The NTR hosts numerous mutations linked to skeletal (RyR1) and cardiac (RyR2) myopathies, highlighting its potential as a therapeutic target. Here, we constructed two biosensors by labeling the mouse RyR2 NTR at domains A, B, and C with FRET pairs. Using fluorescence lifetime (FLT) detection of intramolecular FRET signal, we developed high-throughput screening (HTS) assays with these biosensors to identify small-molecule RyR modulators. We then screened a small validation library and identified several hits. Hits with saturable FRET dose–response profiles and previously unreported effects on RyR were further tested using [3H]ryanodine binding to isolated sarcoplasmic reticulum vesicles to determine effects on intact RyR opening in its natural membrane. We identified three novel inhibitors of both RyR1 and RyR2 and two RyR1-selective inhibitors effective at nanomolar Ca2+. Two of these hits activated RyR1 only at micromolar Ca2+, highlighting them as potential enhancers of excitation–contraction coupling. To determine whether such hits can inhibit RyR leak in muscle, we further focused on one, an FDA-approved natural antibiotic, fusidic acid (FA). In skinned skeletal myofibers and permeabilized cardiomyocytes, FA inhibited RyR leak with no detrimental effect on skeletal myofiber excitation–contraction coupling. However, in intact cardiomyocytes, FA induced arrhythmogenic Ca2+ transients, a cautionary observation for a compound with an otherwise solid safety record. These results indicate that HTS campaigns using the NTR biosensor can identify compounds with therapeutic potential.The N-terminal region (NTR) of ryanodine receptor (RyR) channels is critical for the regulation of Ca Defe.2 MDa chopathies , 7, 8, 9opathies , 12, 13.opathies , many ofopathies . The undopathies , 18, 19.2+ leak in skeletal muscle SR membrane vesicles and mechanically skinned muscle fibers  platform based on fluorescence-labeled RyR regulators FK506 binding protein 12.6 (FKBP) and calmodulin (CaM) to monitor binding of these modulators to RyR , 21. Usie fibers , 21 and e fibers . Howevere fibers . An attre fibers . HoweverA)   is composed of three domains  and is important for the functional regulation of RyR channels . The regFig. 1A) , 26. NTRFig. 1A) , 27. TheFig. 1A) . Very reFig. 1A) . In addiFig. 1A) , 30. ManB), then used an FLT plate reader to measure changes in FLT-detected FRET efficiency as an indication of structural changes in the NTR due to small-molecule binding. Initial hit compounds obtained through FLT-FRET HTS of a small library were further validated using a dose–response format of the same FRET assay, and this was followed by functional assays (ryanodine-binding measurements) using full-length RyR in SR membrane vesicles of skeletal and cardiac muscles to further confirm their effects on RyR channels. The most promising hit was further evaluated on RyR leak in rat skeletal muscle fibers and mouse cardiomyocytes.Given the critical role of the NTR in the regulation of RyR, we have engineered NTR constructs for use as FRET biosensors for an HTS platform to identify small molecules that interact with the NTR and allosterically regulate the activity of full-length RyR. We introduced donor–acceptor FRET pairs at appropriately distanced sites in different domains of the NTR B, then uA (left panel), the similar CD spectra for WT and AC-NTR indicate that the Ala replacement of seven native Cys and addition of two Cys at K30 and K441 did not grossly alter the secondary structure of NTR. The BC-NTR has a marginally different CD spectrum relative to WT- and AC-NTR. CD spectra of the R420Q-NTR mutant and corresponding AC- and BC biosensor constructs also indicate that the engineered mutations did not alter the secondary structure content versus R420Q . However, the CD spectrum of R420Q-NTR shows a clear difference relative to WT-NTR. Previously, conformational changes had been observed for the R420Q mutant relative to WT  . Interprobe distances of the two biosensors were determined using previously described methods of multiexponential analysis of the FLT-detected FRET measurements , and R383C/K441C (termed BC-NTR). We used CD to evaluate the impact of mutations and labeling on the overall secondary structure of NTRs. As shown in Figure S1ve to WT . The CD Fig. 1B) . Based ourements  31) and andA  is sensitive to small ligands. Multiexponential analysis of the FLT-detected FRET measurements was performed as previously described  for an assay driven by the AC or BC construct, respectively , CPNQ had the strongest effect (ΔFLT ≈0.35 ns), which was similar to corbadrine, albeit the latter had lower potency (∼3-fold higher EC50). Both GANT61 and oltipraz M2 decreased FLT but did not reach saturation within the tested concentration range, indicating a lower potency compared with corbadrine and CPNQ. Among the hits that increased FLT , FA and tacrolimus exhibited a stronger effect than the other two compounds in this group (ΔFLT ≈0.1 ns). Temsirolimus and Ro 90-7501 produced smaller increases in FLT, but with a higher potency (EC50 <1 μM). Temsirolimus and the related tacrolimus (aka FK506) are known ligands of FKBP, and they belong to a group of compounds thought to indirectly influence the RyR function, by preventing FKBP-RyR binding  indicate that these compounds may also directly interact with the RyRs, a hypothesis to be explored in future studies. Tacrolimus and Ro 90-7501 had already been identified as RyR regulators in our previous HTS campaigns (via a FRET HTS assay that uses full-length RyRs), and their RyR activatory and inhibitory effects (respectively) have been reported (3H]ryanodine-binding assays (shown below).Individual FRET dose–response curves of the eight remaining hits, along with their chemical structures, are shown in Fig. 4 binding . Our resreported , 21. The3H]ryanodine-binding assays using SR membrane vesicles isolated from pig skeletal and cardiac muscle. This is a well-established method that is often used to determine the activity of RyR channel in their native membrane environment  and high Ca2+ (30 μM), corresponding to the resting and contracting muscle conditions, respectively. This is to glean early insight in the potential effect of a hit on the resting RyR leak, as well as on RyR activation during EC coupling.To functionally evaluate the hits that had a clear FRET dose–response profile and previously unreported effects on RyR function, we performed ryanodine-binding analyses to determine the dose-dependent effects of the novel FRET hits . All discussed compounds show biphasic responses, with inhibition by submicromolar concentration gradually turning into activation at the highest concentrations tested  or by nonspecific inhibition caused by colloidal aggregation of ligands at high concentrations , meaning that these dose–response profiles may represent RyRs that are not in complex with the same set of binders or carrying different posttranslational modifications.For skeletal SR (RyR1) at 30 nM Cas tested . Maximums tested . The bips tested . This matrations , 45, amo2+  versus nanomolar (0.03 μM) for RyR1.For cardiac SR (RyR2) at 30 nM Ca2+   . This ismary HTS .2+ leak using a confocal microscopy readout of enclosed transverse-tubule [Ca2+] in rat mechanically skinned skeletal muscle fibers  and 1 mM tetracaine (an RyR blocker) were exchanged, respectively. As shown in Figure 6A, in the presence of 10 μM FA in bath solution, the steady-state [Ca2+]t-sys decreased, indicating inhibition of RyR1 leak by FA. The recovery of the t-system fluorescence after washout with caffeine and reintroduction of standard solution indicates that FA can be washed out of the fiber. Figure 6B compared normal t-system Ca2+ uptake and t-system [Ca2+] that reduced [Ca2+] by 10 μM FA and tetracaine. This suggests that high FA inhibits RyR1 leak.As described in previously published work, in a typical experiment , the con2+ transients were generated in single rat muscle fibers in the presence of DMSO (0.1%), 10 μM FA, and 1 mM tetracaine. Interestingly, 10 μM FA did not have an effect on Ca2+ transient amplitude. As a comparison the application of tetracaine, which is a full RyR blocker, significantly decreased the electrically induced Ca2+ events. Ideally, therapeutically targeting and reducing excessive RyR1 Ca2+ leak should not be accompanied with a reduction in normal muscle contraction. These results show that FA can reduce RyR1-mediated Ca2+ leak without a significant effect on normal EC coupling.In addition to the effect of FA on RyR1 leak, we also explored its potential influence on EC coupling in rat skeletal muscle . Electri2+]i = 50 nM) to assess Ca2+ spark activity, a relatively direct measure of SR Ca2+ leak, during 6 min exposures to increasing [FA] (A–C). In repeated measures of Ca2+ spark frequency over [FA], we did not detect significant differences between [FA], relative to 0 nM FA , though the data suggest a possible minor trend for reduced Ca2+ spark frequency (meancell slope = −0.082 sparks/100 μm/s/log10(μM)) with increasing [FA] (p = 0.115) (B). However, repeated measures of Ca2+ spark signal mass , a readout of the amount of Ca2+-spark mediated SR Ca leak, was decreased significantly at 1 μM FA (p = 0.011) and 10 μM FA (p = 0.001), relative to 0 nM FA. Moreover, Ca2+ spark signal mass exhibited a progressively decreasing trend (meancell slope = −0.336 F/F0 × μm × s/log10(μM)) with increasing [FA] (p = 0.0007), indicating progressively smaller release events (C). Together, the Ca2+ spark data suggest that FA suppresses SR Ca2+ leak in a dose-dependent manner primarily by limiting SR Ca2+ release during individual events.We first used saponin-permeabilized mouse ventricular cardiomyocytes  B. Howevee events C. Togeth2+ transients in intact mouse ventricular cardiomyocytes during pacing at 1 Hz. In most cardiomyocytes, even at very low [FA] (0.01 μM), we observed some diminution of Ca2+ transient amplitudes and an increased occurrence of arrhythmogenic Ca2+ activity (A). These Ca2+ events appeared as isolated spontaneous Ca2+ transients or runs of automaticity in the absence of pacing, Ca2+ waves, or persistent diastolic Ca2+ elevation (A and B). These events were almost never seen in control conditions  but were seen in the majority of cardiomyocytes  exposed to FA across all concentrations tested (0.01–10 μM). Thus, while FA may indeed have a quieting effect on RyR2, as inferred from the Ca spark measurements, it may also have side effects that increase myocyte Ca2+ loading or otherwise promote automaticity.We also measured Caactivity A. These levation , A and B2+]ER observed in HEK cells expressing human RyR2  that is sterically coupled to the channel pore regulation. The NTR is also one of the “hot-spots” of disease-associated mutations of RyR  ryanopathies to high-prevalence degenerative syndromes. Here, we used the FRET-labeled NTR to detect compound binding, as this is a domain that is critically involved in RyR function. Specifically, NTR assembles into a tetrameric vestibule A that iss of RyR , 27, 29.s of RyR , which aTo exploit the unique structural features and functional