Cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape generation is regulated is not fully resolved. Here, we report a synergy between shape-generating processes in the cell interior and the external organization and composition of the cell-surface glycocalyx. Mucin biopolymers and long-chain polysaccharides within the glycocalyx can generate entropic forces that favor or disfavor the projection of spherical and finger-like extensions from the cell surface. A polymer brush model of the glycocalyx successfully predicts the effects of polymer size and cell-surface density on membrane morphologies. Specific glycocalyx compositions can also induce plasma membrane instabilities to generate more exotic undulating and pearled membrane structures and drive secretion of extracellular vesicles. Together, our results suggest a fundamental role for the glycocalyx in regulating curved membrane features that serve in communication between cells and with the extracellular matrix. (A) The native and synthetic mucin biopolymers that were genetically encoded and used throughout this work. (B) Quantification of membrane tube density in epithelial cells. Mucin polymers induce dramatic tubularization compared to wild-type (Control) cells and compared to a similarly sized biopolymer composed of EGF-like repeats from Notch1 and the Muc1 transmembrane anchor with GFP reporter (EGF-repeats GFP-DCT) cells. Number of cells analyzed is shown on the x axis for each condition. Box notches here and elsewhere indicate 95% confidence intervals. The number of tandem repeats (TRs) are indicated in Muc1 constructs. (C) Scanning electron microscopy (SEM) images of cells expressing the indicated biopolymer. (D) Labelled glycans and membrane morphologies resolved with single-molecule localization microscopy in Muc1-42TR DCT-expressing cells before and after mucin backbone digestion with the StcE mucinase. Images are shown as 2D color-coded histograms of localizations with 32 nm bin width. (E) Representative confocal images of GUVs with and without anchorage of recombinant Podocalyxin. (F) (Left) Cartoons of Muc1 GFP-DCT polymers of varying length. (Right) Flow cytometry data showing similar cell-surface expression levels of the mucins using a GFP-binding nanobody, n = 3, >40,000 cells per population. (G) Representative SEM images of cells expressing mucins with a varying number of TRs. (H) Quantification of membrane tube density for cells expressing the indicated mucins, significance compared to 42TR. ***p < 0.001; ns, not significant (post-hoc Student's two-tailed t test). See also Figure S1.

All cells in the human body are covered by a dense layer of sugars and the proteins and lipids to which they are attached, collectively termed the "glycocalyx." For decades, the organization of the glycocalyx and its interplay with the cellular state have remained enigmatic. This changed in recent years. Latest research has shown that the glycocalyx is an organelle of vital significance, actively involved in and functionally relevant for various cellular processes, that can be directly targeted in therapeutic contexts. This review gives a brief introduction into glycocalyx biology and describes the specific challenges glycocalyx research faces. Then, the traditional view of the role of the glycocalyx is discussed before several recent breakthroughs in glycocalyx research are surveyed. These results exemplify a currently unfolding bigger picture about the role of the glycocalyx as a fundamental cellular agent.

Therapeutic nanoparticles can be directed to cancer cells by incorporating selective targeting ligands. Here, we investigate the epidermal growth factor receptor (EGFR)-mediated endocytosis of gene carriers (polyplexes) either targeted with natural EGF or GE11, a short synthetic EGFR-binding peptide. Highly sensitive live-cell fluorescence microcopy with single particle resolution unraveled the existence of two different uptake mechanisms; EGF triggers accelerated nanoparticle endocytosis due to its dual active role in receptor binding and signaling activation. For GE11, an alternative EGFR signaling independent, actin-driven pathway is presented.

Glyco-immune checkpoint receptors, molecules that inhibit immune cell activity following binding to glycosylated cell-surface antigens, are emerging as attractive targets for cancer immunotherapy. Defining biologically relevant ligands that bind and activate such receptors, however, has historically been a significant challenge. Here, we present a CRISPRi genomic screening strategy that allowed unbiased identification of the key genes required for cell-surface presentation of glycan ligands on leukemia cells that bind the glyco-immune checkpoint receptors Siglec-7 and Siglec-9. This approach revealed a selective interaction between Siglec-7 and the mucin-type glycoprotein CD43. Further work identified a specific N-terminal glycopeptide region of CD43 containing clusters of disialylated O-glycan tetrasaccharides that form specific Siglec-7 binding motifs. Knockout or blockade of CD43 in leukemia cells relieves Siglec-7-mediated inhibition of immune killing activity. This work identifies a potential target for immune checkpoint blockade therapy and represents a generalizable approach to dissection of glycan–receptor interactions in living cells.