The mammalian glycocalyx is a complex meshwork on the extracellular side of the plasma membrane, consisting of various sugars and the proteins and lipids to which they are attached. Despite its undisputed relevance in numerous cellular processes, its detailed architecture and its changes upon shifts in cell state have remained enigmatic. There are three main reasons for this elusive nature: First, the glycocalyx exhibits high complexity that is not directly encoded in the genome which makes conventional methods of biochemistry hard to apply. Second, it contains a huge number of structurally similar building units which impedes specific labeling. Finally, its axial thickness in the range of hundreds of nanometers, i.e. too small to be resolved with conventional light microscopy.<br/>In our work, we combined specific bioorthogonal labeling, targeting fluorophores to two central building units of the glycocalyx, sialic acids and GalNAc, with quantitative super-resolution microscopy. With this, we were able to extract for the first time specific insights into the organization of the mammalian glycocalyx. As in previous work, a huge array of tiny glycocalyx-covered tubular membrane structures are observed, and our additional resolution increases our ability to image the tubules, and many now appear hollow. We find that the glycocalyx exhibits organization at the nanoscale, with sialic acids extending further out into the extracellular space than GalNAc, confirming a long-held assumption. We also show that both sugars are not uniformly distributed, exhibiting their highest density located some distance away from the membrane.<br/>Finally, we investigated the change in glycocalyx morphology when a cancer phenotype is present. We show that the glycocalyx height increases upon epithelial-to-mesenchymal transition, a key step in cancer development. Furthermore, we reveal that a single oncogenic mutation in the Ras gene is sufficient to cause an increase in glycocalyx height. In the light of recent findings that link increased glycocalyx bulk to increased cell adhesion and survival, our findings highly suggest a fundamental link between the glycocalyx state and the earliest stages of cancer progression.

Quantitative super-resolution microscopy reveals the architecture of the mammalian glycocalyx and its changes during cancer progression

256th National Meeting (2018)

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