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

Peckys, Diana B.; Jonge, Niels de

doi:10.3791/53186

Cited by 18 publications

(38 citation statements)

References 32 publications

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“…The interaction of ErbB2 with actin was reported previously in several studies; for instance, activated ErbB2 was found to be spatially correlated with actin-rich membrane protrusions, like peripheral ruffles or filopodia-like protrusions, in ErbB2 overexpressing cells (Brandt et al, 1999;Chung et al, 2016;Jeong et al, 2016). HER2 homodimers were found to preferentially reside in membrane ruffles (Peckys and de Jonge, 2015a).…”

Section: Introductionsupporting

confidence: 58%

“…images were analyzed as collected at two different membrane regions. For the first group, STEM images were collected in cell areas identified by the presence of the fluorescence signal for actin-GFP and peripheral membrane ruffles identified in DIC mode (Supplementary Figure S1), as described elsewhere (Peckys and de Jonge, 2015a). Here, we found a higher density of QD-labels compared to cell areas with a low content of actin ( Table 1).…”

Section: Distribution Of Membranous Erbbs In Relation To Actin-gfpmentioning

confidence: 72%

“…In order to distinguish ErbB2 proteins forming homodimers from ones that were randomly grouped at closer vicinities, the label position data was statistically analyzed using the pair correlation function, g(r) (Peckys and de Jonge, 2015a;Stoyan et al, 1993). This function,…”

Section: Statistical Methods To Determine Functional State Of Erbb2mentioning

confidence: 99%

“…Finally, cells were washed three times in PBS-BSA (1%) and subjected to fluorescence imaging. Further details of the protocol, including control experiments to test for unspecific binding of QDs, are reported elsewhere (Peckys and de Jonge, 2015b).…”

Section: Labeling Of Erbb2mentioning

confidence: 99%

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Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

Dahmke

Trampert

Weinberg

et al. 2020

Preprint

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Epidermal growth factor receptor 2 (ErbB2) is found overexpressed in several cancers, such as gastric, and breast cancer, and is, therefore, an important therapeutic target. ErbB2 plays a central role in cancer cell invasiveness, and is associated with cytoskeletal reorganization. In order to study the spatial correlation of single ErbB2 proteins and actin filaments, we applied correlative fluorescence microscopy (FM), and scanning transmission electron microscopy (STEM) to image specifically labeled SKBR3 breast cancer cells. The breast cancer cells were grown on microchips, transformed to express an actin-green fluorescent protein (GFP) fusion protein, and labeled with quantum dot (QD) nanoparticles attached to specific anti-ErbB2 Affibodies. FM was performed to identify cellular regions with spatially correlated actin and ErbB2 expression. For STEM of the intact plasma membrane of whole cells, the cells were fixed and covered with graphene. Spatial distribution patterns of ErbB2 in the actin rich ruffled membrane regions were examined, and compared to adjacent actin-low regions of the same cell, revealing an association of putative signaling active ErbB2 homodimers with actin-rich regions. ErbB2 homodimers were found absent from actin-low membrane regions, as well as after treatment of cells with Cytochalasin D, which breaks up larger actin filaments.In both latter data sets, a significant inter-label distance of 36 nm was identified, possibly indicating an indirect attachment to helical actin filaments via the formation of heterodimers of ErbB2 with epidermal growth factor receptor (EGFR). The possible attachment to actin filaments was further explored by identifying linear QD-chains in actin-rich regions, which also showed an inter-label distance of 36 nm.

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Section: Introductionsupporting

confidence: 58%

Section: Distribution Of Membranous Erbbs In Relation To Actin-gfpmentioning

confidence: 72%

Section: Statistical Methods To Determine Functional State Of Erbb2mentioning

confidence: 99%

Section: Labeling Of Erbb2mentioning

confidence: 99%

See 2 more Smart Citations

Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

Dahmke

Trampert

Weinberg

et al. 2020

Preprint

Self Cite

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“…A heating element (23) or cooling capability (24) can be integrated, and the silicon nitride surfaces can be patterned or chemically modified to enable reactions with species in solution (25)(26)(27)(28). Careful assembly procedures and cleaning are necessary (29). The sample holder is a key part of the liquid cell experiment, and its function extends beyond simply holding the cell securely.…”

Section: The Rapidly Developing Liquid Cell Microscopy Techniquementioning

confidence: 99%

Opportunities and challenges in liquid cell electron microscopy

Ross

2015

Science

511

438

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Advances in seeing small things Electron microscopes, particularly those with aberration correction, can view materials at the subnanometer scale. Additional improvements make it possible to obtain images at lower electron doses, thus minimizing the damage to the sample. However, for a number of materials, particularly those of biological origin, samples need to be imaged in solution. Ross reviews recent advances that have made it possible to do liquid cell electron microscopy, which opens up the possibility of studying problems such as the changes inside a battery during operation, the growth of crystals from solution, or biological molecules in their native state. Science , this issue p. 10.1126/science.aaa9886

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Linear Chains of HER2 Receptors Found in the Plasma Membrane Using Liquid-Phase Electron Microscopy

Parker

Trampert

Tinnemann

et al. 2018

Biophysical Journal

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The spatial distribution of the human epidermal growth factor 2 (HER2) receptor in the plasma membrane of SKBR3 and HCC1954 breast cancer cells was studied. The receptor was labeled with quantum dot nanoparticles, and fixed whole cells were imaged in their native liquid state with environmental scanning electron microscopy using scanning transmission electron microscopy detection. The locations of individual HER2 positions were determined in a total plasma membrane area of 991 mm 2 for several SKBR3 cells and 1062 mm 2 for HCC1954 cells. Some of the HER2 receptors were arranged in a linear chain with interlabel distances of 40 5 7 and 32 5 10 nm in SKBR3 and HCC1954 cells, respectively. The finding was tested against randomly occurring linear chains of six or more positions, from which it was concluded that the experimental finding is significant and did not arise from random label distributions. Because the measured interlabel distance in the HER2 chains is similar to the 36-nm helix-repetition distance of actin filaments, it is proposed that a linking mechanism between HER2 and actin filaments leads to linearly aligned oligomers.

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

Cited by 18 publications

References 32 publications

Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

Opportunities and challenges in liquid cell electron microscopy

Linear Chains of HER2 Receptors Found in the Plasma Membrane Using Liquid-Phase Electron Microscopy

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