Dynamic Control of Functional Coacervates in Synthetic Cells

Abstract

Membrane-less compartments formed via liquid− liquid phase separation (LLPS) are regulated dynamically via enzyme reactions in cells. Giant unilamellar vesicles (GUVs) provide a promising chassis to control, mimic, and understand the LLPS process; however, they are challenging to construct. Here, we engineer the dynamic assembly and disassembly of LLPS compartments using complex coacervates as models inside synthetic cells. Semipermeable GUVs constructed with defined lipid composition encapsulate the biomolecules, including enzymes required to regulate coacervates. Assembly and disassembly of coacervates are triggered in independent systems by the diffusion of substrates through the membrane into the vesicle lumen. The coupling of enzyme networks in a single synthetic cell system allows for reversible and out-of-equilibrium regulation of coacervates. The functional properties of the coacervates are revealed by sequestering biomolecules, including drugs and enzymes. GUVs, with functional LLPS compartment assembly, open avenues in constructing programmable autonomous synthetic cells with membraneless organelles. The coacervate-in-vesicle platform has significant implications for understanding LLPS regulation mechanisms in cells.

Instructing Liquid‐Liquid Phase Separation Inside Membranous Protocells

“…[17,24] Likewise, it has been demonstrated that the passive diffusion of ATP directly across the lipid membrane can induce the formation of coacervate microdroplets. [25] Alternatively, protein pores can be introduced into the lipid bilayer to facilitate the diffusion of specific substances. For example, Deshpande et al [26] reconstructed the membrane-spanning α-hemolysin within the lipid bilayer to create pores with a diameter of approximately 1.4 nm.…”

“…These substrates penetrate the lipid bilayer, initiating an enzymatic reaction that produces ATP and pyruvate, leading to the complexation of ATP and PLL into coacervates. [25] In another study by Dekker et al, [26] giant vesicles were loaded with polynucleotide phosphorylase (PNPase), RNA oligomer and spermine. Uridine diphosphate (UDP) diffused into the vesicles through α-hemolysin pores, initiating the chain growth of encapsulated RNA oligomers catalyzed by PNPase.…”

“…These compounds can complex with oppositely charged polyelectrolytes to form coacervate microdroplets within proteinosomes [17,24] . Likewise, it has been demonstrated that the passive diffusion of ATP directly across the lipid membrane can induce the formation of coacervate microdroplets [25] . Alternatively, protein pores can be introduced into the lipid bilayer to facilitate the diffusion of specific substances.…”

Engineering semi-permeable giant liposomes

“…The semipermeable nature of the DOTAP-incorporated GUVs facilitated the cyclic assembly and disassembly of condensates inside the vesicle for over 6 cycles. 36 In summary, the current strategy provides an easier alternative to creating semi-permeable membranes for smaller molecules compared to membrane protein reconstitution. The incorporation of DOTAP induces size-selective semi-permeability in giant vesicles.…”

“…S9, ESI †). DOTAP-incorporated GUV system has allowed for dynamic and reversible assembly of coacervates inside vesicles 36 without the need for membrane proteins. The DOTAP GUVs with PLL and enzymes encapsulated allow the transport of enzyme substrates such as phosphoenolpyruvate, glucose, ATP, and ADP across their membrane.…”