We have shown previously that MICU1 mutations cause increased resting mitochondrial Ca2+ concentration ([Ca2 +]m). We now explore the functional consequences of MICU1 mutations in patient derived fibroblasts in order to clarify the underlying pathophysiology of this disorder. We propose that deregulation of mitochondrial Ca2+ uptake through loss of MICU1 raises resting [Ca2+]m, initiating a futile Ca2+ cycle, whereby continuous mitochondrial Ca2+ influx is balanced by Ca2+ efflux through the sodium calcium exchanger (NLCXm). Thus, inhibition of NCLXm by CGP-37157 caused rapid mitochondrial Ca2+ accumulation in patient but not control cells. We suggest that increased NCLX activity will increase sodium/proton exchange, potentially undermining oxidative phosphorylation, although this is balanced by dephosphorylation and activation of pyruvate dehydrogenase (PDH) in response to the increased [Ca2+]m. Consistent with this model, while ATP content in patient derived or control fibroblasts was not different, ATP increased significantly in response to CGP-37157 in the patient but not the control cells. In addition, EMRE expression levels were altered in MICU1 patient cells compared to the controls. The MICU1 mutations were associated with mitochondrial fragmentation which we show is related to altered DRP1 phosphorylation. Thus, MICU1 serves as a signal–noise discriminator in mitochondrial calcium signalling, limiting the energetic costs of mitochondrial Ca2+ signalling which may undermine oxidative phosphorylation, especially in tissues with highly dynamic energetic demands. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.Loss of function mutations of the protein MICU1, a regulator of mitochondrial Ca •2+]m).Loss of MICU1 protein expression in human fibroblasts increases resting mitochondrial calcium concentration  by activating PDH phosphatase, consequently dephosphorylating PDH.Increased [Ca•MICU1 leads to modifications of the MCU complex composition and mitochondrial fragmentation.Loss of  These include contraction in skeletal, cardiac and smooth muscle, secretion from all cell types, while Ca2+ signals play key roles in learning and memory, in cell migration, and triggering the earliest phases of development following fertilisation of the oocyte. It has been clear since the pioneering work of Lehninger, Attardi, Carafoli, Deluca and Crompton that mitochondria have a huge capacity to accumulate calcium ions (Ca2+) 2+ into mitochondria mediated by mitochondrial Ca2+ uptake pathways Calcium signalling is fundamental to much of cell physiology, as a rise in cytosolic calcium ion concentration  both have metabolic consequences. A rise in [Ca2+]c will drive an increase in ATP consumption, but simultaneously stimulates the malate-aspartate shuttle, ARALAR, driving an increase in intramitochondrial NADH that stimulates respiration and increases the rate of ATP synthesis 2+]m, which stimulates the activity of the three rate limiting enzymes of the TCA cycle, each of which is modulated by Ca2+, again increasing the rate at which reduced NADH is generated by the cycle, and so driving an increased rate of ATP synthesis 2+ signalling reflects a simple and elegant mechanism that serves to balance an increased rate of ATP provision to match the increased demand that inevitably accompanies the processes driven by the Ca2+ signal – an increase in work through activation of contraction, secretion, migration, or gene expression.The accumulation of Ca2+ uptake also drives cell death under conditions of cellular Ca2+ overload, as supraphysiological mitochondrial Ca2+ accumulation can trigger opening of a large conductance pore in the inner mitochondrial membrane, the mitochondrial permeability transition pore (mPTP) 2+ induced cell death has been most extensively characterised in ischaemia reperfusion injury in the heart Mitochondrial Ca2+ homeostasis within the mitochondrial matrix is maintained through Ca2+ uptake and efflux pathways. The primary mechanism for Ca2+ efflux that normally maintains a low matrix Ca2+ concentration ([Ca2+]m) is the Na+/Ca2+ exchanger, recently identified as NCLX 2+ was first observed in the 1960s, the molecular identity of the channel that mediates Ca2+ import into mitochondria was identified only recently as the well conserved mitochondrial Ca2+ uniporter (MCU) MCU knockout (MCU KO) mice are smaller than littermates, and show a reduced power and reduced activity on a treadmill but otherwise the phenotype is very mild. The conditional knockout in the heart shows a reduced capacity to respond to increased drive Ca2+ concentration [Ca2+]c2+ sensing EF hands, which allow MICU2 to form dimers upon binding to Ca2+. The two proteins form a heterodimer via a disulphide bond and salt bridge 2+]c levels, sensed by the EF hands in the