Pressure-Jump Kinetics of Liquid–Liquid Phase Separation: Comparison of Two Different Condensed Phases of the RNA-Binding Protein, Fused in Sarcoma

Abstract

The RNA-binding protein fused in sarcoma (FUS) undergoes liquid–liquid phase separation (LLPS) both in vivo and in vitro. Self-assembled liquid droplets of FUS transform into reversible hydrogels and into more irreversible and toxic aggregates. Although LLPS can be a precursor of irreversible aggregates, a generic method to study kinetics of the formation of LLPS has not been developed. Here, we demonstrated the pressure-jump kinetics of phase transition between the 1-phase state and FUS-LLPS states observed at low pressure (<2 kbar, LP-LLPS) and high pressure (>2 kbar, HP-LLPS) using high-pressure UV/vis spectroscopy. Absorbance (turbidity) changes were reproduced repeatedly using pressure cycles. The Johnson–Mehl–Avrami–Kolmogorov theory was used to understand droplet formation occurring via nucleation and growth. The Avrami exponent n, representing the dimensionality of growing droplets, and the reaction rate constant k were calculated. The HP-LLPS formation rate was ∼2-fold slower than that of LP-LLPS. The Avrami exponent obtained for both LLPS states could be explained by diffusion-limited growth. Nucleation and growth rates decreased during LP-LLPS formation (n = 0.51), and the nucleation rate decreased with a constant growth rate in HP-LLPS formation (n = 1.4). The HP-LLPS vanishing rate was ∼20-fold slower than that of LP-LLPS. This difference in vanishing rates indicates a stronger intermolecular interaction in HP-LLPS than in LP-LLPS, which might promote transformation into irreversible aggregates in the droplets. Further, direct transition from HP-LLPS to LP-LLPS was observed. This indicates that interconversion between LP-LLPS and HP-LLPS occurs in equilibrium. Formation of reversible liquid droplets, followed by phase transition into another liquid phase, could thus be part of the physiological maturation process of FUS-LLPS.

Mechanism underlying liquid-to-solid phase transition in fused in sarcoma liquid droplets

“…Our previous high-pressure studies showed that interconversion among the two LLPS states and the one-phase state occurs in equilibrium, with the formation and disappearance of the LLPS states being reversible, whereas the formation and disappearance rates of HP-LLPS are 2- and 20-fold slower than those of LP-LLPS, respectively. 16,17 In the present study, we found that the liquid-to-solid phase transition that occurs over tens of hours under the LP-LLPS condition occurs within tens of minutes for HP-LLPS. Moreover, the droplets in HP-LLPS tend to strongly form clusters, followed by non-spherical aggregates, similar to that with the patient-type FUS variant.…”

“…S1, ESI†). 17 In contrast, the disappearance of the droplets was incomplete when the time spent in HP-LLPS was increased to 30–120 min (Fig. 2B).…”

“…16 The pressure-jump kinetics of the phase transition between the one-phase state and the two LLPS states using high-pressure ultraviolet-visible absorption (UV-vis) spectroscopy have shown that both LLPS states are reversible, such that they can be formed and collapsed repeatedly. 17 These data indicate that interconversion among the two LLPS states and the one-phase state occurs in equilibrium. However, as the pressure gradually changes, the absorbance in the depressurization process behaves differently depending on the pressurization process.…”

“…His-tagged TEV protease was prepared as described previously. 17 The His-tagged TEV protease was added at a final concentration of 10 mg mL À1 to 5 mM MBP-FUS protein in a buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM Mg(OAc) 2 , 20 mM Zn(OAc) 2 , and 2 mM DTT, which was then incubated at 30 1C for at least 3 h. MBP-tagged FUS in which green fluorescent protein (GFP) was fused at the C-terminus was also prepared for the FRAP experiments (i.e., MBP-FUS-GFP, see the following). The MBP domain was cleaved using His-tagged TEV protease in a similar manner.…”

“…According to the pressure-temperature LLPS phase diagram of FUS reported in previous studies, 16,17 two types of FUS liquid condensates exist, LLPS that is stable at o2 kbar (LP-LLPS) and LLPS that is stable at 42 kbar (HP-LLPS), each with distinct partial molar volumes. The partial molar volume of the system decreases in the order LP-LLPS, one-phase, followed by HP-LLPS, indicating that HP-LLPS is more condensed than LP-LLPS.…”