SIMULTANEOUS SINGLE-CELL QUANTITATIVE ANALYSIS OF THE UPR PATHWAYS THROUGH THE USE OF FLUORESCENT REPORTERS IN CULTURED CELLS

SANCHEZ G; IGAZ LM; CHARIF S; COLMAN-LERNER A; COTARELO M; MUAYA M; BLAUSTEIN M

Parana, Entre Rios

Congreso; LIV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2018

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

The Unfolded Protein Response (UPR) is acellular stress signaling cascade essentially triggered by the accumulation ofmisfolded proteins in the Endoplasmic Reticulum (ER). Three mechanisticallydistinct pathways (IRE1, PERK and ATF6) make up this collective response aimedat restoring homeostasis. Evidence hasbeen reported about the UPR being linked with the development of malignanttumors and neurodegenerative diseases.In order to characterize the UPR dynamics, we employed a set of fluorescent reportersthat allows us to monitor the activation of the UPR in human single cells andin real time. We have previously characterized novel reporters for the ATF6 andIRE1 pathways, and we describe here the design of a complementary 5?uORF ATF4-mCherry reporter for the PERKpathway. We have also developed a protocol for automated imaging, segmentation and tracking of single cells, which allowed us to perform a simultaneous quantitative analysis of the activation of the three UPR pathways.Interestingly, activation of the UPR in patients suffering from frontotemporal lobar degeneration (FTLD)and amyotrophic lateral sclerosis (ALS) has been proposed to be linked to the toxicity of Tar DNA bindingProtein-43 (TDP-43), the main component of intracellular inclusions related tothese diseases. Here we show,using our reporters, that TDP-43 regulates the unfolded protein response. These findings will contribute tounderstand the etiology of TDP-43 proteinopathies.