Hence, such broad field of action within eukaryotic cells requires a high level of structural and functional adaptation. Therefore, mitochondria are constantly moving and undergoing fusion and fission processes, changing their shape and their interaction with other organelles. Moreover, mitochondrial activity gets fine‐tuned by intra‐ and interorganelle H+, K+, Na+, and Ca2+ signaling. In this review, we provide an up‐to‐date overview on mitochondrial strategies to adapt and respond to, as well as affect, their cellular environment. We also present cutting‐edge technologies used to track and investigate subcellular signaling, essential to the understanding of various physiological and pathophysiological processes.Mitochondria are as highly specialized organelles and masters of the cellular energy metabolism in a constant and dynamic interplay with their cellular environment, providing adenosine triphosphate, buffering Ca This review provides an up‐to‐date overview of the intra‐ and interorganelle signaling within mitochondria, highlighting their role as highly specialized working units and signaling hubs. Moreover, this review presents cutting‐edge technologies to track the kinetics and dynamics of these molecular processes, which are essential to the understanding of various physiological and pathophysiological processes. For instance, super‐resolution microscopy enables us to follow morphological changes within mitochondria under assorted conditions. CJcristae junctionCMcristae membraneDRP1dynamin‐related protein 1dSTORMdirect stochastic optical reconstruction microscopyEMREessential MCU regulatorETCelectron transport chain2FADHflavin adenine dinucleotideFRETFörster resonance energy transferGEgenetically encodedGPS2G protein pathway suppressor 2IMMinner mitochondrial membraneIP3Rinositol triphosphate receptorLETM1leucine zipper‐EF‐hand‐containing transmembrane protein 1LITLight‐inducible tetheringMAMsmitochondria‐associated ER membranesMCUmitochondrial calcium uniporterMFN1mitofusin 1MFN2mitofusin 2MICOS complexmitochondrial contact site and cristae organizing complexMICU1mitochondrial calcium uptake 1MICU2mitochondrial calcium uptake 2mPTPmitochondrial permeability transition poremtUPRmitochondrial unfolded protein responseNADHnicotinamide adenine dinucleotideOMA1metalloprotease‐related protein 1OMMouter mitochondrial membraneOPA1optic atrophy 1OXPHOSoxidative phosphorylationPALMphoto‐activated localization microscopyPEphosphatidylethanolaminePSphosphatidylserineROSreactive oxygen speciesSIMstructured illumination microscopySTEDstimulated emission depletionTMRMtetramethylrhodamine methyl esterTOMtranslocase of the outer membraneUCP2uncoupling protein 2UCP3uncoupling protein 3VDACvoltage‐dependent anion channelYME1LATP‐dependent metalloproteasemΔψMitochondrial membrane potential2+, and contribution to various cellular signaling pathways, was supplemented with highly dynamic structural and functional adaptations ++2+2+ transfer in restricted subdomains Mitochondria have undergone a drastic transition from free‐living bacteria to fundamental compartments within eukaryotic cells 2+ signaling The mitochondrial network is a highly specialized working unit capable of undergoing dynamic adaptation in order to meet metabolic needs and to allow internal and external signaling Mitochondrial fission is mainly mediated by the cytosolic dynamin‐related protein 1 (DRP1). It binds to the OMM and constricts the mitochondrion, an action facilitated by its GTPase activity Mitochondrial fusion is mainly driven by the proteins optic atrophy 1 (OPA1) and dynamin‐like protein mitofusin 1 and 2 (MFN1 and MFN2) de novo generated mitochondrial protrusions formed by members of the kinesin family, like the protein kinesin‐1 heavy chain along the cellular tubulin cytoskeleton 2+ or apoptotic signals across mitochondria m) of neighboring mitochondria Recently, intermitochondrial signaling by either nanotubes or direct interaction via intermitochondrial junctions has been intensively studied. These intermitochondrial contact sites have a high electron density and are coordinated pairs of cristae oriented orthogonally to the OMM in two adjoined mitochondria 2+ levels 2+ uptake 1 (MICU1) is involved in CJ stabilization, maintenance of IMM potential, and cytochrome c restriction to the cristae lumen. The Ca2+ sensing ability of MICU1 and its thereby affected quaternary structure might influence the CJ stability and permeability OF1‐ATP synthase and the respiratory chain complexes to the CM, leading to a closed compartment of the cristae lumen OF1‐ATP synthase and respiratory chain complexes with lateral pH gradients in vitro experiments have shown that low pH causes cristae invaginations of the IMM based on the electrostatic induced negative curvature of the outer CM leaflet OF1‐ATP synthase in cristae formation and morphology The IMM is morphologically separated into two compartments, divided by the cristae junction (CJ): (a) the inner boundary membrane directly facing the inner leaflet of the OMM; and (b) the cristae membrane (CM), forming the protrusions and invaginations of the IMM into the mitochondrial matrix While the current knowledge about the ultrastructure of mitochondria relies mainly on electron microscopy data, recent advancements in fluorescence microscopy, in particular super‐resolution microscopy techniques like structured illumination microscopy (SIM), stimulated emission depletion (STED), or photo‐activated localization microscopy (PALM), enable researchers to visualize and investigate mitochondrial ultrastructure in living cells.OF1‐ATP synthase direct stochastic optical reconstruction microscopy (dSTORM) is an alternative to immunogold labeling and electron microscopy for analysis of submitochondrial localization of proteins like the Fm, such as Mitotracker GreenFM and tetramethylrhodamine methyl ester (TMRM), to the IMM. By using these techniques, cristae structure can be resolved and dynamic rearrangement of the IMM can be investigated due to high temporal resolution 2+ release decreased IMM kinetics exclusively in mitochondrial areas in close proximity to the ER, pointing to a Ca2+‐regulated mechanism of IMM rearrangement 2+ uniporter (MCU) and UCP2 upon ER‐Ca2+ release form the entire IMM into the inner boundary membrane, which was visualized recently using SIM However, dSTORM is not suitable for live cell imaging and PALM approaches in live cells do not reach the necessary temporal resolution necessary to analyze dynamics of the IMM. Therefore, STED The STED technique has been used to visualize spiral MICOS‐cluster arrays along the inner boundary membrane of yeast and human mitochondria, making an estimation of mitochondrial microarchitecture in combination with the use of immunogold labeling possible 2).The mitochondria's core working unit is the mitochondrial respiratory chain located in the IMM within the cristae 2 as hydrogen molecules, to the ETC via complex I and II, the electron transport through complex I to complex IV is coupled to proton pumping from the mitochondrial matrix into the locally restricted cristae lumen. This process causes a negative charge in the matrix and a positive charge in the IMS resulting in an electrochemical gradient, used for the proton transport back from the IMS into the mitochondrial matrix through ATP synthase (complex V). The released energy is, finally, utilized by FOF1‐ATP synthase to gain the cellular energy carrier, ATP, by phosphorylation of adenosine diphosphate These reducing equivalents serve as electron donors for the electron transport chain (ETC), consisting of NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), ubiquinone, cytochrome bc1 complex (complex III), and cytochrome c and cytochrome c oxidase (complex IV). After transfer of electrons, derived from the NADH and FADHOF1‐ATP synthase. Usage of these so‐called ATeams sensors enabled tracking of ATP levels ranging from 7.4 µm to 3.3 mm in different cellular compartments in real time Besides a broad range of biochemical approaches such as western blot, ROS, and ATP assays, there are specific measurements for mitochondrial bioenergetics available. Based on a polarographic oxygen electrode measuring the concentration and consumption of oxygen before and after injection of various substrates, the Oroboros O2k has been in use since the 1990s. This machine offers the possibility to analyze cells, tissues as well as isolated mitochondria with high resolution. Furthermore, sequential injection and titration of compounds inhibiting different complexes of the mitochondrial respiratory chain or boosting maximal mitochondrial activity can be done during the ongoing measurement. However, the Oroboros O2k is not suitable for high‐throughput screenings as only two samples can be analyzed at the same time + has a unique role. It is well known that the concentration of protons (H+) needs to be tightly regulated to preserve essential functions on a single cell level as well as in organisms + in the intermembrane space is essential for building up a driving force to activate the ATP generating F1/F0 ATP synthase, while re‐entering the mitochondrial matrix m, representing a charge or electrical gradient Mitochondrial activity and function are not solely controlled by mitochondrial structure, but also by various fine‐tuning mechanisms, including homeostasis of ions Fig. . In mitom is not only essential for the generation of ATP ++−2+2+ homeostasis and mitochondrial Ca2+ overload results in increased permeability of the IMM to protons, decreasing Δψm as well as the mitochondrial pH gradient The Δψm lead to severe mitochondrial dysfunctions and have been associated with various disease conditions, including cancer m linked to increased glycolytic rates and resistance to regulated cell death m than the correspondent healthy, noncancerous cell types m was linked to increased tumorigenicity and malignancy of cancer stem cells, linked to development and (re)occurrence of malignant tumors, while cells with lower Δψm were more prone to differentiation. Since reduction of Δψm by rapamycin decreased tumorigenicity significantly in these cells, targeting Δψm might be a potential strategy to prevent development of malignant tumors Since the mitochondrial metabolism is tightly regulated, it is not surprising that dysregulations and sustained changes of the Δψmito and Δψm and the most prominent candidates will be presented below.Nowadays, a huge variety of indicators and GE sensors is available to determine pHmito indicators, visualizing and characterizing