In situ, reversible coacervate formation within lipid vesicles represents a key step in the development of responsive synthetic cellular models. Herein, we exploit the pH responsiveness of a polycation above and below its pK_a, to drive liquid–liquid phase separation, to form single coacervate droplets within lipid vesicles. The process is completely reversible as coacervate droplets can be disassembled by increasing the pH above the pK_a. We further show that pH‐triggered coacervation in the presence of low concentrations of enzymes activates dormant enzyme reactions by increasing the local concentration within the coacervate droplets and changing the local environment around the enzyme. In conclusion, this work establishes a tunable, pH responsive, enzymatically active multi‐compartment synthetic cell. The system is readily transferred into microfluidics, making it a robust model for addressing general questions in biology, such as the role of phase separation and its effect on enzymatic reactions using a bottom‐up synthetic biology approach.

In the field of bottom‐up synthetic biology, lipid vesicles provide an important role in the construction of artificial cells. Giant unilamellar vesicles (GUVs), due to their membrane's similarity to natural biomembranes, have been widely used as cellular mimics. So far, several methods exist for the production of GUVs with the possibility to encapsulate biological macromolecules. The inverted emulsion‐based method is one such technique, which has great potential for rapid production of GUVs with high encapsulation efficiencies for large biomolecules. However, the lack of understanding of various parameters that affect production yields has resulted in sparse adaptation within the membrane and bottom‐up synthetic biology research communities. Here, we optimize various parameters of the inverted emulsion‐based method to maximize the production of GUVs. We demonstrate that the density difference between the emulsion droplets, oil phase, and the outer aqueous phase plays a crucial role in vesicle formation. We also investigated the impact that centrifugation speed/time, lipid concentration, pH, temperature, and emulsion droplet volume has on vesicle yield and size. Compared to conventional electroformation, our preparation method was not found to significantly alter the membrane mechanical properties. Finally, we optimize the parameters to minimize the time from workbench to microscope and in this way open up the possibility of time‐sensitive experiments. In conclusion, our findings will promote the usage of the inverted emulsion method for basic membrane biophysics studies as well as the development of GUVs for use as future artificial cells.

Significance: The biogenesis of a replicative vacuole is an essential step of Coxiella burnetii infections and involves the hijack of several host membrane trafficking pathways. Here we describe Coxiella vacuolar protein B (CvpB) as a Coxiella effector that interacts with phosphoinositides on host cell membranes and manipulates phosphatidylinositol 3-phosphate [PI(3)P] metabolism for optimal Coxiella -containing vacuole (CCV) development. This is achieved by perturbing the activity of the phosphatidylinositol 5-kinase PIKfyve, leading to an enrichment of PI(3)P on CCV membranes, which is required for the autophagy machinery to mediate CCV homotypic fusion. The importance of this process is highlighted by a homotypic fusion defect between CCVs in cells infected with CvpB Coxiella mutants, which translates into an attenuated virulence in the insect model Galleria mellonella .

HIV-1 Gag protein assembles at the plasma membrane of infected cells for viral particle formation. Gag targets lipids, mainly PI(4,5)P2, at the inner leaflet of this membrane. Here, we address the question whether Gag is able to trap specifically PI(4,5)P2 or other lipids during HIV-1 assembly in the host CD4⁺ T lymphocytes. Lipid dynamics within and away from HIV-1 assembly sites were determined using super-resolution microscopy coupled with scanning fluorescence correlation spectroscopy in living cells. Analysis of HIV-1–infected cells revealed that, upon assembly, HIV-1 is able to specifically trap PI(4,5)P2 and cholesterol, but not phosphatidylethanolamine or sphingomyelin. Furthermore, our data showed that Gag is the main driving force to restrict the mobility of PI(4,5)P2 and cholesterol at the cell plasma membrane. This is the first direct evidence highlighting that HIV-1 creates its own specific lipid environment by selectively recruiting PI(4,5)P2 and cholesterol as a membrane nanoplatform for virus assembly.

The self-assembly of HIV-1 Gag polyprotein at the inner leaflet of the cell host plasma membrane is the key orchestrator of virus assembly. The binding between Gag and the plasma membrane is mediated by specific interaction of the Gag matrix domain and the PI(4,5)P₂ lipid (PIP₂). It is unknown whether this interaction could lead to local reorganization of the plasma membrane lipids. In this study, using model membranes, we examined the ability of Gag to segregate specific lipids upon self-assembly. We show for the first time that Gag self-assembly is responsible for the formation of PIP₂ lipid nanoclusters, enriched in cholesterol but not in sphingomyelin. We also show that Gag mainly partition into liquid-disordered domains of these lipid membranes. Our work strongly suggests that, instead of targeting pre-existing plasma membrane lipid domains, Gag is more prone to generate PIP₂/Cholesterol lipid nanodomains at the inner leaflet of the plasma membrane during early events of virus assembly.

Microfluidic production of giant lipid vesicles presents a paradigm-shift in the development of artificial cells. While production is high-throughput and the lipid vesicles are mono-disperse compared to bulk methods, current technologies rely heavily on the addition of additives such as surfactants, glycerol and even ethanol. Here we present a microfluidic method for producing biomimetic surfactant-free and additive-free giant unilamellar vesicles. The versatile design allows for the production of vesicle sizes ranging anywhere from ~10 to 130 µm with either neutral or charged lipids, and in physiological buffer conditions. Purity, functionality, and stability of the membranes are validated by lipid diffusion, protein incorporation, and leakage assays. Usability as artificial cells is demonstrated by increasing their complexity, i.e., by encapsulating plasmids, smaller liposomes, mammalian cells, and microspheres. This robust method capable of creating truly biomimetic artificial cells in high-throughput will prove valuable for bottom-up synthetic biology and the understanding of membrane function.