Mapping Dynamic Protein Clustering with AIEgen-Active Chemigenetic Probe.

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Protein clustering is vital for cell fate and homeostasis, but tracking its dynamics is challenging due to reversible behavior and microenvironment variations.
• Existing probe chemistries face limitations in detecting subtle changes in protein clustering and its associated microenvironment.
• Understanding protein clustering dynamics is crucial for deciphering cellular processes and disease mechanisms.

Purpose of the Study:

• To develop a novel chemigenetic probe for ultra-sensitive tracking of protein clustering dynamics.
• To enable real-time monitoring of protein clustering and disassembling in response to cellular stress.
• To demonstrate the probe's utility in revealing therapy-induced endoplasmic reticulum stress and facilitating drug screening.

Main Methods:

• A bilateral-tailored chemigenetic probe was designed, conjugating an amphiphilic aggregate-induced emission luminogen (AIEgen) QMSO3Cl to a genetically fused protein tag.
• The probe operates in a dark state until it interacts with the protein-of-interest (POI), ensuring low background interference.
• The probe exhibits a significant fluorescence "lighting-up" (67.5-fold increase) upon protein clustering, enabling sensitive detection.

Main Results:

• The chemigenetic probe demonstrated an ultra-high signal-to-noise ratio for monitoring protein clustering dynamics.
• The probe successfully tracked the clustering/disassembling of inositol requiring enzyme 1 (IRE1) under acute and chronic endoplasmic reticulum (ER) stress in living cells.
• The study showcased the probe's ability to analyze microviscosity changes, clustering dynamics, and cluster morphology in a 3D context.

Conclusions:

• The developed chemigenetic probe provides a powerful tool for mapping dynamic protein clustering in real-time within living cells.
• This approach significantly advances the study of cell homeostasis, endoplasmic reticulum stress, and drug discovery.
• The probe design strategy offers a promising avenue for future research in molecular and cellular dynamics.