Real-time visualization of STAT activation in live cells using genetically encoded biosensors

Signal transducer and activator of transcription (STAT) is a family of key transcriptional regulators in immune, epithelial and mesenchymal cells. Aberrant STAT activity is associated with malignancy, autoimmunity and immunodeficiency. The STAT signaling pathways are very attractive drug targets; however, validated tools to monitor real-time activation of STATs are lacking. Here, we developed a class of highly sensitive genetically encoded STAT biosensors, termed STATeLights, which allowed direct and continuous detection of STAT activity in live cells with high spatiotemporal resolution. Using human STAT5A, we demonstrate the versatility of STATeLight5A to quantify the activation of wild-type STAT5 versus disease-associated STAT5 mutants and to precisely select compounds targeting the STAT5 signaling pathway. Moreover, STATeLight5A also facilitated real-time tracking of STAT5 activation in human primary CD4⁺ T cells. Collectively, our biosensors open up unprecedented possibilities of studying STAT biology and druggability in various cellular contexts.