Improved IRE1 and PERK Pathway Sensors for Multiplex Endoplasmic Reticulum Stress Assay Reveal Stress Response to Nuclear Dyes Used for Image Segmentation

In response to a variety of insults the unfolded protein response (UPR) is a major cell program quickly engaged to promote either cell survival or if stress levels cannot be relieved, apoptosis. UPR relies on three major pathways, named from the endoplasmic reticulum (ER) resident proteins IRE1α, PERK, and ATF6 that mediate response. Current tools to measure the activation of these ER stress response pathways in mammalian cells are cumbersome and not compatible with high-throughput imaging. In this study, we present IRE1α and PERK sensors with improved sensitivity, based on the canonical events of xbp1 splicing and ATF4 translation at ORF3. These sensors can be integrated into host cell genomes through lentiviral transduction, opening the way for use in a wide array of immortalized or primary mammalian cells. We demonstrate that high-throughput single-cell analysis offers unprecedented kinetic details compared with endpoint measurement of IRE1α and PERK activity. Finally, we point out the limitations of dye-based nuclear segmentation for live cell imaging applications, as we show that these dyes induce UPR and can strongly affect both the kinetic and dynamic responses of IRE1α and PERK pathways.