The landscape of cell-surface signaling is 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). Employing the robustness and versatility of LLPS, we decided to engineer LLPS-based tools for precisely manipulating CSPs. 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.

细胞表面信号传导的前景极其复杂。对能够操纵细胞表面蛋白 (CSP) 的时空分布以分解信号的稳健工具的需求量很大。一些 CSP 通过多价驱动的液-液相分离 (LLPS) 进行调节。利用 LLPS 的稳健性和多功能性，我们决定设计基于​​ LLPS 的工具来精确操作 CSP。我们通过将多价模块化相分离支架对与 CSP 粘合剂融合，生成了膜束缚 LLPS 系统。支架对的相分离、膜上 CSP 的伴随区室化以及 CSP 的簇依赖性信号输出需要一个或两个支架的膜募集。我们还设计了正交相分离系统，将 CSP 分离到相互排斥的隔间中。工程化的相分离系统可以稳健地聚集单个 CSP，共同聚集两个或多个 CSP，或将不同的 CSP 分离到细胞表面的不同隔室中。这些工具将能够高精度地剖析复杂的细胞信号传导景观。