Yazar "Graier, Wolfgang F." seçeneğine göre listele

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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.
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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.
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    Probing subcellular iron availability with genetically encoded nitric oxide biosensors
    (MDPI (Multidisipliner Digital Publishing Institute), 2022) Sevimli, Gülşah; Alston, Amy E.; Funk, Felix; Flühmann, Beat; Malli, Roland; Graier, Wolfgang F.; Eroğlu, Emrah
    Cellular iron supply is required for various biochemical processes. Measuring bioavailable iron in cells aids in obtaining a better understanding of its biochemical activities but is technically challenging. Existing techniques have several constraints that make precise localization difficult, and the lack of a functional readout makes it unclear whether the tested labile iron is available for metalloproteins. Here, we use geNOps; a ferrous iron-dependent genetically encoded fluorescent nitric oxide (NO) biosensor, to measure available iron in cellular locales. We exploited the nitrosylation-dependent fluorescence quenching of geNOps as a direct readout for cellular iron absorption, distribution, and availability. Our findings show that, in addition to ferrous iron salts, the complex of iron (III) with N,N’-bis (2-hydroxybenzyl)ethylenediamine-N,N’-diacetic acid (HBED) can activate the iron (II)-dependent NO probe within intact cells. Cell treatment for only 20 min with iron sucrose was also sufficient to activate the biosensor in the cytosol and mitochondria significantly; however, ferric carboxymaltose failed to functionalize the probe, even after 2 h of cell treatment. Our findings show that the geNOps approach detects available iron (II) in cultured cells and can be applied to assay functional iron (II) at the (sub)cellular level.
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    The preamble to the free radical biology and medicine virtual special issue on “Targeting genetic biosensors to intracellular signaling pathways”
    (Elsevier Inc., 2022) Eroğlu, Emrah; Graier, Wolfgang F.; Malli, Roland
    Studying biological signaling pathways is key to understanding the various processes within a cell. The science of biological redox regulation is a rapidly growing research area with implications for various disciplines, including physiology, cell biology, and clinical medicine. Given the role that oxidative stress plays in numerous disease states, the area of redox regulation is receiving increasing attention from other life science disciplines. Balancing the beneficial and harmful effects of free radicals is an essential aspect of life. A disrupted redox balance in pathological states demonstrates the biological relevance of redox regulation and its implications for various signaling and metabolic processes. The transition that redox research undergoes these days is particularly exciting because the information from different fields and independent approaches are coming together, painting a wholly new and meaningful picture. Therefore, this is a good window of opportunity to summarize the most critical (redox)-tools and their multiparametric applications for signaling pathways and metabolic activities. This special issue brings together novel experimental model systems, informative biosensors, chemogenetic and nanobody approaches to address the redox-dependent relationship between cellular activities allowing for a more holistic approach to understanding coordinated reactions of a range of molecules in a cell.