Characterization of the unfolded protein response through the use of fluorescent reporters in cultured single cells and in disease models

MARÍA COTARELO; MARIELA VEGGETTI; ALEJANDRO COLMAN-LERNER; LIONEL MULLER IGAZ; MATÍAS BLAUSTEIN

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

The Unfolded Protein Response (UPR) is a cellular stress signaling cascade essentially triggered by the accumulation of misfolded proteins in the Endoplasmic Reticulum (ER). Three mechanistically distinct pathways (IRE1, PERK and ATF6) make up this collective response aimed at restoring homeostasis. However, if this objective is not met within a logical time span, the UPR triggers apoptosis. This dichotomy of outcomes makes determining the cell?s fate a real enigma which could be solved through a clear characterization of the UPR dynamics.In order to do this, we employed a set of fluorescent reporters that allows us to monitor the activation of the UPR in human single cells and in real time. We have previously characterized novel reporters for the ATF6 and IRE1 pathways, and we describe here the design of new reporters for the PERK pathway. We have also developed a protocol for automated imaging and segmentation of cells as well as for quantitative analysis of UPR activation. We aim to study the activation dynamics of the three UPR pathways in single cells in order to shed light into the cell decision-making mechanisms involved in cell death and survival. Interestingly, some evidences show that the UPR might be associated with neurodegenerative diseases: activation of UPR in patients suffering from frontotemporal lobar degeneration and amyotrophic lateral sclerosis has been linked to the toxicity of Tar DNA binding Protein-43 (TDP-43), the main component of intracellular inclusions related to these diseases. Confirmation of this integrated network would open the possibility of designing new therapies targeted to patients suffering from neurodegeneration.