metabolic changes within mitochondria 2+ indicator camgaroo was substituted by a portion of aequorin comprising only two EF hands, resulting in a Ca2+ ‐insensitive probe with an estimated pKa of 8.5, excitation at 498 nm, and emission at 522 nm.mtAlpHi was described as one of the first encoded pHmito sensors is SypHer 2O2 sensor Hyper 2O2, Ca2+, and PO3−. SypHer proved perfectly suitable for the detection of cytosolic as well as mitochondrial pH values due to a pKa of 8.71, a 20‐fold increase upon switching from pH 7 to 10, and a fourfold increase in the range between pH 7 and 8. Furthermore, SypHer has been also used in simultaneous measurements in combination with other sensors performing two‐ or multi‐color imaging One of the most commonly used GE pHmito has been challenging, but was finally accomplished a few years ago. While dyes like BCECF m on the cationic triphenylphosphonium group. Additionally, the benzyl chloride was thought to undergo nucleophilic substitution with reactive thiols of mitochondrial proteins, ensuring mitochondrial localization even upon acidification or membrane depolarization The development of small chemical fluorescent dyes to specifically monitor pHm.While several GE pH sensors have been developed in the last decade, to the best of our knowledge, there is no GE sensor available for measuring Δψm are small fluorescent dyes based on a lipophilic cation structure like TMRM, tetramethylrhodamine ethyl ester (TMRE) m‐dependent accumulation within mitochondria. The excitation and emission wavelength of these monochromatic dyes are quite similar, with excitation at 555 nm and emission at 570 nm for TMRM and excitation at 549 nm and emission at 574 nm for TMRE ex: 488 nm) is a ratiometric dye, existing as green fluorescent monomer at depolarized membrane potential (λem: 530 nm) and forming red aggregates (λem: 595 nm) at hyperpolarized membrane potential m by, for instance, H2O2 or disturbed equilibrium between monomers and aggregates. All these dyes are differently permeant and require specifically adjusted loading protocols dependent on the respective cell type m measurements possible Frequently used indicators for semiquantitative analysis of Δψ+) are essentially involved in various processes and represent the most abundant intracellular cation. The cytosolic K+ concentration is important for the maintenance of the cell's membrane potential, works as cofactor for various enzymes, and regulates cell volume as well as endo‐ and exocytosis. Also, mitochondrial functions rely on an intact mitochondrial K+ (Kmito+) homeostasis. While cytosolic K+ concentration is close to 140 mm in vital cells, the concentration of Kmito+ ranges between 20 and 60 mmPotassium ions  channels, and via ATP‐sensitive K+ channels located in the IMM (mitoKATP). Besides, voltage gated K+ (Kv) channels and Ca2+ activated K+ (KCa) channels are located within the IMM ΔψATP, in particular, has been extensively studied. The generation of high levels of ATP by FOF1‐ATP synthase causes a decrease in Δψm and leads to a decreased flux of K+ across the mitoKATP channels into the matrix, probably preventing devastating mitochondrial depolarization. However, under conditions of oxidative stress, increased levels of ROS activate mitoKATP channels, dissipating Δψm and counteracting further ROS production. The interplay between K+ and H+ becomes evident when considering the presence of K+/H+ transporters (KHX) in the IMM, transporting K+ from and H+ into the mitochondrial matrix. One of these transporters is the leucine zipper‐EF‐hand‐containing transmembrane protein 1 (LETM1), facilitating K+ extrusion from the mitochondrial matrix and, thereby, also modulating Na+ and Ca2+ homeostasis The role of mitoK+ assist in modulating the transmembrane H+ gradient, altering ATP production, but may also promote the controlled re‐generation of the H+ gradient toward the IMM via the ETC ATP channel blockers such as glibenclamide, widely used for the treatment of type 2 diabetes, was found to ameliorate ischemia reperfusion injury in the brain, kidney, intestine, and lungs. The effect was associated with a modulation of the oxidative stress caused by releasing ischemia, after starting reperfusion Kmito+ homeostasis.High levels of matrix KKmito+ with Δψm and H+, Kmito+ is fundamentally affected by mitochondrial Ca2+ homeostasis. The most prominent example of Ca2+ activated K+ channels represents the large conductance KCa (BKCa) channel, found in the plasma membrane of excitable cells such as neurons and skeletal muscle cells. Several types of KCa channels could be also found in the IMM in various cell types, including the mitochondrial localized large conductance KCa (mitoBKCa) in LN299 human glioma cells 2+ levels as well as by changes in the Δψm, leading to K+ fluctuations across the membrane. While the pore vestibule of mitoBKCa faces the intermembrane space, the Ca2+ sensitive domain is located in the mitochondrial matrix, indicating that activation of mitoBKCa requires elevation of mitochondrial matrix Ca2+Ca channels at the plasma membrane, failed to affect mitoBKCa, raising the question whether mitoBKCa and BKCa are structurally possibly unrelated to each other Besides the interplay of Ca channel openers like NS1619 + uptake twofold, halved the size of a myocardial infarct in guinea pig hearts after reperfusion of ischemic regions. These results suggest the participation of mitoBKCa channels against necrotic and apoptotic cell injury after ischemic tissue damage, possibly by modulation of the mitochondrial respiratory chain 2+]mito overload, maintenance of Δψm, and/or normalization of ROS levels Ca channel opener, also exhibited protective effects on neuronal cells + flux from the extracellular space into the mitochondrial matrix in isolated rat brain mitochondria, causing Δψm depolarization and reduced ROS production Ca channel activator NS11021 was reported to inhibit glutamate‐induced oxidative stress by attenuating ER stress and mitochondrial dysfunction Ca by single‐channel recordings. While this compound boosted oxygen consumption rate in isolated rat brain mitochondria, it exhibited cytotoxic effects by increase of cytoplasmic Ca2+ concentration and consequent activation of calpains in intact neuronal HT22 cells Ca channel modulating compounds.Notably, application of compounds acting as plasma membrane BK+ homeostasis, scientists have been eager to find ways to investigate cellular and mitochondrial K+ levels. Kmito+ dynamics are frequently measured via patch‐clamp approaches using isolated mitochondria or mitoblasts Kmito+ dynamics within single living cells is provided by fluorescent indicators + binding to the sensors results in an alteration of their fluorescence depending on the K+ concentration + measurements have been developed + will in future deepen our understanding of subcellular and particularly mitochondrial K+ homeostasis Considering the importance of intact cellular K2+ homeostasis, mitochondria, in turn, also affect the ion's intricate role as a widespread signaling molecule within the cell. Previously thought to function primarily as a regulator of cytosolic Ca2+, it was later determined that Camito2+ influx and efflux machinery are geared more toward control of the organelle´s own Ca2+ levels Camito2+ concentration are known to have an effect on cellular ATP production 2+ movement. For example, the mitochondrion is known to preferentially take up Ca2+ released by the ER Camito2+ uptake, specifically, has multiple significant consequences pertaining to proper overall function; Ca2+ being sequestered into the matrix ultimately has an effect on local and more widespread cellular Ca2+ signals. Homeostasis between this organelle´s Ca2+ stores and the rest of the cell is crucial, as an overload of intraluminal Ca2+ can lead to the initiation of the apoptotic pathway While mitochondrial function and activity is heavily dependent on Ca2+ overload through the release of multiple proteins from their lumen into the cytosol 2+ overload, mitochondria release this protein into the cytosol, where it stimulates the formation of a caspase‐activating complex otherwise known as the apoptosome. The apoptosome's overall function of activating killer caspases ultimately results in the death of the cell 2+ is again vital. It is known that the close positioning of mitochondria to large sources of Ca2+  allows for significant accumulation of the ion inside the mitochondrial matrix upon physiological Ca2+ release. This increase in matrix Ca2+ levels, in turn, affects mitochondrial metabolism through stimulation of mitochondrial effector molecules such as particular dehydrogenases of the Krebs cycle which are functionally dependent on Ca2+, ultimately leading to ATP production 2+, and the existence of a Camito2+ uniporter, has been reported on since the 1960s Camito2+ uptake occurs primarily through MCU activity, and this uptake is highly selective but has a low apparent KD for Ca2+.Mitochondria are able to activate the intrinsic apoptotic pathway following Ca2+ levels Camito2+ uptake threshold, which is higher than that of the MCU Camito2+ levels 2+ uptake and was also reported to vary across different tissues and organs 2+ uptake at higher Ca2+ concentrations, whereas LETM1 was shown to influence a potentially different uptake mechanism, likely more pronounced when Ca2+ levels are lower 2+Camito2+ uptake is whether there are different uptake pathways operating in relation to low versus high cellular Ca2+ concentration sources, as well as what the physiological implications of these potential different uptake pathways may be. Another interesting phenomenon that awaits clarification is the spatial resolution of Camito2+ uptake, namely whether it occurs along the full length of the IMM, or whether this activity is restricted to certain membrane regions. Important to consider when discussing uptake of Ca2+ into the mitochondrial matrix is the organelle's ability to balance overall charge through extrusion mechanisms. In the case of the mitochondrion, Ca2+ influx is mainly kept in check through an exchange with other ions. The major protein playing a role in this process was identified to be Na+/Ca2+ exchanger (NCLX) + that accumulates in the matrix following the exchanger's activity. This is proposed to be achieved by members of the Na+/H+ exchanger family, consisting of multiple plasma membrane and organellar transporters 2+ movement, though its exact function in this capacity is as yet unclear. It has been proposed to act as a Ca2+/H+ antiporter, which