Analysis of signaling pathways dynamics in animal single cells

MATÍAS BLAUSTEIN; ALICIA GRANDE; ALEJANDRO COLMAN-LERNER

Campana, Buenos Aires, Argentina

Workshop; Exciting Biologies: Biology in Balance; 2009

Cardiovascular Research Center at MGH, Fondation IPSEN y Cell Press

Given the relevance of cellular signaling in the development and function of animal cells, it is not surprising that its dysfunction is associated with cancer. We decided to study the ERK, AKT and STAT5a signaling pathways, which are activated simultaneously by ErbB receptor ligands, such as EGF or HRG. To do this, we are developing fluorescent-based relocalization reporters to characterize quantitatively dynamic aspects of signaling systems such as signal strength, speed of signal transmission and cell-to-cell variation in pathway activation. These reporters enable us to measure the activation of these pathways at the same time in single live cells. Initial characterization of our reporters already showed interesting observations. In the majority of the cells, while AKT1 co-localizes with ERK2 in the cytoplasm and the nucleus, AKT2 is found only in the cytoplasm. In unstimulated mammary cells a small population exhibits spontaneous polarized membrane localization of AKT. Stimulation with EGF triggers translocation of AKT1 and STAT5a to the cell nucleus. Intriguingly, in these conditions AKT2 relocalizes to a novel perinuclear localization in mammary cells. In stark contrast, in the highly transformed HEK293T cells, the AKT1 and AKT2 reporters are localized at plasma membrane, indicating strong activation of these pathways. In order to better understand both normal and cancer cells, and to develop effective therapies to combat signaling based diseases, we are trying to determine the importance of each pathway for cell survival or death, to analyze cell-to-cell variation and to compare these parameters in normal and cancer cells.