The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen presenting cells (APC)1. T cell signaling is initiated at these contacts when surface-expressed antigen receptors (TCR) recognize peptide fragments (antigens) of pathogens bound to Major Histocompatibility Complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction between T cells and APCs, known as the immunological synapse (IS)3, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft2. T cell recognition of pMHC and the adhesion ligand Intercellular Adhesion Molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse (IS)4–5, which is characterized by central accumulation of TCR5, adjacent to a secretory domain3, both surrounded by an adhesive ring4–5. Although accumulation of TCR at the IS center correlates with T cell function4, this domain is itself largely devoid of TCR signaling activity5–6, and is characterized by an unexplained immobilization of TCR-pMHC complexes relative to the highly dynamic IS periphery4–5. Here we show that centrally accumulated TCR is located on the surface of extracellular microvesicles that bud at the IS center. Tumor susceptibility gene 101 (TSG101)6 sorts TCR for inclusion in microvesicles, while vacuolar protein sorting 4 (VPS4) 7–8 mediates scission of microvesicles from the T cell plasma membrane. The HIV polyprotein GAG co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCR from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These microvesicles deliver transcellular signals across antigen-dependent synapses by engaging cognate pMHC on APC.

Integrin-cadherin cross talk is an important aspect of cell function. We explored this signaling using substrates micropatterned with islands of fibronectin surrounded by E-cadherin, capturing the segregation of these signals in normal tissue. While MDCK cells were able to concurrently form adhesive structures with these two proteins, engagement of fibronectin by MCF-7 cells, an adenocarcinoma cell line, inhibited response of these cells to E-cadherin. We further demonstrated that this inhibition is rigidity dependent; on soft elastomer substrates with Young's modulus in the range of tens of kiloPascals, MCF-7 cells were able to engage both integrin and cadherin ligands.

dFunctional convergence of CD28 costimulation and TCR signaling is critical to T-cell activation and adaptive immunity. These receptors form complex microscale patterns within the immune synapse, although the impact of this spatial organization on cell signaling remains unclear. We investigate this cross talk using micropatterned surfaces that present ligands to these membrane proteins in order to control the organization of signaling molecules within the cell-substrate interface. While primary human CD4 ؉ T cells were activated by features containing ligands to both CD3 and CD28, this functional convergence was curtailed on surfaces in which engagement of these two systems was separated by micrometer-scale distances. Moreover, phosphorylated Lck was concentrated to regions of CD3 engagement and exhibited a low diffusion rate, suggesting that costimulation is controlled by a balance between the transport of active Lck to CD28 and its deactivation. In support of this model, disruption of the actin cytoskeleton increased Lck mobility and allowed functional T-cell costimulation by spatially separated CD3 and CD28. In primary mouse CD4؉ T cells, a complementary system, reducing the membrane mobility increased the sensitivity to CD3-CD28 separation. These results demonstrate a subcellular reaction-diffusion system that allows cells to sense the microscale organization of the extracellular environment. Spatial organization plays important roles in cell signaling, governing a wide range of functions, including migration, polarization, and morphogenesis. A striking example at subcellular scales has emerged in the immune synapse (IS), a small (ϳ70-m 2 ) area of contact between a lymphocyte and an antigen-presenting cell (APC) which serves as a platform that focuses and modulates cell-cell communication. The archetypal IS formed between a T cell and an APC contains a central supramolecular activation cluster (cSMAC) of T-cell receptor (TCR)-pMHC complexes surrounded by a peripheral supramolecular activation cluster (pSMAC) with LFA-1-ICAM-1 (1-3). The interfaces of different T-cell-APC pairings exhibit variations on this "bullseye" pattern (4-10), and manipulation of IS structure modulates T-cell activation (11-13), suggesting that microscale organization contributes to the language of cell-cell communication. However, the concept that signaling can be modulated at such scales places stringent requirements on the dynamics of intracellular signaling molecules (14-17), and experimental examples of such mechanisms, particularly within the small dimensions of the IS, have been elusive.We focus here on spatially resolved, microscale cell signaling in the context of CD28 costimulation. When bound by CD80 or CD86, typically presented by an APC in conjunction with pMHC, CD28 augments TCR signaling and is essential for full activation of naive T cells. A role of spatial organization in this signaling was established by experiments in which CD28 was engaged outside the IS, a trans-costimulation configuration representing the action...

