Interfacial Assembly of Bacterial Microcompartment Shell Proteins in Aqueous Multiphase Systems.

Compartments are a fundamental feature of life, based variously on lipid membranes, protein shells, or biopolymer phase separation. Here, this combines self-assembling bacterial microcompartment (BMC) shell proteins and liquid-liquid phase separation (LLPS) to develop new forms of compartmentalization. It is found that BMC shell proteins assemble at the liquid-liquid interfaces between either 1) the dextran-rich droplets and PEG-rich continuous phase of a poly(ethyleneglycol)(PEG)/dextran aqueous two-phase system, or 2) the polypeptide-rich coacervate droplets and continuous dilute phase of a polylysine/polyaspartate complex coacervate system. Interfacial protein assemblies in the coacervate system are sensitive to the ratio of cationic to anionic polypeptides, consistent with electrostatically-driven assembly. In both systems, interfacial protein assembly competes with aggregation, with protein concentration and polycation availability impacting coating. These two LLPS systems are then combined to form a three-phase system wherein coacervate droplets are contained within dextran-rich phase droplets. Interfacial localization of BMC hexameric shell proteins is tunable in a three-phase system by changing the polyelectrolyte charge ratio. The tens-of-micron scale BMC shell protein-coated droplets introduced here can accommodate bioactive cargo such as enzymes or RNA and represent a new synthetic cell strategy for organizing biomimetic functionality.

Kinetic control of shape deformations and membrane phase separation inside giant vesicles.

A variety of cellular processes use liquid-liquid phase separation (LLPS) to create functional levels of organization, but the kinetic pathways by which it proceeds remain incompletely understood. Here in real time, we monitor the dynamics of LLPS of mixtures of segregatively phase-separating polymers inside all-synthetic, giant unilamellar vesicles. After dynamically triggering phase separation, we find that the ensuing relaxation-en route to the new equilibrium-is non-trivially modulated by a dynamic interplay between the coarsening of the evolving droplet phase and the interactive membrane boundary. The membrane boundary is preferentially wetted by one of the incipient phases, dynamically arresting the progression of coarsening and deforming the membrane. When the vesicles are composed of phase-separating mixtures of common lipids, LLPS within the vesicular interior becomes coupled to the membrane's compositional degrees of freedom, producing microphase-separated membrane textures. This coupling of bulk and surface phase-separation processes suggests a physical principle by which LLPS inside living cells might be dynamically regulated and communicated to the cellular boundaries.

Formation and properties of liposome-stabilized all-aqueous emulsions based on PEG/dextran, PEG/Ficoll, and PEG/sulfate aqueous biphasic systems.

Vesicle-stabilized all-aqueous emulsion droplets are appealing as bioreactors because they provide uniform encapsulation via equilibrium partitioning without restricting diffusion in and out of the interior. These properties rely on the composition of the aqueous two-phase system (ATPS) chosen for the emulsion and the structure of the interfacial liposome layer, respectively. Here, we explore how changing the aqueous two-phase system from a standard poly(ethyleneglycol), PEG, 8 kDa/dextran 10 kDa ATPS to PEG 8 kDa/Ficoll 70 kDa or PEG 8 kDa/Na2SO4 systems impacts droplet uniformity and partitioning of a model solute (U15 oligoRNA). We also compare liposomes formed by two different methods, both of which begin with multilamellar, polydisperse vesicles formed by gentle hydration: (1) extrusion, which produced vesicles of 150 nm average diameter, and (2) vortexing, which produced vesicles of 270 nm average diameter. Our data illustrate that while droplet uniformity and stability are somewhat better for samples based on extruded vesicles, extrusion is not necessary to create functional microreactors, as emulsions stabilized with vortexed liposomes are just as effective at solute partitioning and allow diffusion across the droplet's liposome corona. This work expands the compositions possible for liposome-stabilized, all-aqueous emulsion droplet bioreactors, making them amenable to a wider range of potential reactions. Replacing the liposome extrusion step with vortexing can reduce time and cost of bioreactor production with only modest reductions in emulsion quality.

Aqueous Emulsion Droplets Stabilized by Lipid Vesicles as Microcompartments for Biomimetic Mineralization.

Mineral deposition within living cells relies on control over the distribution and availability of precursors as well as the location and rates of nucleation and growth. This control is provided in large part by biomolecular chelators, which bind precursors and regulate their availability, and compartmentalization within specialized mineralizing vesicles. Biomimetic mineralization in self-assembled lipid vesicles is an attractive means of studying the mineralization process, but has proven challenging due to vesicle heterogeneity in lamellarity, contents, and size across a population, difficulties encapsulating high and uniform precursor concentrations, and the need to transport reagents across an intact lipid bilayer membrane. Here, we report the use of liposome-stabilized all-aqueous emulsion droplets as simple artificial mineralizing vesicles (AMVs). These biomimetic microreactors allow the entry of precursors while retaining a protein catalyst by equilibrium partitioning between internal and external polymer-rich phases. Small molecule chelators with intermediate binding affinity were employed to control Ca(2+) availability during CaCO3 mineralization, providing protection against liposome aggregation while allowing CaCO3 formation. Mineral deposition was limited to the AMV interior, due to localized production of CO3(2-) by compartmentalized urease. Particle formation was uniform across the entire population of AMVs, with multiple submicrometer amorphous CaCO3 particles produced in each one. The all-aqueous emulsion-based approach to biomimetic giant mineral deposition vesicles introduced here should be adaptable for enzyme-catalyzed synthesis of a wide variety of materials, by varying the metal ion, enzyme, and/or chelator.