implies a function in Ca2+ extrusion +/H+ antiporter, thereby influencing Ca2+ uptake and/or extrusion via secondary means These effects are achieved through MCU regulators. Essential MCU regulator (EMRE) was shown to be a core component of the uniporter complex and is essential for MCU mediated‐ MICU 2+ homeostasis is clearly a complex and wide‐ranging process; therefore, the techniques required are also spread across multiple disciplines. In particular, the use of various sensors to study intracellular Ca2+, as well as more specific indicators able to determine the ion's movement to, within and from the mitochondrion itself, provide the quickest insight into the intricate web of Ca2+ signals constantly present throughout the cell.Mitochondrial Ca2+ measurement can generally be clustered into chemically engineered fluorophores and GE FPs.Probes for overall intracellular Ca2+ sensors specific to mitochondria and other organelles has reached new heights. Sensors used to measure general intracellular Ca2+ movement, including the ubiquitously employed cytosolic Ca2+ sensor Fura‐2 2+, dependent on the desired experimental read‐out. Choosing the best‐suited Ca2+ sensor necessitates consideration of factors such as the probe´s original form and modification required for expression in cells, its affinity for Ca2+, and its spectral properties, among others , a main disadvantage of chemical sensors for organelle‐specific Ca2+ measurement is the lack of controlled localization once loaded into target cells. Use of such probes will therefore not guarantee mitochondria‐exclusive expression and Ca2+ monitoring. Chemical fluorophores are thus mainly suitable for drawing indirect conclusions on Camito2+ signaling as it pertains to the rest of the cell.Briefly, chemical indicators, compounds which change their fluorescence properties following binding to Ca2+ specifically over other divalent cations, among other advantages 2+ research related to crosstalk between mitochondria and the rest of the cell; for example, investigation into the mechanism whereby the ER and mitochondria interact through mitochondria‐associated ER membranes (MAMs) commonly employs the cytosolic Ca2+ sensor Fura‐2 to draw conclusions on Ca2+ signaling between the two organelles 2+ movement, but has its own purported disadvantages. For example, Lee et al. 2+, combined with increased background fluorescence as leaked dye binds to Ca2+ present in surrounding medium. Fluo‐3's close relative, Fluo‐4, proves similar in structure and other properties, but exhibits increased fluorescence and range for Ca2+2+2+ into the mitochondria. Due to these properties, the rhodamine‐based AM esters have been employed in the literature as mitochondrial‐Ca2+ selective indicators 2+ dyes, only cellular incubation prior to experiments is required. However, Rhod‐2 is known to diffuse out of the mitochondria and into the cytosol after a relatively short period of time, making longer experiments unreliable insofar as accurate mitochondrial Ca2+ measurement goes. These are but a few examples, and, importantly, each of the outlined tools come with their own advantages and drawbacks. Nevertheless, together, they exemplify the broad range of sensors currently available for cytoplasmic Ca2+ measurement.Fura‐2 is a classic example of a ratiometric sensor designed for such purposes. Compared to what was available prior to its characterization, Fura‐2 offered ~ 30× increased fluorescence signals and improved ability to bind Caet al. (2001) with the use of a cameleon indicator targeted to each of the cytosol, mitochondrial matrix, and ER lumen.Among those sensors that are continuously evolving are the new generation of GE fluorescent sensors; compounds comprised of a FP fused to some form of sensing polypeptide. In most cases, the FP is bound to a protein that undergoes a conformational change in response to substrate binding. These sensors are generally considered advantageous over their chemical counterparts due to their substrate specificity, and the fact that their GE nature prevents variance in probe uptake across different cells Camito2+ levels specifically has provided new insights into Camito2+ fluxes and their regulation.Development of tools for accurate measurement of 2+ sensor that is currently widely used. It is excited with blue light (430 nanometers—nm) and exhibits dual emission at 480 and 535 nm. The ratio of the 480 nm emission to the FRET signal (535 nm) provides investigators with insight into and enhanced understanding of basal Camito2+ levels as well as its fluctuations For example, 4mtD3cpV is a FRET‐based ratiometric Ca2+ sensor, which can be combined with cytosolic Fura‐2 dye, is 4mtD1GO‐CAM 2+ in the mitochondria while simultaneously employing Fura‐2 to measure cytosolic Ca2+. As Fura‐2 has a wide excitation–emission spectrum, combined measurement with 4mtD1GO‐CAM proves extremely useful for investigation into the spatiotemporal fluxes of Ca2+. In addition, as both the Fura‐2 dye and mtD1GO‐CAM sensor are ratiometric, accurate observation of basal Ca2+ levels is also possible Another FRET‐based ratiometric Ca2+ sensors of the GECO series provide a good opportunity to measure Ca2+ levels simultaneously in different compartments KD for Ca2+ (340 nm) and has an excitation wavelength of 394 nm and dual emission of 455 and 511 nm 2+ levels in two different organelles or mitochondrial compartments simultaneously with almost no spectral overlap.Ca2+ in this organelle and throughout the cell.From this simple examination of a handful of the more prominent cytosolic and mitochondria‐specific sensors in use today, it is clear that investigators have no lack of options when it comes to studying the intricate movements of CaAlthough mitochondria are equipped with their own circular deoxyribonucleic acid containing 37 genes , almost all mitochondrial proteins are encoded by the nuclear genome, making constant communication between mitochondria and the nucleus indispensable Dually targeted proteins, localizing to the nucleus as well as to mitochondria, are used as communication indicators for mtUPR (retrograde) signaling. As mitochondrial protein import strongly affects cell viability, the transport of these proteins, visualized by tagging them with a FP, can be used as an indicator of mitochondrial fitness Interestingly, cytosolic proteins prone to aggregation are imported into mitochondria for degradation. This translocation of misfolded or aggregated proteins from the cytosol to mitochondria, associated with increased mitochondrial stress levels, can be visualized using split FP techniques 2+ between mitochondria and the ER Contact between mitochondria and the ER occurs at very specialized junctions stabilized by MAMs, forming locally restricted signaling hubs to restrict and protect the transfer of lipids and Caet al. (2017) have designed split‐GFP‐based contact site sensors (SPLICS), fusing the GFP1–10 moiety to an OMM targeting signal and the GFP11 β‐strand to an ER leading peptide and varying the linker length between the targeting signals and the split‐FP to visualize narrow (approximately 8–10 nm) and wide (approximately 40–50 nm) distances between the ER and mitochondria. As soon as the split‐FPs are in close vicinity due to the interactions between proteins fused to each fragment, they from a full fluorescent FP et al. have developed a split‐FP approach to study mitochondrial–ER interaction based on a YFP Venus by fusing the n‐terminal fragment of Venus128 to a mouse ER–protein and targeting the c‐terminal fragment of Venus to the OMM via the n‐terminal leading peptide of TOMM20. As a result, alterations in the MAM structure in response to ER stress, starvation, and protein level changes could be detected Different approaches have been developed to explore the physical and functional sides of ER‐mitochondria tethering. The current state‐of‐the‐art technique to visualize MAMs is electron microscopy, making quantification of the distance between the membranes of the ER and mitochondria, as well as the number of contact sites, possible et al. et al. et al. exploited the FKBPFRB heterodimerization system by fusing FKBP to an OMM targeting signal and combining FRB with an ER retention signal to enable rapamycin inducible tethering between the two organelles. By using this approach, tethering of the ER and mitochondria results in an increase of already in close apposition located contact sites, rather than creating new organelle contacts Recently, Ding Furthermore, recently developed light‐inducible tethering systems allow the induction of ER–mitochondrial interaction, which facilitates the functional study of ER–mitochondrial contacts in situ tool, enable the detection of endogenous proteins, protein modifications, and protein interactions with high specificity and sensitivity by using antibodies to detect two unique protein targets 2+ imaging has offered a very nice approach to determine Ca2+ exchange between mitochondria and the ER Besides various techniques based on cutting‐edge microscopy, an IP3R–VDAC proximity ligation assay has been developed for the quantification of ER–mitochondria interplay. Proximity ligations assays, as an As presented in the current review, mitochondria are versatile organelles within eukaryotic cells, which deliver utilizable energy and function as signaling hubs, communicating with various cellular compartments to maintain their own function but also to provide their service. Their structure and shape as well as their activity undergo dynamic changes in order to fulfill their tasks under various conditions in different cellular subdomains. As discussed in this review, investigation into mitochondrial function advanced alongside cutting‐edge technologies, enabling us to gain new insight into subcellular signaling processes. Further development of these methods, as well as completely new technological strategies, will most likely further broaden our understanding in the upcoming years. This might potentially yield the path to unveiling subcellular processes causing still‐incurable diseases and, thereby, help to develop novel treatment strategies. Through that example, it is obvious that constructive teamwork between people with different expertise is increasingly important and is urgently needed to meet the upcoming health‐related challenges in an aging society.The authors declare no conflict of interest.CTM, JR, GZ, SB, HB, ZK, and BG contributed chapters to the manuscript. CTM together with WFG and RM planned the manuscript's structure and content.


