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.

Automated summary

The study introduced a novel dipyrene probe Pyr-A to characterize liquid-liquid phase separation (LLPS) of biomolecules, shedding light on the physicochemical properties of phase-separated condensates. Using fluorescent microscopic imaging, the research investigated protein droplets and centrosomes, showing that their hydrophobic and viscous properties increased with size and maturation, respectively. Overall, Pyr-A proved to be a valuable tool for studying LLPS and enhancing understanding of biological functions underlying phase separation.