Epidermal growth factor receptor subunit locations determined in hydrated cells with environmental scanning electron microscopy

Abstract

Imaging single epidermal growth factor receptors (EGFR) in intact cells is presently limited by the available microscopy methods. Environmental scanning electron microscopy (ESEM) of whole cells in hydrated state in combination with specific labeling with gold nanoparticles was used to localize activated EGFRs in the plasma membranes of COS7 and A549 cells. The use of a scanning transmission electron microscopy (STEM) detector yielded a spatial resolution of 3 nm, sufficient to identify the locations of individual EGFR dimer subunits. The sizes and distribution of dimers and higher order clusters of EGFRs were determined. The distance between labels bound to dimers amounted to 19 nm, consistent with a molecular model. A fraction of the EGFRs was found in higher order clusters with sizes ranging from 32–56 nm. ESEM can be used for quantitative whole cell screening studies of membrane receptors, and for the study of nanoparticle-cell interactions in general.

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

“…EGFRs are thus not randomly oriented but a significant fraction of them resides at this preferred distance. From an approximate molecular model 6 (Figure 3 inset) we estimate that the center-to-center distance between the QD and the EGF binding pocket amounts to ~14 nm, and center-to-center distance between the two QDs attached to a EGFR dimer to be ~27 nm (this value is likely to vary by a few nanometers due to the flexibility of the linker). The preferred label distance is thus consistent with the expected label distance for the EGFR dimer within the precision of the method.…”

“…Note that this analysis is not part of this protocol and the procedure was described elsewhere 6,7 . g(x) is a measure of the chance of a particle to be found within a certain radial distance x from a reference particle.…”

“…Note that the electron dense QD core appears somewhat smaller and rounder than the cores of QDs emitting at 655 nm, consistent with their smaller core size 22 of ~5 nm. a length scale of a few nanometers so that its stoichiometry can be studied within the context of individual, intact cells [4][5][6][7] . This is not possible with (super resolution) light microscopy 23 because its resolution is insufficient to distinguish molecules engaged in a protein complex.…”

“…Moreover, the thickness of the water layer may vary between cells. It is advisable to monitor the presence of the water layer using the gaseous electron detector above the specimen, as described elsewhere 6 . Cellular regions should only be imaged once or twice to avoid damage.…”

“…A new approach was introduced recently, to image whole cells with labeled proteins in liquid state using STEM [1][2][3] , or correlative fluorescence microscopy and STEM [4][5][6][7] . This methodology is capable of studying the spatial distribution of membrane proteins and protein complexes including their stoichiometry at the single molecule level in the intact plasma membranes of whole cells.…”