Studying Macromolecular Composition in Cell–Cell Interfaces Using 3D Membrane Reconstitution Systems

ABSTRACT During direct communication between two cells, the plasma membranes of each cell serve as a platform for ligand‐receptor interaction initiating downstream signaling cascades. In immune cell signaling, this cell–cell interface – the immune synapse – is highly spatiotemporally organized. Multiple stimulatory and co‐stimulatory signals need to be integrated over time to ensure proper immune cell function. This process is still not fully understood given the vast complexity of interactions between proteins, lipids, glycocalyx and associated cortical actin cytoskeleton. Here, we presented a fully artificial model system to study the interface between two vesicles and a semi‐artificial one between a live cell and a vesicle to reconstitute 3D contacts. We investigated the distribution and reorganization of immune cell proteins at artificial and semi‐artificial contacts. We show the enrichment and depletion of different proteins in the synapse and how different peptides with varying affinity presented by the same MHC class I affect T cell activation. We further explored the distribution of glycocalyx elements and showed differential partitioning of different sugar moieties in the interface. While we focused on the T cell interface here, our model systems are powerful tools to study the distribution and reorganization of lipids, proteins and glycocalyx components at any cell–cell contact.