Erdoğan, Yusuf CeyhunPilic, JohannesGottschalk, BenjaminYiğit, Esra NurZaki, Asal GhaffariÖztürk, GürkanEroğlu, EmrahOkutan, BegümSommer, Nicole G.Weinberg, Annelie M.Schindl, RainerGraier, Wolfgang F.Malli, Roland2024-11-042024-11-042024Erdoğan, Y. C., Pilic, J., Gottschalk, B., Yiğit, E. N., Zaki, A. G., Öztürk, G. ... Malli, R. (2024). Development of a dual reporter system to simultaneously visualize ca2+ signals and ampk activity. ACS Sensors, 9(9), 4680-4689. http://dx.doi.org/10.1021/acssensors.4c010582379-3694http://dx.doi.org/10.1021/acssensors.4c01058https://hdl.handle.net/20.500.12511/12845In this study, we introduce a new separation of phases-based activity reporter of kinase (SPARK) for AMP-activated kinase (AMPK), named AMPK-SPARK, which reports the AMPK activation by forming bright fluorescent clusters. Furthermore, we introduce a dual reporter system, named GCaMP-AMPK-SPARK, by incorporating a single-fluorescent protein (FP)-based Ca2+ biosensor, GCaMP6f, into our initial design, enabling simultaneous monitoring of Ca2+ levels and AMPK activity. This system offers the essential quality of information by single-channel fluorescence microscopy without the need for coexpression of different biosensors and elaborate filter layouts to overcome spectral limitations. We used AMPK-SPARK to map endogenous AMPK activity in different cell types and visualized the dynamics of AMPK activation in response to various stimuli. Using GCaMP-AMPK-SPARK, we revealed cell-to-cell heterogeneities in AMPK activation by Ca2+ mobilization. We anticipate that this dual reporter strategy can be employed to study the intricate interplays between different signaling networks and kinase activities.enAttribution 4.0 Internationalinfo:eu-repo/semantics/openAccessAMPK ReporterCa2+ BiosensorFluorescence MicroscopyGcampMultiplexingPhase Separation-Based Activity Reporter Of KinaseSPARKArticle994680468910.1021/acssensors.4c01058Q10012959993000012-s2.0-8520176390139167044Q1