Development of a Dual Reporter System to Simultaneously Visualize Ca2+ Signals and AMPK Activity

Erdoğan Y. C., Pilic J., Gottschalk B., Yiğit E. N., Zaki A. G., Öztürk G., ...More

ACS Sensors, vol.9, no.9, pp.4680-4689, 2024 (SCI-Expanded) identifier

  • Publication Type: Article / Article
  • Volume: 9 Issue: 9
  • Publication Date: 2024
  • Doi Number: 10.1021/acssensors.4c01058
  • Journal Name: ACS Sensors
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Chemical Abstracts Core, Compendex, EMBASE, INSPEC, MEDLINE
  • Page Numbers: pp.4680-4689
  • Keywords: AMPK reporter, Ca2+ biosensor, fluorescence microscopy, GCaMP, multiplexing, phase separation-based activity reporter of kinase, SPARK
  • Istanbul Medipol University Affiliated: Yes

Abstract

In 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.