Mapping Dynamic Protein Clustering with AIEgen‐Active Chemigenetic Probe

Abstract Protein clustering/disassembling is a fundamental process in biomolecular condensates, playing a crucial role in cell fate decision and cellular homeostasis. However, the inherent features of protein clustering, especially for its reversible behavior and subtle microenvironment variation, present significant hurdles in probe chemistry for tracking protein clustering dynamics. Herein, we report a bilateral‐tailored chemigenetic probe, in which an “amphiphilic” aggregate‐induced emission luminogen (AIEgen) QMSO 3 Cl is covalently conjugated to a protein tag that is genetically fused to protein‐of‐interest (POI). Prior to target POI, the “amphiphilic” AIE‐active QMSO 3 Cl achieves a completely dark state in both aqueous biological environment and lipophilic organelles, thereby ensuring an ultra‐low intrinsic background interference. Upon reaching POI, the combination of synthetic molecule and genetically encoded protein allows for protein clustering‐dependent ultra‐sensitive response, with a substantial lighting‐up fluorescence (67.5‐fold) as protein transitions from disassembling to clustering state. Such ultra‐high signal‐to‐noise ratio enables to monitor the dynamic and fate of inositol requiring enzyme 1 (IRE1) clustering/disassembling under both acute and chronic endoplasmic reticulum (ER) stress in living cells. For the first time, we have demonstrated the use of chemigenetic probe to reveal therapy‐induced ER stress and screen drugs in a three‐dimensional scenario: microviscosity change, clustering dynamic, and cluster morphology. This chemigenetic probe design strategy would greatly facilitate the advancement of mapping protein dynamics in cell homeostasis and medicine research.