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Mapping Core Components of Membrane-less Organelles in Living Cells by Phase-APEX2 Proximity Labeling

Chen, Z.; Chen, Y.; Ding, H.; Huang, M.; Chen, W.; Fu, B.; Wang, M.; Zhang, L.; Qin, W.; Li, P.

2025-12-17 cell biology
10.64898/2025.12.17.694854 bioRxiv
Show abstract

Membrane-less organelles (MLOs), formed via liquid-liquid phase separation, are crucial for organization of biomacromolecules and cellular processes, yet their dynamic nature challenges compositional analysis. Conventional proximity labeling methods lack the specificity to overcome high background and systematic biases inherent in studying these condensates. Here we introduce Phase-APEX2, a three-input AND-gate strategy that dramatically enhances labeling specificity. By integrating a split-APEX2 system with a light-controlled H2O2 generator, we developed Phase-APEX2 platforms for two application contexts: Phase-APEX2-MS for proteomics and Phase-APEX2-seq for genomics. Our analyses revealed not only the core interactomes of the nucleolus and stress granules with high accuracy but also uncovered distinct "molecular grammars" governing their assembly and a surprising link between nucleolar dynamics and the transcriptional regulation of mitosis-related genes. This highly specific and adaptable platform opens new avenues for exploring the composition and function of diverse biomolecular condensates.

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