importance of the NTR, and the well-established high-precision FLT detection of FRET, we constructed two NTR biosensors by substituting Cys at two residues located in A and C, or B and C subdomains of the Cys-light NTR, and labeled these Cys pairs with fluorescent probes suitable as FRET donor and acceptor . The twoB) for further testing. The list of hits was further narrowed down to eight compounds based on FRET dose–response curves , or dantrolene  should be further pursued through studies using cell and animal models of skeletal and cardiac myopathies. In situ, cellular assays of RyR leak reinforce the in vitro results, as shown before. However, potentially deleterious effects with intact cardiomyocytes indicate that further safety studies are necessary for FA. Further studies will be necessary to determine the mechanisms of action of these new effectors and pursue medicinal chemistry development toward an RyR-targeted drug. Notable limitations of the NTR biosensor are typical to target-focused approaches, whereby a particular biosensor system can never identify all the ligands that may interact with the target, or that the Hit functional effects are often unpredictable. Nevertheless, we are encouraged about the outcomes of this study and plan future HTS screening of the NTR biosensor against a larger chemical library, to accelerate the discovery of RyR-targeted therapeutics.In summary, we developed an early-stage RyR-targeted drug discovery method exploiting high-throughput FLT-detection of FRET within an RyR2 NTR construct labeled with donor and acceptor, to identify small-molecule Escherichia coli Rosetta (DE3) pLacI cells at 18 °C and purified with a protocol adapted from previously published work (The NTR of mouse RyR2 (residues 1–547) was cloned and expressed as previously described . The DNAhed work . An amylhed work .The 1536-well assay plates were prepared as described in our previous reports . The LOP−, time-correlated single-photon counting measurement of fluorescence lifetime (FLT) was used. Time-resolved fluorescence decays were recorded after excitation with a 481-nm subnanosecond pulsed diode laser . Emitted light was selected using a 517 ± 10 nm filter (Semrock) and detected with a PMH-100 photomultiplier (Becker-Hickl). FLT waveforms of the donor only and donor–acceptor-labeled samples were acquired in media containing 60 nM biosensor, 10 mM HEPES, 250 mM KCl, pH 7.4 at 21 °C and analyzed to determine FRET distance relationships, as described in our previous publications , due to the presence of acceptor (τDA), using the following equation:HTS measurements were conducted in a prototype top-read FLT plate reader , which reads each 1536-well plate in ∼3 min , 64. AF4+ and σ− are the SDs of the positive and negative control τDA, respectively; μ+ and μ− are the means of the positive and negative control τDA, respectively. A value of Z′ ≥ 0.5 indicates an excellent HTS assay, while 0.5>Z′ ≥ 0 indicates an adequate assay  relative to negative control (0.1% DMSO) and measurement precision, as indexed by the Z′ factor :(2)Z′=1−te assay .2+ solution at a flow rate of 10 μl/min. The NTR at a concentration of 30 μg/ml in 10 mM HEPES, 150 mM KCl, pH 7.4, was immobilized on flow cell 2; flow cell 1 was left blank to serve as a reference surface. To collect kinetic binding data, the hits in the running buffer  were injected over the two flow cells at concentrations of 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 μM at a flow rate of 30 μl/min and at a temperature of 25 °C. The complex was allowed to associate and dissociate for 60 and 60 s, respectively. The surfaces were regenerated with a 120 s injection of 350 mM EDTA. Data were collected at a rate of 1 Hz. The data were fit using the global data analysis option available within Biacore S200 Evaluation 1.0 software.The SPR experiments were performed using a BIACORE S200  equipped with a NTA sensor chip. NTR with His and MBP tags (>90% pure based on SDS-PAGE) was immobilized using affinity of the His tag to the sensor surface. The surfaces of the sensor chip were activated with 0.5 mM NiCrude sarcoplasmic reticulum membrane vesicles were isolated from porcine longissimus dorsi muscle and porcine cardiac left ventricle tissue by differential centrifugation of homogenized tissue, as established previously . Skeleta2 , 0.1 mg/ml of BSA, and 20 mM K-PIPES (pH 7.0). Nonspecific [3H]ryanodine binding to SR was assessed by addition of 40 μM nonradioactively labeled ryanodine. Maximal [3H]ryanodine binding was assessed by addition of 5 mM adenylyl-imidodiphosphate (AMP-PNP), and in the case of CSR, 20 mM caffeine was added. These control samples were each loaded over four wells per plate. Binding of [3H]ryanodine  was determined following a 3 h incubation at 37 °C and filtration through grade GF/B glass microfiber filters (Brandel Inc) using a Brandel Harvester. In 4 ml of Ecolite scintillation cocktail , [3H]ryanodine retained on the filter was counted using a Beckman LS6000 scintillation counter.In 96-well plates, skeletal SR membranes  and cardiac SR membranes  were preincubated with 1% DMSO or hit compound for 30 min at 22 °C in a solution containing 150 mM KCl, 5 mM GSH, 1 μg/ml Aprotinin/Leupeptin, 1 mM EGTA, and 65 μM or 1.02 mM CaClvia CO2 exposure, and the extensor digitorum longus (EDL) was rapidly excised. Once dissected, muscles were pinned to Sylgard set in a Petri dish containing paraffin oil.All experimental methods using rodents were approved by the Animal Ethics Committees at The University of Queensland and were performed in accordance with the relevant guidelines and regulations. Five-month-old Wistar rats  were euthanized by asphyxiation 2+ movements across the t-system membrane, images were continuously recorded in xyt mode with an aspect ratio of 256 × 512, with the long aspect of the image parallel with that of the preparation. Temporal resolution of imaging in this mode where the fluorescence signal was within the borders of the fiber was 0.8 s.Bundles of fibers were isolated and exposed to an internal solution containing rhod-5N, as described previously . After 12+]t-sys as previously described . This chronically opened the RyR, thus thoroughly depleting the SR of Ca2+ and consequent depletion of t-system Ca2+ occurred via the activation of SOCE. Alternatively, the application of 200 nM [Ca2+]cyto standard internal solution allowed the uptake of Ca2+ and the [Ca2+]t-sys reached a steady state that was partially dependent on Ca2+ leaking through RyRs into the tight junctional space between the t-system and SR membranes . FA (10 μM), tetracaine (1 mM), and DMSO control (0.1%) were added to the standard internal solution bathing the skinned fibers, at known concentrations, to assess their influence on RyR Ca2+ leak.T-system rhod-5N fluorescence was converted to  of the Tyrode’s solution was then raised to 0.5 mM in stepwise fashion, and cardiomyocytes were kept at room temperature in this solution until use. All animal handling and laboratory procedures were in accordance with the approved protocols of the University of California, Davis Institutional Animal Care and Use Committee conforming to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health .Mouse ventricular cardiomyocytes were isolated enzymatically from hearts of male C57BL/6J mouse  as previously described with minor modifications . Briefly2+ activity was imaged via confocal microscopy  in the line scan mode . Ca2+ sparks were recorded from ventricular cardiomyocytes permeabilized with saponin (50 μg/ml) for 1 min in internal solution containing (in mM) 10 HEPES, 1 EGTA, 1 free MgCl2 (with free [Ca2+] adjusted to 50 nM using MaxChelator), 120 K-aspartate, 10 reduced glutathione, 5 ATP, 5 phosphocreatine di-Na, creatine phosphokinase 5 U/ml, and dextran  8% at pH 7.20. Cardiomyocytes were immediately washed with saponin-free internal solution and then superfused with internal solution (50 nM Ca2+free) containing the Ca2+-sensitive indicator, Fluo-4 pentapotassium salt . Ca2+ spark parameters were analyzed over a 10 s period from each line scan recording. Ca2+ transients and diastolic Ca2+ release events were recorded from intact ventricular cardiomyocytes loaded for 30 min at room temperature with Fluo-4 AM  and Pluronic F-127  followed by wash and de-esterification for 30 min in Tyrode’s solution containing (in mM) NaCl 140, KCl 4, CaCl2 1.8, MgCl2 1, HEPES 5, Na-HEPES 5, and glucose 5.5 at pH 7.40. To evoke Ca2+ transients, intact cardiomyocytes were paced at 1 Hz in a field stimulation chamber (Warner Instruments) at room temperature. Ca2+ activity was assessed in paired fashion before and after incubating cardiomyocytes with fusidic acid (FA) for 6 min at each concentration. ImageJ was used for image processing and analysis, and Ca2+ sparks were analyzed using the SparkMaster plugin  one-way ANOVA with Geisser–Greenhouse correction followed by the Dunnett’s multiple comparison post-hoc test was used for normally distributed data to determine individual differences in matched responses. The nonparametric Friedman one-way ANOVA with Dunn’s multiple comparison post-hoc test was used for non-normally distributed data to determine individual differences in matched responses. To determine dose-dependent trends in Ca2+]ER load-leak balance in HEK293 cells was previously described : K-aspartate 100; KCl 15; KH2PO4 5; MgATP 5; EGTA 0.35; CaCl2 0.22; MgCl2 0.75; HEPES 10; dextran  2%, pH 7.2. Free [Ca2+] and [Mg2+] were set at 100 nM and 1 mM respectively. After 3 min of saponin incubation, the permeabilized cells were perfused with saponin-free experimental solution.The method for measuring of [Caescribed . Briefly2+]ER load-leak balance in permeabilized HEK293 cells were monitored using a laser scanning confocal microscope  equipped with a 40× oil objective lens (N.A. =1.3). R-CEPIA1er was excited using the 543-nm line of a He–Ne laser, and fluorescence was observed at >580 nm. 2D images (512 × 512 pixels) were collected every 5 s at a scanning speed of 3 ms/line for each measurement.Changes in [Ca2+]ER signal. Then, the indicated drug concentration was applied for another 5 min. Caffeine (10 mM) was used at the end of each measurement to induce a complete depletion of [Ca2+]ER and obtain the Fmin value. Fmax was determined upon application of 2 μM ionomycin and 10 mM Ca2+. Changes in [Ca2+]ER were calculated according to [Ca2+]ER = (F−Fmin)/(Fmax−Fmin).Cells were perfused with the experimental solution for 3 min to ensure a steady [Cat test or one-way ANOVA followed by Tukey’s post-hoc test, as indicated, where p < 0.05 was considered significant. EC50 values were derived from the fits to Hill equations.Errors are reported as the standard error of the mean (SE), except when noted. Statistical significance was determined by Student’s All data are contained within the manuscript or supporting information.This article contains D. D. T. and R. L. C. hold equity in and serve as executive officers for Photonic Pharma LLC. These relationships have been reviewed and managed by the University of Minnesota. Photonic Pharma had no role in this study.


