Pressure and Temperature Phase Diagram for Liquid–Liquid Phase Separation of the RNA-Binding Protein Fused in Sarcoma

Abstract

Liquid–liquid phase separation (LLPS) of proteins and nucleic acids to form membraneless cellular compartments is considered to be involved in various biological functions. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vivo and in vitro. Here, we investigated the effects of pressure and temperature on the LLPS of FUS by high-pressure microscopy and high-pressure UV/vis spectroscopy. The phase-separated condensate of FUS was obliterated with increasing pressure but was observed again at a higher pressure. We generated a pressure–temperature phase diagram that describes the phase separation of FUS and provides a general understanding of the thermodynamic properties of self-assembly and phase separation of proteins. FUS has two types of condensed phases, observed at low pressure (LP-LLPS) and high pressure (HP-LLPS). The HP-LLPS state was more condensed and exhibited lower susceptibility to dissolution by 1,6-hexanediol and karyopherin-β2 than the LP-LLPS state. Moreover, molecular dynamic simulations revealed that electrostatic interactions were destabilized, whereas cation−π, π–π, and hydrophobic interactions were stabilized in HP-LLPS. When cation−π, π–π, and hydrophobic interactions were transiently stabilized in the cellular environment, the phase transition to HP-LLPS occurred; this might be correlated to the aberrant enrichment of cytoplasmic ribonucleoprotein granules, leading to amyotrophic lateral sclerosis.

ALS-linked FUS mutations dysregulate G-quadruplex-dependent liquid–liquid phase separation and liquid-to-solid transition

“…The aggregate was further confirmed to be in a solid-phase state because it was resistant to 1,6-hexanediol, an agent known to disrupt liquid-phase condensates ( Fig. S9 ) ( 44 , 45 ). This transition was not observed in the absence of RNA or with the addition of randomized RNA ( Figs.…”

“…S9 ). The reactions were incubated in the presence or absence of blocking DNA for 30 min at 25 °C as in standard method, then 1,6-hexanediol was added and observed 15 min later as described previously ( 45 ). Phase separation assay with FUS and the long promiscuous RNA was performed using wild-type FUS (2 μM) and various concentrations of long randomized RNA (68 nt) ( 8 ), and turbidity was measured after incubating for 30 min at 25 °C.…”

Molecular insights into the effect of hexanediol on FUS phase separation

“…Aliphatic alcohols such as 1,6-hexanediol (1,6-HD), 2,5-hexanediol (2,5-HD), 1,4-butanediol (1,4-BD) and 1,5-pentanediol (1,5-PD) have been extensively used as FUS hydrogel melting (Lin et al , 2016) and phase separation prevention (Berkeley et al , 2021) (Li et al , 2021), agents. We sought to determine how a series of different alkanediols, including also 1,2-hexanediol (1,2-HD) and 1,2-cyclohexanediol (1,2-CHD), alters the capacity of the purified isolated low-complexity (LC) domain (residues 1-163) of FUS to form liquid droplets in vitro .…”

Structure-dependent recruitment and diffusion of guest proteins in liquid droplets of FUS

“…This different recruitment property between folded and disordered guest proteins in FUS droplets was attributed to the voids formed in the network of host FUS molecules inside the droplets. The voids in FUS droplets have been confirmed 36 . When the molecular size of the folded guest protein is larger than the size of the void, the guest protein should be excluded from the void (Fig.…”

“…Accordingly, void exclusion would compensate for the large intermolecular interactions expected in large folded proteins, resulting in the size-independent uptake of folded proteins. The voids in FUS droplets were supported by the pressure-mediated dissolution of droplets 36 . Overall, the apparent size-independent uptake in the FUS droplets is interpreted as a trade-off between intermolecular interactions and size exclusion from droplet voids (see details in “ Discussion ” section).…”