Development of\na Dual Reporter System to Simultaneously\nVisualize Ca<sup>2+</sup> Signals and AMPK Activity

In\nthis study, we\nintroduce a new separation of phases-based activity\nreporter of kinase (SPARK) for AMP-activated kinase (AMPK), named\nAMPK-SPARK, which reports the AMPK activation by forming bright fluorescent\nclusters. Furthermore, we introduce a dual reporter system, named\nGCaMP-AMPK-SPARK, by incorporating a single-fluorescent protein (FP)-based\nCa<sup>2+</sup> biosensor, GCaMP6f, into our initial design, enabling\nsimultaneous monitoring of Ca<sup>2+</sup> levels and AMPK activity.\nThis system offers the essential quality of information by single-channel\nfluorescence microscopy without the need for coexpression of different\nbiosensors and elaborate filter layouts to overcome spectral limitations.\nWe used AMPK-SPARK to map endogenous AMPK activity in different cell\ntypes and visualized the dynamics of AMPK activation in response to\nvarious stimuli. Using GCaMP-AMPK-SPARK, we revealed cell-to-cell\nheterogeneities in AMPK activation by Ca<sup>2+</sup> mobilization.\nWe anticipate that this dual reporter strategy can be employed to\nstudy the intricate interplays between different signaling networks\nand kinase activities.