Plasma membrane organization during EGFR signaling: a FRET-based analysis

Since two decades it has been suggested that the plasma membrane is organized into lipid-separated domains called lipid rafts. A number of functions have been attributed to these domains, including spatially separating or combining functionally linked proteins. Proteins such as the Epidermal Growth Factor Receptor (EGFR) are found to be localized in membrane domains, as well as effector molecules downstream in the signal transduction cascade. The investigation of these structures has mainly been performed by highly invasive techniques such as biochemical analysis, but lacks data on the situation in intact cells. Chapter 2 of this thesis describes the use of Förster Resonance Energy Transfer (FRET) to study the presence and dynamics of membrane domains containing EGFR. To study this receptor we developed fluorescent nanobodies, monovalent domains from cameloid heavy-chain only antibodies. FRET-FLIM analysis revealed that EGFR resides in a subclass of nanodomains including the ganglioside GM1, and is absent in domains composed of GM1 and GPI-linked GFP. Activation of EGFR results in the coalescence of the two domains, suggesting the formation of signaling platforms. Lipid domains are often suggested to promote the local clustering of their constituents. Therefore, a part of this work describes the development of a novel approach to study the oligomerization state of the domain components in intact cells. This technique, confocal time-resolved fluorescence anisotropy imaging microscopy (CTRFAIM), is based on FRET between identical fluorophores (homo-FRET). The anisotropy, defined as the degree of polarization of the fluorescence, is directly related to the degree of oligomerization. In this thesis, a simplification of this approach is described by applying time-gated data acquisition (chapter 3 and 4). An inducible FKBP dimerization and oligomerization system was developed to correlate the anisotropy value to the oligomerization state. Chapter 4 describes an evaluation of different modes of data acquisition. When compared to steady-state anisotropy, CTR-FAIM shows an improved dynamic range of anisotropy values. A further improvement can be obtained with two-photon excitation, although this improvement is diminished by significant higher photobleaching (chapter 4). When subjected to CTRFAIM analysis, the domain components in our study were indeed found to be clustered. By controlled photobleaching, the lipid raft probe GPI-GFP was found to form small nanoclusters of 1-5 molecules. Also EGFR was found to organize into oligomers, depending on the activation state of the receptor. The CTRFAIM data reveal pre-existing dimers of EGFR in the plasma membrane of resting cells. After stimulation with EGF, the receptor oligomerizes in a kinase- and phosphotyrosine-dependent manner, forming clusters of 3 or more receptors (chapter 5). Furthermore, induced receptor clustering enhances receptor internalization speed, which suggests a stimulatory role for EGFR oligomerization in the internalization process. In conclusion, these data suggest the formation of larger signaling platforms in the plasma membrane upon EGFR activation.