Fluorescence based microviscosity mapping in membraneless organelles

Abstract Membraneless organelles (MLOs) are cellular biomolecular condensates formed by liquid-liquid phase separation. Their biological functions are intimately linked to their material properties including viscosity. Condensate viscosity is determined by the size, shape, concentration and molecular interactions between MLOs components. It impacts the diffusion of MLOs constituents and the selective permeability of the condensate, thereby regulating the rate of biochemical reactions. Viscosity modifications associated with liquid-to-gel transition of the condensates are related to pathologies. Current experimental approaches for characterizing the material properties of cellular condensates remain limited. In this study, we report the use of BODIPY-based molecular rotors, in combination with fluorescence lifetime imaging microscopy (FLIM), to monitor the microviscosity of cellular MLOs directly in living cells. The fluorescence lifetime of BODIPY derivatives increases with the viscosity of their microenvironment, enabling quantitative assessment of microviscosity within condensates. HaloTag technology was employed to specifically label MLO components. Our findings reveal that the nucleolus exhibits higher viscosity than the surrounding nucleoplasm and that microviscosity varies across nucleolar sub-compartments. Furthermore, nucleolar reorganization induced by inhibition of rRNA synthesis results in a measurable increase in microviscosity. Finally, we demonstrate that the microviscosity of stress granules is lower than that of the nucleolus. Overall, presented results demonstrate the strong potential of the BODIPY based molecular rotors as a versatile and powerful tools for probing the material properties of cellular MLOs.