Revealing Stress Granule Compositional Heterogeneity through Antibody-Guided Proximity Labeling

Stress granules (SGs), transient nonmembranous cytoplasmic condensates that formed in response to cellular stresses, require precise characterization to unravel their cell-type and stressspecific protein compositions. This study introduced a G3BP1 antibody-guided proximity labeling (Ab-PL) method to explore the composition and diversity of SGs, overcoming the challenges of traditional enzyme-mediated proximity labeling techniques across various cell types, especially for the immune cells. Application of Ab- PL to HeLa and RAW264.7 cells under heat shock (HS), sodium arsenate (AS), and sodium chloride stress (SS) revealed two categories of SG proteins: “SG-core” and “SG-shell,” characterized by their different abilities to undergo phase separation. The core proteins form the SG scaffold with strong self-segregation, while shell proteins are dynamically recruited based on the type of stress. Cell- and stress-specific SG proteins were also identified, highlighting compositional heterogeneity. Intriguingly, unique nuclear-cytoplasmic shuttling behaviors of SG components were observed under varying conditions, uncovering over 10 novel SG proteins, including REXO4, RBM28, and OGFR. This study provides a versatile tool for SG analysis across diverse cell types and offers insights into SG heterogeneity, which has potential implications for human diseases, paving the way for future studies on RNA metabolism, ribosome assembly, and immune regulation.