Transient pH changes drive vacuole formation in enzyme–polymer condensates

Membraneless organelles are essential for cellular function. These biomolecular condensates often exhibit complex morphologies in response to biological stimuli. In vitro condensate models help elucidate how these multiphase assemblies form and their possible functions. Here we use such a model to investigate the formation of hollow internal regions, or vacuoles, within condensates in response to a pH change. Fast rates of pH decrease and larger droplet sizes promote vacuole development within the condensates. We show that vacuole formation is a non-equilibrium process driven by the diffusion-limited exchange of condensate components during a rapid pH change. We develop a physics-based model that describes how associative phase-separating systems respond to rapid changes in external conditions, specifically pH. Our qualitative model agrees with 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 model illustrates a mechanism of vacuole formation in associative condensates and provides insights into the regulation of multiphase condensates in vivo.