Epidermal growth factor (EGF) signalling regulates normal epithelial and other cell growth, with EGF receptor (EGFR) overexpression reported in many cancers. However, the role of EGFR clusters in cancer and their dependence on EGF binding is unclear. We present novel single-molecule total internal reflection fluorescence microscopy of (i) EGF and EGFR in living cancer cells, (ii) the action of anti-cancer drugs that separately target EGFR and human EGFR2 (HER2) on these cells and (iii) EGFR–HER2 interactions. We selected human epithelial SW620 carcinoma cells for their low level of native EGFR expression, for stable transfection with fluorescent protein labelled EGFR, and imaged these using single-molecule localization microscopy to quantify receptor architectures and dynamics upon EGF binding. Prior to EGF binding, we observe pre-formed EGFR clusters. Unexpectedly, clusters likely contain both EGFR and HER2, consistent with co-diffusion of EGFR and HER2 observed in a different model CHO-K1 cell line, whose stoichiometry increases following EGF binding. We observe a mean EGFR : EGF stoichiometry of approximately 4 : 1 for plasma membrane-colocalized EGFR–EGF that we can explain using novel time-dependent kinetics modelling, indicating preferential ligand binding to monomers. Our results may inform future cancer drug developments.

Epidermal growth factor (EGF) signaling regulates normal cell development, however EGF receptor (EGFR) overexpression is reported in several carcinomas. Despite structural and biochemical evidence that EGF-EGFR ligation activates signaling through monomer-dimer transitions, live cell mechanistic details remain contentious. We report single-molecule multispectral TIRF of human epithelial carcinoma cells transfected with fluorescent EGFR, and of CHO-K1 cells containing fluorescent EGFR and HER2, enabling super-resolved localization to quantify receptor architectures and spatiotemporal dynamics upon EGF ligation. Using inhibitors that block binding to EGFR, and time-dependent kinetics modelling, we find that pre-activated EGFR consist predominantly of preformed clusters that contain a mixture of EGFR and HER2, whose stoichiometry increases following EGF activation. Although complicated by EGFR internalization and recycling, our observation of an EGFR:EGF stoichiometry >1 for plasma membrane colocalized EGFR/EGF foci soon after activation may indicate preferential binding of EGF ligand to EGFR monomers, negative cooperativity and preferential ligated-unligated dimerization of monomers.

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Epidermal growth factor (EGF) signalling regulates cell growth, differentiation and proliferation 19 in epithelium and EGF receptor (EGFR) overexpression has been reported in several carcinoma types. 20 Structural and biochemical evidence suggests EGF binding stimulates EGFR monomer-dimer transitions, 21 activating downstream signalling. However, mechanistic details of ligand binding to functional receptors in 22 live cells remain contentious. We report real time single-molecule TIRF of human epithelial carcinoma 23 cells with negligible native EGFR expression, transfected with GFP-tagged EGFR, before and after 24 receptor activation with TMR-labelled EGF ligand. Fluorescently labelled EGFR and EGF are 25 simultaneously tracked to 40nm precision to explore stoichiometry and spatiotemporal dynamics upon 26 EGF binding. Using inhibitors that block binding to EGFR directly, or indirectly through HER2, our 27 results indicate that pre-activated EGFR consists of preformed homoclusters, while larger heteroclusters 28 including HER2 form upon activation. The relative stoichiometry of EGFR to EGF after binding peaks at 29 2, indicating negative cooperativity of EGFR activation. 30 31Main Text: 35 The epidermal growth factor receptor (EGFR) is essential for normal growth and development of epithelial 36 tissues and is a key component in several signaling pathways 1 . Aberrant signal transduction is a primary 37 driver of many epithelial cancers, EGFR upregulation implicated in formation and progression of several 38 carcinomas 2 . Human EGFR or ERBB1, (also denoted 'ErB1'or 'HER1') is a 1,186 amino acid (aa) residue 39 170 kDa molecular weight protein 3 belonging to a family of receptor tyrosine kinase (RTK) receptors with 40 three additional members: ERBB2 ('ErbB2', 'HER2' or 'neu'), ERBB3 ('ErbB3' or 'HER3') and ERBB4 41 ('ErbB4' or 'HER4') expressed predominantly in the plasma membrane of epithelial cells 4 . EGFR has a 42 621aa extracellular region, divided into subdomains I-IV 5 . Domains I and III directly participate in ligand 43 binding 6 , connected via a 23aa hydrophobic transmembrane α-helix to a 542aa cytoplasmic domain 44 containing a 300aa tyrosine kinase 7 . 45 EGFR activation requires ligand binding, receptor-receptor interactions, and full activation of the 46 tyrosine kinase 8 . At least 11 different ligands bind to the EGFR family, four to EGFR including EGF 47 itself 9 . Prior to ligand binding the tyrosine kinase has low catalytic activity. Ligand binding results in full 48 kinase activation through c-lobe interaction of an 'activator' and n-lobe 'receiver' 10 . Subsequent 49 autophosphorylation of intracellular tyrosine residues 11 initiates intracellular reactions ultimately 50 stimulating cellular growth, differentiation and proliferation 12 , terminated by internalization and 51 proteolytic degradation of the receptor-ligand complex 13 . 52The field has detailed insights concerning extracellular and intracellular interactions that contribute 53 to signal transduction, however, there remai...