Table of Contents
Methods Protoc., Volume 2, Issue 1 (March 2019)
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Cover Story (view full-size image) Single-particle tracking has been developed over the past 25 years to investigate molecular [...] Read more. Single-particle tracking has been developed over the past 25 years to investigate molecular dynamics, structure, interactions, and function in the cellular context. We show how single-molecule and ensemble Förster resonance energy transfer (FRET) are used to build a model of epidermal growth factor receptor (EGFR) complexes in cells. EGFRs in cells are labelled with a mixture of donor (green) and acceptor (red) fluorophores. FRET then measures distances between EGFR in complexes. EGFRs are also liganded with EGF labelled with a green fluorescent dye, and the plasma membrane is labelled with a red dye. Ensemble FRET between the dyes measures EGFR-membrane separation, and hence the receptor’s conformation. These data are combined to produce a model for EGFR oligomerization in the cell. Data from Webb et al., Biophys. J. 94, 803 (2008) and Tynan et al., Mol. Cell. Biol. 31, 2241 (2011). View this paper.