Hexokinase 1 forms rings that regulate mitochondrial fission during energy stress.

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.

Development of a Chemogenetic Approach to Manipulate Intracellular pH.

Chemogenetic Operation of iNTRacellular prOton Levels (pH-Control) is a novel substrate-based enzymatic method that enables precise spatiotemporal control of ultralocal acidification in cultured cell lines and primary neurons. The genetically encoded biosensor SypHer3s showed that pH-Control effectively acidifies cytosolic, mitochondrial, and nuclear pH exclusively in the presence of ß-chloro-d-alanine in living cells in a concentration-dependent manner. The pH-Control approach is promising for investigating the ultralocal pH imbalance associated with many diseases.

Genetically Encoded Biosensors Unveil Neuronal Injury Dynamics via Multichromatic ATP and Calcium Imaging.

When a cell sustains damage, it liberates cytosolic ATP, which can serve as an injury signal, affecting neighboring cells. This study presents a methodological approach that employs in vitro axotomy and in vivo laser ablation to simulate cellular injury. Specially tailored biosensors are employed to monitor ATP dynamics and calcium transients in injured cells and their surroundings. To simultaneously visualize extracellular and cytosolic ATP, we developed bicistronic constructs featuring GRABATP1.0 and MaLionR biosensors alongside the calcium sensor RCaMP, enabling multiparametric imaging. In addition to transducing primary neuron cultures, we developed another method where we cocultured dorsal root ganglion neurons together with specialized "sniffer" cell lines expressing the bicistronic biosensors. Exploiting these approaches, we successfully demonstrated the release of ATP from the injured neurons and its extracellular diffusion in response to cellular injury in vitro and in vivo. Axotomy triggered intracellular calcium mobilization not only in the injured neuron but also in the intact neighboring cells, providing new insights into ATP's role as an injury signal. The tools developed in this study have demonstrated remarkable efficiency in unraveling the intricacies of ATP-mediated injury signaling.

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

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.

Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels.

Iron 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.