Early Stages of FUS Droplet Formation via Liquid–Liquid Phase Separation

The phase behavior of complex biomolecular solutions may explain different cellular processes, including the organization of cells by membraneless organelles. The early stages of phase separation are crucial to understanding the underlying mechanism and identifying biomolecules that trigger or drive the transition. Here, we analyze the early events of liquid–liquid phase separation (LLPS) of FUS by multiangle time-resolved static and dynamic light scattering. LLPS was triggered by TEV-catalyzed cleavage of the MBP-tag from FUS-MBP. The light scattering measurements revealed the existence of at least two fractions of FUS-MBP aggregates already prior to the onset of LLPS. The orders of magnitude of the aggregate size in these two fractions are 10 and 100 nm, respectively. LLPS started after an induction period, which depended on the concentration of FUS-MBP. The data from time-dependent light scattering revealed a coalescence of droplets also denoted as a step growth process. A step growth process instead of nucleation and growth via monomer addition suggests that LLPS takes place within the spinodal rather than between the binodal and the spinodal.