A Molecular Recruitment Colocalization Platform for Visualizing Multi-Protein Interactions and Engineering Biomolecular Condensates in Living Cells

Visualizing dynamic protein-protein interactions (PPIs) and phase-separated biomolecular condensates in live cells is crucial for understanding cellular processes. Here, a method is introduced for analyzing both PPIs and condensates, termed molecular recruitment colocalization (MRC), which localizes interacting partners and generates fluorescent signals at designated genomic loci. MRC enables the simultaneous visualization of multi-protein interactions and the preliminary estimation of binding affinities during PPIs. Through MRC, the study can quickly discover and validate new proteins or structural domains capable of forming condensates and can also integrate PPIs to study condensates. Furthermore, the versatility of MRC is demonstrated in phase-separated condensates research: programming phase separation sites to study the interactions and behaviors between different condensates, recruiting target proteins into programmable condensates, obtaining the properties of diverse engineered condensates, and probing the physical properties of condensates within live cells. Therefore, MRC can become a versatile platform for studying both PPIs and biomolecular condensates.

colocalization; condensates; live cell imaging; multi‐protein interactions; phase separation; protein binding ability.