An image-processing protocol to quantify Akt localization in different subcellular compartments

MATÍAS BLAUSTEIN; ESTAFANÍA PIEGARI; ALEJANDRO COLMAN-LERNER; HERNÁN GRECCO

Córdoba

Congreso; Reunión anual SAIB; 2016

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Akt is a kinase involved in a great variety of processes such as cell proliferation, survival and malignant transformation. Several posttranslational modifications (PTMs)as well as numerous subcellular localizations have been reported for Akt recently. However, it is unclear how and why the localization of Akt is regulated, and how this information is encoded in the repertoire of Akt PTMs (?Akt molecular code?). In order to shed light on these questions we studied how Akt variants (WT and mutants) are recruited to different organelles. Qualitatively, we observed differences between Akt mutants that simulate or abolish certain PTMs. In particular, we found that Akt is recruited to Golgi, a localization that has not been previously reported. We found new substrates and PTMs associated to this localization. However, cell-to- cell variability in Akt localization patterns leads to the necessity of improving the methods to quantify robustly the presence of Akt in different subcellular compartments. To this end, we plan to develop a protocol for quantitative measurement of Akt localization and its substrates in the nucleus, cytosol, mitochondria and other organelles, by performing image-processing that combines cellular segmentation in Cell Profiler and co-localization analysis in Matlab. Here we show preliminary measurements and the strategies taken to improve the protocol.