Imaging of single cell responses to ER stress indicates that the relative dynamics of IRE1/XBP1 and PERK/ATF4 signalling rather than a switch between signalling branches determine cell survival.

An accumulation of misfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR) mediated via the activation of three transmembrane proteins IRE1, PERK and ATF6. Signalling through these proteins is aimed at enhancing the ER folding capacity and reducing the folding load. If these processes fail to re-establish protein homeostasis within the ER, then cell death prevails via apoptosis. How the shift from pro-survival to pro-apoptotic signalling is regulated remains unclear with both IRE1 and PERK signalling associated with pro-survival as well as pro-apoptotic signalling. To investigate the temporal activation of IRE1 and PERK in live cells and their relationship to cellular fate, we devised single cell reporters for both ER stress signalling branches. SH-SY5Y neural cells stably expressing these fluorescent protein reporter constructs to monitor IRE1-splicing activity and PERK-mediated ATF4-translation were imaged using single cell and high content time lapse live cell microscopy. We could correlate an early onset and attenuation of XBP1 splicing in the IRE1-reporter cells as cytoprotective. Indeed, silencing of IRE1 expression using shRNA inhibited splicing of XBP1 resulting in an early onset of cell death. In contrast, in the PERK-reporter cells, we observed that a slow rate of ATF4-translation and late re-initiation of general translation coincided with cells which were resistant to ER stress-induced cell death. Interestingly, whereas silencing of PERK did not affect overall levels of cell death in response to ER stress, it did increase sensitivity to ER stressors at early time points following treatment. Our results suggest that apoptosis activation in response to ER stress is not caused by a preferential activation of a single UPR branch, or by a switch from one branch to the other. Rather, our data indicated that the relative timing of IRE1 and PERK signalling determines the shift from cell survival to apoptosis.