Date of Award

2014

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Professor Jochen HM Prehn

Second Supervisor

Dr Caoimhin G Concannon

Abstract

The Endoplasmic Reticulum (ER) provides the environment for the folding and posttranslational modification of all secreted proteins in eukaryotic cells. Disruption of ER homeostasis triggers the unfolded protein response (UPR). Upon accumulation of unfolded proteins three transmembrane proteins IRE1, PERK and ATF6 are activated. Their combined signalling is aimed at reducing the folding load and enhancing ER folding capacity. If these processes fail to re-establish homeostasis the cell undergoes apoptosis. ER-stress was found to contribute to the pathogenesis of a range of different disease, including diabetes and neurodegenerative diseases

Here we sought to investigate the temporal activation patterns of the UPR signal transducers IRE1, PERK and ATF6 and contribution of signalling events initiated by them to cell death decisions in response to ER-stress by employing a single cell imaging approach.

In order to observe the kinetics of UPR signalling, cell lines stably expressing fluorescent protein reporter constructs for the IRE1-, PERK- or ATF6-signalling branch were generated. Monitoring the activation of the UPR-reporters and cell death employing high content time lapse live cell imaging we found that responses to ER-stress suggested a temporal rather than a concentration dependent switch from pro-survival to proapototic signalling. Furthermore our data indicated different activation pattern of IRE1-, PERK- and ATF6-signalling in response to different ER-stress inducing agents.

Single cell analysis of ER-stress responses suggested that early onset and a high rate of IRE1 mediated XBPl-splicing as well as a low translation rate of PERK dependent ATF4 and a late dephosphorylation of elF2a are beneficial for cell outcome. Attenuation of IRE1- and PERKsignalling was observed in both surviving and dying cells, suggesting that neither event is decisive for cellular fate.

Further investigation of the UPR response by silencing expression of IRE1, PERK or ATF6 in the UPR-reporter cell lines, showed that deficiency of the IRE1-XBP1 signalling led to an earlier onset of cell death, while loss of PERK-signalling and translational attenuation increased cell death at an early stage of the experiment. Cells deficient of ATF6-signalling did not show a changed onset or magnitude of cell death. However, our data suggests a role for ATF6 in the attenuation of IRE1- and PERK-activity.

Taken together the live cell time lapse imaging approach employed here yielded new insights into the relationship between activation and attenuation of the UPR and cell death decisions in response to ER-stress.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.

File Size

10.2 MB

Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2014.

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