Stress granules (SGs) are membraneless organelles formed in the cytoplasm by liquid-liquid phase separation (LLPS) of translationally-stalled mRNA and RNA-binding proteins during stress response. Understanding the mechanisms governing SG assembly requires imaging SG formation in real time. Although numerous SG proteins have been identified, the kinetics of their recruitment during SG assembly has not been well established. Here we used live cell imaging and super-resolution imaging to visualize SG assembly in human cells. We found that IGF2BP proteins formed microscopically visible clusters in living cells almost instantaneously after osmotic stress, followed by fusion of clusters and the recruitment of G3BP1 and TIA1. Rapid clustering of IGF2BP1 was reduced in cells pretreated with emetine that stabilizes polysomes on mRNA. The KH3/4 di-domain and an intrinsically disordered region (IDR) of IGF2BP1 were found to mediate its clustering. Super-resolution imaging confirmed the formation of IGF2BP clusters associated with mRNA at 40 s after osmotic stress. In mature SGs, multiple clusters of poly(A) mRNA were found to associate with the periphery and the interior of a dense granule formed by IGF2BP1. Taken together, our findings revealed a novel, multi-stage LLPS process during osmotic stress, in which rapid clustering of IGF2BP proteins initiates SG assembly.

应激颗粒（SGs​​）是在应激反应期间通过翻译固定的mRNA和RNA结合蛋白的液-液相分离（LLPS）在细胞质中形成的无膜细胞器。了解控制SG组装的机制需要实时对SG形成进行成像。尽管已鉴定出许多SG蛋白，但尚未很好地确定其在SG组装过程中募集的动力学。在这里，我们使用活细胞成像和超分辨率成像来可视化人细胞中的SG装配。我们发现，IGF2BP蛋白在渗透压后几乎立即在活细胞中形成了微观可见的簇，随后簇融合并募集了G3BP1和TIA1。依替丁预处理的细胞中IGF2BP1的快速聚簇减少了，后者使多核糖体稳定在mRNA上。发现IGF2BP1的KH3 / 4双结构域和固有无序区（IDR）介导其聚类。超分辨率成像证实了渗透胁迫后40 s与mRNA相关的IGF2BP簇的形成。在成熟的SG中，发现poly（A）mRNA的多个簇与IGF2BP1形成的致密颗粒的外围和内部相关。综上所述，我们的发现揭示了在渗透胁迫期间一个新颖的，多阶段的LLPS过程，其中IGF2BP蛋白的快速聚集启动了SG组装。发现poly（A）mRNA的多个簇与IGF2BP1形成的致密颗粒的外围和内部相关。综上所述，我们的发现揭示了在渗透胁迫期间一个新颖的，多阶段的LLPS过程，其中IGF2BP蛋白的快速聚集启动了SG组装。发现poly（A）mRNA的多个簇与IGF2BP1形成的致密颗粒的外围和内部相关。综上所述，我们的发现揭示了在渗透胁迫期间一个新颖的，多阶段的LLPS过程，其中IGF2BP蛋白的快速聚集启动了SG组装。