Liquid-liquid phase separation (LLPS), especially coacervation, plays a crucial role in cell biology, as it forms numerous membraneless organelles in cells. Coacervates play an indispensable role in regulating intracellular biochemistry, and their dysfunction is associated with several diseases. Understanding of the LLPS dynamics would greatly benefit from controlled in vitro assays that mimic cells. Here, we use a microfluidics-based methodology to form coacervates inside cell-sized (~10 µm) liposomes, allowing control over the dynamics. Protein-pore-mediated permeation of small molecules into liposomes triggers LLPS passively or via active mechanisms like enzymatic polymerization of nucleic acids. We demonstrate sequestration of proteins (FtsZ) and supramolecular assemblies (lipid vesicles), as well as the possibility to host metabolic reactions (β-galactosidase activity) inside coacervates. This coacervate-in-liposome platform provides a versatile tool to understand intracellular phase behavior, and these hybrid systems will allow engineering complex pathways to reconstitute cellular functions and facilitate bottom-up creation of synthetic cells.

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Recently, both the cellular and synthetic biology communities have expressed a strong interest in coacervates, membrane‐less liquid droplets composed of densely packed multivalent molecules that form as a result of spontaneous phase separation. Here, it is studied how FtsZ, a protein that plays a key role in the bacterial division process, remodels coacervates made of polylysine (pLL) and guanosine triphosphate (GTP). It is shown that FtsZ strongly partitions at the surface of the coacervates and induces their disassembly due to the hydrolysis of GTP by FtsZ. Surprisingly, the coacervates are found to promote lateral interactions between FtsZ filaments, inducing the formation of an emanating network of FtsZ bundles that interconnect neighboring coacervates. Under mechanical stress, coacervates are shown to fracture, resulting in profound invaginations along their circumference. The results bring out the potential of coacervates for their use as membrane‐free scaffolds for building synthetic cells as well as are possibly relevant for coacervation in prokaryotic cells.@en