epidermal growth factor (EGF) covalently attached to beads. This was achieved by quantitative imaging of protein reaction states in cells by fluorescence resonance energy transfer (FRET) with global analysis of fluorescence lifetime imaging microscopy (FLIM) data. The rapid and extensive propagation of receptor phosphorylation over the entire cell after focal stimulation demonstrates a signaling wave at the plasma membrane resulting in full activation of all receptors.

Topics: Arsenicals; Carbocyanines; Cell Membrane; Diffusion; Dimerization; Endocytosis; Energy Transfer; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Fluorescence; Fluorescent Dyes; Green Fluorescent Proteins; Humans; Immunoglobulin Fab Fragments; Ligands; Luminescent Proteins; Microscopy, Confocal; Microscopy, Fluorescence; Microspheres; Phosphorylation; Phosphotyrosine; Protein Tyrosine Phosphatases; Signal Transduction; Tumor Cells, Cultured