Mutations in Ubiquilin-2 (UBQLN2), a ubiquitin-binding shuttle protein involved in several protein quality control processes, can lead to amyotrophic lateral sclerosis (ALS). We previously found that wild-type UBQLN2 forms dynamic, membraneless biomolecular condensates upon cellular stress, and undergoes liquid-liquid phase separation in vitro. However, the impact of ALS-linked mutations on UBQLN2 condensate formation in cells is unknown. Here, we employ live-cell imaging with photokinetic analysis to investigate how five patient-derived ALS-linked mutations in UBQLN2 impact stress-induced UBQLN2 condensate assembly and condensate material properties. Both wild-type and mutant UBQLN2 condensates are generally cytoplasmic and liquid-like. However, cells transfected with mutant UBQLN2 contain fewer stress-induced UBQLN2 condensates than those with wild-type UBQLN2. Most strikingly, ectopically expressed P506T UBQLN2 forms the lowest number of stress-induced condensates of all UBQLN2 mutants, and these condensates are significantly smaller than those of wild-type UBQLN2. Fluorescence recovery after photobleaching (FRAP) analysis of UBQLN2 condensates revealed higher immobile fractions for UBQLN2 mutants, especially P506T. P497S and P497H mutations differentially impact condensate properties, demonstrating that the effects of ALS-linked mutations are both position- and amino acid-dependent. Collectively, our data show that disease mutations hinder assembly and alter viscoelastic properties of stress-induced UBQLN2 condensates, potentially leading to aggregates commonly observed in ALS.

中文翻译：

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ALS连锁突变损害UBQLN2应激诱导的细胞中生物分子冷凝物组装

Ubiquilin-2（UBQLN2）的突变是一种遍在蛋白结合的穿梭蛋白，涉及多个蛋白质质量控​​制过程，可导致肌萎缩性侧索硬化症（ALS）。我们以前发现，野生型UBQLN2在细胞应力作用下会形成动态的无膜生物分子缩合物，并在体外进行液-液相分离。但是，尚不知道ALS连锁突变对UBQLN2冷凝物形成的影响。在这里，我们采用活细胞成像和光动力学分析来研究UBQLN2中五个患者衍生的ALS连锁突变如何影响应力诱导的UBQLN2冷凝物组装和冷凝物材料特性。野生型和突变型UBQLN2冷凝物通常都是胞质的和液体状的。然而，与野生型UBQLN2相比，突变UBQLN2转染的细胞含有更少的应激诱导UBQLN2冷凝物。最为引人注目的是，异位表达的P506T UBQLN2在所有UBQLN2突变​​体中形成的应力诱导的冷凝物数量最少，并且这些冷凝物显着小于野生型UBQLN2。UBQLN2缩合物的光漂白（FRAP）分析后的荧光恢复显示UBQLN2突变​​体（尤其是P506T）具有更高的固定部分。P497S和P497H突变对冷凝物特性的影响不同，表明ALS关联突变的影响既取决于位置，也取决于氨基酸。总体而言，我们的数据表明，疾病突变会阻碍应力诱导的UBQLN2冷凝物的组装并改变其粘弹性，从而可能导致在ALS中常见的聚集体。