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  • Quantitative Super-Resolution Microscopy of the Mammalian Glycocalyx.

Quantitative Super-Resolution Microscopy of the Mammalian Glycocalyx.

Developmental cell (2019-05-21)
Leonhard Möckl, Kayvon Pedram, Anish R Roy, Venkatesh Krishnan, Anna-Karin Gustavsson, Oliver Dorigo, Carolyn R Bertozzi, W E Moerner
초록

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 organization 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.

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Sigma-Aldrich
Aminoguanidine hydrochloride, ≥98%
Sigma-Aldrich
Cysteamine, ≥98.0% (RT)
Sigma-Aldrich
Cysteamine, ~95%
Sigma-Aldrich
Glutaraldehyde solution, Grade I, 25% in H2O, specially purified for use as an electron microscopy fixative
Sigma-Aldrich
Catalase from bovine liver, aqueous suspension, 40,000-60,000 units/mg protein (E1%/405)
Sigma-Aldrich
Sodium periodate, ACS reagent, ≥99.8%
Supelco
Copper Sulfate, Pharmaceutical Secondary Standard; Certified Reference Material
Avanti
16:0 azidocaproyl PE, Avanti Research - A Croda Brand 870126P, powder