Ion- and Temperature-Programmable Reconfiguration of Subcompartments in Synthetic Cells

Spatial reorganization of subcompartments is a hallmark of living cells, enabling coordinated metabolism and signaling. Achieving dynamic reconfiguration remains a major challenge in synthetic cell design. Although specific ions play fundamental physiological roles, their relevance in bottom-up synthetic biology has been underexplored. Particularly, magnesium ions (Mg²⁺), essential cofactors and signaling mediators in biological systems, regulate membrane interactions and molecular assemblies. Here, we present a purely Mg²⁺-mediated mechanism that enables reversible subcompartment assembly within synthetic cells. Mg²⁺ mediates adhesion between negatively charged giant unilamellar vesicles (GUVs), serving as synthetic cell chassis, and neutral large unilamellar vesicles (LUVs), mimicking subcompartments. Mg²⁺ coordination bridges opposing membranes form stable subcompartment layers. Lowering Mg²⁺ concentration, e.g., by chelation with EDTA, disrupts adhesion, whereas reintroduction of Mg²⁺ restores it, enabling dynamic and reversible control over membrane organization. The extent of LUV adhesion depends on membrane charge density, lipid phase state, and temperature. Phase separation allows spatially directed subcompartment to specific domains. Notably, adhesion is lost above the LUV phase transition temperature but re-established upon cooling, enabling temperature-programmed reassembly. Together, these findings define a minimal physicochemical framework for dynamic synthetic cell organization through physical stimuli and suggest a primitive lipid-ion mechanism underlying early membrane contact phenomena.