The Spatiotemporal Organization of ErbB Receptors: Insights from Microscopy

Valley, Christopher C.; Lidke, Keith A.; Lidke, Diane S.

doi:10.1101/cshperspect.a020735

Cited by 28 publications

(28 citation statements)

References 63 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Other techniques, for example, cryo TEM, are needed to study the protein structure, and the cellular ultrastructure 15 . Secondly, studying the dynamic interplay of a multiple of proteins in live cell in time-lapse microscope is not feasible on account of radiation damage, and requires state-of-the-art light microscopy 23 .…”

Section: Representative Resultsmentioning

confidence: 99%

“…Studying the spatial arrangement of this membrane receptor in whole cells provides important context information about its function and possible changes thereof [11][12][13][14] . But it remains challenging to probe the distribution of EGFR monomers, dimers, and clusters directly on a (sub-)cellular level with biochemical-or conventional microscopy methods 15 . Our method is capable of visualizing EGFRs with a spatial resolution of a few nanometers such that the locations of proteins in a complex can be determined.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

Peckys

Jonge

2015

JoVE

View full text Add to dashboard Cite

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e., one emitting at 655 nm and at 800 revealed similar characteristic results.

show abstract

Section: Representative Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

Peckys

Jonge

2015

JoVE

View full text Add to dashboard Cite

show abstract

“…This model has been supported by crystal structures of the extracellular domain of EGFR (7,8) as well as by reports of EGFR dimerization after stimulation with EGF. However, numerous studies have also reported the presence of EGFR dimers or even larger oligomers in the membranes of resting cells (9)(10)(11)(12)(13). The presence of preformed EGFR dimers and oligomers indicates that regulation of EGFR signaling is more complex than implied by the model outlined above (Fig.…”

Section: Introductionmentioning

confidence: 92%

“…The existence of preformed dimers, their relative amount in resting cells, and their role in EGFR signaling continue to present open questions and the findings of individual studies differ considerably (9,14). While some authors report negligible amounts of preformed dimers (15), others have found most EGFR molecules in dimeric form (6,9,16,17). Nagy et al (18) observed preformed dimers only at high expression levels of EGFR of ~600,000 receptors per cell.…”

Section: Introductionmentioning

confidence: 99%

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

Yavas

Macháň

Wohland

2016

Biophysical Journal

View full text Add to dashboard Cite

The epidermal growth factor receptor (EGFR) is a prototypical receptor tyrosine kinase involved in cell growth and proliferation and associated with various cancers. It is commonly assumed that after activation by binding of epidermal growth factor to the extracellular domain it dimerizes, followed by autophosphorylation of tyrosine residues at the intracellular domain. However, its oligomerization state before activation is controversial. In the absence of ligands, EGFR has been found in various, inconsistent amounts of monomeric, inactive dimeric, and oligomeric forms. In addition, evidence suggests that the active conformation is not a simple dimer but contains higher oligomers. As experiments in the past have been conducted at different conditions, we investigate here the influence of cell lines (HEK293, COS-7, and CHO-K1), temperature (room temperature and 37 C), and membrane localization on the quantitation of preformed dimers using SW-FCCS, DC-FCCS, quasi PIE-FCCS, and imaging FCCS. While measurement modality, temperature, and localization on upper or lower membranes have only a limited influence on the dimerization amount observed, the cell line and location to periphery versus center of the cell can change dimerization results significantly. The observed dimerization amount is strongly dependent on the expression level of endogenous EGFR in a cell line and shows a strong cell-to-cell variability even within the same cell line. In addition, using imaging FCCS, we find that dimers have a tendency to be found at the periphery of cells compared to central positions.

show abstract

“…and lead to decisions triggering distinct signalling pathway/s is incompletely understood (Bessman et al, 2014;Valley et al, 2014). A key challenge…”

Section: Introductionmentioning

confidence: 99%

Membrane protein stoichiometry studied in intact mammalian cells

Jonge¹

2016

infocus

View full text Add to dashboard Cite

The Spatiotemporal Organization of ErbB Receptors: Insights from Microscopy

Cited by 28 publications

References 63 publications

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

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

Membrane protein stoichiometry studied in intact mammalian cells

Contact Info

Product

Resources

About