Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR–CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR–CD3 complexes. We found that only TCR–CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR–pMHC affinity determined the density of TCR–CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR–CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination

Membrane Compartmentalisation and Endocytosis by Galectin-3 in Mammalian Cells

Galectin-3 is a carbohydrate binding protein that is widely expressed and can be found in tissues and cells of the immune system. At the cell surface, galectin-3 interacts with various cell surface glycoproteins through its carbohydrate recognition domain (CRD) and exhibits multivalent binding properties by assembling into pentamers through its N-terminal domain. Galectin-3 molecules have been implicated in the formation of molecular lattices at plasma membranes and in endocytosis. The aim of this study was to examine the molecular arrangement of galectin-3 on the surface of fibroblasts and Hela cells as well resting and activated T cells, and investigate whether and how galectin-3 is internalized in T cells to generate signalling endosomes. To map the molecular organisation of galectin-3 on the cell surface, direct stochastic optical reconstruction microscopy (dSTORM) and quantitative analysis of galectin-3 and galectin-3 ligands was established. It was found that galectin-3 clustering depended on glycosphingolipids in the plasma membrane and that galectin-3-dependent clustering of known galectin-3 binding partners was sensitive to branched N-acetylglucosamine saccharides that were absent in β-1,6-N-acetylglycosaminyltransferase V (Mgat5)-deficient mouse embryonic fibroblasts (Mgat5-/- MEF). These data supports the concept that galectin-3 compartmentalises the plasma membrane. Next, it was demonstrated with confocal microscopy and flow cytometry that the binding of galectin-3 to the plasma membrane of T cells occurred in a carbohydrate-dependent fashion and led to internalisation. Further it was shown that uptake of galectin-3 was facilitated by different endocytic mechanisms suggesting that galectin-3 participates in various endocytic routes in T cells. Finally, in activated T cells, data is presented to show that galectin-3- positive vesicles were positioned at or near the immunological synapse and co-localised with proteins involved in signalling processes, suggesting that galectin-3 functions in the regulation of T cell signalling. In conclusion, the data presented in this PhD thesis suggest that galectin-3 binding to the cell surface creates distinct membrane domains in a glycosphingolipid- and branched N-glycosylation-dependent manner. In T cells, galectin-3 domains lead to the formation of galectin-3 vesicles that may function as signaling endosomes

The integration of signaling and the spatial organization of the T cell synapse

Engagement of the T cell antigen receptor (TCR) triggers signaling pathways that lead to T cell selection, differentiation and clonal expansion. Superimposed onto the biochemical network is a spatial organization that describes individual receptor molecules, dimers, oligomers and higher order structures. Here we discuss recent findings and new concepts that may regulate TCR organization in naïve and memory T cells. A key question that has emerged is how antigen-TCR interactions encode spatial information to direct T cell activation and differentiation. Single molecule super-resolution microscopy may become an important tool in decoding receptor organization at the molecular level.publishe

Phagocytosis of IgG-coated polystyrene beads by macrophages induces and requires high membrane order

The biochemical composition and biophysical properties of cell membranes are hypothesized to affect cellular processes such as phagocytosis. Here, we examined the plasma membranes of murine macrophage cell lines during the early stages of uptake of immunoglobulin G (IgG)‐coated polystyrene particles. We found that the plasma membrane undergoes rapid actin‐independent condensation to form highly ordered phagosomal membranes, the biophysical hallmark of lipid rafts. Surprisingly, these membranes are depleted of cholesterol and enriched in sphingomyelin and ceramide. Inhibition of sphingomyelinase activity impairs membrane condensation, F‐actin accumulation at phagocytic cups and particle uptake. Switching phagosomal membranes to a cholesterol‐rich environment had no effect on membrane condensation and the rate of phagocytosis. In contrast, preventing membrane condensation with the oxysterol 7‐ketocholesterol, even in the presence of ceramide, blocked F‐actin dissociation from nascent phagosomes and particle uptake. In conclusion, our results suggest that ordered membranes function to co‐ordinate F‐actin remodelling and that the biophysical properties of phagosomal membranes are essential for phagocytosis

Galectin-3 drives glycosphingolipid-dependent biogenesis of clathrin-independent carriers

Several cell surface molecules including signalling receptors are internalized by clathrin-independent endocytosis. How this process is initiated, how cargo proteins are sorted and membranes are bent remains unknown. Here, we found that a carbohydrate-binding protein, galectin-3 (Gal3), triggered the glycosphingolipid (GSL)-dependent biogenesis of a morphologically distinct class of endocytic structures, termed clathrin-independent carriers (CLICs). Super-resolution and reconstitution studies showed that Gal3 required GSLs for clustering and membrane bending. Gal3 interacted with a defined set of cargo proteins. Cellular uptake of the CLIC cargo CD44 was dependent on Gal3, GSLs and branched N-glycosylation. Endocytosis of Î 2 1-integrin was also reliant on Gal3. Analysis of different galectins revealed a distinct profile of cargoes and uptake structures, suggesting the existence of different CLIC populations. We conclude that Gal3 functionally integrates carbohydrate specificity on cargo proteins with the capacity of GSLs to drive clathrin-independent plasma membrane bending as a first step of CLIC biogenesis

Correction for Pageon et al., functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

The authors note that Philip R. Nicovich should be added to the author list between Yuanqing Ma and John S. Bridgeman. Philip R. Nicovich should be credited with contributing new reagents/analytic tools