(442c) Antibody Binding As a Noninvasive Reporter of Cell Surface Heterogeneity and Organization

The mammalian plasma membrane is coated with a dense array of glycoproteins and glycolipids, which mediates ligand, receptor, and macromolecular binding. The glycocalyx is sufficiently crowded to induce membrane curvature in live cells, and in-vitro reconstituted experiments have shown that steric crowding on lipid membranes can also exclude macromolecules like antibodies. Cell surface crowding is highly heterogeneous over nanometer length scales, making it difficult to characterize on live cells, without resorting to destructive techniques like electron microscopy. In this work we tune the extracellular position of engineered molecular antigen sensors and leverage local differences in IgG monoclonal antibody binding avidity to report crowding heterogeneities on live cells. Using in-vitro reconstitution and simulations, we validate the strong sensitivity of our sensors, and capture steric crowding variations with nanometer spatial resolution. Using analytical theory and coarse-grained molecular dynamics simulations, combined with proteomics data, we reconstruct the red blood cell glycocalyx in-silico, and corroborate our sensor measurements. We additionally find stronger IgG binding in raft-like plasma membrane domains on human cancer cells, suggesting that these domains exclude bulky extracellular proteins. Our facile and high-throughput method to quantify spatial crowding heterogeneities on live cell membranes may provide an improved biophysical understanding of the molecular-to-mesoscale spatial organization of the cell surface.