Illuminating Dynamic Receptor Clustering in the Epidermal Growth Factor Receptor Signal Transduction Pathway Using Plasmon Coupling

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Summary – Research Supplement to Promote Diversity in Health-Related Research The organization of EGFR into clusters has shown to play an essential role in controlling and modulating he nature and intensity of cellular signaling but many aspects of this spatial control mechanism remain insufficiently understood. The ability to monitor the structure and dynamics of EGFR clusters in living cells with optical microscopies would go a long way to unravel the mechanisms underlying spatial clustering mediated control of EGFR signaling. Past studies aimed at probing EGFT clustering optically involved the use of quantum dots, fluorescent dyes, and gold nanoparticles. Each of these labels has, however, its own challenges and limitations. Quantum dots blink are cytotoxic, fluorescent dyes bleach and metal nanoparticles (NPs) require relatively large sizes (>40nm) to be detectable in conventional darkfield or total internal reflection microscopy. Although gold NPs have extreme photostabilities, labelling proteins with particle of this size can perturb the structure and function of the molecule. All these challenges make it difficult to study the true dynamics of the membrane protein with conventional techniques. To overcome these problems, we propose here to develop an interferometric scattering microscopy (iSCAT) to study the kinetics of EGFR clustering as well as the stability of EGFR clusters through distance-dependent plasmon coupling signals of NP-labelled EGFR. The interferometric detection approach can still take advantage of the unique photophysical properties of noble metal NPs (these materials don’t blink or bleach) but is compatible with much smaller NP sizes. We will use gold and silver NP labels with diameters as small as 5 nm in this project. Distance-dependent plasmon coupling between the NPs will be used to monitor the association of NP-labelled EGFR monomers into dimers and larger oligomers and their spatial association into clusters. The self-association of the proteins will be indicated by spectral shifts of the plasmon resonance of the NP labels, which can be detected and quantified in the far-field.