Use of fluorescent reporters for the analysis of Akt and UPR activation dynamics in human single cells

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

Congreso; LV Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular - SAIB; 2019

In order to process the information from the extra- and intracellular milieu, cells have evolved a multiplicity of signaling systems. At least 15% of the vertebrate?s genome protein coding capability is linked to receptors, components of signaling systems and transcription regulatory proteins. Our general goal is to study the regulation of cell signaling systems and the key mechanisms of tumor cell decision-making using a mixed approach that combines quantitative fluorescence techniques in individual cells with classical techniques of molecular biology. Particularly, we study the PI3K/Akt pathway - or cell survival pathway - and the Unfolded Protein Response (UPR) pathways. Akt, a serine/threonine protein kinase member of the AGC family, plays a central role in growth, proliferation, glucose uptake, metabolism, angiogenesis, protein translation and cell survival. Not surprisingly, a variety of human cancers exhibit deregulated Akt activity and several mouse models with activated Akt develop cancer. The 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. Tumor cells, however, evade this outcome and exploit the UPR pathways for proliferation and metastasis. Crosstalk between Akt and UPR pathways has been described. Particularly, we have previously shown that Akt is a PERK kinase, influencing UPR activation. Here, we designed and characterized a set of fluorescent reporters for each of these pathways and tested them in cells treated with traditional activators or inhibitors. In the case of UPR, we co-transfected these reporters into different tumor cell lines and were able to follow the activation of all three UPR pathways at the same time in each single cell for the first time. Specifically, we analyzed the pattern of activation of these pathways by automated segmentation of single cells and quantitative measurement of subcellular fluorescence using Cell Profiler. We validated our results by comparing reporters´s behavior with the activation of endogenous counterparts, confirming that our reporters allow us to study these pathways in an accurate and efficient way. Finally, we used our fluorescent reporters for Akt and UPR pathways in order to address whether environmental pollutants linked to cancer onset regulate these pathways.