Dissecting Rate-Limiting Processes in Biomolecular Condensate Exchange Dynamics

An increasing number of biomolecules have been shown to phase-separate into biomolecular condensates — membraneless subcellular compartments capable of regulating distinct biochemical processes within living cells. The speed with which they exchange components with the cellular environment can influence how fast biochemical reactions occur inside condensates and how fast condensates respond to environmental changes, thereby directly impacting condensate function. While Fluorescence Recovery After Photobleaching (FRAP) experiments are routinely performed to measure this exchange timescale, it remains a challenge to distinguish the various physical processes limiting fluorescence recovery and identify each associated timescale. Here, we present a reaction-diffusion model for condensate exchange dynamics and show that such exchange can differ significantly from that of conventional liquid droplets due to the presence of a percolated molecular network, which gives rise to different mobility species in the dense phase. In this model, exchange can be limited by diffusion of either the high- or low-mobility species in the dense phase, diffusion in the dilute phase, or the attachment/detachment of molecules to/from the network at the surface or throughout the bulk of the condensate. Through a combination of analytic derivations and numerical simulations, we quantify the contributions of these distinct physical processes to the overall exchange timescale and predict an experimentally testable scaling relationship between the exchange timescale and condensate size. We discover that the exchange dynamics can be accelerated via a pore-mediated pathway in which molecules pass through the pores of the meshwork and attach/detach directly in the condensate interior. Notably, this pathway leads to a new regime in which the exchange timescale becomes independent of condensate size, which we validate through FRAP experiments on a biosynthetic DNA nanostar system. Our work offers insight into the predominant physical mechanisms driving condensate material exchange, with implications for natural and engineered systems.