A comparison of various ES1 analogs suggested that the 5-nitrofuran (5-NF) ring of ES1 is crucial for this effect. Accordingly, the analog ES24, which conserves the 5-NF domain of ES1, selectively inhibited protein translocation into the ER, displayed the highest potency on ER Ca2+ leakage of ES1 analogs studied and induced Ca2+-dependent cell death. Using small interfering RNA-mediated knockdown of Sec61α, we identified Sec61 complexes as the targets that mediate the gain of Ca2+ leakage induced by ES1 and ES24. By interacting with the lateral gate of Sec61α, ES1 and ES24 likely capture Sec61 complexes in a Ca2+-permeable, open state, in which Sec61 complexes allow Ca2+ leakage but are translocation incompetent.Eeyarestatin 1 (ES1) inhibits p97-dependent protein degradation, Sec61-dependent protein translocation into the endoplasmic reticulum (ER), and vesicular transport within the endomembrane system. Here, we show that ES1 impairs Ca  •2+ in ERES1, ES2, and ES24 deplete Ca•2+ homeostasisESR35 and ES47 do not affect cellular Ca•The most potent eeyarestatin, ES24, comprises only the 5-nitrofuran domain•ES1 and ES24 target Sec61 complexes in ER 2+-permeable, open state. Specifically, ES24 enhances strongly the Sec61-mediated Ca2+ leakage from ER and induces Ca2+-dependent cell death.Gamayun et al. discovered that eeyarestatins (ESs) interfere with closing mechanisms of Sec61 complexes of the ER and, as a “foot in the door,” stabilize Sec61 complexes in a Ca Early st manner . It is n at rest . However at rest . Hence Cgradient . Since ECA pumps . The molucidated , but it m the ER . The corm the ER . Apparennditions . Given t leakage , but, me leakage . Some in leakage . With th leakage , howeverin cellula and in vitro  are small molecules that were initially identified as inhibitors of protein dislocation from ER into the cytosol . Subsequin vitro . The ES1in vitro  and, aftin vitro . Thus, Ein vitro  and, sinembranes , it can 2+ status in cells under treatment with ES1, we measured free Ca2+ concentrations simultaneously in the ER ([Ca2+]ER) and the cytosol ([Ca2+]cyt) in the absence of extracellular Ca2+ to minimize Ca2+ entry. [Ca2+]ER was imaged with the genetically encoded, FRET-based Ca2+ sensor D1ER  and measured the specific effects of ES1 on Ca2+ leakage from the ER.To obtain an integrated view of the Casor D1ER , which wress see , cellulaprotocol . In this2+]cyt immediately after application of ES1. Since we minimized extracellular Ca2+ entry by using EGTA in the bath solution, the increase of [Ca2+]cyt induced by ES1 could in principle have resulted from the outflow of Ca2+ from intracellular Ca2+ stores. It is conceivable that ES1 potentiated the accumulation of ER Ca2+ in cytosol by also inhibiting cellular clearance mechanisms. However, all cells treated with 0.1 and 1 μM ES1 successfully removed the large amounts of Ca2+ that were released by TG into the cytosol . By contrast, [Ca2+]ER began to decrease immediately after application of 1–10 μM ES1 , i.e., the time required for [Ca2+]ER to decrease by 50% of the pre-TG level. Since the TG-induced changes in [Ca2+]cyt likely reflect the Ca2+ leakage, it is expected that a time-dependent decrease of [Ca2+]ER due to enhanced Ca2+ leakage will paradoxically reduce cytosolic responses to TG, as is observed for 10 μM ES1 in 2+]cyt and [Ca2+]ER. Despite the lower [Ca2+]ER levels, the cytosolic responses to TG were higher in cells treated with 10 μM ES1 for 4 min than in those treated with 1 μM ES1 , the [Ca2+]ER levels before TG application (pre-TG [Ca2+]ER) and the half-times of the TG-induced decay in [Ca2+]ER (post-TG t1/2) exhibited a strong dependence on ES1 concentrations in the 17-min treatments  for the effects of ES1 on cytosolic and ER Ca2+ is approximately 1 μM  after 4- and 17-min treatments with 1 μM ES1 .A quantitative analysis of the Caeatments E–1G. Sped by ES1 G. Based ely 1 μM E–1G. Theely 1 μM E reflectely 1 μM A, which M  was approximately two times shorter for ES24 than for ES1 at 10 μM . We performed these experiments in HeLa cells, in which silencing efficiencies of about 80% are routinely achieved  was used as a control. Following our online protocol, HeLa cells were exposed to 10 μM ES1 and ES24, and the amplitude of cytosolic TG responses (post-TG Δ[Ca2+]cyt) was measured as an indicator for Ca2+ leakage , while INS-1 and NALM-6 cells were more prone to the effects of disruption of the Ca2+ homeostasis by TG . Combining the cell viability data for ES24 and TG, we observed a striking correlation between cell viabilities in the presence of ES24 and TG .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Adolfo Cavalié  were cultured in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated FBS. INS-1 cells are derived from rat insulinoma  were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) foetal bovine serum (FBS). In order to obtain cells with homogenous expression levels of the FRET-based CaNA3-D1ER , which wsulinoma  and wereThe compounds ES1, ES2 and ESR35 were synthesized as previously described .1 and 2 with the appropriate conjugated aldehyde. Phenylacetic hydrazide 1 is commercially available and the synthesis of 2 (ESR35) has been reported previously (1H-1H coupling constant (15.9 Hz for ES24 and 14.8 Hz for ES47).ES24 and ES47 were prepared in good yields by condensation of acyl hydrazides eviously . The dou1H NMR spectra were recorded on a Bruker Avance II 500MHz spectrometer. Chemical shifts are referenced to the residual solvent peak. Mass spectra were recorded on a Micromass Platform II (electrospray) spectrometer. Melting points were recorded using a Sanyo Gallenkamp MPD350 heater and are uncorrected.General experimental synthetic reagents and solvents were purchased from Sigma-Aldrich or Alfa Aesar and used as supplied. IR spectra were recorded on a Perkin Elmer 881 spectrometer, an AT1-Matson Genesis Series FTIR spectrometer or a Perkin Elmer Spectrum BX FTIR spectrometer. E)-3-(5-Nitro-2-furyl)acrylaldehyde  was added, under nitrogen, to a solution of phenylacetic hydrazide  in anhydrous CH3OH (4 mL). The reaction mixture was stirred at room temperature overnight and then Et2O was added to allow complete precipitation to occur. The solid was collected by filtration and washed with Et2O and pentane, yielding the title compound  as a yellow solid. Mp 202-204°C; νmax (evaporated film)/cm-1 3137 , 3040 , 2971 , 2857 , 1747 , 1667 , 1587 , 1557 , 1504 ; 1H NMR  δ 4.03 H2), 6.63 H), 6.65 H), 7.15 H), 7.33-7.37 H & Ar-CH), 7.51 H); m/z (-ve ion electrospray) 298 , (+ve ion electrospray) 322 ; found 322.0805, C15H13N3O4Na ([M+Na]+), requires 322.0798.(E)-3-(2-Furanyl)acrylaldehyde  was added, under nitrogen, to a solution of 2  in anhydrous CH3OH (1 mL). The reaction mixture was stirred at room temperature for 2 days and then concentrated under reduced pressure. The residue was recrystallised at room temperature from Et2O /pentane, yielding the title compound  as a pale yellow solid. Mp 213-214.5°C; νmax (solid state)/cm-1 3395 , 3222 , 3145 , 3132 (brw), 3039 , 2979 , 2974 , 2930 , 1699 , 1680 , 1661 , 1633 , 1588 , 1550 , 1512 ; 1H NMR  δ 1.31 H3 or C(20)H3), 1.40 H3 or C(20)H3), 4.41 H2), 5.75 H), 6.10 H), 6.29 H), 6.38 H), 6.59 H), 7.17 , 7.20-7.25 H), 7.40 H), 7.47 , 7.78 , 9.41 , 9.75 ; m/z (−ve ion electrospray) 587 , 10%), 585 , 65%), 583 , 100%); (+ve ion electrospray) 611 , 10%) 609 , 60%), 607 , 100%); , requires 607.1234).