Abstract: Cell surface signaling landscapes are formidably complex. Robust tools capable of manipulating the spatiotemporal distribution of cell surface proteins (CSPs) for dissecting signaling are in high demand. Some CSPs are regulated via multivalency driven liquid-liquid phase separation (LLPS). We generated membrane-tethering LLPS systems by fusing multivalent modular phase separation scaffold pairs with CSP binders. Phase separation of the scaffold pairs, concomitant compartmentalization of CSPs on membranes, and cluster-dependent signaling outputs of CSPs require membrane recruitment of one or both scaffolds. We also engineered orthogonal phase separation systems to segregate CSPs into mutually exclusive compartments. The engineered phase separation systems can robustly cluster individual CSPs, co-cluster two or more CSPs, or segregate different CSPs into distinct compartments on cell surfaces. These tools will enable the dissection of complicated cell signaling landscapes with high precision.
Programming cell surface signaling via phase separation-controlled compartmentalization