Fusarium oxysporum is an important plant pathogen and an emerging opportunistic human pathogen. Germination of conidial spores and their fusion via conidial anastomosis tubes (CATs) are significant events during colony establishment in culture and on host plants and, hence, very likely on human epithelia. CAT fusion exhibited by conidial germlings of Fusarium species has been postulated to facilitate mitotic recombination, leading to heterokaryon formation and strains with varied genotypes and potentially increased virulence. Ca2+ signalling is key to many of the important physiological processes in filamentous fungi. Here, we tested pharmacological agents with defined modes of action in modulation of the mammalian Ca2+ signalling machinery for their effect on germination and CAT-mediated cell fusion in F. oxysporum. We found various drug-specific and dose-dependent effects. Inhibition of calcineurin by FK506 or cyclosporin A, as well as chelation of extracellular Ca2+ by BAPTA, exclusively inhibit CAT induction but not germ-tube formation. On the other hand, inhibition of Ca2+ channels by verapamil, calmodulin inhibition by calmidazolium, and inhibition of mitochondrial calcium uniporters by RU360 inhibited both CAT induction and germ-tube formation. Thapsigargin, an inhibitor of mammalian sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), partially inhibited CAT induction but had no effect on germ-tube formation. These results provide initial evidence for morphologically defining roles of Ca2+-signalling components in the early developmental stages of F. oxysporum colony establishment—most notably, the indication that calcium ions act as self-signalling molecules in this process. Our findings contribute an important first step towards the identification of Ca2+ inhibitors with fungas-specific effects that could be exploited for the treatment of infected plants and humans. Evidence has been obtained suggesting the involvement of Ca2+ in the regulation of morphological and pathogenic events in fungi, such as secretion c forms a Ca2+ signal, which activates downstream signalling pathways through Ca2+-dependent proteins, such as calmodulin. Proteins such as calmodulin undergo conformational changes upon binding to four Ca2+ ions each c dynamics in living cells, pharmacological agents that modulate different components of the Ca2+-signalling machinery in animal cells have been extensively employed to study the role of Ca2+ signalling in filamentous fungi ethane-N,N,N′,N′-tetraacetic acid (BAPTA) chelates extracellular Ca2+ and inhibits the availability of extracellular Ca2+ for influx to cells c) were observed in growing hyphal tips of the wild type and FoCCH1 and FoMID1 mutants in the F. oxysporum strain O-685, which is different from the tomato pathogen F. oxysporum strain 4287 used in this study, although the pattern differed between each of the mutants and the wild type c during staurosporine-induced cell death in N. crassa [2+ ATPase by thapsigargin showed partial inhibitory effects on germination in 1% PDB or CAT induction in fusion medium. Germ-tube formation in fusion media was unaffected by inhibition of ER Ca2+ ATPase with thapsigargin. Our results with thapsigargin indicate that germ-tube formation could take place in the absence of Ca2+ uptake to the ER, albeit with reduced efficiency. A possible explanation is that the transient rise of cytoplasmic Ca2+ quickly falls back to resting levels due to alternative mechanisms involving other Ca2+-storage organelles, such as mitochondria or vacuoles, or by export to the extracellular medium. Germ-tube formation progresses since it is less sensitive to these changes, whereas CAT induction is inhibited, as it is dependent on Ca2+ uptake to the ER.Calmidazolium, an inhibitor of calmodulin, was found to inhibit both germ-tube formation and CAT fusion. CAT formation was again more sensitive to this agent. Deletion of the gene encoding the calmodulin-binding protein HAM-3 was shown to be required for cell fusion , and los defects ,74. We aium spp. . RU360 w. crassa ,48. On t2+ influx through these plasma membrane channels [F. oxysporum. FK506 forms a complex with FKBPs, which, in turn, binds to the regulatory subunit B of calcineurin to inhibit its function [2+ signalling, they are differentially regulated by Ca2+ channels, calmodulin and mitochondrial Ca2+ uniporters.The HACS system involving CCH1 and MID1 has been shown to be sensitive to calcineurin and is therefore expected to have a functional role downstream of Cachannels . The calchannels ,77. Our function . Cyclospfunction . Even thfunction ,78. In o3), germ-tube formation and CAT fusion occur at different time points during germling development [F. oxysporum, commencing ~2 h after incubation starts, whereas CAT fusion begins ~7 h after incubation starts. Our results also indicate the dependency of CAT fusion on a higher extracellular Ca2+ concentration, followed by its intracellular sequestration to mitochondria, as well as more Ca2+ binding to calmodulin when compared to germ-tube formation. Germ-tube formation, followed by continued germ-tube protrusion, increases the cell volume much more significantly than the protrusion of expansion-limited CATs. This might lead to further release of Ca2+ to the medium or alter the ratio of intracellular and extracellular Ca2+ levels. Thus, high extracellular Ca2+ availability following germ-tube formation might be a key prerequisite for CAT induction to occur in Fol. This is different from other filamentous fungi, such as N. crassa, in which CATs and germ tubes can be simultaneously formed by one and the same cell in Vogel’s media without other supplements.CATs were more sensitive to all pharmacological agents tested, which inhibited the process at much lower concentrations compared to germ-tube formation. Even though both cell protrusions do occur in the same medium  their pulsatile nature in growing tips ; (2) thssa tips ; and (3)


PPARG, rs11108094 on USP44, rs6685701 on RPS6KA1 and rs7929543 on AC118942.1 comprise common SNPs susceptible to the two diseases and, together with 64 other co-inherited proxy SNPs, may affect the expression patterns of metabolic genes, such as USP44, METAP2, PPARG, TIMP4 and RPS6KA1, in adipose tissue, skeletal muscle, liver, pancreas and whole blood. Most importantly, a set of 86 AML/T2D common susceptibility genes was found to be significantly associated with metabolic cellular processes, including purine, pyrimidine, and choline metabolism, as well as insulin, AMPK, mTOR and PI3K signaling. Moreover, it was revealed that the whole blood of AML patients exhibits deregulated expression of certain T2D-related genes. Our findings support the existence of common metabolic perturbations in AML and T2D that may account for the increased risk for AML in T2D patients. Future studies may focus on the elucidation of these pathogenetic mechanisms in AML/T2D patients, as well as on the assessment of certain susceptibility variants and genes as potential biomarkers for AML development in the setting of T2D. Detection of shared therapeutic molecular targets may enforce the need for repurposing metabolic drugs in the therapeutic management of AML.Type-2 diabetes mellitus (T2D) is a chronic metabolic disorder, associated with an increased risk of developing solid tumors and hematological malignancies, including acute myeloid leukemia (AML). However, the genetic background underlying this predisposition remains elusive. We herein aimed at the exploration of the genetic variants, related transcriptomic changes and disturbances in metabolic pathways shared by T2D and AML, utilizing bioinformatics tools and repositories, as well as publicly available clinical datasets. Our approach revealed that rs11709077 and rs1801282, on  Type-2 diabetes mellitus (T2D) is a chronic metabolic disorder, nowadays considered a global epidemic, with ever-increasing prevalence and high cardiovascular mortality rates . Metabol+ T cell anti-tumor responses [T2D also has been associated with the development of various types of human neoplasia, including both solid tumors and hematological malignancies . A recenesponses .Among hematological malignancies, acute and chronic leukemias have been associated with a previous history of T2D. A recent meta-analysis of 18 studies involving 10516 leukemia cases within a total of more than 4 million individuals with diabetes showed that the risk for the disease is increased in patients with T2D but not in patients with type 1 diabetes . EspeciaDespite the identified epidemiological association of AML with T2D, the genetic and molecular links between the two disorders remain unclear. The possible existence of common metabolic interferences that may underlie the development and perpetuation of the disease has not yet been investigated. Neither is it known whether these are attributed to aberrations in the genomic, transcriptional, or post-transcriptional level. To this end, we herein investigated a network of common genetic alterations  and co-inherited variants, related mRNA deviations and pathway deregulations in the two conditions, utilizing appropriate bioinformatic tools and publicly available clinical datasets. Priority was given to the identification of gene sets and pathways associated with possible metabolic disturbances, perchance known to be related to T2D, that may control the development of AML. To the best of our knowledge, our results provide the first information regarding common genetic predisposition and connected mechanisms that may lead to the development of AML in the setting of T2D.p-value for the association with either disease of <5 × 10−8, which was set as a threshold of significance. These five SNPs were included in the subsequent analyses of this study as significantly associated with both AML and T2D. Corresponding information on these SNPs is summarized in Data on all SNPs associated with AML or T2D development were downloaded from the NHGRI-EBI Catalog . The numPPARG (peroxisome proliferator-activated receptor gamma) gene, exerting the following p-values: for rs11709077 5 × 10−11 for AML and 2 × 10−36 for T2D, and