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Quantitative Super-Resolution Microscopy of the Mammalian Glycocalyx

preprint
submitted on 26.11.2018 and posted on 27.11.2018 by Leonhard Möckl, Kayvon Pedram, Anish R. Roy, Venkatesh Krishnan, Anna-Karin Gustavsson, Oliver Dorigo, Carolyn Bertozzi, W.E. Moerner

The mammalian glycocalyx is a heavily glycosylated extramembrane compartment found on nearly every cell. Despite its relevance in both health and disease, studies of the glycocalyx remain hampered by a paucity of methods to spatially classify its components. We combine metabolic labeling, bioorthogonal chemistry, and super-resolution localization microscopy to image two constituents of cell-surface glycans, N-acetylgalactosamine (GalNAc) and sialic acid, with 10-20 nm precision in 2D and 3D. This approach enables two measurements: glycocalyx height and the distribution of individual sugars distal from the membrane. These measurements show that the glycocalyx exhibits nanoscale architecture, on both cell lines and primary human tumor cells. Additionally, we observe enhanced glycocalyx height in response to epithelial-to- mesenchymal transition and to oncogenic KRAS activation. In the latter case, we trace increased height to an effector gene, GALNT7. These data highlight the power of advanced imaging methods to provide molecular and functional insights into glycocalyx biology.

Funding

Single-Molecule Imaging for Cell Biology and Super-Resolution Microscopy

National Institute of General Medical Sciences

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Deep Super-localization Microscopy and Effectively Unbleachable Labeling for 4D Nucleomics

National Institute of Biomedical Imaging and Bioengineering

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Chemical Cell Surface Engineering

National Institute of General Medical Sciences

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Metabolic Engineering with Bioorthogonal Chemical Reporters

National Institute of General Medical Sciences

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History

Email Address of Submitting Author

wmoerner@stanford.edu

Institution

Stanford University

Country

USA

ORCID For Submitting Author

0000-0002-2830-209X

Declaration of Conflict of Interest

The authors do not declare any competing interest.

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