Mechanistic insights into EGFR membrane clustering revealed by super-resolution imaging

Abstract

The clustering of membrane receptors such as EGFR is critical for various biological processes, for example cell signaling and tumorigenesis. However, the mechanism involved remains poorly understood. Here, we used a super resolution imaging technique, which has shattered the longstanding resolution barrier of light diffraction, to investigate the distribution of membrane EGFR on apical or basal surfaces of COS-7 cells and on the surface of suspended COS-7 cells. Our data show that more and larger EGFR clusters are detected on the apical surface in comparison with those on the basal surface and this difference is not affected by the EGFR activation state, whereas suspended COS-7 cells exhibit a moderate clustering state and a homogeneous distribution pattern, indicating that the external environment surrounding the cell membrane is the decisive factor in the EGFR clustering pattern. A dual-color dSTORM image reveals the significant colocalization of EGFR and lipid rafts; interestingly MβCD treatment leads to a dramatic decrease of the amount and size of EGFR clusters on both apical and basal surfaces, highlighting a key role of lipid rafts in EGFR cluster formation. Altogether, our results illustrate the distribution pattern of EGFR in polarized cells and uncover the essential role of lipid rafts in EGFR cluster maintenance.

The Epidermal Growth Factor Receptor Forms Location-Dependent Complexes in Resting Cells

“…These values are higher than the cross-correlation amounts measured in untreated cells, demonstrating that EGFR complex formation is to some extent inhibited by partitioning of the receptor into cholesterol-dependent domains. These results are in agreement with a previously published study (63) and are consistent with the findings that EGFR resides in cholesterol-dependent domains (33,64).…”

“…Consistent with our results, Saffarian et al (63) reported an increase of oligomeric EGFR fraction after cholesterol depletion. Other studies have reported decreased EGFR clustering upon cholesterol depletion (62,64); however, the EGFR clusters described in those studies are much larger and most likely of a different nature than the mobile complexes investigated here. In addition, the disruption of the actin cytoskeleton by LAT-A treatment had no effect on the EGFR complex fraction.…”

Galectin-3, a Druggable Vulnerability for KRAS-Addicted Cancers

“…The redundancy in receptors capable of mediating KRAS clustering may explain why matrix-adherent cells can easily switch dependence between pathways when one is inhibited by a given targeted therapeutic. However, in the absence of matrix adhesion, epithelial cell surface receptors poorly cluster, as has been demonstrated for EGFR [7]. Because the ability of a cell to exhibit anchorage independent growth is a hallmark of tumor progression and metastasis [8, 9], we propose KRAS-containing signaling complexes that form in non-adherent cells as therapeutic targets to reverse anchorage-independence, thereby containing the spread of malignant cells.…”

Artifacts in single-molecule localization microscopy

“…Especially, two-dimensional imaging of intrinsically threedimensional membrane structures like microvilli, filopodia, overlapping membranes and vesicles with high local emitter densities is prone to generate artifacts. To judge the quality and reliability of super-resolution images, the can even give quantitative information about the distribution of proteins and the ratio of molecules residing inside and outside of subcellular compartments such as plasma membrane domains (Williamson et al 2011;Lando et al 2012;Bar-On et al 2012;Puchner et al 2013;Ehmann et al 2014;Letschert et al 2014;Löschberger et al 2014;Saka et al 2014;Honigmann et al 2014;Fricke et al 2014;Gao et al 2015;Chen et al 2015).…”