An RNF4-Based Tool for Tracking Subcellular Localization of PolySUMOylation During Cellular Stress.

SUMOylation is a rapid and dynamic process that orchestrates the switch between complex assembly and disassembly and between protein stabilization and turnover, making it particularly suitable for regulating stress responses. While proteomic methodologies exist for analyzing SUMOylated proteins under stress conditions, methods/tools for visualizing polySUMOylation dynamics have not been established. Here, we develop a polySUMOylation tracking tool by fluorescently labeling the polySIM domains derived from RNF4, which can reliably track polySUMO location and relate polySUMOylation levels to puncta number and intensity under various stress conditions, such as serum starvation, oxidative stress, and genotoxic stress. Furthermore, we extend its utility for tracking polySUMOylation across multiple cellular contexts in both control and stressed states. Collectively, this tracking tool enables deeper investigation of polySUMOylation dynamics and advances our understanding of how polySUMOylation regulates cellular processes in stress responses and disease pathogenesis.