intermembrane space 2+]c at which MCU will open, keeping MCU closed at low [Ca2+]c – at concentrations found at rest in the cytosol - while the channel opens at [Ca2+]c above 2–3 μM showing a cooperative increase in uptake as Ca2+ concentrations increase as described in the earliest studies of mitochondrial Ca2+ uptake 2+ uptake through its interaction with both MCU and MICU1 2+ sensor has been identified in the complex regulation of the MCU 2+-induced cell death was discovered The MCU complex consists of the MCU in association with several proteins which are thought to play a regulatory role, and some of which show variation in expression in different tissues MICU1 were described, showing fatigue and lethargy amongst other symptoms 2+ uptake, resulting in increased mitochondrial Ca2+ load, but surprisingly, did not reveal significant consequences on oxidative phosphorylation or membrane potential, consistent with reports from studies in cell lines as well as in vivoMCU KO mouse, a whole body knockout of MICU1 in the mouse has been reported to result in a high probability of perinatal lethality in two independent studies The functional consequences of altered MICU1 expression were characterised initially by knockout or overexpression in cell lines MICU1 expression in patient derived fibroblasts. Whole exome-sequencing of the patients reported by Logan et al. revealed a homozygous mutation at a splice acceptor site, c.1078-1G > C in MICU1 in 11 of the 15 individuals and at a splice donor site, c.741 + 1G > A in the remaining 4 patients. Experiments were carried out in fibroblasts obtained from two of the patients with the c.1078/1G > C mutation (referred to below as ΔMICU1) and from age matched controls.In the present study, we have further investigated the functional consequences of loss of 2+ uptake even at resting [Ca2+]c is balanced by Ca2+ efflux through the NLCX, in turn driving increased activity of the sodium proton exchanger. We propose that, as a consequence, an increase in proton influx across the inner membrane would undermine the proton-motive force to drive ATP synthesis by the ATP synthase. We provide evidence for the existence of a futile mitochondrial Ca2 + cycle in patient derived fibroblasts and to show that this cycle impairs ATP synthesis through oxidative phosphorylation.We here propose a mechanism which could explain a bioenergetic deficiency in the patients suggesting that increased Ca22.1Human fibroblasts were obtained from patient and control skin samples, from a previously published report 2. Where galactose conditions are indicated, fibroblasts were cultured in zero glucose DMEM with 4 mM L-glutamine (Invitrogen), 10% FBS (Invitrogen), 1 mM sodium pyruvate (Sigma), 0.1% w/v (5.5 mM) galactose  and 1% PS (Invitrogen).Cells were grown in Dulbecco's modified Eagle's medium (DMEM: 4.5 g/L glucose and pyruvate) containing 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin  at 37 °C in 5% CO2.2g at 4 °C and protein concentrations determined using Pierce BCA assay (Thermo Scientific).Following relevant drug treatment and/or media changes, fibroblasts were washed with PBS, scraped and centrifuged. Cell pellets were then lysed in RIPA buffer ) for 30 min on ice. Samples were subsequently centrifuged at 16,000 When using antibodies for detecting phosphorylation, 15–40 μg of protein was boiled at 95 °C for 5 mins in NuPAGE 4X LDS sample buffer (Invitrogen) containing 5% β-mercapethanol. Proteins were separated using 4–12% NuPAGE Bis-Tris gels (Invitrogen) with MOPS running buffer (Invitrogen) and transferred onto nitrocellulose membranes using NuPAGE transfer buffer (Invitrogen) supplemented with 20% methanol. Membranes were washed with TBS-T and blocked with 3% BSA in TBS-T for 1 h at RT, followed by overnight incubation with primary antibody. Following 3 × 10 min washes in TBS-T, membranes were incubated with secondary antibody solution for 1–1.5 h at RT. After 3 × 5 min washes in TBS-T, the membranes were developed using Amersham ECL reagent  and imaged with a ChemiDoc system (BioRad). Densitometry analysis was carried out using ImageJ. For detecting EMRE expression levels, 50 μg of protein was boiled and processed as above, the only differences being the use of 12% NuPAGE Bis-Tris gels, polyvinylidene fluoride (PVDF) membrane and 5% milk in TBS-T as the blocking buffer.Primary antibodies used were anti-C22orf32 Antibody (C-12) , anti-DRP1 , anti-phospho DRP1 (Ser637) , anti-LC3b , anti-β-actin , anti-PDH E1α  and anti-phospho PDH E1α (Ser293) . Secondary antibodies used were anti-mouse and anti-rabbit (both from Thermo Scientific and diluted 1:4000).In order to assess the PDH state and minimise variability relating to substrate supply, protein samples were made approximately 2 h after refreshing the culture