Enhanced receptor–clathrin interactions induced by N -glycan–mediated membrane micropatterning

Abstract

Glycan-protein interactions are emerging as important modulators of membrane protein organization and dynamics, regulating multiple cellular functions. In particular, it has been postulated that glycan-mediated interactions regulate surface residence time of glycoproteins and endocytosis. How this precisely occurs is poorly understood. Here we applied single-molecule-based approaches to directly visualize the impact of glycan-based interactions on the spatiotemporal organization and interaction with clathrin of the glycosylated pathogen recognition receptor dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). We find that cell surface glycan-mediated interactions do not influence the nanoscale lateral organization of DC-SIGN but restrict the mobility of the receptor to distinct micrometer-size membrane regions. Remarkably, these regions are enriched in clathrin, thereby increasing the probability of DC-SIGN-clathrin interactions beyond random encountering. N-glycan removal or neutralization leads to larger membrane exploration and reduced interaction with clathrin, compromising clathrin-dependent internalization of virus-like particles by DC-SIGN. Therefore, our data reveal that cell surface glycan-mediated interactions add another organization layer to the cell membrane at the microscale and establish a novel mechanism of extracellular membrane organization based on the compartments of the membrane that a receptor is able to explore. Our work underscores the important and complex role of surface glycans regulating cell membrane organization and interaction with downstream partners.pathogen receptor DC-SIGN | clathrin-dependent endocytosis | STED nanoscopy | cell membrane compartmentalization | protein glycosylation

The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells

“…3D). In the case of truly interacting nanoclusters, their separation distances should remain small (close to the nanocluster size) for periods longer than random encounter events (22,37). As expected, CytoD treatment of α-GalCer-loaded WT-hCD1d and α-GalCer-loaded TD-hCD1d showed significantly shorter separation distances over time compared with the untreated pulsed WT-hCD1d control (Fig.…”

“…Time-dependent membrane exploration maps of iNKT-TCR-QD655-labeled hCD1d (WT and TD) with respect to actin were obtained as described in the work by TorrenoPina et al (37). In brief, we first obtained dual-color TIRFM images of iNKT-TCR-QD655 and Lifeact-GFP at a frame rate of 60 Hz for a total observation time of 1,200 frames.…”

Nanoclustering as a dominant feature of plasma membrane organization

“…As shown in the enlarged regions of the direct stochastic optical reconstruction microscopy (dSTORM) image (top right), nanoclusters appear to be in close proximity to each other. The cartography map (bottom left) represents the reconstructed molecular positions (blue dots) obtained from single-particle tracking (SPT) movies of several DC-SIGN nanoclusters as they explore the cell membrane (Torreno-Pina et al, 2014). This map demonstrates that DC-SIGN nanoclusters explore restricted areas of ,1 mm, which is consistent with static dSTORM images.…”

“…The DC-SIGN CRD could bind to glycosaminoglycans or glycosyl moieties of ECM proteins, or to transmembrane proteoglycans that link to the ECM or to TRAPs, which might be directly or indirectly linked to the membraneapposed cytoskeleton. More recently, we found that DC-SIGN nanoclusters further organize into larger compartments of ,1 mm in size that are maintained by interactions between the receptor glycosylation motif and surface galectins (Torreno-Pina et al, 2014) (Fig. 3).…”

Variation in Carbohydrates between Cancer and Normal Cell Membranes Revealed by Super‐Resolution Fluorescence Imaging

“…The resulting “glycoproteins” or “glycolipids” contain abundant carbohydrates on the extracellular side of the cell membranes and play integral roles in cell–cell and cell–matrix interactions by regulating cell adhesion, cell trafficking, and signal transduction 1, 2, 3. Abnormal glycosylation is associated with severe diseases, including tumorigenesis, malignant differentiation, and cancer metastasis and development 4, 5, 6…”