bioRxiv2022
Number and Brightness analysis (N&B) is a fluorescence spectroscopy technique to quantify protein oligomerization. Accurate results, however, rely on a good knowledge of non-fluorescent states of the fluorescent labels, especially of fluorescent proteins (FP), which are widely used in biology. FPs have been characterized for confocal but not camera-based N&B, which allows in principle faster measurements over larger areas. Here, we calibrate camera-based N&B implemented on a total internal reflection fluorescence (TIRF) microscope for various fluorescent proteins by determining their propensity to be fluorescent. We then apply camera-based N&B in live CHO-K1 cells to determine the oligomerization state of the epidermal growth factor receptor (EGFR), a transmembrane receptor tyrosine kinase that is a crucial regulator of cell proliferation and survival with implications in many cancers. EGFR oligomerization in resting cells and its regulation by the plasma membrane microenvironment is still under debate. Therefore, we investigate the effects of extrinsic factors, including membrane organization, cytoskeletal structure, and ligand stimulation, and intrinsic factors, including mutations in various EGFR domains, on the receptors oligomerization. Our results demonstrate that EGFR oligomerization increases with removal of cholesterol or sphingolipids, or the disruption of GM3-EGFR interactions, indicating raft association. However, oligomerization was not significantly influenced by the cytoskeleton. Mutations in either I706/V948 residues or E685/E687/E690 residues in the kinase and juxtamembrane domains, respectively, led to a decrease in oligomerization, indicating their necessity for EGFR dimerization. Finally, EGFR phosphorylation is oligomerization-dependent involving the extracellular domain (550-580 residues). Coupled with biochemical investigations, camera-based N&B indicates that EGFR oligomerization and phosphorylation is the outcome of several molecular interactions involving the lipid content and structure of the cell membrane and multiple residues in the kinase, juxtamembrane, and extracellular domains. STATEMENT OF SIGNIFICANCENumber and brightness (N&B) analysis is a powerful tool to determine protein association but is mostly conducted in confocal microscopes. This work determines the brightness and fluorescence probability of a range of fluorescent proteins for camera-based N&B on a total internal reflection microscope, demonstrating that with proper calibration different fluorescent proteins provide the same answers on oligomerization within the margins of error. This camera-based approach allows measuring N&B values across whole cell basal membranes up to an area of ~1,000 m2 simultaneously. N&B is then used in combination with biochemical assays to investigate the oligomerization and activation of the epidermal growth factor receptor (EGFR), a prototypical receptor tyrosine kinase with importance in cell signalling, division and survival and implicated in various cancers. The results indicate that EGFR oligomerization and activation is governed by an interplay between membrane structure and composition and key amino acid residues of EGFR that span the extracellular to the intracellular domains.