Programmable Coacervate–Membrane Interactions Direct Internal and Collective Organization in Membranized Protocells

ABSTRACT In eukaryotic cells, membraneless organelles reorganize through regulated interactions with the plasma membrane and its underlying cortex, where cytoskeletal coupling and inner‐leaflet biochemistry tune condensate positioning, wetting, and function. Recreating such adaptive, cortex‐mediated control in synthetic systems remains a challenge, requiring a chassis that combines interfacial programmability with the mechanical resilience necessary to withstand the osmotic and electrostatic stresses of bottom‐up assembly. Here, we introduce polysaccharidosomes (P‐somes); semipermeable, mechanically robust protocells that function as membrane‐programmable chassis for directing coacervate–membrane coupling. By establishing a thin, cortex‐like protein layer on the inner membrane leaflet via template‐directed assembly, we demonstrate that in situ protein succinylation enables precise tuning of surface charge and coacervate–membrane wetting. Together with the systematic variation of membrane building blocks, this platform allows for fine control over coacervate wetting, morphology, and spatial organization. The uptake of external DNA adds a second tier of regulation: on nonpassivated membranes, DNA‐reconfigured coacervates generate interfacial protrusions that bridge neighboring P‐somes to promote tissue‐like clustering, whereas on passivated membranes, they coalesce into a single, nonwetting, nucleus‐like droplet centered within the lumen. This membrane–cortex‐inspired framework integrates mechanical resilience with chemical programmability, providing a scalable route to constructing membranized protocells with self‐organizing interiors and emergent collective behaviors.