Graphene Liquid Enclosure for Single-Molecule Analysis of Membrane Proteins in Whole Cells Using Electron Microscopy.
Dahmke, Indra N
Peckys, Diana B
American Chemical Society (ACS)
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Dahmke, I. N., Verch, A., Hermannsdörfer, J., Peckys, D. B., Weatherup, R., Hofmann, S., & de Jonge, N. (2017). Graphene Liquid Enclosure for Single-Molecule Analysis of Membrane Proteins in Whole Cells Using Electron Microscopy.. ACS Nano, 11 (11), 11108-11117. https://doi.org/10.1021/acsnano.7b05258
Membrane proteins govern many important functions in cells via dynamic oligomerization into active complexes. However, analytical methods to study their distribution and functional state in relation to the cellular structure are currently limited. Here, we introduce a technique for studying single-membrane proteins within their native context of the intact plasma membrane. SKBR3 breast cancer cells were grown on silicon microchips with thin silicon nitride windows. The cells were fixed, and the epidermal growth factor receptor ErbB2 was specifically labeled with quantum dot (QD) nanoparticles. For correlative fluorescence- and liquid-phase electron microscopy, we enclosed the liquid samples by chemical vapor deposited (CVD) graphene films. Depending on the local cell thickness, QD labels were imaged with a spatial resolution of 2 nm at a low electron dose. The distribution and stoichiometric assembly of ErbB2 receptors were determined at several different cellular locations, including tunneling nanotubes, where we found higher levels of homodimerization at the connecting sites. This experimental approach is applicable to a wide range of cell lines and membrane proteins and particularly suitable for studies involving both inter- and intracellular heterogeneity in protein distribution and expression.
Cell Line, Tumor, Humans, Graphite, Silicon Compounds, Receptor, erbB-2, Membrane Proteins, Neoplasm Proteins, Microscopy, Electron, Quantum Dots, Lab-On-A-Chip Devices, Single Molecule Imaging
Engineering and Physical Sciences Research Council (EP/K016636/1)
European Commission Horizon 2020 (H2020) Marie Sk?odowska-Curie actions (656870)
External DOI: https://doi.org/10.1021/acsnano.7b05258
This record's URL: https://www.repository.cam.ac.uk/handle/1810/275562