(35S Protein Labelling Mix containing [35S] methionine (Perkin Elmer) , ∼2.5% (v/v) amino acids minus methionine (Promega), 9% (v/v) in vitro transcribed mRNA (290 ng/μL stock) encoding residues 17 to 232 of the short form of human HLA class II histocompatibility antigen gamma chain (Ii) and 10% (v/v) inhibitor/DMSO pre-treated rough microsomes for 20 min at 30°C. The short form of Ii was kindly provided by Dr. B. Dobberstein . Following translation, puromycin was added to 0.1 mM and samples incubated for 5 min at 30°C to ensure ribosomal release. The membrane-associated fraction of the reaction was isolated by centrifugation  through an 80 μL high-salt cushion 2, 50 mM Hepes-KOH, pH 7.9) and the membrane pellet suspended in 30 μL SDS sample buffer  SDS, 10% (v/v) glycerol; pH 7.6; 1 M dithiothreitol). Samples were denatured at 70°C for 5 min prior to resolution by SDS-PAGE . Gels were incubated in fix mix  for 5 min, dried (BioRad Gel Dryer) for 2 h at 65°C and exposed to a phosphore imaging plate for 24 h. Radiolabelled products were visualized using a Typhoon FLA-7000 . Quantitative analysis was performed with AIDA v5.0 (Raytest Isotopenmeβgeräte) whereby the signal intensity of Ii-2 Gly/Ii was used as a read-out for total translocation and expressed relative to the DMSO control to determine the mean relative ER processing from 3 independent experiments. Data is presented as mean ± SEM.Nuclease-treated canine pancreatic microsomes were treated with 250 μM ESR35, ES1, ES2, ES24, ES47 (from 1 mM stock solutions in DMSO), or an equal volume of DMSO, and incubated on ice for 1 h before inclusion in translocation analyses. Cell-free translation (20 μL) was performed in nuclease-treated rabbit reticulocyte lysate (Promega) in the presence of EasyTag EXPRESS Experiments performed whit HeLa cells cultured to ∼80% confluent. DMEM was aspirated from each well 16 h after cell plating and cells treated with 8 μM ES1, ES2, ES24, ESR35 and ES47 (from 10 mM stock solutions in DMSO), or an equal volume of DMSO, in 1 mL fresh DMEM for 8 h. Upon treatment completion, DMEM was aspirated, cells washed twice with PBS  and solubilized in 100 μL SDS sample buffer. Samples were denatured for 16 h at 37°C, resolved by SDS-PAGE  and proteins transferred to a PVDF membrane in transfer buffer  at 300 mA for 2.5 h at ambient temperature. The membrane was blocked in Casein blocking buffer (Sigma) diluted in TBS  for 1 h at ambient temperature prior to primary antibody (FK2 mouse (Enzo Life Sciences) and β-actin (Abcam)) incubation in Casein blocking buffer diluted 1:10 in TBST (TBS supplemented with 0.1% Tween-20) for 16 h at 4°C. After washing with TBST (4 x 10 min), the membrane was then incubated with a fluorescent secondary antibody for 1 h at ambient temperature. Following washing with TBST (4 x 10 min), samples were visualized using an Odyssey CLx Imaging System (LICOR Biosciences). Ubiquitin signals were normalized relative to the intensity of the β-actin loading control and normalized signals expressed relative to the DMSO solvent control. Data is shown as mean ± SEM from 6 independent experiments.SEC61A1 was performed in HeLa cells as previously described  or SEC61A1#5 siRNA  or scramble siRNA  using HiPerFect Reagent (Qiagen). After 24 h, cells were transfected for a second time. Ca2+ imaging experiments were performed 96 h after beginning of transfection. Silencing efficiencies were evaluated by Western blot analysis using the respective antibodies and an anti-β-actin-antibody from mice  or with 30 μM scr RNA  using HiPerFect (Qiagen). Ca2+ imaging experiments were performed 24 h after transfection. SERCA2 expression levels were detected with a SERCA2 antibody .Silencing of escribed . Brieflyrom mice . For sil2+]ER and [Ca2+]cyt were imaged using D1ER and FURA-2, respectively. D1ER is a genetically encoded FRET-based Ca2+ sensor that comprises two fluorescent proteins, CFP and citrine, and two sensing proteins, a calmodulin-binding peptide derived from skeletal muscle myosin light chain kinase and calmodulin . Accordingly, D1ER and FURA-2 reported changes in [Ca2+]ER and [Ca2+]cyt, respectively, which arose from Ca2+ mobilisation within internal cell compartments. Ca2+ imaging experiments were performed with cells that were plated on poly-L-lysine-coated cover slips and cultured for 48-72 h.ER and [Ca2+]cyt, respectively /(Rmax-R)) ) see , in whicibed see . HeLa ceibed see .2+ imaging by adding 2X solutions to the bath at a ratio of 1:1 to avoid problems arising from slow mixing. The final DMSO concentration in bath was 0.01-0.05 % v/v. During Ca2+ imaging, a base line was routinely recorded before applying ES compounds in order to determine the status of [Ca2+]cyt and [Ca2+]ER. In the so-called “online” protocol, ES compounds were applied at 3 min while imaging was running. Mock treatment represents the application of 0.01–0.05% v/v DMSO in bath solution. Following 1, 4 and 17 min exposures to eeyarestatin compounds, TG was applied in bath to unmask the Ca2+ leakage from ER. In experiments with D1ER and FURA-2, 5-10 HEK D1ER cells were imaged simultaneously, while 30-50 HeLa cells were imaged per experiment with FURA-2. Time courses of [Ca2+]ER and/or [Ca2+]cyt were constructed for each experiment and parameters such as the area under the curve and amplitude of TG responses were measured for each single cell. Ca2+ imaging experiments were performed with 3-5 different batches of HeLa and HEK D1ER cells. Data has been collected in 6-12 independently experiments repeated for each experimental setting and is shown as mean ± SEM.Stock solution of ES compounds were prepared by dissolving the compounds ES1, ES2, ES24, ESR35 and ES457 in DMSO to obtain concentrations of 10-100 mM. Stock were maintained frozen at -20°C. Similarly, 1 mM stocks of thapsigargin were prepared in DMSO. Just before experiments, stocks of TG and ES compounds were diluted to a 2X concentration in the bath solution, i.e., 2 times greater than the desired final concentration. Routinely, TG and ES compounds were applied to the cells during Ca6 cells/ml in the respective culture medium and treated with ES1, ES24 and ESR35 at concentrations between 1-20 μM for 24 h. Similarly, cells were treated with 1 μM thapsigargin for 24 h. The DMSO concentration was 0.1 % in TG-containing medium and below 0.02 % in medium with ES compounds. Following treatment, cells were incubated with PBS and Guava ViaCount reagent (1:10) for 5 min in the dark at room temperature. Cell viability was measured by flow cytometry with Guava easyCyte 8HT (Merck Millipore). Viability measurements were performed as duplicates in 3 independent experiments per treatment with 5000 cells/measurement. The cell viability of cells exposed to ES1, ES24 and ESr35 was normalised with respect to the viability of non-treated cells. For cells exposed to TG, cell viability was normalised to 0.1 % DMSO controls. Normalised viability data is presented as mean ± SD.HEK, HeLa, INS-1 and NALM-6 cells were plated in culture dishes at densities between 0.25-0.50⋅10P61619). The crystal structure of canine Sec61α (PDB: 3JC2) was selected for homology modelling of human Sec61α. Both sequences share 99.8% sequence identity. The 3JC2 structure shows Sec61α in an open conformation but is lacking structural information for the helical plug region (residue 63 to 69). Since in the open conformation, the plug is believed to be outside of the translocation pathway, we assumed that it does not form relevant interaction with the substrates. Homology modelling was carried out using the MODELLER 9.17 package. The plug region was modelled and optimised as a loop structure. Subsequently, the homology model was subjected to energy minimization, using the NAMD package, to relax side chain atoms. The resulting model (3JC2 and PDB: 5A6U) and two other membrane protein structures: a GPCR (PCB: 3P0G) and an ABC transporter (PDB: 3G5U). The analysis from ProSa-web showed that the score of the homology model is within the range of scores typically found in experimentally determined structures of protein chains. The structural model of Sec61 α carrying the T86M mutation was generated by Swiss-Pdb Viewer and subsequent minimization with NAMD.The protein sequence of human Sec61α isoform 1, which contains 476 amino acids, was retrieved from the UniProtKB database . In the second docking step, the size of the grid box was scaled down based on the population of the most stable binding positions of the ligand. In a finer run, more stringent parameters  were used.The protonation states of protein residues and partial charges of ligands were assigned by the in vitro and in cellula experiments as well as in Ca2+ imaging experiments are described in Quantification procedures used in vitro and in cellula data with respect to DMSO controls was determined using Tukey’s and Dunnett’s multiple comparison tests, respectively. Statistical significance of the Ca2+ imaging data was assessed with the two-sample Kolmogorov-Smirnov test. Statistical significance is given as n.s., non-significant; *, P < 0.05; **, P < 0.01 and ***, P < 0.001.The statistical significance of In vitro data was analysed with AIDA. Matlab was used in the analysis of Ca2+ imaging data. Homology modelling and docking analysis were performed with Modeller 9.17, NAMD, Pro-Sa-web and AutoDock4.


