title: Detection and quantification of endoplasmic reticulum stress in living cells using the fluorescent compound, Thioflavin T.
abstract: The endoplasmic reticulum (ER) plays a central role in the co- and post-translational modification of many proteins. Disruption of these processes can lead to the accumulation of misfolded proteins in the endoplasmic reticulum - a condition known as endoplasmic reticulum stress. In recent years, the association of endoplasmic reticulum stress with a number of disease pathologies has increased interest in the study of this condition. Current methods to detect endoplasmic reticulum stress are indirect and retrospective. Here we describe a new method to detect and quantify endoplasmic reticulum stress in live cells using Thioflavin T (ThT), a small molecule that exhibits enhanced fluorescence when it binds to protein aggregates. We show that enhanced ThT-fluorescence correlates directly with established indicators of unfolded protein response activation. Furthermore, enhanced ThT-fluorescence can be detected in living cells within 20 min of application of an endoplasmic reticulum stress-inducing agent. ThT is capable of detecting endoplasmic reticulum stress induced by distinctly different conditions and compounds, in different cultured cell types as well as in mouse tissue samples. Pre-treatment with a potent endoplasmic reticulum stress-reducing agent, 4-phenylbutyric acid, mitigates the enhanced ThT signal. This new tool will be useful in future research investigating the role of protein misfolding in the development and/or progression of human diseases.
authors.affiliation: Thrombosis and Atherosclerosis Research Institute, McMaster University, Hamilton, ON, Canada.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Beriault
authors.forename: Geoff H
authors.initials: GH
authors.lastname: Werstuck
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000378:metabolism / Q000473:pathology*; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000473:pathology*; D059865:Endoplasmic Reticulum Stress; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology*; D005456:Fluorescent Dyes; D056945:Hep G2 Cells; D006801:Humans; D015151:Immunoblotting; D008099:Liver / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D018345:Mice, Knockout; D017510:Protein Folding; D055503:Protein Multimerization; D013844:Thiazoles; D056811:Unfolded Protein Response
chemical_list: D052160:Benzothiazoles; D005456:Fluorescent Dyes; D013844:Thiazoles; C009462:thioflavin T
keywords: Detection; Endoplasmic reticulum stress; Thioflavin T; Unfolded protein response