for rs1801282 5 × 10−11 for AML and 2 × 10−19 for T2D. Another common SNP, the rs6685701, is found in the gene encoding for the ribosomal protein S6 kinase A1 (RPS6KA1) and exhibits a significant association with AML (p = 6 × 10−18) and T2D (p = 1 × 10−08). USP44 (Ubiquitin Specific Peptidase 44) also bears an SNP (rs11108094) significantly related to both AML and T2D development . Last, rs7929543, located in AC118942.1 (NADPH oxidase 4 pseudogene), is also significantly associated with both AML (p = 7 × 10−09) and T2D (p = 2 × 10−09). It is important to note that all SNPs are in non-coding regions except SNP rs1801282 which is a missense variant in PPARG, also known as Pro12Ala. The more common C allele encodes for the Pro amino acid at the SNP position [Two of these SNPs  lie in the position .PPARG gene, were found to affect the mRNA expression levels of SYN2 (synapsin II) in the skeletal muscle : 0.35 and 0.36 for rs11709077 and rs1801282, respectively) (GATA3 transcription factor in whole blood (z-score = −4.54)  (NES = −0.21) in visceral adipose tissue (USP44 was associated with decreased expression of METAP2 (methionine aminopeptidase 2) in subcutaneous and visceral adipose tissue  (RPS6KA1 negatively affects its own expression levels (NES: −0.099), while rs7929543  on AC118942.1 is positively associated with the expression levels of RP11-347H15.5 (clone-based (Vega) gene) (NES: 0.53) , we searched for eQTLs through the GTex and Blood eQTL Browser databases ,22. All l muscle  and wholl muscle . In the ctively) , whereasctively) . In addi= −4.54)  and of Te tissue . The rs1ctively) . FinallyS: 0.53) .2) of LD greater than 0.8. Data are shown in USP44, METAP2, PPARG, TIMP4, FOLH1 (folate hydrolase 1), AC118942.1 and RPS6KA1 genes were identified; some of them were detected as proxies for more than one of the five common SNPs. Through this analysis, it was also revealed that two of the common AML/T2D susceptibility genes  on the PPARG gene were mutual proxy SNPs  acting upon them, affected by AML or T2D-specific SNPs in-disease target tissues, we searched through the GTex and eQTL Browsers. Analysis using Venn diagrams identified AML- or T2D-specific SNPs/eQTLs in certain susceptibility genes in adipose, muscle tissue, liver, pancreas and/or whole blood , GATA3, METAP2, RP11-347H15.5, RPS6KA1, SYN2, TIMP4. The corresponding protein–protein interaction (PPI) network is depicted in 3K-Akt and insulin signaling, as well as pathways deposited as related to AML   .Differently colored nodes designate various genes/proteins involved in one or more pathways. Edges represent protein–protein associations—either known interactions, predicted interactions or other associations. All regulated pathways revealed in this analysis are included in CAPN10, CDK5, CDKN2A, IGF2BP2, KCNQ1, THADA, TSPAN8) and explored their mRNA levels in peripheral blood samples from AML- versus non-cancerous individuals utilizing RNAseq data and the TNMplot web tool [CAPN10, CDK5, CDKN2A, IGF2BP2 and THADA, as well as significantly decreased levels of KCNQ1 and TSPAN8, were found in 151 AML patients compared to 407 normal individuals tested . The percentage (%) of AML samples that displayed up- or downregulated expression for each of the above genes, at each of the four quantile cut-off values , as well as the specificity (the ratio of the number of AML samples to the sum of AML and non-cancerous samples over or below each given cut-off), are depicted in The second aim of the study was to investigate the possible deregulation of T2D-related metabolic mechanisms in AML patients. To this end, we selected a panel of genes previously reported to be deregulated in T2D patients  , while the rest of the deregulated genes have not been identified to bear AML-related SNPs. Investigation for their proxies revealed three proxy SNPs  for rs10832134 and five proxy SNPs  for rs12576156, all of them in KCNQ1. No proxies were found for rs11523905 (data not shown). Out of these, the proxy SNP rs12574553  consists of an eQTL for KCNQ1; the minor allele leads to the downregulation of mRNA levels in whole blood [To search for AML-specific SNPs on these deregulated genes, we used data obtained from the NHGRI-EBI Catalog of GWAS. It was found that rs10832134 , rs12576156 (11:2477588) and rs11523905 (11:2477029) variants lie in the Today, there is a well-accepted epidemiological link between T2D and cancer development . HoweverPPARG gene, the first gene reproducibly associated with T2D [PPARG with AML, it is worth mentioning that the protein is implicated in the TGF-beta and mTOR signaling pathways, both associated with cancer development [PPARG negatively affect the expression of SYN2 (Synapsin II) in skeletal muscle and in whole blood  in visceral adipose tissue. The TIMP family has been associated with several cancers [GATA3 in whole blood. GATA3 is a transcription factor with a multi-faceted role in hematopoiesis [Another common SNP, which is a missense variant rs1801282, was found to negatively regulate the expression of the tissue inhibitor of metalloproteinases 4 , was found to be associated with its lower expression levels in visceral adipose tissue. The protein belongs to the family of serine/threonine kinases that govern various cellular processes, and it acts downstream of ERK (MAPK1/ERK2 and MAPK3/ERK1) signaling [RPS6KA1 has been implicated in impaired glucose homeostasis in β-pancreatic, muscle and liver cells [Rs6685701, located in the gene encoding for the ribosomal protein S6 kinase A1  was also recognized as a common susceptibility variant for AML and T2D, which acts as an eQTL downregulating the expression of METAP2 (methionyl aminopeptidase 2) in subcutaneous and adipose tissue. The USP44 protein is implicated in protein metabolism and ubiquitin-mediated proteasome-dependent proteolysis. More importantly, METAP2 is involved in the metabolism of fat-soluble vitamins [AC118942.1 pseudogene was identified as an eQTL influencing the expression of the RP11-347H15.5 pseudogene in visceral adipose tissue. The involvement of this deregulation in possible pathogenetic processes for both diseases might be part of the complex underlying genetic–molecular mechanisms.The rs11108094 in vitamins . Its inhvitamins . On the vitamins , which rPPARG, SYN2, TIMP4, GATA3, RPS6KA1, USP44, METAP2, AC118942.1, RP11-347H15.5) in disease-target tissues. A new eGene added to the panel was DHHS, which is downregulated in whole blood by SNPs on RP11-347H15.5. The gene encodes for the dehydrodolichyl diphosphate synthase subunit and is involved in pathways of protein metabolism and in N-glycan biosynthesis [To describe the network of genetic variants’ inheritance more extensively, we developed a panel of 64 unique proxy SNPs associated with the five common AML/T2D ones . Interesynthesis . HoweverNext, we identified a panel of 86 common AML/T2D susceptibility genes using the GWAS NHGRI-EBI Catalog . Several3K-Akt and insulin signaling pathways, were also among the ontologies significantly regulated by the AML/T2D gene set. Analysis also revealed an association with a pathway category deposited as “Acute Myeloid Leukemia”, which refers to ERK, PI3K and JAK-STAT signaling and transcription regulation pathways including mutated RUNX1 and the fusion genes AML1-ETO, PML-RARA and PLZF-RARA [Choline metabolism is another pathway that emerged through gene set enrichment analysis. Indeed, its upregulation in malignant transformation is well described , while tLZF-RARA .THADA (thyroid adenoma-associated protein), IGF2BP2 (insulin-like growth factor 2 mRNA binding protein 2), CDKN2A (cyclin-dependent kinase inhibitor 2A) and CDK5 (cyclin-dependent kinase 5) were upregulated, while levels of KCNQ1 (potassium voltage-gated channel subfamily Q member 1) were downregulated in the peripheral blood of AML patients compared to normal subjects. IGF2BP2, CDKN2A, CDK5 and KCNQ1 are known to be implicated in the mass development, proliferation, and insulin secretory function of β-cells, and in metabolic processes in T2D-affected tissues [THADA, despite its susceptibility to T2D, there are no data yet related to its involvement in the disease’s pathogenesis and/or metabolic pathways [CAPN10  shows increased whereas TSPAN8 (Tetraspanin 8) exhibits decreased mRNA levels in AML versus non-cancerous individuals, a trend opposite to what was observed in T2D versus healthy subjects. CAPN10 plays important roles in the translocation of glucose transporter 4 (GLUT4), secretion of insulin and apoptotic processes in pancreatic cells [TSPAN8 has been described as a prognostic indicator for patients with certain solid tumors [Finally, exploration through clinical datasets revealed that certain T2D-related genes, previously shown to be deregulated in T2D individuals , also ex tissues ,20,55,56pathways . Howeverpathways . CAPN10 ic cells , while Td tumors ,59, but PPARG) or different mechanisms (such as GATA3) and (iii) common susceptibility genes can regulate metabolic pathways, which may be implicated in the pathogenetic mechanisms leading to the development of the two disorders. It should be noted, however, that the study has certain limitations, including that it exclusively analyzed in silico data and the fact that other parameters affecting the gene expression, such as epigenetic mechanisms, were not explored. Moreover, in the case of certain genes and their SNPs, i.e., those of PPARG and GATA3, their specific implication in AML and/or T2D development is not well documented. Therefore, it is yet difficult to provide a plausible explanation regarding their possible impact as risk factors for AML in the context of T2D. Lastly, it needs to be clarified that, although some of the reported SNPs are associated with certain genes involved in AML (such as RPS6KA1 and METAP2), the latter are not considered driver genes for AML initiation.In summary, this study provides, for the first time, evidence for a strong genetic network that is related to aberrations in metabolic processes and molecular pathways, shared between AML and T2D. Even though the metabolic vulnerability of AML cells and aberrant metabolic pathways observed in AML patients ,60 have Despite these limitations, significant evidence emerging from this study can be further explored in future basic and clinical studies. For example, the common susceptibility genes revealed can be evaluated for their potential to serve as prognostic biomarkers of AML development in cohorts of T2D individuals. Moreover, in depth exploration of the described metabolic pathways and involved genes may lead to a better understanding of the pathogenetic basis of the increased risk for AML development observed in individuals with T2D. Finally, detailed investigation of the common therapeutic targets identified may suggest that repurposing of metabolic drugs  could be exploited as novel therapeutic strategies to enhance the anti-leukemic armamentarium.Our study was performed in two axes. (A) Detection of common genetic variants and deregulated pathways in T2D and AML: We first created a panel of SNPs associated with AML or T2D, upon an in-depth search in the NHGRI-EBI Catalog of published GWAS , to detehttp://bioinformatics.psb.ugent.be/webtools/Venn/) (May 2021). A genome-wide statistically significant p-value lower than or equal to 5 × 10−8 was applied to detect the SNPs that were significantly associated with the diseases. Data regarding the prevalence of the SNPs of interest in the general population were obtained from the gnomAD browser [The panels of total susceptibility genes specific for AML and T2D were developed upon an in-depth search in the NHGRI-EBI GWAS Catalog . All pop browser .2) of linkage disequilibrium (LD); proxy SNPs are considered those having ≥80% possibility of coinheritance with the tag SNP, which equals to a R2 value ≥ 0.8, and (b) the combined recombination rate (cM/Mb) from HapMap; the recombination rate is the rate at which the association between the two loci is changed. It combines the genetic (cM) and physical positions (Mb) of the marker by an interactive plot.Proxy SNPs of disease susceptibility SNPs of interest were detected utilizing the LDLink tool . LDLink Expression quantitative trait loci (eQTLs), which explain variations in mRNA expression levels, related to the SNPs of interest were explored utilizing the GTEx portal and the Blood eQTL Browser ,22. AnalAnalysis through the STRING  and Kyotp-value of significance and the fold-change between groups. Other information included (a) the percentage (%) of AML samples that exerted up- or downregulated expression of query genes compared to non-cancerous samples, at each of the four quantile cut-off values , and (b) the specificity, defined as the ratio of the number of AML samples to the sum of AML and non-cancerous samples over or below each given cut-off.To explore possible variations in the mRNA expression levels of previously described T2D-deregulated genes  in patie


Mitochondria-ER contact sites (MERCS) are known to underpin many important cellular homoeostatic functions, including mitochondrial quality control, lipid metabolism, calcium homoeostasis, the unfolded protein response and ER stress. These functions are known to be dysregulated in neurodegenerative diseases, including Parkinson’s disease (PD), Alzheimer’s disease (AD) and amyloid lateral sclerosis (ALS), and the number of disease-related proteins and genes being associated with MERCS is increasing. However, many details regarding MERCS and their role in neurodegenerative diseases remain unknown. In this review, we aim to summarise the current knowledge regarding the structure and function of MERCS, and to update the field on current research in PD, AD and ALS. Furthermore, we will evaluate high-throughput screening techniques, including RNAi vs CRISPR/Cas9, pooled vs arrayed formats and how these could be combined with current techniques to visualise MERCS. We will consider the advantages and disadvantages of each technique and how it can be utilised to uncover novel protein pathways involved in MERCS dysfunction in neurodegenerative diseases. Mitochondria can form contacts with the ER to regulate vital cellular homoeostatic functions.3R), voltage-dependent anion channel (VDAC), glucose-related protein 75 (GRP75) and deglycase (DJ-1) act as a tetramer to regulate Ca2+ homoeostasis. Mitofusin-2 (MFN2) and mitofusin-1 (MFN1) can act as a physical tether, control Ca2+ homoeostasis and regulate mitochondrial morphology changes. Vesicle-associated membrane protein B (VAPB) and protein tyrosine phosphatase-interacting protein-51 (PTPIP51) are physical tethers and can impact Ca2+ homoeostasis, while B-cell receptor-associated protein-31 (BAP-31) can regulate apoptosis.A range of mitochondria-ER contact site (MERCS) tethering proteins maintain these contacts and facilitate their functions. Inositol 1,4,5-triphosphate receptor , Parkinson’s disease (PD) and amyloid lateral sclerosis (ALS).Pooled or arrayed high-throughput screening can be used in conjunction with CRISPR/Cas9 or RNAi to identify novel disease-relevant pathways in many neurodegenerative diseases.The gold standard for visualising MERCS is electron microscopy and super-resolution techniques, but these require vast quantities of sample or image processing and so are not suitable for high-throughput screening.A range of split fluorescent protein constructs have been developed, which can be used to visualise MERCS in real time, and are suitable for high-throughput screening.What are the molecular mechanisms that govern MERCS dysfunction in neurodegenerative diseases such as AD, PD and ALS? Is there an overarching disease mechanism related to MERCS?2+ indicators be adapted and optimised for high-throughput screening and which of them will provide the most accurate and efficient readout?How can electron microscopy, super resolution, split FP techniques and Ca2+) homoeostasis, unfolded protein response (UPR), ER stress and mitochondrial quality control (MQC), all of which have been implicated in neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral scoliosis (ALS) ), which can be achieved through entry of Ca2+ into the mitochondria via MERCS. It has been proposed that Ca2+ exits the ER via IP3R and enters the mitochondria through VDAC, producing a Ca2+ hotspot [2+], allowing the threshold for fast Ca2+ transfer into the mitochondria matrix via the highly selective low-affinity Ca2+ channel MCU in the IMM [2+-dependent [2+ shuttling through MERCS can decrease ATP production and the oxygen consumption rate [2+ entry into the mitochondria is required, as elevated mitochondria matrix Ca2+ can result in increased mitochondria membrane permeability and damage to the mitochondria itself. This process is mediated by the mitochondrial permeability transition pore (mPTP), a non-specific high-conductance, voltage- dependent channel. The activation of this channel causes the mitochondrial membrane to become permeable to proteins of up to 1,500 Da, causing swelling and OMM rupture, allowing the release of pro-apoptotic factors from the mitochondria, uncoupling of oxidative phosphorylation and increased reactive oxygen species (ROS) [Ca hotspot . This ho the IMM , 34. Thiependent . Perturbion rate , 37. Howes (ROS) , 39.There are a range of MQC mechanisms that protect against mitochondrial insult. These include the regulation of mitochondria morphology, mitochondria microtubule dynamics and mitophagy . An earl2+ from the ER to the mitochondria through MERCS is required for both starvation and PINK1/Parkin-induced mitophagy [Mitophagy, the bulk degradation of mitochondria, is also linked to MERCS. Pre-autophagosome markers ATG14L and ATG5, key autophagy components, are localised to MERCS upon starvation- induced mitophagy . Studiesitophagy . Furtheritophagy . This de2v uptake into the mitochondria, increasing ATP production and providing energy for the chaperone machinery to aid in the folding of proteins, thereby preventing ER stress and apoptosis [MERCS has also been connected with ER stress and UPR, an intracellular signalling pathway that is activated by the accumulation of unfolded proteins in the ER . Under bpoptosis . These ipoptosis ; howeverpoptosis . Interespoptosis , with PEpoptosis . More repoptosis . MITOL cpoptosis . These sAPP and PS1/2 are the principal genes associated with familial AD [PS1, PS2 or APP result in increased MERCS proximity and enhanced lipid metabolism [2v homoeostasis [Drosophila, forced expression of an artificial linker, that increases MERCS in vivo, rescues locomotion and prolongs the survival of AD models [AD studies were among the first to demonstrate a link between neurodegeneration and MERCS. The key pathologic hallmarks of AD are aberrant protein aggregates of hyperphosphorylated Tau and extracellular plaques of β-amyloid (Aβ). Aβ40 and Aβ42 are the main component of extracellular plaques and are the result of abnormal cleavage of amyloid precursor protein (APP) by Presenilin 1 (PS1) and Presenilin 2 (PS2), active components of the γ-secretase complex. ilial AD . PS1 andilial AD . In addiilial AD , 59. Muttabolism , 61. In eostasis , while feostasis . In DrosD models . StudiesD models . This le2+ flux and ATP production [2+ transfer to mitochondria in HeLa cells [There is growing evidence that MERCS also plays an important role in PD pathogenesis. Firstly, α-Synuclein, the most common component of Lewy bodies, a hallmark of PD, can localise to MERCS, with WT α-Synuclein associating more with MERCS than PD-related mutations ; howeveroduction . Others La cells , 69. Thi3R in a tetramer complex, and is thought to facilitate Ca2+ homoeostasis, as well as acting as a functional tether. Ablation of DJ-1 decreases MERCS association and alters Ca2+ handling, which can be rescued with WT but not PD-related mutant DJ-1 [PINK1 and Parkin, two genes mutated in autosomal recessive PD, acting together to regulate MQC mechanisms, have been found to localise to MERCS under mitophagy induction [2+ signalling and ATP production [PARK2- knockout mice and patient cells showed an increase in the proximity of the ER and mitochondria and altered Ca2+ flux [Drosophila models [Secondly, familial linked PD genes have also been associated with MERCS. As described previously, the PD-related protein DJ-1 has been shown