A light-switching pyrene probe to detect phase-separated biomolecules

Biomolecules may undergo liquid-liquid phase separation (LLPS) to spatiotemporally compartmentalize and regulate diverse biological processes. Because the number of tools to directly probe LLPS is limited (ie. FRAP, FRET, fluorescence microscopy, fluorescence anisotropy, circular dichroism, etc.), the physicochemical traits of phase-separated condensates remain largely elusive. Here, we introduce a light-switching dipyrene probe (Pyr-A) that forms monomers in either hydrophobic or viscous environments, and intramolecular excimers in aqueous solutions. By exploiting their distinct fluorescence emission spectra, we used fluorescent microscopic imaging to study phase-separated condensates formed by in vitro protein droplets and membraneless intracellular organelles (centrosomes). Ratiometric measurement of excimer and monomer fluorescence intensities showed that protein droplets became hydrophobic and viscous as their size increased. Moreover, centrosomes became hydrophobic and viscous during maturation. Our results show that Pyr-A is a valuable tool to characterize LLPS and enhance our understanding of phase separation underlying biological functions.