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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.

ABSTRACT: 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.

SUBMITTER: Walter F

PROVIDER: S-EPMC4532775 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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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.

Walter F F Schmid J J Düssmann H H Concannon C G CG Prehn J H M JH

Cell death and differentiation 20150130 9

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 rem ...[more]

PMID: 25633195

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Project description:The unfolded protein response (UPR) is a conserved cellular response to the accumulation of proteinaceous material in endoplasmic reticulum (ER), active both in health and disease to alleviate cellular stress and improve protein folding. Multiple epiphyseal dysplasia (EDM5) is a genetic skeletal condition and a classic example of an intracellular protein aggregation disease, whereby mutant matrilin-3 forms large insoluble aggregates in the ER lumen, resulting in a specific 'disease signature' of increased expression of chaperones and foldases, and alternative splicing of the UPR effector XBP1. Matrilin-3 is expressed exclusively by chondrocytes thereby making EDM5 a perfect model system to study the role of protein aggregation in disease. In order to dissect the role of XBP1 signalling in aggregation-related conditions we crossed a p.V194D Matn3 knock-in mouse model of EDM5 with a mouse line carrying a cartilage specific deletion of XBP1 and analysed the resulting phenotype. Interestingly, the growth of mice carrying the Matn3 p.V194D mutation compounded with the cartilage specific deletion of XBP1 was severely retarded. Further phenotyping revealed increased intracellular retention of amyloid-like aggregates of mutant matrilin-3 coupled with dramatically decreased cell proliferation and increased apoptosis, suggesting a role of XBP1 signalling in protein accumulation and/or degradation. Transcriptomic analysis of chondrocytes extracted from wild type, EDM5, Xbp1-null and compound mutant lines revealed that the alternative splicing of Xbp1 is crucial in modulating levels of protein aggregation. Moreover, through detailed transcriptomic comparison with a model of metaphyseal chondrodysplasia type Schmid (MCDS), an UPR-related skeletal condition in which XBP1 was removed without overt consequences, we show for the first time that the differentiation-state of cells within the cartilage growth plate influences the UPR resulting from retention of a misfolded mutant protein and postulate that modulation of XBP1 signalling pathway presents a therapeutic target for aggregation related conditions in cells undergoing proliferation.

Dataset Information

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.

Publications

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.

Similar Datasets

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