to interact with VDAC, GRP75 and IPant DJ-1 . Furthernduction . Inducednduction . PINK1 induction . Overexpoduction . Howevera2+ flux , a resula models . More rea models . These dDrosophila and human fibroblasts, where loss of Parkin or ubiquitination-preventing mutations in MFN2, reduced MERCS, implying that PINK1/Parkin can regulate MERCS through MFN2 [2+ transport through MERCS [RHOT1, the gene that encodes Miro, in PD patients. All four novel RHOT1 mutations showed attenuated Ca2+ handling, autophagy and a decrease in MERCS in patient-derived fibroblasts [Many PINK1- and Parkin-associated proteins have also been implicated in MERCS, including MFN2 and Miro. MFN2 (a key tether in MERCS) can be phosphoubiquitinated in a PINK1-/Parkin- dependent manner under induction of mitophagy, and degraded, resulting in an uncoupling of the ER from the mitochondria . This waugh MFN2 . Miro, augh MFN2 , 76. Preugh MFN2 . Howevergh MERCS . A more roblasts , 80. ThuSOD1, C9ORF72, FUS and TARDBP occurring most frequently [VAPB, have been associated with altered signalling at MERCS. Specifically, mutations in VAPB cause an autosomal-dominant form of ALS and increase the affinity to its binding partner PTPIP51. This was shown to correlate with increased Ca2+ transfer into the mitochondria [2+ uptake into the mitochondria and ATP production [2+ transfer by binding IP3R in MERCS) to familial ALS [ALS is a fatal neurodegenerative disease. Familial forms of ALS account for 10% of all cases with mutations in equently . Many ofchondria . Furtherchondria , 14, cauoduction , 14. Othlial ALS  as loss lial ALS . Mouse mlial ALS , 84. Furlial ALS . These dFunctional genomics link gene sequence to function and have been key in understanding molecular mechanisms of disease. Combined with genetic screening approaches, functional genomics is a powerful tool. The introduction of RNAi and CRISPR/Cas9 has revolutionised genetic screening due to ease of use, high efficiency, reliability and cost-effectiveness , 87. HerFor the last decade, RNAi has been the principal approach for functional genomic screening. RNAi is a conserved endogenous pathway that uses the ribonuclease DICER to cut double-stranded RNA into small-interfering RNA (siRNA). The siRNA utilises base complementation in conjunction with RNA-induced silencing complex (RISC) to degrade mRNA, reducing protein levels . ArtificTwo principal screening strategies are used: arrayed and pooled Fig. . In an aOwing to its small size and high complexity, studying MERCS is difficult, but recent technological advances can be coupled with HTS and offer new opportunities for investigation.EM is the gold standard for visualising MERCS as it can resolve the close apposition of the ER and mitochondria membranes (10–30 nm); however, EM requires that samples must be fixed and processed, making it difficult to collect multiple images and thus inappropriate for HTS. Super-resolution techniques have also been used to visualise MERCS, including stimulated emission depletion (STED), photo-activation localisation microscopy (PALM), stochastic optical reconstruction microscopy (STORM) and structural illumination microscopy (SIM) –103. HowA highly sensitive approach, termed in situ Proximity Ligation Assay (PLA), has been used to investigate endogenous protein interactions  and optiLike PLA, other methods that rely on construct proximity are split FP systems, including biomolecular fluorescence complementation (BiFC), double-dimerising GFP (ddGFP), fluorescence resonance emission transfer (FRET) and bioluminescence resonance emission transfer (BRET).Typically, in BiFC, a FP is split into two non-fluorescent fragments that are fused to the proteins of interest. With regard to MERCS, they are tethered to either resident ER or mitochondrial proteins. If the two membranes come into close proximity, then the two complementary fragments of the FP will reassemble, restoring the fluorescence of the chromophore . A rangeThe ddGFP system has also been used to investigate MERCS and involves the reversible binding of two non-fluorescent, “dark”, ddGFP monomers, ddGFPa and ddGFPb, to produce a fluorescent heterodimer. ddGFPa is mutated (I11L and S14A and K163G) such that the chromophore is destabilised and quenched, while ddGFPb completely lacks a chromophore. When ddGFPa heterodimerises with ddGFPb, complementation occurs and a fluorescent heterodimeric complex is formed , 122. ThFRET is a collision-free, but distance-dependent, process that involves the transfer of energy from one excited donor fluorophore to a suitable acceptor fluorophore . FRET fl2+ flux, could allow for different avenues of MERCS investigation. Many tools can be used to visualise intracellular Ca2+, including bioluminescent proteins such as aequorin, which upon binding of Ca2v, undergo a conformational change, oxidation of coelenterazine and emission of a photon at 470 nm, chemical Ca2+ indicators and dyes that change their emitted fluorescence when Ca2+ is bound or genetically encoded Ca2+ indicators (GECI), which contain Ca2+ binding protein Calmodulin, which, with the binding of Ca2+, changes the fluorophore environment resulting in fluorescence128. Previously, the combination of cytosolic (Fluro4) and mitochondria (Rhod2) fluorescent dyes has been used to investigate Ca2+ flux and functionality of MERCS in vitro [2+ indicators require dye loading, increasing workload. Aequorin, however, has a high signal-to-noise ratio, a wide dynamic range and does not require an external illumination avoiding phototoxicity, but the recharge process, via coelentrazine, is slow and thus makes it suboptimal for HTS [Functional readouts, such as Cain vitro , 128. Siin vitro , 129. Th for HTS . GECIs h for HTS . Further for HTS  and cyto for HTS  were rec for HTS . Thus, G for HTS , 134.2+ into the mitochondria from the ER. However, MERCS has a variety of functions, not only Ca2+ homoeostasis, and this approach may exclude potential regulators of other functional subtypes of MERCS2.Overall, MERCS has been implicated in a range of neurodegenerative diseases, but many questions still remain unanswered. A range of different tools have been developed to study MERCS in a range of circumstances. The microscopy-based techniques, such as EM, SEM, SIM and STORM, are remarkable in their ability to visualise MERCS on the nm scale, but require specialised equipment and can only be conducted on a small number of samples; therefore, they are not suitable for screening. Both PLA and split FP use fluorescence as a readout and the proximity of membrane-bound ER and mitochondrial proteins to visualise MERCS. A downside of PLA is that it requires fixation and has multiple incubation steps, meaning it cannot be used as HTS but would likely be useful in the validation of hits. Unlike PLA, split FP can easily be conducted on a large scale as it has limited sample or data processing, specialised equipment is not needed and stable cell lines can be engineered, which dramatically reduces cost and labour. The range of split FP comes with its own advantages and disadvantages: BiFC has the simplest readout, clearest signal and least noise-associated analysis, unlike ddGFP, FRET or BRET. The advantage of FRET/BRET probes is that they do not self-assemble and are reversible, as they do not require protein folding. BRET also has reduced phototoxicity and bleaching, thereby decreasing noise previously seen with FRET. GECIs may also be suitable for investigating MERCs, these sensors do not rely on proximity of either the mitochondria or ER, but the influx of Ca2+ + flux and so are also suitable for investigating MERCS. The combination of both proximity-based and functional (Ca2+ indicators) screening approaches would be highly beneficial in understanding both the physical and functional role of MERCS in neurodegenerative diseases.Overall, BRET probes would make a good choice for HTS of MERCS, but screening using the other FP systems  would also allow for rapid discovery of novel proteins involved with MERCS regulation in neurodegenerative disease with relative ease. Furthermore, GECIs have been shown to be successful in high-throughput screening to investigating Ca


Cells of a multicellular organism express a variety of cell-surface receptors and channels that trigger an increase of the intracellular Ca2+ concentration upon stimulation. The elevated Ca2+ concentration is not uniformly distributed within the cytoplasm but is organized in subcellular microdomains with high and low concentrations of Ca2+ at different locations in the cell. Ca2+ ions are stored and released by intracellular organelles that change the concentration and distribution of Ca2+ ions. A major function of the rise in intracellular Ca2+ is the change of the genetic expression pattern of the cell via the activation of Ca2+-responsive transcription factors. It has been proposed that Ca2+-responsive transcription factors are differently affected by a rise in cytoplasmic versus nuclear Ca2+. Moreover, it has been suggested that the mode of entry determines whether an influx of Ca2+ leads to the stimulation of gene transcription. A rise in cytoplasmic Ca2+ induces an intracellular signaling cascade, involving the activation of the Ca2+/calmodulin-dependent protein phosphatase calcineurin and various protein kinases . In this review article, we discuss the concept of gene regulation via elevated Ca2+ concentration in the cytoplasm and the nucleus, the role of Ca2+ entry and the role of enzymes as signal transducers. We give particular emphasis to the regulation of gene transcription by calcineurin, linking protein dephosphorylation with Ca2+ signaling and gene expression.Ca At rest, the intracellular cytosolic Ca2+ concentration is estimated to be at 100 nM while the extracellular milieu has a Ca2+ concentration of approximately 1 mM. Thus, opening of voltage-gated or ligand-gated Ca2+ channels in the plasma membrane leads to an influx of Ca2+ ions into the cell. In addition, stimulation of Gαq-coupled receptors triggers the activation of intracellularly located IP3 receptors and results in an influx of Ca2+ ions from the endoplasmic reticulum into