BPS2026 – Aqueous transient phase separated model systems and their interplay with actin

A eukaryotic cell’s cytoplasm is a crowded viscoelastic environment with about 20% of the volume occupied by proteins. In this crowded environment, volume-exclusion effects can lead to aqueous transient phase separation (ATPS), and this liquid-liquid phase separation is increasingly recognized as a key process in the cells’ internal, membrane-less organization of proteins and metabolites. To study ATPS and the interplay between aqueous phases and cytoskeletal proteins, we utilized an in vitro phase-separated model system using two polymers, polyethylene glycol (PEG) and dextran (DEX). While entropically mixed at low-concentrations, PEG and DEX are immiscible at high-concentrations and form two separate aqueous phases. In this work, using small unilamellar vesicles (SUVs) and aqueous polymers, we make liposome-stabilized droplets that stabilize DEX-rich droplets in a PEG-rich environment, generating Pickering-like emulsions. Using confocal fluorescence microscopy, we confirm that this method generates a stable ATPS system with DEX droplets a few 10s of μm in size. Bulk rheology confirms that both the PEG- and DEX-rich phases behave as Newtonian fluids. Upon incorporating monomeric skeletal muscle actin into the system, microscopy shows that both the monomeric and polymeric forms of actin prefer the DEX-rich phase. After adding polymerization buffer to the droplets, we also observe destabilization and coalescence of DEX-rich droplets, suggesting that the formation of a filamentous biopolymer network can affect the stability of ATPS domains. Bulk rheology confirms the formation of a viscoelastic entangled actin network in our systems. Interestingly, G-actin in the DEX-rich phase exhibits shear thickening, suggesting that the increased crowding due to ATPS in the DEX-rich phase can lead to localized protein interaction and polymerization, even under otherwise unfavorable polymerization conditions. Our results demonstrate ATPS as a mechanism both influencing and influenced by cytoplasmic protein interactions and organization.