Exploring Endoplasmic Reticulum Dysfunction on Protein Phase Separation Using Viscosity-Sensitive Fluorescent Lifetime Probe

Degenerative diseases are closely associated with protein phase transitions. Endoplasmic reticulum (ER), the primary site of protein synthesis, experiences homeostasis imbalance as the key trigger of the protein phase transition. Effective tools to monitor ER microenvironment changes are crucial for investigating protein phase behavior. In this work, we developed a set of viscosity-sensitive probes based on dicyanomethylene-4H-pyran (namely, VisDCM probes) with dual response of fluorescence intensity and fluorescence lifetime to local viscosity changes. Computational analysis demonstrated that fluorescence activation of VisDCM probes is due to the restricted accessible conical intersection mechanism under specific viscosity. Dual-color probes targeting the ER and protein of interest were designed. They revealed how ER stress regulates TDP-43 protein phase separation via Ca²⁺ signaling. <i>In vitro</i> experiments exhibited that TDP-43 phase separation is Ca²⁺-dependent. Increased Ca²⁺ promotes TDP-43 liquid-liquid phase separation and aggregation. Finally, fluorescence lifetime imaging was applied to map ERS-induced microenvironment changes. In summary, this work provides a novel toolbox to visualize protein phase transitions as well as highlights Ca²⁺ role in TDP-43 phase separation and aggregation, offering insights and potential therapies for degenerative diseases.