the cytosol . Even lysosomes, often viewed as a “terminal end” of intracellular pathways, are involved in intracellular Ca2+ signaling ,372+ act complex C. Elk-1 2+. This family of proteins is involved in the regulation of developmental programs of different vertebrate tissues [N-terminal transcriptional activation domain (TAD), a regulatory domain  and a C-terminal DNA-binding domain  and for the protein phosphatase calcineurin  metal center. In the absence of Ca2+ ions, calcineurin is inactive, due to a binding of the autoinhibitory domain to the catalytic center. An influx of Ca2+ ions from the external milieu into the cytosol (via activation of cell-surface-located Ca2+ channels) or an influx of Ca2+ ions from the endoplasmic reticulum into the cytosol (following stimulation of Gαq-coupled receptors) leads to the activation of calcineurin. The regulatory B-subunit is tightly bound to calcineurin A, whereas calmodulin binding is Ca2+ dependent. Activation of calcineurin requires the binding of Ca2+ ions to calcineurin B and the Ca2+-dependent binding of a Ca2+/calmodulin complex to calcineurin A. The binding of calmodulin to the calmodulin-binding domain triggers the displacement of the autoinhibitory domain, leading to the activation of the enzyme [C-terminal autoinhibitory domain and a portion of the calmodulin binding site. It is constitutively active and does not need Ca2+ ions for its activation. The small regulatory calcineurin B-subunit  and cyclosporin A, used for the treatment of organ rejection in transplanted patients, function as potent calcineurin inhibitors when they are bound to the immunophilins FKBP12 or cyclophilin A, respectively . However folding ,60. In aion pore ,62. Cyclion pore . Tacroliion pore  that is ion pore . RCAN1 bion pore ,66.2+ channels, activation of nitric oxide synthase, and regulation of the Na+, K+-ATPase and others. Here, we focus our attention on the regulation of gene transcription by calcineurin-mediated protein dephosphorylation.Calcineurin-induced dephosphorylation of proteins is connected with several biological functions, including inhibition of neurotransmission, desensitization of postsynaptic Ca2+ concentration. Calcineurin is activated by a rise in cytosolic Ca2+ concentration, which can be generated in various microdomains of the cell. A rise in cytoplasmic Ca2+ may be the result of a Ca2+ influx through Ca2+ channels of the plasma membrane  or the endoplasmic reticulum (ionotropic IP3 receptors) [2+ ions from mitochondria. Prolonged Ca2+ release from the mitochondria of sensory neurons facilitates nuclear transport of NFAT in neurons [2+ levels. N-terminus of the regulatory region, encompasses the consensus sequence PxIxIT, as depicted in 2+ concentrations.The best studied substrates of calcineurin are the transcription factors of the NFAT family that are regulated by the cytoplasmic Caceptors) ,67,68. C neurons . The NFA neurons ,56,70,712+ rise in the cytoplasm and presence of active calcineurin. The hypothesis that a rise in nuclear Ca2+ activates NFAT4 [2+ is accompanied by a rise of activated calcineurin in the nucleus, because NFAT proteins must be kept in a dephosphorylated state to sustain nuclear expression. It has been proposed that calcineurin is transported in the nucleus together with NFAT4 where it continues to dephosphorylate NFAT4 [2+ activates NFAT4, a direct gene transcription assay is needed to clearly show the upregulation of NFAT-regulated gene transcription under these conditions.Activation of NFAT proteins requires a Caes NFAT4  implies te NFAT4 . Howeverte NFAT4 . It woulte NFAT4  is built2+/calmodulin-dependent protein kinase IV, activates CREB-regulated transcription, whereas dephosphorylation by protein phosphatases 1 and 2A inactivates the protein. CREB is not a substrate for calcineurin, but is indirectly regulated by this phosphatase. 2+ activates CREB-controlled gene transcription  and subsequently triggers an inactivation of CREB . Calcineurin is therefore part of a negative feedback loop of Ca2+-initiated transcription of CREB target genes.NFAT transcription factors are active in the dephosphorylated state. Thus, dephosphorylation by calcineurin induces activation of NFAT-controlled transcription. In contrast, several transcription factors are activated by phosphorylation. Accordingly, dephosphorylation is connected with inactivation of gene transcription by these transcription factors. The prototype of a phosphorylation-regulated transcription factor is CREB. Phosphorylation by several protein kinases, in particular PKA and Ca2+-activated calcineurin catalyzes the dephosphorylation of TORC2, allowing its translocation into the nucleus. TORC2 binds to the bZIP domain of CREB and functions as a coactivator [2+.Alternatively, calcineurin has been described as a positive regulator of CREB. According to this scenario, calcineurin dephosphorylates the transducer of regulated CREB activity-2 (TORC2) which is sequestered in the cytoplasm via binding to 14-3-3 proteins. Cactivator . Our exp2+ and calmodulin [The ternary complex factor protein Elk-1 has been identified as a major substrate for calcineurin ,81. Elk-lmodulin . Using plmodulin . In neurlmodulin .The connection between calcineurin activity and Elk-1-regulated gene transcription was highlighted in several transgenic mouse models . It has 2+ ions function as second messengers in cells and regulate multiple intracellular functions. Stimulus-regulated gene expression is one of the important functions of elevated Ca2+ concentrations. Stimulation of plasma membrane receptors or ion channels leads to a rise in intracellular Ca2+, but also the release of Ca2+ from intracellular stores such as the endoplasmic reticulum, mitochondria and lysosomes. Specialized subcellular Ca2+ microdomains orchestrate the specificities of intracellular Ca2+ signals. Due to the uptake of Ca2+ ions into intracellular Ca2+ stores and their export by Ca2+ pumps, the duration and periodicity of Ca2+ transients may be very different, resulting in different signaling events. Ca2+ activates a variety of intracellular enzymes that are used as signal transducers and for prolonging the duration of the Ca2+ signal. While the Ca2+ concentration may be reduced in the cell by active Ca2+ pumps and transporters, the Ca2+-activated enzymes remain active and execute the functions initiated by a local Ca2+ rise. A particularly interesting Ca2+-regulated enzyme is the phosphatase calcineurin that can either activate or inhibit Ca2+-responsive transcription factors. Thus, calcineurin links Ca2+-induced protein dephosphorylation with the regulation of gene expression.Ca


The management of patients with relapsed or refractory (R/R) acute myeloid leukaemia (AML) remains a challenge with few reliably effective treatments. Chidamide, a new selective HDAC inhibitor, has demonstrated some effectiveness in AML patients. Herein, we reported three patients with R/R AML who were unresponsive to venetoclax plus azacitidine (VA) but were successfully treated with VA when chidamide was added to the regimen. MCL1 is one of the anti-apoptotic proteins. Chidamide targets the MCL1 protein, which may permit venetoclax resistance when upregulated. We determined MCL1 protein expression in different AML cell lines, and chidamide could downregulate MCL1 expression in venetoclax resistance AML cells. In general, our experience showed that the chidamide/VA combination could improve the condition of R/R AML patients who are resistant to VA. Formally evaluating this regimen in R/R AML patients may be meaningful. AML is a disease caused by the blocked differentiation of myeloid haematopoietic stem cells and the clonal proliferation of primitive or immature myeloid cells in the bone marrow (BM). With current chemotherapies, approximately 10% to 40% of newly diagnosed AML patients cannot achieve complete remission (CR), and more than 50% of AML patients will ultimately relapse  regimen and were successfully salvaged by chidamide in combination with venetoclax and azacitidine. The regimen was safe and effective. This may provide a new choice for relapsed or refractory AML patients with resistance to VA.ASXL1, CEBPA, JAK2, and RUNX1. None of the 41 gene fusions were detected by using multiple RT–PCR assay  regimen [cytarabine 2 g/m2 over 3 h every 12 h on day1–3.] as consolidation therapies. However, the remission duration only lasted for 11 months. In November 2020, relapsed BM morphology was detected, with 17.5% blasts concurrent with molecular aberration recurrence. Therefore, relapsed AML was diagnosed. Initially, venetoclax combined with azacitidine  was administered as a salvage therapy, an effective regimen recommended for the treatment of R/R AML patients who are ineligible for intensive salvage chemotherapy, but progressive disease was observed. Then, his treatment plan switched to a chidamide combined with venetoclax plus azacitidine regimen , and CR was achieved after one course of therapy. The patient treatment process is shown in Patient 1: A 65-year-old male was diagnosed with AML in January 2020 with the manifestation of leukocytosis, thrombocytopenia and anaemia. BM aspiration revealed a hypercellular BM with 50% blasts. Flow cytometry showed the immunophenotype of myeloid blasts. Cytogenetics revealed a complex karyotype, and a molecular panel identified aberrations in DNMT3A and IDH2. The patient achieved CR after 1 cycle of the IA regimen  and received 6 courses of the HiDAC regimen [cytarabine 2 g/m2 over 3 h every 12 h on day1–3.] as consolidation therapies while not adopting allogeneic haematopoietic stem cell transplantation. The patient experienced his first relapse 2 years after first remission with a 9% immature cell level in the BM and was treated with the CAG  regimen for 3 cycles, resulting in a second CR in March 2019. A second relapse occurred 20 months later. The patient began the VA regimen [venetoclax once daily  and azacitidine 75 mg/m2 day1-7.], but no response was observed after 2 courses of therapies. Finally, the patient received a chidamide combined with venetoclax plus azacitidine regimen  as salvage therapy as described above. The patient achieved his third CR. Treatment process is shown in Patient 2: A 57-year-old male was diagnosed with AML in July 2016. Investigation revealed a pancytopenia. BM examination showed a hypercellular marrow with 65% myeloid blasts. Flow cytometry analysis showed the immunophenotype of myeloid blasts. Cytogenetics revealed del . Different AML cell lines represent the AML FAB classifications accordingly. KG1A, Kasumi-1, NB4, and OCI-AML3 and U937, SKM-1, and MV4–11 represent M1, M2, M3, M4 and M5, respectively. As shown in These results suggest that chidamide plus venetoclax and azacitidine can be safely administered to R/R AML patients and show promising efficacy when patients previously failed the VA regimen. Chidamide is an epigenetic regulator targeting HDAC1, 2, 3, 10 that shows a potential role in altering the imbalance of anti- and pro-apoptotic proteins. To the best of our knowledge, this is the first report of implementing a chidamide combined with a venetoclax plus azacitidine regimen and enabling patients to achieve CR after one course. We suggest that this strategy be considered in patients who are resistant to venetoclax and those who are unfit for intensive chemotherapy. Investigating this combination regimen in R/R AML patients may be meaningful in future planned trials.