Light-activated assembly of connexon nanopores in synthetic cells

During developmental processes and wound healing, activation of living cells occurs with spatiotemporal precision and leads to rapid release of soluble molecular signals, allowing communication and coordination between neighbors. Non-living systems capable of similar responsive release hold great promise for information transfer in materials and site-specific drug delivery. One non-living system that offers a tunable platform for programming release is synthetic cells. Encased in a lipid bilayer structure, synthetic cells can be outfitted with molecular conduits that span the bilayer and lead to material exchange. While previous work expressing membrane pore proteins in synthetic cells demonstrated content exchange, user-defined control over release has remained elusive. In mammalian cells, connexon nanopore structures drive content release and have garnered significant interest since they can direct material exchange through intercellular contacts. Here, we focus on connexon nanopores and present activated release of material from synthetic cells in a light-sensitive fashion. To do this, we re-engineer connexon nanopores to assemble after post-translational processing by a protease. By encapsulating proteases in light-sensitive liposomes, we show that assembly of nanopores can be triggered by illumination, resulting in rapid release of molecules encapsulated within synthetic cells. Controlling connexin nanopore activity provides an opportunity for initiating communication with extracellular signals and for transferring molecular agents to the cytoplasm of living cells in a rapid, light-guided manner.