Single-molecule imaging of EGFR signalling on the surface of living cells.
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.
Sako
Y
Y
Department of Physiology and Biosignalling, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita 565-0871, Japan.
Minoghchi
S
S
Yanagida
T
T
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002232:Carbocyanines; D002277:Carcinoma / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D019281:Dimerization; D004735:Energy Transfer / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D010766:Phosphorylation / Q000187:drug effects; D012235:Rhodamines; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D002232:Carbocyanines; D005456:Fluorescent Dyes; D012235:Rhodamines; C089187:cyanine dye 3; D004815:Epidermal Growth Factor; C005358:tetramethylrhodamine; D066246:ErbB Receptors; D002118:Calcium