Compartmentalization is a key feature of living systems. Cells are separated from their environment by a membrane, and intracellular compartments are widely used to carry out the biochemistry upon which life relies. Biomolecular condensates are transient intracellular compartments formed when molecules within the cytoplasm undergo a condensing phase transition. The transition produces a region within the cytoplasm that’s typically denser and/or more viscous than the surrounding fluid and the transition is often referred to as liquid-liquid phase separation (LLPS). Molecules co-located within a condensate can more readily react with one another and biologists have learned that the formation and eventual dissolution of biomolecular condensates is ubiquitous across life. While much has been learned about the functions facilitated by these transient organelles, there are many open questions about how the basic physics of LLPS is impacted by the complex, heterogeneous cellular environment within which LLPS occurs. This grant funds work by Jennifer Ross and Jennifer Schwarz, professors of experimental and theoretical physics, respectively, at Syracuse University to study how two specific features of the intracellular microenvironment—the presence of an enzyme-driven ‘active bath’ that modifies the local energy landscape, and the presence of viscoelastic polymers that modify the local entropy landscape—influence the formation and dissolution of protein condensates. The phrase ‘active bath’ refers to a fluid that has been perturbed from its equilibrium thermal state by some type of activity that leads to fluid regions with local fluctuations (e.g. position fluctuations of water molecules) that exceed those associated with the fluid’s overall (equilibrium) thermal state. In this project, the relevant activity is ‘background’ enzyme reactions; chemical reactions that do not directly involve the proteins that condense during LLPS, but which may nonetheless influence LLPS. The cellular entropy feature to be explored by Ross and Schwarz is the presence of a cytoskeleton, a network of viscoelastic (i.e. both viscous and elastic) protein filaments that act to constrain the motion of molecules within a cell via crowding. The team will create cytoskeletal-like networks of varying density and stiffness by using the cytoskeletal biopolymers actin and tubulin. Experiments will vary both the overall polymer density and the actin-to-tubulin ratio. Temperature and condensing-polymer concentration are two key parameters that will be used to experimentally characterize the LLPS phase transition, and Ross and Schwarz plan to study two types of condensing proteins and—for each type of condensing protein—two types of phase transition.