Pressure-Jump Kinetics of Liquid-Liquid Phase Separation: Comparison of Two Different Condensed Phases of the RNA-Binding Protein, Fused in Sarcoma
Pressure-Jump Kinetics of Liquid-Liquid Phase Separation: Comparison of Two Different Condensed Phases of the RNA-Binding Protein, Fused in Sarcoma
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DOI：
10.1021/jacs.1c07571
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发表时间：
2021-12-01
影响因子：
15
通讯作者：
Yoshizawa, Takuya
中科院分区：
文献类型：
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作者：
Kitahara, Ryo;Yamazaki, Ryota;Yoshizawa, Takuya
融合于肉瘤中的RNA结合蛋白（FUS）在体内和体外均经历液-液相分离（LLPS）。FUS的自组装液滴转化为可逆的水凝胶，并转化为更不可逆和有毒的聚集体。虽然LLPS可以是不可逆聚集体的前体，但尚未开发出研究LLPS形成动力学的通用方法。在这里，我们证明了在低压（2千巴，HP-LLPS）使用高压UV/维斯光谱观察到的1-相态和FUS-LLPS态之间的相变的压力跳跃动力学。使用压力循环重复再现吸光度（浊度）变化。Johnson-Mehl-Avrami-Kolmogorov理论用于理解通过成核和生长发生的液滴形成。计算了Avrami指数n和反应速率常数k。HP-LLPS的形成速率类似于LP-LLPS的2倍慢。两种LLPS状态下的Avrami指数可以用扩散限制生长来解释。在LP-LLPS形成过程中，成核和生长速率降低（n = 0.51），而在HP-LLPS形成过程中，成核速率以恒定的生长速率降低（n = 1.4）。HP-LLPS的消失速度比LP-LLPS慢约20倍。这种消失率的差异表明HP-LLPS中的分子间相互作用比LP-LLPS中的分子间相互作用更强，这可能促进液滴中不可逆聚集体的转化。此外，观察到从HP-LLPS到LP-LLPS的直接转变。这表明LP-LLPS和HP-LLPS之间的相互转化在平衡中发生。可逆液滴的形成，随后相转变成另一液相，因此可能是FUS-LLPS生理成熟过程的一部分。
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, 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 similar to 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 similar to 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.