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee of the First Affiliated Hospital of Soochow University. The patients/participants provided their written informed consent to participate in this study.B-RW and C-LW collected, analyzed data and wrote the manuscript. Q-CQ contributed to methodology and investigation. S-BL and S-LX performed research and interpreted data. T-MW, JW and Y-YL helped in the collection of samples/data. S-SG, YQ, and X-DS performed the experiments. S-LX and ZL conceived and designed the study. All authors contributed to the article and approved the submitted version.This work was supported by the grants from the National Natural Science Foundation of China (grant No. 81970138), Translational Research Grant of NCRCH (grant No. 2020ZKMB05), Jiangsu Province “333” project, Social Development Project of the Science and Technology Department of Jiangsu (Grant No. BE2021649) and Gusu Key Medical Talent Program (grant No. GSWS2019007).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


During the transition from a quiescent to a fully activated state, Ca2+ microdomains characterized by reduced spatial and temporal extents are observed in the junctions between the plasma membrane (PM) and the endoplasmic reticulum (ER). Such Ca2+ responses can also occur in response to T cell adhesion to other cells or extracellular matrix proteins in otherwise unstimulated T cells. These non-TCR/CD3-dependent Ca2+ microdomains rely on d-myo-inositol 1,4,5-trisphosphate (IP3) signaling and subsequent store operated Ca2+ entry (SOCE) via the ORAI/STIM system. The detailed molecular mechanism of adhesion-dependent Ca2+ microdomain formation remains to be fully elucidated. We used mathematical modeling to investigate the spatiotemporal characteristics of T cell Ca2+ microdomains and their molecular regulators. We developed a reaction-diffusion model using COMSOL Multiphysics to describe the evolution of cytosolic and ER Ca2+ concentrations in a three-dimensional ER-PM junction. Equations are based on a previously proposed realistic description of the junction, which is extended to take into account IP3 receptors (IP3R) that are located next to the junction. The first model only considered the ORAI channels and the SERCA pumps. Taking into account the existence of preformed clusters of ORAI1 and STIM2, ORAI1 slightly opens in conditions of a full ER. These simulated Ca2+ microdomains are too small as compared to those observed in unstimulated T cells. When considering the opening of the IP3Rs located near the junction, the local depletion of ER Ca2+ allows for larger Ca2+ fluxes through the ORAI1 channels and hence larger local Ca2+ concentrations. Computational results moreover show that Ca2+ diffusion in the ER has a major impact on the Ca2+ changes in the junction, by affecting the local Ca2+ gradients in the sub-PM ER. Besides pointing out the likely involvement of the spontaneous openings of IP3Rs in the activation of SOCE in conditions of T cell adhesion prior to full activation, the model provides a tool to investigate how Ca2+ microdomains extent and interact in response to T cell receptor activation.Ca In particular, Ca2+ controls many processes that are essential for an adaptive immune response such as transcriptional activation, proliferation, differentiation or secretion of cytokines  and subsequent activation of IP3R  has been shown to be also involved through its action on type 1 ryanodine receptor (RYR1) (3R and RyR1 release Ca2+ from the endoplasmic reticulum (ER), which results in a significant decrease of the luminal Ca2+ concentration and thereby stimulates capacitative or store operated Ca2+ entry (SOCE)  and 2 (STIM2) located in the ER membrane , thereby creating a 10-20 nm gap of cytosol between the two membranes  is non-linear and generally described by a decreasing Hill function of CS, with a KD for half activation of the order of ~200 µM when it depends on the dissociation of Ca2+ from STIM1 and of ~400 µM when it depends on the dissociation of Ca2+ from STIM2 . These small signals are more than four times less frequent than those occurring in the first seconds following stimulation and are shorter [44 ± 4 ms, computed from the data of , integrin evoked IP3 signaling and subsequent SOCE via the ORAI/STIM system . The sub-PM ER is embedded in a cubic cytosolic domain of 400 nm x 400 nm that includes the ER-PM junction itself, represented as a cylindrical sub-domain of 200 nm in diameter that extends 15 nm from the ER membrane (ERM) up to the PM. The rest of the cube corresponds to the cytosol adjacent to the junction  and in the sub-PM ER (sC). These concentrations obey the diffusion equationsThe model describes the evolutions of CaC and DS stand for the Ca2+ diffusion coefficient in the cytosol and in the ER, respectively. Default values for these coefficients are 220 and 10 μm2/s  instead of using Green’s functions as it was done in McIvor et al. , we used the finite element method (FEM) and simulation software COMSOL Multiphysics 5.5 , the time of residence of an ion in the junction is of the order of 0.045 ms. Because of this small residence time and of the activity of SERCA pumps, average numbers of free Ca2+ ions in the microdomain evaluated on the basis of the simulated concentrations thus remain smaller than one.Non-TCR/CD3-dependent Ca2+ signals created by the simultaneous opening of five ORAI1 channels are thus not compatible with the average amplitude of 300 nM observed experimentally. This indicates that even in the unlikely situation of a simultaneous opening of the five ORAI1 channels of the junction in conditions of a full ER, the amount of Ca2+ entering in the simulated junction is too small to account for experimental observations (Anim. S1). Indeed, SERCA pumps, which may in principle attenuate the Ca2+ increase, have a very limited activity in these conditions. The maximal Ca2+ concentration at the ER mouth of simulated pumps does not exceed 402 μM. In conclusion, simulations of the Ca2+ fluxes in and out the PM-ER junction show that the spontaneous opening of ORAI1 channels in a conductance state that corresponds to a full ER cannot account for the non-TCR/CD3-dependent Ca2+ microdomains observed in T cells.The Ca2+ increases thus probably involve an ER-related component. RYRs are not significantly involved in the formation of non-TCR/CD3-dependent microdomains in T cells . Clearly, two sources of Ca2+ contribute to this Ca2+ event: first, Ca2+ released by nearby IP3Rs can diffuse in the junction as discussed below. Second, the local depletion at the luminal extremity of the IP3R activates ORAI1 opening and Ca2+ influx from the extracellular medium. Because in the luminal region surrounding an open IP3R, ER Ca2+ drops to 330 μM, closest ORAI1 channels open half-maximally . However, IP3Rs are not in the junction itself and the contribution of ER Ca2+ in Ca2+ microdomains is rather limited, as attested by simulations that do not consider Ca2+ entry . Changes in the value of the diffusion coefficient much affects the Ca2+ profile near the mouth of the channel . Simulations thus predict that if Ca2+ diffusion inside the ER is fast (DS = 110 μm2/s), non-TCR/CD3-dependent Ca2+ microdomains could occur in the absence of extracellular Ca2+ or in T cells that do not express ORAI1, which does not agree with experimental observations . Actually, clustering of the ORAI1 channels could increase the amplitude of the microdomain if either Ca2+ diffusion in the junction or Ca2+ pumping was affected by the inter-channel distance. Since Ca2+ increases near ORAI1 channels always remain highly localized in both situations, diffusion is not much affected. SERCA pump activity associated with an active microdomain based on clustered or non-clustered ORAI1 channels are shown in 2+ fluxes through SERCA pumps when IP3Rs and ORAI1 channels are open are clearly not much sensitive to the conditions, whatever the value of the Ca2+ diffusion coefficient in the ER. Interestingly, pumps are not saturated in contrast to the situation encountered with a highly depleted ER  in order to match experimental conditions, as it was done by Maccari et al.  has a drastic effect on the Ca2+ microdomain in the junction. Agreement with observations could only be reached when considering Ds = 10 μm2/s, i.e. a value that is ~20 times smaller than the value reported for the cytoplasm. This smaller value could result from molecular crowding in the lumen  , by the Joachim-Herz-Stiftung (Hamburg), Infectophysics Consortium , by NCL-Stiftung Hamburg (to AG), the Hamburg Ministry of Science, Research and Equality , and by University Medical Center Hamburg-Eppendorf . Research in the AG labs is also supported by EU project INTEGRATA - DLV-813284. GD is Research Director at the FNRS.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.