Studying Macromolecular Composition in Cell–Cell Interfaces Using 3D Membrane Reconstitution Systems doi-10-17044-scilifelab-30540932-0 https://doi.org/10.17044/SCILIFELAB.30540932 Swedish National Data Service Svensk nationell datatjänst Landing page Studying Macromolecular Composition in Cell–Cell Interfaces Using 3D Membrane Reconstitution Systems doi-10-17044-scilifelab-30540932-0 https://doi.org/10.17044/SCILIFELAB.30540932 Sezgin, Erdinc Ragaller, Franziska Swedish National Data Service Svensk nationell datatjänst Landing page It contains data sets from the publication, with the details for citation provided in the README file. Please cite this item as: F. Ragaller, A. M. Schneider, E. Sjule, R. Sun, X. Han, L. Andronico, E. Jenkins, M. Dustin, A. Achour, E. Sezgin DOI: 10.17044/scilifelab.30540932 It contains raw microscopy images, figures and excel sheets of the data. Abstract During direct communication between two cells, the plasma membranes of each cell serve as a platform for ligand-receptor interaction initiating downstream signalling cascades. In immune cell signalling, 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 major histocompatibility complex (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 distribution and reorganization of lipids, proteins and glycocalyx components at any cell-cell contact. Data usage Researchers are welcome to use the data contained in the dataset for any projects. Please cite this item upon use or when published. We encourage reuse using the same CC BY 4.0 License. Data Content Excel files for graphs lsm or czi files for raw microscopy images tif files for processed microscopy images svg files for figures Software to open files .xlsx - Microsoft excel .czi - Fiji (https://imagej.net/Fiji.html#Downloads) .tif - Fiji (https://imagej.net/Fiji.html#Downloads) .svg - Inkscape Access to data through an external actor. Åtkomst till data via extern aktör.