Transient pH changes drive vacuole formation in enzyme-polymer condensates

Intracellular membraneless organelles formed by the phase separation of biomolecules are essential for cellular functioning. These biomolecular condensates often exhibit complex morphologies in response to biological stimuli. In vitro condensate models help elucidate the mechanism of formation and the associated function of these hierarchical assemblies. Here, we use an in vitro model to investigate the formation of hollow internal regions, or vacuoles, within the condensate interior in response to a pH change. Our experimental system is a pH-responsive complex coacervate formed by the anionic glucose oxidase enzyme phase separating with the weak polycation, DEAE-dextran. Fast rates of pH decrease and larger droplet sizes trigger vacuole development within the coacervates. We show that the emergence of vacuoles is a non-equilibrium process caused by the diffusion-limited exchange of condensate components during a fast pH change. We develop a theoretical model that captures how a phase-separating system responds dynamically to changes in system conditions, particularly pH. Our qualitative phase diagram aligns with our experimental results, showing that rapid pH changes shift the phase boundaries, triggering spinodal decomposition and inducing vacuole formation within the condensates. Our pH-sensitive in vitro coacervate model provides a platform to modulate the internal structure of ternary phase separating systems and gain insights into the mechanisms controlling condensate organization in vivo .