Cell-Sized Confinement Initiates Phase Separation of Polymer Blends and Promotes Fractionation upon Competitive Membrane Wetting

Abstract

Biomolecular condensates driven by liquid–liquid phase separation (LLPS) have received attention as novel activity regulators of living organisms. In intracellular LLPS, an important question is what type of biomolecules form condensates under what conditions. Recently, LLPS condensates have been reported to regulate the membrane structure upon wetting. However, the opposite possibility, i.e., whether membrane wetting regulates the LLPS, remains unexplored. Using variously sized droplets of short polyethylene glycol (PEG) and long dextran blends encapsulated with a lipid membrane, we demonstrate that membrane wetting regulates LLPS in cell-sized spaces and alters the equilibrium state. In small droplets, the two-phase region expands beyond the bulk, and the degree of fractionation increases as the droplet size decreases. We explain the space-size-dependent LLPS from the higher wettability of short PEG than dextran. This shows that cell-sized confinement can regulate LLPS upon competition for membrane wettability among various molecules, rendering this LLPS principle feasible in living cells.

Cell-Size Space Regulates the Behavior of Confined Polymers: From Nano- and Micromaterials Science to Biology

“…This phenomenon indicates that CSE shifts the critical point to the lower concentrations and expands the two-phase region. A similar phenomenon was observed for PEG and dextran blends . Furthermore, the degree of separation into PEG- and dextran-rich phases increases as the droplet size decreases.…”

“…The polymer blends confined in cell-sized space are expected to exhibit different phase behavior than the bulk system. This phenomenon has been analyzed for binary polymer blends of PEG and biopolymers such as PEG and gelatin, PEG and BSA, PEG and DNA, and PEG and dextran, where the hydrophilic polymer PEG behaves as a demixing agent for the biopolymers. Figure a shows the CSE on the pattern formation of gelatin and PEG in a two-phase region in the bulk .…”

“…A similar phenomenon was observed for PEG and dextran blends. 54 Furthermore, the degree of separation into PEGand dextran-rich phases increases as the droplet size decreases. This implies that CSE alters the system's free energy.…”

Cell-size space effects on phase separation of binary polymer blends

“…However, since there is no significant difference in the characteristic length of PEG and dextran, unlike the above system, the origin may be a hydrodynamic effect. Thus, although the Next, we discuss our investigations of CSE on the enthalpydriven phase separation of the binary blends of PEG6k and BSA (Watanabe et al 2020), or dextran with an average molecular weight of 500,000 (Dextran500k) (Watanabe et al 2022). To minimize direct membrane interaction with the polymers used in these studies, we employed a zwitterionic lipid, phosphatidylcholine (PC), to cover the droplet surface.…”

“…This is because the large interfacial tension at the W/O interface stabilizes the system and the outer oil phase prevents molecular transport through the membrane. From our studies using droplets, we found that the small space comparable to a cell significantly alters the various behaviors of encapsulated molecules and we termed this effect the cell-size space effect (CSE) (Yanagisawa, Watanabe et al 2022 ). Here, we review both our and others’ published observations of the CSE on phase separation.…”