Liquid-liquid phase separation in artificial cells

Abstract

Liquid-liquid phase separation (LLPS) in biology is a recently appreciated means of intracellular compartmentalization. Because the mechanisms driving phase separations are grounded in physical interactions, they can be recreated within less complex systems consisting of only a few simple components, to serve as artificial microcompartments. Within these simple systems, the effect of compartmentalization and microenvironments upon biological reactions and processes can be studied. This review will explore several approaches to incorporating LLPS as artificial cytoplasms and in artificial cells, including both segregative and associative phase separation.

Keywords: aqueous two-phase system; coacervate; droplet; synthetic cytoplasm.

Figure 1.

Examples of membraneless organelles in living cells. (a) Fluorescence imaging of the protein LAF-1 within P granules show their liquid-like behaviour via coalescence. (b) Coalescence and separation of X. laevis nucleoli. (c) Distribution of various membraneless organelles throughout HeLa cell. Images adapted from [–26]. Image in (c) licensed under Creative Commons (CC BY).

Figure 2.

Categories of coexisting liquid–liquid systems studied as model cytoplasms for artificial cells. (a) Non-stabilized dispersion of an aqueous phase within another aqueous phase. (b) Surfactant-stabilized w/o emulsion droplets that contain two aqueous phases. (c) Giant lipid vesicles with aqueous phase-separated interiors. (d) Lipid vesicle-stabilized all-aqueous emulsion droplet. In these illustrations, green and blue represent distinct aqueous phases, while yellow indicates oil.

Figure 3.

Overview of segregative and associative phase separation. (a) Scheme of segregative phase separation of two polymers, where each is primarily localized in one phase. (b) Phase diagram of PEG/dextran ATPS showing phase-transition binodal at varying temperatures. (c) Scheme of associative phase separation of two polymers, where both are distributed within the same, concentrated phase. (d) Simulation of coacervate phase dependence as a function of charge periodicity (τ), salt concentration and polymer concentration. Images adapted from [44,45]. Image in (d) licensed under Creative Commons (CC BY).

Figure 4.

Examples of bulk two-phase systems and reactions contained within. (a) Scheme of non-stabilized aqueous phase dispersed within another aqueous phase. (b) Reaction progress of hammerhead ribozyme (HHL) within PEG/dextran ATPS with varying phase volumes. Ratios of PEG : dextran phase volume are 1 : 0 (black circles), 1 : 5 (blue squares), 1 : 12.5 (red diamonds), 1 : 50 (blue triangles) and 1 : 100 (green inverted triangles). (c) Production of resorufin at interface of PEG/citrate ATPS demonstrating interfacial reaction. (d) Release of BSA-FITC from PDDA/PAA coacervate due to unfolding upon addition of urea. Scale bars are 5 µm. Images adapted from [57,88,89]. Image in (d) adapted with permission from [89]. Copyright 2016 American Chemical Society.

Figure 5.

Examples of phase-separated w/o emulsion droplets stabilized via surfactant and their use as artificial cells. (a) Scheme of surfactant-stabilized w/o emulsion droplet containing a phase-separated solution. (b) Aqueous three-phase system w/o emulsion droplets containing PEG (blue), dextran (green) and ficoll (red). Scale bar is 50 µm. (c) Microfluidic production of PEG/dextran droplet within an oil phase showing control over internal aqueous mixing via flow rate. Scale bar is 100 µm. (d) Production of mYPet (green) within phase-separated droplets containing PEG and dextran (blue). Scale bar is 25 µm. (e) Assembly of produced FtsZ within dextran-rich phase and at lipid-coated interface of PEG/dextran droplets. (f) Increase in transcription rate when carried out within coacervate droplets compared to a single-phase solution. Images adapted from [–122]. Image in (f) licensed under Creative Commons (CC BY).

Figure 6.

Examples of phase-separated GVs as artificial cells. (a) Scheme of GV containing a phase-separated solution. (b) Overview of gentle hydration of lipid film with a PEG/dextran ATPS. Bottom shows final droplet with lipid membrane (red) surrounding phases of PEG and dextran (green). Scale bar is 10 µm. (c) Osmotically induced budding of lipid-coated ATPS droplets. Separate lipid domains of PEGlyated lipids (green) and non-PEGlyated lipids (red) surround the PEG-rich and dextran-rich phases, respectively. A protein, soyabean agglutinin (blue), partitions into the dextran-rich phase. Scale bar is 10 µm. (d(i,ii)) Osmotically induced shrinkage of PEG/dextran droplets causes phase separation and formation of lipid nanotubes (red with arrowheads). (iii) xz view of droplet shows lipid nanotubes at aqueous–aqueous interface. (iv) Image from arrowhead in (iii) shows lipid nanotubes at interface. All scale bars are 15 µm. (e) Scheme of coacervate-containing liposome production via microfluidics. Bottom shows fusion of polylysine/ATP coacervates after droplet formation. Images adapted from [,–144]. Image in (b) Copyright (2005) National Academy of Sciences. Image in (e) licensed under Creative Commons (CC BY).

Figure 7.

Examples of liposome-stabilized Pickering emulsions of phase-separated droplets. (a) Scheme of liposome stabilization of dispersed aqueous phase within continuous aqueous phase. (b) Phase-separated droplets of dextran-rich phase (green) within PEG-rich phase, stabilized by liposomes (red). Scale bar is 25 µm. (c) Cartoon of biomineralization within liposome-stabilized droplets. Right is image of mineral formation within droplets surrounded by liposomes (red). Scale bar is 10 µm. (d) Liposome (red) assembly around coacervate droplets comprised spermine and polyU (blue). Images adapted from [67,138,164]. Image in (c) reprinted with permission from [138]. Copyright 2015 American Chemical Society.