BPS2026 – Light-activated transbilayer coupling of protein clusters at the living cell membrane

Cell membranes serve as dynamic interfaces that regulate the bidirectional flow of information between the intra- and extracellular environments. While transmembrane receptors have long been considered indispensable to membrane-bound functional structures, recent evidence reveals alternative biophysical mechanisms of transmembrane communication that rely on the spatial reorganization of proteins and lipids. We previously discovered a phenomenon termed transbilayer protein coupling, where protein condensates on one side of the membrane colocalize with those on the opposite side in the absence of transmembrane proteins, using a reconstituted membrane system. Could such coupling also occur in living cells? To address this question, we developed a live-cell platform that enables controlled formation of protein clusters on both the cytoplasmic and extracellular sides of the plasma membrane. On the cytoplasmic side, we employed an optogenetic tool to induce membrane-bound protein clusters upon light activation. On the extracellular side, we introduced exogenous ligands that selectively bind to a model receptor. Remarkably, we observed that cytoplasmic clusters colocalized with extracellular clusters, demonstrating transbilayer protein coupling in living cells, even in the absence of a transmembrane domain. Importantly, this coupling required phase-separating disordered regions on proteins on both sides of the membrane, highlighting protein condensation as the key driver of transbilayer communication. Moreover, coupling occurred independently of the receptor's lipid phase preference. These findings reveal that protein condensation can mediate transbilayer communication in living cells, expanding our understanding of signal propagation across membranes. Furthermore, we demonstrated that artificially triggered transbilayer coupling can be exploited to engineer synthetic modes of transmembrane communication, offering potential applications for targeted therapeutic strategies.