Genetically-encoded phase separation sensors for intracellular probing of biomolecular condensates.

Biomolecular condensates are dynamic membraneless compartments with enigmatic roles across intracellular phenomena. Intrinsically-disordered proteins (IDPs) often function as condensate scaffolds, fueled by their liquid-liquid phase separation (LLPS) dynamics. Intracellular probing of these condensates relies on live-cell imaging of IDP-scaffolds tagged with fluorescent proteins. Conformational heterogeneity in IDPs, however, renders them uniquely sensitive to molecular-level fusions, risking distortion of the native biophysical properties of IDP-scaffolds and their assemblies. Probing epidermal condensates in mouse skin, we recently introduced genetically encoded LLPS-sensors that circumvent the need for molecular-level tagging of skin IDPs. The concept of LLPS-sensors involves a shift in focus from subcellular tracking of IDP-scaffolds to higher-level observations that report on the assembly and liquid-dynamics of their condensates. Towards advancing the repertoire of intracellular LLPS-sensors, here we demonstrate biomolecular approaches for the evolution and tunability of epidermal LLPS-sensors and assess their impact in early and late stages of intracellular LLPS dynamics. Benchmarking against scaffold-bound fluorescent reporters, we found that tunable ultraweak scaffold-sensor interactions are key to 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.