Genetically-Encoded Phase Separation Sensors EnableHigh-Fidelity Live-Cell Probing of Biomolecular Condensates

Biomolecular condensates are membraneless compartments with enigmatic roles across intracellular phenomena. Intrinsically disordered proteins (IDPs) often function as condensate scaffolds, fueled by liquid–liquid phase separation (LLPS) dynamics. Intracellular probing of condensates relies on live-cell imaging of IDP-scaffolds tagged with fluorescent proteins. Conformational heterogeneity in IDPs, however, renders them uniquely susceptible to artifacts from tagging. Probing epidermal condensates in skin, we recently introduced genetically-encoded LLPS-sensors that circumvent the need for molecular-level tagging of skin IDPs. Departing from subcellular tracking of IDP-scaffolds, LLPS-sensors report on the assembly and liquid-like dynamics of their condensates. Here, we demonstrate biomolecular approaches for the evolution and tunability of epidermal LLPS-sensors and assess their impact in the early and late stages of intracellular phase separation. Benchmarking against scaffold-bound fluorescent reporters, we discovered that tunable ultraweak scaffold–sensor interactions uniquely enable the sensitive and innocuous probing of nascent and established biomolecular condensates. Our LLPS-sensitive tools pave the way for the high-fidelity intracellular probing of IDP-governed biomolecular condensates across biological systems.