Reactive oxygen species (ROS) are key regulators of protein aggregation in numerous aging-related diseases, yet their impact on biomolecular condensates remains poorly understood. Here, using quantitative measurements of condensate viscoelasticity, we show that ROS solidifies condensates in a dose-dependent manner both in vitro and in living cells. Excitation of fluorescently tagged proteins triggers rapid protein crosslinking in a condensate-specific manner, leading to ∼10,000-fold increases in condensate viscosity. This effect is universal across a wide range of proteins and fluorophores and does not require the presence of cysteine. In cells, excitation of GFP-tagged stress granules results in a viscosity increase that can be buffered by the cytoplasm. In contrast, condensate viscosity increases permanently upon excitation of stress granules extracted to the extracellular environment. Moreover, protein crosslinking depends on the spatial distribution of ROS, where condensates can shield proteins from ROS added externally. Our results reveal that redox level directly mediates protein crosslinking in a condensate-specific manner, thus providing new mechanistic insight into how phase separation interferes with protein aggregation and disease progression.
Wang et al. (Sun,) studied this question.