Multicomponent Supported Membrane Microarray for Monitoring Spatially Resolved Cellular Signaling Reactions

Cells sense biochemical as well as mechanical signals from their microenvironment by engaging multiple receptors. In many cases, multiple receptors operate in concert, and it can be misleading to attempt to isolate a single ligand–receptor interaction. Supported lipid membranes are employed to reconstitute a number of cell receptor systems. Efforts are also made to fabricate membrane microarrays presenting multiple ligands in a spatially segregated manner. However, such membrane multiplexing methods are generally limited by complex instrumentation and scalability. Here, a straightforward method is presented to produce centimeter-scale membrane microarrays displaying multiple, spatially segregated membrane-anchored protein ligands suitable for live single-cell studies. The method is based on stochastic membrane fusion and subsequent diffusion-mediated mixing of their lipid content. The result is the delivery of membrane contents into spatially segregated membrane corrals. Utilizing the technology developed here, this work probes the recruitment of an adaptor protein, Shc1, to epidermal growth factor receptor (EGFR) and EphA2 receptors and shows that activation of EGFR results in a decrease in the recruitment of protein to activate EphA2 same cell.