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    Development of a dual reporter system to simultaneously visualize ca2+ signals and ampk activity
    (2024) Erdoğan, Yusuf Ceyhun; Pilic, Johannes; Gottschalk, Benjamin; Yiğit, Esra Nur; Zaki, Asal Ghaffari; Öztürk, Gürkan; Eroğlu, Emrah; Okutan, Begüm; Sommer, Nicole G.; Weinberg, Annelie M.; Schindl, Rainer; Graier, Wolfgang F.; Malli, Roland
    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.
  • Küçük Resim
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    Hexokinase 1 forms rings that regulate mitochondrial fission during energy stress
    (2024) Pilic, Johannes; Gottschalk, Benjamin; Bourgeois, Benjamin; Habisch, Hansjörg; Koshenov, Zhanat; Oflaz, Furkan Enes; Erdoğan, Yusuf Can; Miri, Seyed Mohammad; Yiğit, Esra Nur; Aydın, Mehmet Şerif; Öztürk, Gürkan; Eroğlu, Emrah; Shoshan Barmatz, Varda; Madl, Tobias; Graier, Wolfgang F.; Malli, Roland
    Metabolic enzymes can adapt during energy stress, but the consequences of these adaptations remain understudied. Here, we discovered that hexokinase 1 (HK1), a key glycolytic enzyme, forms rings around mitochondria during energy stress. These HK1-rings constrict mitochondria at contact sites with the endoplasmic reticulum (ER) and mitochondrial dynamics protein (MiD51). HK1-rings prevent mitochondrial fission by displacing the dynamin-related protein 1 (Drp1) from mitochondrial fission factor (Mff) and mitochondrial fission 1 protein (Fis1). The disassembly of HK1-rings during energy restoration correlated with mitochondrial fission. Mechanistically, we identified that the lack of ATP and glucose-6-phosphate (G6P) promotes the formation of HK1-rings. Mutations that affect the formation of HK1-rings showed that HK1-rings rewire cellular metabolism toward increased TCA cycle activity. Our findings highlight that HK1 is an energy stress sensor that regulates the shape, connectivity, and metabolic activity of mitochondria. Thus, the formation of HK1-rings may affect mitochondrial function in energy-stress-related pathologies.