Molecules associated with the outer surface of living cells exhibit complex, non-Brownian patterns of diffusion. In this report, supported lipid bilayers were patterned with nanoscale barriers to capture key aspects of this anomalous diffusion in a controllable format. First, long-range diffusion coefficients of membrane-associated molecules were significantly reduced by the presence of the barriers, while short-range diffusion was unaffected. Second, this modulation was more pronounced for large molecular complexes than for individual lipids. Surprisingly, the quantitative effect of these barriers on long-range lipid diffusion could be accurately simulated using a simple, continuum-based model of diffusion on a nanostructured surface; we thus describe a metamaterial that captures the properties of the outer membrane of living cells.The outer surface of cells presents a complex, nanostructured, yet fluid environment that controls the movement of signaling proteins. The lateral movement of many membrane biomolecules, including transmembrane or tethered proteins as well as lipids themselves, can be interpreted as being free and isotropic within compartments of the cell membrane measuring tens to hundreds of nanometers in scale [1][2][3][4][5][6] . These compartments are delineated by semipermeable barriers that arise from interactions between the plasma membrane, underlying cytoskeleton, and associated proteins [6][7][8] . Fluctuations in these structures allow biomolecules to occasionally cross between compartments, allowing long-range, but comparatively slow, transport over the cell surface. More formally, transport along the membrane is an anomalous, non-Brownian process that can be characterized by two diffusion coefficients, one that describes short-range motion within an individual compartment and a second, smaller, effective diffusion coefficient that is associated with long-range motion over many barriers. The extent to which these values differ is dependent on the spacing and properties of the barriers as well as the diffusing molecule. Emerging models suggest significant impacts of this behavior on cell signaling 2, 9, 10 , but experimental systems for testing these hypotheses are not widely available. In this report, we capture this anomalous diffusion by nanopatterning supported lipid bilayers with barriers to lipid diffusion using a geometry that captures the semipermeable nature of those posed to be present in living cells. As is posed by models of these interactions, we aim to gain control over long-range diffusion, while maintaining local, isotropic diffusion associated with a membrane in the absence of such barriers. We demonstrate that these nanopatterned barriers give rise to different short-and long-range diffusion coefficients of lipids and membrane-associated proteins, and provide a quantitative model of this diffusion that suggests specific aspects of membrane structure at the sub-micrometer level. The basic substrate supported lipid bilayer system consists of a phospholipid membrane...

Supported lipid bilayers capture the fluidity and chemical properties of cellular membranes. In this report, we introduce a method for creating surfaces that contain multiple, aligned regions of supported membranes of different compositions at scales of micrometers and smaller. This method uses the design of a diffusional barrier to increase the resolution that can be achieved directly using traditional bilayer patterning techniques, such as laminar flow. We demonstrate the use of this platform for presenting ligands to the T Cell Receptor and LFA-1 that are tethered to separate, closely juxtaposed regions of bilayer, capturing an important aspect of the natural organization observed between T cells and Antigen Presenting Cells. Our results present a novel platform for the study of spatial separation of extracellular ligands and its impact on cell signals.Substrate supported lipid bilayers (SLBs) capture the fluidity of cellular membranes in vitro, providing a powerful tool for investigating protein mobility in cell signaling [1][2][3][4][5][6][7][8][9] . This system has been applied most prominently to studies of T lymphocyte function 1,2 ; the SLB mimics an antigen presenting cell (APC) by presenting tethered proteins to receptors on the T cell. The receptor/ligand signaling clusters that form within the small (5-10 μm diameter) area of contact between T cell and SLB organize into complex patterns capturing the natural T cell/APC interface, a region termed the "immune synapse" (IS). As a specific example, these patterns include a concentric bulls-eye configuration in which T Cell Receptor (TCR) and LFA-1 clusters localize to the center and periphery, respectively, of the IS [10][11][12][13] . Surprisingly, this configuration emerges from a more transient structure, in which LFA-1 clusters are in the center of the IS, surrounded by TCR; notably, this rearrangement would not be possible in the absence of ligand mobility provided by the SLB. The factors that drive the inversion of this structure and other dynamics of the IS, as well as their impacts on cell function, are the topic of current research. Recent studies have shown that patterning the engagement of receptors on the T cell using surface-immobilized ligands modulates cell responses including migration and cytokine lk2141@columbia.edu. Supporting Information Available: Experimental procedures. [14][15][16] . However, a system that provides similar control while retaining the lateral mobility that is essential for IS dynamics remains elusive; intermixing of ligands hinders the ability to precisely define biomolecular layout. Moreover, membrane topology and convergence of downstream signaling pathways complicates interpretation of cell function when ligands are locally mixed. The ability to present multiple, membrane-tethered ligands to T cells within the IS while minimizing the background presence of other ligands would greatly accelerate understanding of the IS. NIH Public AccessTowards this goal, we introduce a simple approach for aligning mult...

The fluidity of cellular membranes imparts lateral mobility of proteins across the cell surface. To understand the impact of lateral mobility on cell-cell communication, a protein consisting of the extracellular recognition domains of E-cadherin was associated with the surface of silica beads by either tethering to a bead-supported lipid bilayer or direct adsorption, resulting in laterally mobile and immobile presentations of this protein. These beads were then seeded onto the upper surface of MDCK cells. Functional engagement of these beads was compared by measurement of Rac1 recruitment around the bead. Lateral mobility enhanced recognition of E-cadherin, promoting cell response to the beads at lower per-area concentrations than their immobilized counterparts. A more complete understanding of how lateral mobility of membrane-associated proteins influences molecular recognition, and potentially other downstream responses, could provide new strategies for the design of materials and devices intended to capture the architecture of natural tissues.

Using high resolution optical and electron microscopy, we show that centrally accumulated T cell receptors (TCR) at in the center of the immunological synapse (IS) is located on the surface of extracellular microvesicles that bud at the IS center. An early endosomal‐sorting complex required for transport (ESCRT) sorts TCR for inclusion in microvesicles, while terminal ESCRT components mediate scission of microvesicles from the T cell plasma membrane. The HIV polyprotein GAG co‐opts this process for budding of virus‐like particles. B cells bearing cognate pMHC receive TCR from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. These microvesicles deliver transcellular signals across antigen‐dependent synapses by engaging cognate pMHC on APC.

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