GATA2, a zinc finger transcription factor predominantly expressed in hematopoietic cells, acts as an essential regulator of hematopoietic stem cell generation, survival and functionality. Loss and gain of GATA2 expression has been implicated in myelodysplastic syndrome and acute myeloid leukemia (AML) yet the precise biological impact of GATA2 expression on human AML cell fate decisions remains ambiguous. Herein, we performed large-scale bioinformatics that demonstrated relatively frequent GATA2 overexpression in AML patients as well as select human AML (or AML-like) cell lines. By using shRNAi to target GATA2 in these AML cell lines, and an AML cell line expressing normal levels of GATA2, we found that inhibition of GATA2 caused attenuated cell proliferation and enhanced apoptosis exclusively in AML cell lines that overexpress GATA2. We proceeded to pharmacologically inhibit GATA2 in concert with AML chemotherapeutics and found this augmented cell killing in AML cell lines that overexpress GATA2, but not in an AML cell line expressing normal levels of GATA2. These data indicate that inhibition of GATA2 enhances chemotherapy-mediated apoptosis in human AML cells overexpressing GATA2. Thus, we define novel insights into the oncogenic role of GATA2 in human AML cells and suggest the potential utilization of transient GATA2 therapeutic targeting in AML. Exemplifying its role as a crucial regulator of HSC behavior, perturbations of GATA2 expression have been observed in AML4. GATA2 loss of function mutations cause immunodeficiency syndromes that progress to myelodysplastic syndrome and AML on acquisition of secondary mutations7. Conversely, overexpression of GATA2 has been implicated in the pathogenesis of pediatric and adult AML8 and low-level overexpression of GATA2 immortalizes murine BM in vitro without leading to myeloid neoplasia in vivo9. As of yet, the precise biological impact of GATA2 overexpression on human AML cell fate decisions remains unclear. In this study, we therefore explore the requirement for GATA2 expression in human AML cells.Life-long hematopoiesis is sustained by bone marrow (BM) resident, multi-potent hematopoietic stem cells (HSCs). Extrinsic signals conveyed from the BM niche alongside a cell intrinsic transcriptional program driven by transcription factors (TFs) are crucial for HSC homeostasis and normal hematopoiesis. Chromosomal translocations as well as epigenetic and genetic alterations of TF activity perturb HSC homeostasis leading to the development of acute myeloid leukemia (AML). GATA2, a zinc finger TF predominantly expressed within the hematopoietic system, is an essential, level-dependent regulator of HSC generation, survival and functionalityTHP1, HL60, K562, and NOMO1 were cultured in RPMI supplemented with 10% FBS. K-7174 (Bioquote limited) and Etoposide  were dissolved in DMSO. Ara-C  was dissolved in PBS. Inhibitor assays were performed for 48 hours.Colony forming cell (CFC) assay was performed by plating 2,000 THP1 cells into methylcellulose H4434 (STEMCELL technologies) following the manufacturer’s instructions.10 and ArrayExpress11 to yield a case/control AML cohort hybridized to the same array (Affymetrix Human Genome U133 Plus 2.0 GeneChip). AML cohort (n = 2611), control cohort: (n = 77) from GEO  and ArrayExpress (E-MTAB-3444). Raw Affymetrix data were downloaded in raw CEL format and imported into an in-house analysis pipeline written in R (version 3.1.1) using Bioconductor12 packages from limma13, affy14 and oligo15. Data were normalised using RMA and differentially expressed genes/transcripts were identified using limma “best practice”, and p-values were corrected for multiple testing using Benjamini-Hochberg . Samples were then run through 2 bespoke R scripts to enable visualization per gene, where the WGNCA package16 was used to convert probe-level data to gene-level data. These datasets were then used to produce boxplots.AML and control patient datasets were downloaded from GEOBone marrow, peripheral blood or leukapheresis samples from patients diagnosed with AML/MDS  were diluted in H2O, and then mixed with calcium chloride (Sigma). This mix was added drop-wise to 2x hepes buffered saline  and after 15′ incubation, added to the media of HEK293T cells at 70% confluency in a 10-cm dish. Supernatant containing lentiviruses was collected at 48 hours after transfection, passed through a 0.45 μm filter (Sigma), snap freezed in dry ice, and stored at minus 80 °C.Calcium phosphate method was used to generate lentiviruses+ cells.Lentiviruses encoding shGATA2 (31–34) (Genecopoeia) linked to GFP or a GFP control vector were bound to the retronectin-coated wells and AML cells were bound to the virus-retronectin-coated plate by centrifugation. Eight hours later, AML cells were transferred to another plate and expanded for 5 days before sorting GFP−ΔΔCT method of relative quantification18.RNA extraction was carried out with the RNAeasy Plus Micro Kit (Qiagen) according to the manufacturer’s instructions. cDNA was made using the QuantiTec RT Kit (Qiagen) according to the manufacturer’s instructions. Real time quantitative PCR (RT-qPCR) was performed in a QuantStudio® 7 Flex Real-Time PCR System (Applied biosystems) using Taqman (Applied Biosystems) method. Differences in input cDNA were normalised against the housekeeping gene GAPDH (Hs02758991_g1) and the mRNA expression levels of GATA2 (Hs00231119_m1) were determined by the 210 μg of protein extracts were separated in a SDS-PAGE gel, and transferred to PVDF membranes overnight. Membranes were incubated overnight with anti-human GATA2  and detected by a HRP-conjugated anti-mouse IgG secondary antibody and enhanced chemiluminescent (ECL) reagents . Beta-actin or GAPDH was used as a loading control.19. To assess the cell cycle status, AML cells were stained with DAPI (5 μg/mL) and 0.1% NP40 (Sigma). Live cells were enumerated by staining with propidium iodide (20 μg/mL) (Sigma). Data were analysed using FlowJo 10.0.8  software and graphed using GraphPad Prism 7 . For intracellular flow cytometry, AML cells were fixed with 1% PFA (Thermofisher) for 12′ on ice. Cells were permeabilised with 2% BSA (Thermofisher) and 0.1% Triton-X (Sigma) for 20′ on ice, washed, and stained for 30′ on ice in the dark with anti-human GATA2-PE (IC2046P) (R&D systems). Cells were then washed twice with 2% BSA PBS and analysed in a BD LSR Fortessa IV (BD).To measure apoptosis, Annexin V assay (BioLegend) was performed as described previouslyData presented as mean ± SEM. Significant differences were calculated using Mann–Whitney U test or ONE-WAY ANOVA.We initially sought to uniformly evaluate GATA2 expression in a large cohort of AML patients. AML (n = 2611) and control (n = 77) patient datasets were downloaded from Gene Expression Omnibus (GEO) to create a case/control cohort hybridised to the same array (Affymetrix Human Genome U133 Plus 2.0 GeneChip) and analyzed through R using bio-conductor packages, where data was normalized using Robust Multi-array Average (RMA). We found that GATA2 expression was higher in AML patients compared to healthy controls (BM MNCs) and was overexpressed in 25% of AML samples Fig. . GATA2 o8). In agreement with results in GATA2 KD THP1 cells, we observed an impact on cell proliferation and survival in HL60 and K562 cells following GATA2 KD  and Etoposide (VP16). By incubating THP1, HL60 and K562 cells with K-7174, a pharmacological inhibitor of GATA2lls Fig. . HL60 cells Fig. . In contlls Fig. . While klls Fig. , complet23). Yet GATA2 inhibition did not impact NOMO1 cells, which exhibit normal GATA2 expression. It is possible that persistent GATA2 inhibition could alter the proliferative and survival capacity of AML cells with normal GATA2 expression. Notably, the extent of impact of GATA2 knockdown in leukemic cells appears to be independent of basal GATA2 overexpression level , which may reflect differential dose-dependent transcriptional requirements for GATA2 in myeloid leukemia subtypes or in pediatric versus adult patients. We propose transient, pharmacological inhibition of GATA2, in collaboration with standard chemotherapeutics, as a novel therapeutic strategy in AML cases where GATA2 is overexpressed. That effects were observed in cell lines derived from patients with pediatric AML (THP1) or adult AML (HL60) suggests that inhibiting GATA2 will be clinically effective in both pediatric and adult AMLs that overexpress GATA2. Alternatively, as opposed to directly targeting GATA2, it may feasible to therapeutically target the GATA2 transcriptional network in these AML settings. Given that relative GATA2 expression increases in AML patients after chemotherapy23, beneficiaries of this therapy could extend beyond those patients who overexpress GATA2 at diagnosis. GATA2 mediated therapeutic strategies should also focus on human AML leukemic stem cells, where GATA2 expression is up-regulated24 and where inducing cell death would be an attractive strategy to eliminate the provenance of leukemic cell growth.In this report, we identified that GATA2 mediates regulation of cell proliferation and survival in human AML cells overexpressing GATA2. This inference is consistent with independent observations in the GATA2 overexpressing AML cell line KG-1 Fig.  and 23. Supplementary Information


