One strategy that circumvents these nagging complications is normally to determine systems that faithfully imitate immune system cell interactions, but allow complexity to become dialled-in as needed. allow intricacy to become dialled-in as required. Here, we present an program which makes usage of artificial vesicles that imitate essential areas of immune system cell areas. Using this system, we began to explore the spatial distribution of signalling molecules (receptors, kinases and phosphatases) and how this changes during the initiation of signalling. The GUV/cell system offered here is expected to be widely relevant. reconstitution, Model membranes, Giant unilamellar vesicles INTRODUCTION Dynamic cellCcell contacts govern the activation and effector functions of immune cells. Communication occurs through membrane protein interactions on opposing surfaces, whereby surface-presented antigens and ligands are recognised by key immune cell receptors. This induces intracellular signalling cascades that lead, eventually, to the formation of an immunological synapse, which comprises a spatiotemporally regulated supramolecular cluster of proteins at the interface between the cells (Dustin and Baldari, 2017; Dustin and Choudhuri, 2016). Quantitative investigation of the receptors and their molecular behaviour at the cellular contact is essential in order to understand how immune cells integrate activating and inhibitory signals, allowing decisions about whether/when to respond (Dustin and Groves, 2012; Kamphorst et al., 2017). Studying these factors in physiological systems is usually, however, challenging because of the topographical complexity and transient nature of immune cellCcell contacts. In addition, surface protein dynamics and organisation can be influenced by a variety CZC-25146 of factors such as proteinCprotein or proteinClipid interactions, the activity of the cortical actin cytoskeleton and the barrier properties of the glycocalyx, which makes it challenging to identify the exact role of each component (Chernomordik and Kozlov, 2003; Cho and Stahelin, 2005; Lemmon, 2008; Ritter et al., 2013). To this end, minimal systems with controllable complexity are essential tools for unravelling the molecular biology of cellCcell contact. The most basic systems for reconstituting immune cell interactions are planar substrates coated with immobile antibodies or purified biological ligands (Bunnell et al., 2001). Glass-supported lipid bilayers (SLBs) reconstituted with mobile proteins acting as surrogate antigen-presenting cell (APC) surfaces capture additional features of physiological T cellCAPC interfaces (Dustin et al., 2007). Advantages of SLBs include being able to control protein variety and density, and a two-dimensional format that allows advanced optical imaging of the contact. Accordingly, SLBs have been used extensively to study immune cell activation (Bertolet and Liu, 2016; CZC-25146 Dustin et al., 2007; Lever et al., 2016; Lopes et al., 2017; Zheng et al., 2015). However, use of solid supports and SLBs also has several disadvantages. First, the small hydration layer (1C2?nm) between the bilayer and the underlying support is insufficient to completely de-couple the support’s influence on reconstituted proteins: the glass support restricts diffusion of the molecules in the membrane plane, mostly in an unpredictable manner, thereby affecting the membrane dynamics significantly (Przybylo et al., 2006; Sezgin and Schwille, 2012) and influencing cell behaviour (Snchez et al., 2015). Second, the solid glass support imposes rigidity around the lipid membrane. Although it varies, the stiffness of immune cell membranes is known to be several orders of magnitude lower than that of SLBs, that is, 0.1C1?kPa versus 1?MPa for SLBs (Bufi et al., 2015; Rosenbluth et al., 2006; Saitakis et al., 2017), and it has been shown that substrate stiffness influences B- and T-cell migration, synapse formation and signalling (Judokusumo et al., 2012; Martinelli et al., 2014; Natkanski et al., 2013; Schaefer and Hordijk, 2015; Shaheen et al., 2017; Tabdanov et al., 2015; Zeng et al., 2015). Third, the necessarily large area and planar nature of SLBs (i.e. centimetres) mean that they are poor mimics of the topological constraints experienced by cells system. (A) Depiction of supported lipid bilayers and free-standing vesicles. (B) Plan showing the cellCvesicle conversation. (C) Molecules of interest for this study, drawn to level based on structure determinations (Chang et al., 2016). (D) Example bright field (top) and fluorescence (bottom) images of CD2+ JurkatCCD58+ GUV contact (image size 50?m50?m). (E) Diffusion analysis of fluorescently labelled lipids and proteins in GUVs and SLBs. (F) Lipid packing of IB2 GUVs of varying composition revealed by a GP map (image size 40?m40?m). (G) Quantification of the GP. (H) Diffusion analysis of fluorescently labelled pMHC on GUVs composed of different lipids. Student’s GUV-based system to investigate the principles of protein spatial organisation at cellCcell contacts in three sizes. We used a 1G4 TCR-expressing Jurkat T cell collection to study the formation of contacts between cells and vesicles presenting the His-tagged proteins shown in Fig.?1C, using the NTA-His coupling method depicted in Fig.?1B. These proteins were: (1) the pMHC recognised by the CZC-25146 1G4 TCR (i.e. a peptide derived from the tumour antigen NY-ESO; Chen et al., 2005); (2) CD58, which is the ligand of the small adhesion protein CD2;.