# Interactions between Phase-Separated Liquids and Membrane Surfaces
## 2 Physical Properties of Phase-Separating Molecules and Biomolecular Condensates
### Stimulus Responsiveness of Biomolecular Condensates  
Because liquid-liquid phase separation occurs in a precise thermodynamic regime, biomolecular condensate formation and dissolution is intrinsically responsive to stimuli that shift the system in and out of these regimes. However, how biomolecular condensates respond to stimuli depends on the nature of the intermolecular interactions that generate them (Figure 1). Temperature, naturally, affects the thermodynamics of phase separation. In Flory-Huggins theory, phase separation is enthalpically driven, and so theoretically simple polymer condensates have an upper critical solution temperature (UCST) above which phase separation will not occur [25]. However, many phase-separating systems instead have a lower critical solution temperature (LCST) below which phase separation does not occur. These polymer condensates are often entropically driven by the release of water from hydrophobic polymer surfaces [26]. Hydrophobic residue-rich proteins such as elastin-like polypeptides and the protein BuGZ phase separate with increased temperature, and indeed BuGZ mutants replacing aromatic residues with the polar residue serine require much higher temperature and concentration to phase separate [26; 27]. Ddx4 condensates on the other hand, which are driven by electrostatic interactions, form at lower temperatures and dissolve upon heating [8].  
Figure 1: Trends in stimulus responsiveness of phase-separated condensates. (**a**) Condensation of proteins driven by polar and/or charged residues typically generates condensates (**b**) that phase separate at low temperatures and low salt concentrations; (**c**) condensation of proteins driven by hydrophobic interactions frees some water molecules; this typically generates condensates (**d**) that phase separate at high temperatures and high salt concentrations. There are exceptions depending on the precise thermodynamics of the polymer phase separation; for example, FUS LC phase separates at low temperatures but at high salt concentrations [19].  
Like temperature, ionic strength can either enhance or inhibit phase separation depending on the nature of intermolecular interactions. LAF-1, like Ddx4, undergoes phase separation driven by electrostatic interactions, and both experience an inhibitory effect from salt--increased salt concentrations increase the critical protein concentration for phase separation [6,8]. Proteins such as tropoelastin and FUS, which form biomolecular condensates due to hydrophobic interactions, can \"salt out\" of solution in a way that promotes liquid-liquid phase separation at elevated salt concentrations [19,28,29]. It should be noted that though FUS's low-complexity domain (FUS LC) undergoes hydrophobic residue-mediated phase separation that is enhanced by ionic strength, it is lower temperatures rather than higher that promote its phase separation [19]. These principles of stimulus responsiveness based on the type of intermolecular interactions are only guidelines and the specific thermodynamics of phase separation vary from case to case.