Revealing Protein Aggregates under Thapsigargin-Induced ER Stress Using an ER-Targeted Thioflavin

Abstract

Endoplasmic reticulum-thioflavin T <b>(ER-ThT</b>), a thioflavin T-based fluorescent chemosensor, was developed to detect protein aggregates in the endoplasmic reticulum (ER) and was applied to live cells under various forms of ER stress. Upon dithiothreitol (DTT)-induced reductive denaturation of lysozyme and albumin, the intensity was increased in a protein concentration-dependent way, following a nonfluorescent lag phase. <b>ER-ThT</b> detects protein aggregates rather than unfolded proteins in solution, and the protein aggregation can be visualized in the presence of lipid membranes or native proteins. Within live HeLa cells, <b>ER-ThT</b> is localized in the ER and its fluorescence was dramatically increased upon ER stress induction by DTT, Thapsigargin, or Brefeldin A. Moreover, in the presence of ER stress modulators (tauroursodeoxycholic acid, trimethylamine <i>N</i>-oxide, or 4-phenylbutyric acid), also known as chemical chaperones, the fluorescence under Thapsigargin treatment was suppressed to the level of the control group. Thus, <b>ER-ThT</b> is capable of detecting the accumulation of protein aggregates under ER stress in living cells and acts as an in vitro screening tool for ER stress modulators, putative prodrugs against ER-related proteopathy. Overall, the results strongly suggest that protein aggregation is intricately involved in the activation of the unfolded protein response following ER stress.