Quantum dot ligands provide new insights into erbB/HER receptor–mediated signal transduction

Abstract

The erbB/HER family of transmembrane receptor tyrosine kinases (RTKs) mediate cellular responses to epidermal growth factor (EGF) and related ligands. We have imaged the early stages of RTK-dependent signaling in living cells using: (i) stable expression of erbB1/2/3 fused with visible fluorescent proteins (VFPs), (ii) fluorescent quantum dots (QDs) bearing epidermal growth factor (EGF-QD) and (iii) continuous confocal laser scanning microscopy and flow cytometry. Here we demonstrate that EGF-QDs are highly specific and potent in the binding and activation of the EGF receptor (erbB1), being rapidly internalized into endosomes that exhibit active trafficking and extensive fusion. EGF-QDs bound to erbB1 expressed on filopodia revealed a previously unreported mechanism of retrograde transport to the cell body. When erbB2-monomeric yellow fluorescent protein (mYFP) or erbB3-monomeric Citrine (mCitrine) were coexpressed with erbB1, the rates and extent of endocytosis of EGF-QD and the RTK-VFP demonstrated that erbB2 but not erbB3 heterodimerizes with erbB1 after EGF stimulation, thereby modulating EGF-induced signaling. QD-ligands will find widespread use in basic research and biotechnological developments.

Apical and lateral cell protrusions interconnect epithelial cells in live Drosophila wing imaginal discs

“…Molecules associated with protrusions include Sevenless, a receptor for the Boss ligand (Tomlinson et al, 1987); Delta, a Notch ligand (De Joussineau et al, 2003); Scabrous, a signaling molecule (Chou and Chien, 2002); and Tkv, a receptor for Dpp (Hsiung et al, 2005) on cellular protrusions in Drosophila and members of the erbB/HER family, receptors for EGF and related ligands, on extensions in mammalian cells (Lidke et al, 2004). We extend these findings by showing that Tkv-GFP can also localize to apical and lateral cell protrusions.…”

Signal transduction of erbB receptors in trastuzumab (Herceptin) sensitive and resistant cell lines: Local stimulation using magnetic microspheres as assessed by quantitative digital microscopy

“…Six carcinoma cell lines, SKBR-3, JIMT-1(18), A431, an A431-derived cell line stably transfected with the erbB2-mYFP fusion gene (A4-erbB2-mYFP F4 line, expressing erbB2 with a C-terminal monomeric YFP fusion) (36), HeLa, and erbB1 transfected Chinese hamster ovary and MCF7 cells were used. Cells were cultured in a humidified atmosphere with 5% CO 2 at 37°C in Dulbecco's Minimal Essential Medium (Sigma, Schnelldorf, Germany) supplemented with 10% fetal calf serum (Sigma) and antibiotics.…”

The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

“…We chose this cell-receptor system to test our calibration beads because there is a commercially available biotinylated EGF ligand, and the receptor system has already been characterized with Qdots (39). In addition, A431 cells are a good model system to study the interaction of epidermal cells with a naturally occurring multivalent ligand, namely human papillomaviruses labeled with Qdots (vide infra, cf.…”