Spatio-temporal cell signaling dynamics: towards an Akt fingerprint

MATIAS BLAUSTEIN; DANIELA CHINY BARRIONUEVO; ALAN BUSH; JOSÉ CLEMENTE; ALEJANDRO COLMAN-LERNER

Bariloche

Simposio; The Third South American Symposium in Signal Transduction and Molecular Medicine - SISTAM; 2015; 2015

SISTAM

Cell-to-cell variability (CCV), also known as phenotypic heterogeneity, is a commonly observed phenomenon in normal and pathological conditions. Cancer is perhaps the most challenging situation in which to investigate what information is contained in CCV and how to use this information in therapy. Deregulation of Akt has been widely linked to cancer. This kinase orchestrates many biological functions including cell proliferation and survival. Our leading hypothesis is that the information contained in the expression level, posttranslational modifications, activity and specific subcellular localization of each Akt isoform in each cell may explain and predict differential Akt target specificity, CCV in cell fate choices as well as in anti-tumor drug sensitivity. As an initial approach to test this hypothesis, we have developed immunofluorescence-based technics and designed fluorescent reporters to study CCV in the spatio-temporal dynamics of the Akt pathway. Here, we describe a protocol for automated imaging and quantitative measurement and analysis of the localization and phosphorylation profiles of Akt and its substrates. The combined dataset obtained with this suite of techniques defines what we call the ?Akt fingerprint?. We found novel subcellular compartments were Akt is recruited and we are now analyzing the molecular mechanisms that govern relocalization to these places. Our results indicate that the newly described Akt destinations are associated to specific cross-talks with other signaling pathways, such as the Unfolded Protein Response (UPR), a cellular stress signaling cascade linked to the Endoplasmic Reticulum (ER), which is also deregulated during cancer. In the long term, we hope that analysis of Akt substrates by phosphorylation and localization profiles as well as classification of cancer cell lines by Akt fingerprint can lead us to understand complex cell and tumor behaviors that escape human intuition.