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

Erdoğan Y. C., Pilic J., Gottschalk B., YİĞİT E. N., Zaki A. G., ÖZTÜRK G., ...Daha Fazla

ACS Sensors, 2024 (SCI-Expanded) identifier

  • Yayın Türü: Makale / Tam Makale
  • Basım Tarihi: 2024
  • Doi Numarası: 10.1021/acssensors.4c01058
  • Dergi Adı: ACS Sensors
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Chemical Abstracts Core, Compendex, EMBASE, INSPEC, MEDLINE
  • Anahtar Kelimeler: AMPK reporter, Ca2+ biosensor, fluorescence microscopy, GCaMP, multiplexing, phase separation-based activity reporter of kinase, SPARK
  • İstanbul Medipol Üniversitesi Adresli: Evet

Özet

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.