Activation of the EphA2 receptor tyrosine kinase by ephrin-A1 ligands presented on apposed cell surfaces plays important roles in development and exhibits poorly understood functional alterations in cancer. We reconstituted this intermembrane signaling geometry between live EphA2-expressing human breast cancer cells and supported membranes displaying laterally mobile ephrin-A1. Receptor-ligand binding, clustering, and subsequent lateral transport within this junction were observed. EphA2 transport can be blocked by physical barriers nanofabricated onto the underlying substrate. This physical reorganization of EphA2 alters the cellular response to ephrin-A1, as observed by changes in cytoskeleton morphology and recruitment of a disintegrin and metalloprotease 10. Quantitative analysis of receptor-ligand spatial organization across a library of 26 mammary epithelial cell lines reveals characteristic differences that strongly correlate with invasion potential. These observations reveal a mechanism for spatio-mechanical regulation of EphA2 signaling pathways.Mammalian cells exhibit marked sensitivity to physical aspects of their environment, such as compliance (1), texture (2), and geometry (3). Tensional homeostasis between and within cells contributes to proper cell differentiation, development, and, ultimately, survival (4). Because most cellular decision making occurs via chemical processes, understanding the coupling between physical forces and chemical signaling networks is of fundamental importance. Focal adhesions, which consist of protein assemblies organized at sites where cell-surface integrin receptors bind extracellular matrix ligands, are the most widely studied interface for tensile force transduction (5). However, the majority of membrane receptors are not associated with focal adhesions. The mechanisms (and even existence) of chemomechanical regulatory coupling in these systems remain largely unknown.It is becoming clear that spatial organization of cell surface receptors can regulate associated signal transduction pathways (6-9). An important corollary is that mechanical forces acting on ligands can influence receptor spatial organization and, correspondingly, signaling (10-12). Juxtacrine signaling, in which receptor and ligand reside in apposed cell membranes, represents an important class of intercellular communication where physical restriction of ligand spatial organization and movement is evident (6,13). Here, we reconstitute the juxtacrine signaling geometry between live cells expressing the EphA2 receptor tyrosine kinase and supported membranes displaying laterally mobile ephrin-A1 ligand.EphA2 is implicated and functionally altered in a number of cancers. In particular, 40% of human breast cancers overexpress the receptor (14). Upon binding to natively membraneanchored ephrin-A1, EphA2 undergoes dimerization, transphosphorylation of the cytoplasmic domains, recruitment of a molecular complex with SHC and GRB2 adaptor proteins, and subsequent activation (15). EphA2 activation stimul...

T cells react to extremely small numbers of activating agonist peptides. Spatial organization of T-cell receptors (TCR) and their peptide-major histocompatibility complex (pMHC) ligands into microclusters is correlated with T-cell activation. Here we have designed an experimental strategy that enables control over the number of agonist peptides per TCR cluster, without altering the total number engaged by the cell. Supported membranes, partitioned with grids of barriers to lateral mobility, provide an effective way of limiting the total number of pMHC ligands that may be assembled within a single TCR cluster. Observations directly reveal that restriction of pMHC content within individual TCR clusters can decrease T-cell sensitivity for triggering initial calcium flux at fixed total pMHC density. Further analysis suggests that triggering thresholds are determined by the number of activating ligands available to individual TCR clusters, not by the total number encountered by the cell. Results from a series of experiments in which the overall agonist density and the maximum number of agonist per TCR cluster are independently varied in primary T cells indicate that the most probable minimal triggering unit for calcium signaling is at least four pMHC in a single cluster for this system. This threshold is unchanged by inclusion of coagonist pMHC, but costimulation of CD28 by CD80 can modulate the threshold lower.cell biophysics | cell patterning | immune synapse