Genetically-Encoded Phase Separation Sensors Enable High-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.