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...

physical inputs, both internal and external to a cell, can directly alter the spatial organization of cell surface receptors and their associated functions. Here we describe a protocol that combines solid-state nanolithography and supported lipid membrane techniques to trigger and manipulate specific receptors on the surface of living cells and to develop an understanding of the interplay between spatial organization and receptor function. While existing protein-patterning techniques are capable of presenting cells with well-defined clusters of protein, this protocol uniquely allows for the control of the spatial organization of laterally fluid receptor-ligand complex at an intermembrane junction. a combination of immunofluorescence and single-cell microscopy methods and complementary biochemical analyses are used to characterize receptor signaling pathways and cell functions. the protocol requires 2–5 d to complete depending on the parameters to be studied. In principle, this protocol is widely applicable to eukaryotic cells and herein is specifically developed to study the role of physical organization and translocation of the EphA2 receptor tyrosine kinase across a library of model breast cancer cell lines.