Insect OR/Orco dimers are nonselective cation channels that pass also calcium which leads to elevated calcium levels after OR activation. CALX exhibits an anomalous regulation in comparison to its homolog in mammals sodium/calcium exchanger, NCX: it is inhibited by increasing intracellular calcium concentration [Ca2+]i. Thus, CALX mediates only Ca2+ efflux, not influx. The main goal of this study was to elucidate a possible role of this protein in the olfactory response. We first asked whether already described NCX inhibitors were capable of blocking CALX. By means of calcium imaging techniques in ex-vivo preparations and heterologous expression systems, we determined ORM-10962 as a potent CALX inhibitor. CALX inhibition did not affect the odor response but it affected the recovery of the calcium level after this response. In addition, CALX controls the calcium level of OSNs at rest.CALX, the Na Taking into account that the Ca2+ concentration outside the cells is around 10,000-fold higher than in the interior , a special class of olfactory receptors, have evolved in winged insects probably as a response to the challenge of detecting airborne odors  and the sodium/calcium exchanger (NCX). ATP-driven calcium pumps bind Ca2+ with high affinity but display a low turnover rate, NCX extrudes Ca2+ with a >10-fold higher turnover rate compared to PMCA pumps , e.g., during an action potential, the NCX is likely to be more efficient in extruding calcium than the PMCA.There are two main Ca2+ ion for the uptake of three Na+ first described in Nicoll et al.  mode, it exports one Cal et al. . During 2+ overload in a situation of ischemia/reperfusion injury  expression vector as previously described  using an Amaxa 4D-Nucleofector  with the SF Cell Line 4D-Nucleoefector X Kit . After electroporation, cells were cultured on poly-L-lysine  coated coverslips at a density of ~3 × 105 cells per well (24 well plates). For experiments cells were exposed to normal bath solution .Drosophila melanogaster flies with genotype w;UAS-GCaMP6f/Cyo;Orco-Gal4/TM6B were reared under a 12 h light: 12 h dark cycle at 25°C on conventional agar medium. For experiments, antennae of 4–8 days old females were excised and prepared as described in Mukunda et al.  or Na+ free Ringer solution , 130; HCl, 10; KCl, 5; MgCl2·6H2O, 2; Ca, 2; and sucrose, 30; pH = 7.3. Thereafter the funiculus was cut allowing access to the OSNs for experiments. Antennae were immersed in solution during the experiments.a et al. . Briefly2+]i. Imaging experiments of cells were conducted 24 h after electroporation. Cells were incubated in Opti-MEM medium (Gibco) and loaded with 5 μM Fura-2 acetoxymethyl ester  for 30 min at room temperature. After wash, cells were kept during the experiment in bath solution. Free intracellular Ca2+ concentration ([Ca2+]i) was calculated according to [Ca2+]i = Keff(R − Rmin)/(Rmax − R), where Rmin and Rmax were determined as described in Mukunda et al. , coupled to an epifluorescence microscope . A water immersion objective  was used controlled by an imaging control unit . Fluorescence images were acquired using a cooled CCD camera controlled by TILLVision 4.5 software (TILL Photonics). Experiments lasted 20 min with a sampling interval of 5 s. One-hundred microliter of the different chemicals were applied via pipette in proximity of the objective. VUAA1 was applied at a concentration of 25 μM, KB-R7943 at 100 μM, SEA 0400 at 0.1 μM and ORM-10962 at 1 μM. Samples were continuously perfused with bath solution in the perfusion/recording chamber  during the experiments. TillVision software  was used to subtract background fluorescence and to define regions of interest (ROI) characterized by a change in the phenyl]ethyl ester carbamimidothiotic acid, monomethanesulfonate) from Cayman Chemical . SEA-0400 methoxy]phenoxy]-5-ethoxyaniline) was purchased from ApexBio Tech LLC . ORM-10962 ([2-(4-hydroxy-piperidin-1-yl)-N-(6-((2-phenylchroman-6yl)oxy)pyridin-3-yl)acetamide]) was kindly provided by Orion Corporation . All chemicals were dissolved in DMSO to yield a stock solution. When used as control DMSO was dissolved 1:1000.Female flies between 4 days and 8 days old were collected. First, the head was removed and prefixed in 4% PFA  +0.1% Triton for 10 min on ice. For preparation of the antenna, the 3rd segment was excised and fixed with 4% PFA  +0.1% Triton for 2 h on ice. Antenna were washed with phosphate-buffered saline solution containing 0.1% Triton X-100 (PBST) for 3 × 20 min. Antennae were blocked in normal goat serum (NGS) in PBST (PBST-NGS) for 60 min at room temperature and then incubated with the primary antibodies in PBST-NGS for 2 days at 4°C. After 4 × 15 min wash at room temperature, antennae were incubated with secondary antibodies in PBST-NGS for 2 days at 4°C. Finally, antennae were washed again for 3 × 20 min and mounted in Vectashield . Primary antibodies used: mouse anti-GFP , rabbit anti-CALX . Secondary antibodies used: goat anti mouse Alexa 488- and goat anti rabbit Alexa 546 .We prepared female flies (4 and 8 days old) according to Hsiao et al. . BrieflyImages were acquired on a cLSM 880  using a 40× water immersion objective . Images were obtained at 0.10–0.10 μm intervals at 1592 × 1592 pixel resolution for the antennae overview and at 0.12–0.12 μm intervals at 512 × 512 pixel resolution for the detailed section. Confocal images were adjusted for contrast and brightness by using LSM Image Browser 4.0 (Carl Zeiss) and Adobe Photoshop CS.t-test or Unpaired t-test. The decay was calculated and normalized in percentage during washing periods. Each washing period entails 150 s.Statistical analyses were performed using Prism 4 . Data are given as mean ± SEM (standard error of the mean) and were analyzed using Paired 2+-permeable, any receptor activation causes a Ca2+ influx. To ensure a fast and reliable Ca2+ handling after OR activation, we expected a high expression of CALX in the outer dendrites of OSNs, where ORs are expressed. To demonstrate this, we performed CALX immunostaining in the Drosophila antenna. Figure Drosophila eyes. The immunolocalization of CALX in photoreceptors cells had been previously shown by Wang et al.  compared to that of the second response . Our first candidate for CALX inhibition, KB-R7943, was already reported to block activation of ORs in the mosquito, and our results further supported this finding in Drosophila OSNs. The intensity of the response decreased from 114.2 ± 16.3 to 25.51 ± 9 after VUAA1 25 μM in the presence of KB-R7943 .In order to elucidate a possible role of CALX in the odor response, it was first necessary to determine a selective inhibitor that did not affect OR function. To determine if the previously described NCX inhibitors may act on ORs, we performed experiments using an Drosophila Orco was expressed in Human Embryonic Kidney cells (HEK293) and the change in [Ca2+]i was monitored with fura 2. The average increase in [Ca2+]i decreased from 418.66 ± 47 to 146.67 ± 13.46 nM under VUAA1 100 μM conditions . To exclude the possibility of Orco adaptation after a robust first response at VUAA1 100 μM, we repeated the experiments using a moderate stimulation of VUAA1 50 μM. Under this condition, the average increase in [Ca2+]i also decreased significantly from 214.02 ± 16.94 to 135.03 ± 4.11 nM . Hereby we confirmed that KB-R7943 is blocking Orco and therefore is not suitable to investigate a possible role of CALX in the odor response.To confirm the blocking of Orco by KB-R7943, we then performed experiments in heterologous expression system. Drosophila OSNs with SEA 0400 at 10 μM to test for a negative effect on Orco. In presence of SEA 0400 (10 μM), there was no significant difference between the intensity of the first response (140.6 ± 22.9) and the intensity of the second . Hence, SEA 0400 has no blocking effect on ORs and is a putative candidate for investigating the role of CALX in the odor response.Our second candidate, SEA 0400, is a benzyloxyphenyl derivative, as KB-R7943, but was reported to be a much more potent NCX inhibitor .We then focused our attention in the last and most recently described NCX inhibitor: ORM-10962. This compound has been reported to be a new selective inhibitor of NCX in its reverse and forward mode at 1 μM  these compounds do not act on CALX; or (ii) CALX does not affect the maximum of the response. However, as shown in Figures 2+ level recovery, the decay of the response was calculated and normalized in percentage during washing periods  = 8; Figure t-test; ns, p > 0.05; n(SEA) = 8). However, we observed a significantly lower decay, 45% compared to 74% under control conditions, after application of ORM-10962  = 7). To further test the influence of CALX in the decay of the Ca2+ levels, we performed the control experiments under Na+ free conditions  and the second (29%) decay in Na+ free conditions  = 8, n(VUAA1Na+ free = 8)). Furthermore, the decay in Na+ free conditions is comparable to the decay in presence of ORM . These results point out that CALX plays a major role as a calcium extrusion mechanism and that it is involved in restoring Ca2+ levels after an odor response. It also provides a strong hint about the effectiveness of ORM-10962 as CALX inhibitor.We thus focused our attention on the decay of the response. To evaluate the contribution of CALX to Cas Figure . Under ts Figure  are sign2+ extrusion mechanism, we would expect an increase in the [Ca2+]i over time. For analysis, we compared the calcium levels at the end of the recording (900 s) between treatments and control  = 9, n(SEA) = 9; Figure 2+]i at 900 s (44.9 ± 8.8) compared to control conditions  = 9, n(ORM) = 8). When we performed the control experiments under Na+ free conditions we observed an increase in the [Ca2+]i over time  and in the presence of ORM 1 μM (44.9 ± 8.8), t-test; p > 0.05; ns, not significant, n(DMSONa+ free) = 7, n(ORM) = 8). However, there is a significant difference between the quantified [Ca2+]i between control and Na+ free conditions at 900 s  = 7, n(DMSO) = 9).To confirm ORM-10962 as a putative CALX inhibition, we performed experiments under resting conditions in the antenna preparation. In case of blocking the main Cae Figure . This isDrosophila Na+/Ca2+exchanger CALX without side effects on the co-receptor Orco and that CALX seems to play no role in the modulation of the odor response. However, according to our Na+ free and resting conditions experiments, CALX controls the Ca2+ level of OSNs at rest and is involved in shaping the recovery after odor stimulation.Taken together, our results point out that ORM-10962 is capable of blocking the 2+ pumps or exchangers. Sensory cascades operating through rapid Ca2+-mediated signaling seem to rely on Na+/Ca2+ exchange mechanisms. For example, Drosophila photoreceptor cells are very sensitive to perturbations in the Na+/Ca2+ exchange activity mediated by CALX  olfactory neurons. They argued that because of its lower affinity to calcium and its voltage dependent properties, NCX’s efficiency will decline with depolarization of the neurons during an odor response. Their evidence suggests that both Ca2+ transporters contribute to re-establish resting Ca2+ levels in the cilia following olfactory responses. However, our results suggest that in Drosophila, CALX plays a more important role in maintaining calcium homeostasis (Figure 2+ binding protein, modulates Drosophila odorant receptor function through Orco (Mukunda et al., 2+ regulatory mechanisms in Drosophila olfactory transduction.The experiments with the last candidate for inhibition of CALX, ORM-10962, indicated that there was no negative effect in the Orco-response Figures . By contd Figure . This ins Figure . The imp Figures . Under ts Figure . Calmodu+/Ca2+ exchangers are important for the dynamics of the olfactory response (Jung et al., Drosophila olfactory neurons both under resting conditions and after enhanced activity.In conclusion, in the current study we identified ORM-10962 as potent CALX inhibitor. As in other organisms, where NaDrosophila melanogaster was performed in Germany where the research on invertebrates does not require a permit from a committee that approves animal research.This study on the vinegar fly DW and LH-L designed the experiments. LH-L conducted the experiments and the