Simultaneous quantitative analysis of the unfolded protein response through the use of fluorescent reporters in cultured single cells

MARÍA COTARELO; SANTIAGO CHARIF; GONZALO SÁNCHEZ; MARIANA SUAYA; ALEJANDRO COLMAN-LERNER; LIONEL MULLER IGAZ; MATÍAS BLAUSTEIN

Paraná

Congreso; LIV Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2018

SAIB

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 a complementary 5?uORF ATF4-mCherry reporter for the PERK pathway. We have also developed a protocol for automated imaging, segmentation and tracking of single cells, which allow us to perform a simultaneous quantitative analysis of the three UPR pathways and to understand the decision-making mechanisms involved in UPR-regulated cell death or survival. Interestingly, some evidences show that the UPR might be associated with neurodegenerative diseases: activation of UPR in patients suffering from frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) has been linked to the toxicity of Tar DNA binding Protein-43 (TDP-43), the main component of intracellular inclusions related to these diseases. We are currently evaluating if TDP-43 regulates the unfolded protein response, a process that modifies global protein synthesis. These findings will contribute to understand the etiology of TDP-43 proteinopathies.