Changing complexity into simplicity: visualization of endoplasmic reticulum autophagy employing a self-deliver viscosity sensor by lifetime imaging

Abstract

Endoplasmic reticulum autophagy (ER-phagy) is a highly specialized form of autophagy that maintains cellular homeostasis by sweeping damaged or excessive endoplasmic reticulum segments. Dysregulation of ER-phagy has profound implications for normal physiological activities and is closely associated with various diseases. Therefore, accurately visualizing ER-phagy process is imperative. Herein, we successfully developed a novel ER-targeted viscosity sensor (YKI) for conveniently and accurately visualizing ER-phagy by lifetime imaging. Compared with commercial ER Tracker Green and ER Tracker Red that required specific combination incubation reagent to stain the endoplasmic reticulum of live cell, YKI could directly target endoplasmic reticulum by the self-delivery way and without leakage, which profited from the hydrophobic interaction between the long alkyl chain of YKI and the lipid bilayer of the ER. And it could also specifically target the endoplasmic reticulum of fixed cells. Since YKI was sensitive to viscosity, it displayed short fluorescence lifetime in endoplasmic reticulum but exhibited longer fluorescence lifetime upon co-delivery with ER into lysosomes. As a consequence, YKI could achieve accurate and convenient visualization of ER-phagy alone via lifetime imaging. This work provided a powerful tool for studying ER-phagy and could promote the development of related fields.