analysis. LH-L, DW and BH wrote the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Calcium transients in thin astrocytic processes can be important in synaptic plasticity, but their mechanism is not completely understood. Clearance of synaptic glutamate leads to increase in astrocytic sodium. This can electrochemically favor the reverse mode of the Na/Ca-exchanger (NCX) and allow calcium into the cell, accounting for activity-dependent calcium transients in perisynaptic astrocytic processes. However, cytosolic sodium and calcium are also allosteric regulators of the NCX, thus adding kinetic constraints on the NCX-mediated fluxes and providing for complexity of the system dynamics. Our modeling indicates that the calcium-dependent activation and also calcium-dependent escape from the sodium-mediated inactive state of the NCX in astrocytes can form a positive feedback loop and lead to regenerative calcium influx. This can result in sodium-dependent amplification of calcium transients from nearby locations or other membrane mechanisms. Prolonged conditions of elevated sodium, for example in ischemia, can also lead to bistability in cytosolic calcium levels, where a delayed transition to the high-calcium state can be triggered by a short calcium transient. These theoretical predictions call for a dedicated experimental estimation of the kinetic parameters of the astrocytic Na/Ca-exchanger. Initial IP3 production is attributed to activation of astrocytic G protein-coupled receptors to glutamate (mGluRs), with subsequent production of diacylglycerol and IP3 by phospholipase Cβ. The importance of this pathway has been challenged by questioning the existence of mGluRs in adult astrocytes  channels, astrocytic ionotropic receptors to glutamate, voltage-gated Ca2+ channels and NCX working in Ca2+ entry mode . The opposite exchange direction (“reverse mode”) becomes energetically favorable at high enough [Na+]i and depolarized membrane potential. For example, in the cardiomyocytes, the NCX switches between the modes during the contraction cycle  binds Na+ ions (E2·3Na+) and changes its conformation to the inward-facing binding site (E1·3Na+), from where it can either release Na+ to acquire the substrate-free inward-facing conformation (E1) or undergo transition to Na+-bound inactive conformation I1 in a Na+-dependent manner; the protein in E1 state can bind a Ca2+ ion (E1·Ca2+) or switch to substrate-free inactive state I2; the inward-facing Ca2+-bound state (E1·Ca2+) flips to the outward-facing Ca2+-bound state (E2·Ca2+), and dissociation of Ca2+ closes the cycle. All the transitions are reversible and going rough the cycle in the clockwise direction corresponds to “reverse” (Ca2+-influx) mode. Transitions from I1 and I2 are facilitated by cytosolic Ca2+. Thus, NCX activity is allosterically regulated by cytosolic Na+ and Ca2+  as a product of maximal flow JEΔ (2)–(3), and gating variables n and h corresponding to deinactivation dependent on occupancy of the high- and low-affinity Ca2+ binding sites. The JEΔ factor is described as a simplification of the normalized net reaction rate of the ping-pong bi-bi cyclic reaction scheme. In essence, JEΔ models the main kinetic cycle shown in Figure h and n correspond to the fraction of active enzyme in the exchange with I1 and I2 states, respectively.We follow the general approach of earlier works by Weber et al.  and GinsKM· are the corresponding Michaelis constants, η ∈ 0 … 1 is the position of the energy barrier of the enzyme in the membrane electric field relative to membrane width, and ksat parameter controls the saturation of Jncx at negative potentials; these parameters are as in Weber et al. –(7):The allosteric factor of Weber et al.  and Ginsa et al.  reportedn et al. , where th negatively depends on [Na+]i, but the extent of this influence is scaled by [Ca2+]i with cooperativity coefficients HNa and HCa, correspondingly (6). We also acknowledge the dependence of τh on [Ca2+]i reported in Matsuoka et al. –(3), and for simplicity, all Ca2+ equilibrating mechanisms, including Ca2+ buffering in the excessive buffer approximation, are lumped into a single linear term setting resting Ca2+ concentration and equilibration timescale τCa. The second equation of the model is the kinetics of the h gating variable (5).where dh/dt = 0 (h-nullcline), and 2+-nullcline). Any point on this plane corresponds to some state of the model, a combination of specific [Ca2+]i and h values. Dynamics of the model starting from some initial conditions will be a line on this plane, called phase trajectory. Because 2+-nullcline, all trajectories will cross it vertically. Correspondingly, all trajectories must cross the h-nullcline horizontally. Nullcline intersections are equilibrium points because here both variables don't change: Phase plane h-nullcline (defined by h = h∞) shows only subtle dependence on [Na+]i, the Ca2+-nullcline is more parameter-sensitive. First, it has a discontinuity when JEΔ=0 which impliesWhile Jncx vanishes, the 2+ range and slope of the Ca2+-nullcline considerably depend on the +]i, and Vm.Because when +]i for any reasonable +]i, there is a single and stable equilibrium point. However, the appropriate combination of high [Na+]i, depolarized Vm and +]i  the Ca2+–nullcline shows a discontinuity within a physiological range of resting [Ca2+]i and a single equilibrium point. Increasing [Na+]i to 80 mM tilts the Ca2+–nullcline, and drives the discontinuity to very high values of [Ca2+]i. It leads to appearance of two stable equilibrium points: one at low [Ca2+]i and one at higher [Ca2+]i. All trajectories starting from the purple region of the phase plane will converge to the low–Ca2+ state, while all trajectories starting from the yellow region will converge to the high–Ca2+ state.Consequently, at low [NaVm and [Na+]i shape the Ca2+-nullcline, a smaller increase in [Na+]i would lead to a similar nullcline configuration at a more depolarized Vm. Maps of the highest stable [Ca2+]i and regions of Vm–[Na+]i combinations leading to bistability for different + and Ca2+ modulation of the NCX leads to bistability in the system with NCX shaping the Ca2+ exchange.Because both +]i and [Ca2+]i as dynamical variables and an additional controlled Ca2+-flux + also with a controlled Na influx We next turn to a simple point model with both i responds to the onset of 2+]i due to Na+-dependent drop in h. A short Ca2+ stimulating pulse is widened by NCX in the Na+-dependent manner due to a regenerative Ca+ influx, where rising [Ca2+]i deinactivates NCX (increases h) with a positive feedback. Eventually, a high enough increase in [Na+]i is sufficient to lead to a sustained high–Ca2+ state. Note,that activation of NCX by a Ca2+ pulse also leads to a drop in [Na+]i level. The same Ca2+ pulse given without Na+ leads only to a small Na+ transient due to NCX working in the direct mode.Model space is summarized in Figure n Figure , the syss Figure  resultins Figure . Here 202+ elevation in a spatially distributed system? We tested this conjecture in a minimalistic one-dimensional model describing a section of a thin astrocytic process lacking internal Ca2+ stores. One-dimensional space is discretized into elements 0.35 μm long. Each element contains the same local dynamics as in the point model with an additinal diffusion of Na+ and Ca2+ between the elements. Only the central (x = 0) element experienced an external Ca2+-pulse and elements from x = 0 to x = 10μm. on the one side experienced a short Na+ pulse. Ca2+ could diffuse in both directions from the central position, but [Na+]i profile was uneven, leading to anisotropic amplification and spreading of the initial Ca2+ pulse as compared to linear summation of the [Ca2+]i changes caused by Ca2+ and Na+ pulses given separately (Supplementary Figure Will the same mechanism allow for spreading waves of Ca+ influx caused by neurotransmitter uptake, if happening under resting [Ca2+]i-conditions, beside a small elevation in [Ca2+]i, can “prime” the NCX by simultaneously electrochemically favoring Ca2+ influx via the reverse mode and inactivating NCX by Na+-dependent transition to I1. In this state, a localized Ca2+ surge, e.g., via spontaneous opening of a TRP channel, will be amplified nonlinearly by the NCX rescued form the inactive conformation by Ca2+. In contrast, under resting membrane potential and low [Na+]i, localized Ca2+ transients will be reduced by NCX working in the direct mode. This attenuation can be small if a large portion of the NCX is already inactivated at resting [Na+]i, but can be substantial in sub-normal [Na+]i conditions.We argue that Na2+ transients, account for switching between low-Ca2+ and high-Ca2+ states and even spatially spreading zones of elevated Ca2+. The extent and duration of Na+ elevations are dependent on the timescale of Na+ clearance from the cytoplasm by the Na/K-pump. In hippocampal slices (Langer et al., + elevations were observed on a similar timescale to the presented in simulations. On this timescale the NCX-mediated Ca2+ influx can prime IP3 receptors on ER membranes in the astrocytic branchlets and branches in a fashion, similar to that proposed in cardiomyocytes, where the reverse mode of NCX is supposed to participate in excitation–contraction coupling (Shattock et al., 2+ dynamics of the astrocyte. This, especially in view of apparent colocalization between NCX and IP3-receptors in plasma membrane-ER junctions (Blaustein et al., 2+ dynamics and the NCX-mediated Ca2+ influx. It is also expected that under a metabolically challenged state, when the Na/K-pump turnover is slowered, there will be higher Na+ elevations and correspondigly higher NCX-mediated Ca2+ influx. Corroborating this prediction, a recent study by Gerkau et al. (+ loading and increased Ca2+ transients in astrocytes and neurons under chemical ischemia; blocking the reverse mode of NCX increased Na+ loading and dramatically reduced Ca2+ activity. Pathologically high synaptic activity is also expected to both cause higher Na+ accumulation and locally depolarize perisynaptic astrocyte processes due to increased Our modeling predicts that NCX can increase amplitude and duration of Cau et al.  demonstr2+ signaling to justify a dedicated experimental testing for the predicted nonlinear interaction between NCX and Ca2+ under high-Na+ and low-Na+ conditions.The proposed model is primarily based on experimental data for cardiac NCX1 isoform (Matsuoka et al., https://zenodo.org/record/1218115.Model implementation and parameter values used in the simulations are supplemented as a Jupyter (Kluyver et al., AB: model formulation, preparing the figures, writing the paper; AV: model simulations, preparing the figures; DV: model simulations, preparing the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.