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Öğe A co-culture-based multiparametric imaging technique to dissect local H2O2 signals with targeted HyPer7(MDPI, 2021) Seçilmiş, Melike; Altun, Hamza Yusuf; Pilic, Johannes; Erdoğan, Yusuf Ceyhun; Çokluk, Zeynep; Ata, Büşra Nur; Sevimli, Gülşah; Zaki, Asal Ghaffari; Yiğit, Esra Nur; Öztürk, Gürkan; Malli, Roland; Eroğlu, EmrahMultispectral live-cell imaging is an informative approach that permits detecting biological processes simultaneously in the spatial and temporal domain by exploiting spectrally distinct biosensors. However, the combination of fluorescent biosensors with distinct spectral properties such as different sensitivities, and dynamic ranges can undermine accurate co-imaging of the same analyte in different subcellular locales. We advanced a single-color multiparametric imaging method, which allows simultaneous detection of hydrogen peroxide (H2O2) in multiple cell locales (nucleus, cytosol, mitochondria) using the H2O2 biosensor HyPer7. Co-culturing of endothelial cells stably expressing differentially targeted HyPer7 biosensors paved the way for co-imaging compartmentalized H2O2 signals simultaneously in neighboring cells in a single experimental setup. We termed this approach COMPARE IT, which is an acronym for co-culture-based multiparametric imaging technique. Employing this approach, we detected lower H2O2 levels in mitochondria of endothelial cells compared to the cell nucleus and cytosol under basal conditions. Upon administering exogenous H2O2, the cytosolic and nuclear-targeted probes displayed similarly slow and moderate HyPer7 responses, whereas the mitochondria-targeted HyPer7 signal plateaued faster and reached higher amplitudes. Our results indicate striking differences in mitochondrial H2O2 accumulation of endothelial cells. Here, we present the method's potential as a practicable and informative multiparametric live-cell imaging technique.Öğe 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, RolandIn 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.Öğe 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, RolandMetabolic 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.Öğe Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels(Elsevier B.V., 2022) Sevimli, Gülşah; Smith, Matthew J.; Akgül Çağlar, Tuba; Bilir, Şükriye; Seçilmiş, Melike; Altun, Hamza Y.; Yiğit, Esra N.; Yang, Fan; Keeley, Thomas P.; Malli, Roland; Öztürk, Gürkan; Mann, Giovanni E.; Eroğlu, EmrahIron is an essential metal for cellular metabolism and signaling, but it has adverse effects in excess. The physiological consequences of iron deficiency are well established, yet the relationship between iron supplementation and pericellular oxygen levels in cultured cells and their downstream effects on metalloproteins has been less explored. This study exploits the metalloprotein geNOps in cultured HEK293T epithelial and EA.hy926 endothelial cells to test the iron-dependency in cells adapted to standard room air (18 kPa O2) or physiological normoxia (5 kPa O2). We show that cells in culture require iron supplementation to activate the metalloprotein geNOps and demonstrate for the first time that cells adapted to physiological normoxia require significantly lower iron compared to cells adapted to hyperoxia. This study establishes an essential role for recapitulating oxygen levels in vivo and uncovers a previously unrecognized requirement for ferrous iron supplementation under standard cell culture conditions to achieve geNOps functionality.Öğe Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels (vol 53, 102319, 2022)(Elsevier, 2022) Sevimli, Gülşah; Smith, Matthew J.; Akgül Çağlar, Tuba; Bilir, Şükriye; Seçilmiş, Melike; Altun, Hamza Y.; Yiğit, Esra N.; Yang, Fan; Keeley, Thomas P.; Malli, Roland; Öztürk, Gürkan; Mann, Giovanni E.; Eroğlu, EmrahThe authors regret that the cited reference list in the online PDF of this article contains a duplicate reference (Ref 34 and incomplete Ref 56). Ref 56 should read as follows.Öğe Probing intracellular potassium dynamics in neurons with the genetically encoded sensor lc-LysM GEPII 1.0 in vitro and in vivo(2024) Groschup, Bernhard; Calandra, Gian Marco; Raitmayr, Constanze; Shrouder, Joshua; Llovera, Gemma; Zaki, Asal Ghaffari; Burgstaller, Sandra; Bischof, Helmut; Eroğlu, Emrah; Liesz, Arthur; Malli, Roland; Filser, SeverinNeuronal activity is accompanied by a net outflow of potassium ions (K+) from the intra- to the extracellular space. While extracellular [K+] changes during neuronal activity are well characterized, intracellular dynamics have been less well investigated due to lack of respective probes. In the current study we characterized the FRET-based K+ biosensor lc-LysM GEPII 1.0 for its capacity to measure intracellular [K+] changes in primary cultured neurons and in mouse cortical neurons in vivo. We found that lc-LysM GEPII 1.0 can resolve neuronal [K+] decreases in vitro during seizure-like and intense optogenetically evoked activity. [K+] changes during single action potentials could not be recorded. We confirmed these findings in vivo by expressing lc-LysM GEPII 1.0 in mouse cortical neurons and performing 2-photon fluorescence lifetime imaging. We observed an increase in the fluorescence lifetime of lc-LysM GEPII 1.0 during periinfarct depolarizations, which indicates a decrease in intracellular neuronal [K+]. Our findings suggest that lc-LysM GEPII 1.0 can be used to measure large changes in [K+] in neurons in vitro and in vivo but requires optimization to resolve smaller changes as observed during single action potentials.Öğe 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, EmrahCellular 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.Öğe 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, RolandStudying 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.