media. Culture plates were also snap frozen at − 80 °C before scraping to minimise any subsequent kinase and phosphatase activity. Western blotting for phosphorylated PDH (pPDH) was carried out first and then the same membrane was washed and re-probed overnight for total PDH. The proportion of pPDH was then expressed as average intensity of pPDH band / average intensity of total PDH band. For the DCA experiments, plated cells were treated with 0, 2.5 and 5 mM sodium dichloroacetate, 98% (DCA) (347795) concentrations  respectively for two hours prior to whole cell solubilisation.2.32PO4, 1.25 mM; NaHCO3, 4 mM; CaCl2, 1.2 mM; MgCl2, 1.2 mM; HEPES, 10 mM) at room temperature for 30 min. Prior to imaging, the dye was washed off and the solution was replaced with recording buffer. Images were acquired on a Zeiss 700 CLSM  using a 40 × objective and a 37 °C heated stage.Cells were plated one day before imaging on 22-mm glass coverslips in 6-well plates . Cells were incubated with 5 μM rhod-FF AM  dyes supplemented with 0.002% pluronic acid, in recording buffer  O2 consumption. Data were acquired and analysed using the DatLab 5 software and each of the respiratory states was defined as the average value over a region of stabilised signal.Oxygen consumption rates were measured using the Oroboros Oxygraph-2K . The sensors in each chamber were calibrated in the respiration medium, prior to the experiment. Cells were trypsinised and resuspended at 1 million cells/mL in DMEM buffered with 20 mM HEPES, supplemented with 5.5 mM glucose , 2 mM glutamine and 1 mM pyruvate. The cellular suspension was maintained at 37 °C and stirred at 750 rpm. Drug additions were performed using Hamilton syringes. Once resting rate had stabilised, 10 μM histamine was added to induce a Ca2.5ATP was measured using the CellTiter-Glo Luminescent Cell Viability Assay  protocol. This protocol is based on the principle that bioluminescence is produced when the enzyme luciferase catalyses the reaction between luciferin (both present in the assay buffer) and ATP present in the cell. The luminescent signal is proportional to the amount of ATP present. Cells were seeded in white 96 well plates  and the next day were incubated with one of the following treatments for 1 h at 37 °C: 1 μL/mL DMSO, 5 μM oligomycin A, 1 mM iodoacetic acid (IAA), 10 μM CGP-37157 or 10 mM 2-deoxyglucose (DG). The cells were allowed to equilibrate at RT before incubating with CellTiter-Glo® Reagent (Promega) for 10 min. Luminescence values proportional to ATP content were measured in a plate reader  using a luminescence optic with 3 mm diameter light guide. Each condition was carried out with a minimum sample size of 3 per replicate.2.6t-tests were applied to test significance to a P value of 0.05. Where the means of three or more independent groups were being compared, one-way analysis of variance (ANOVA) was used. When the effect of two different independent variables was being measured e.g. cell line and drug treatment, two-way ANOVA was used. When several comparisons between groups were being made, appropriate post hoc tests were used to correct for multiple testing.Statistical analysis was performed using Prism 6 (GraphPad software). Values are presented as mean ± standard error. N numbers indicate number of independent repeat experiments unless otherwise indicated. Where the means of two independent groups were being compared e.g. control group v ΔMICU1 group, two tail 33.1MICU1 showed an increase in resting [Ca2+]m, an increased rate of mitochondrial Ca2+ uptake in response to stimulation but no change in peak Ca2+ accumulation 2+ homeostasis might give rise to the disorder seen in the children, we considered whether loss of MICU1 might increase susceptibility to Ca2+ induced cell death. Experiments using thapsigargin to promote Ca2+ induced cell death failed to show any significant difference between thapsigargin induced death in controls or in ΔMICU1 cells (data not shown), although rates of thapsigargin induced cell death in fibroblasts were very low.We have shown previously that fibroblasts from patients with mutations in Furthermore, it has been suggested that MICU1 knockout in cell models increases rates of ROS generation, which might contribute to increased cell death 2+ cycle. In patients, increased mitochondrial Ca2+ uptake at rest through a loss of the threshold, ‘gatekeeping’ function of MICU1 raises [Ca2+]m. Increased matrix [Ca2+] will inevitably activate the NCLX, promoting Ca2+ efflux from the matrix. The concomitant increase in Na+ flux will in turn stimulate the Na+/H+ exchange (NHX), compromising the proton gradient available for ATP production , consistent with the previous studies reporting that mitochondria are Ca2+ loaded at rest 2+]m started to rise in the patient cells, increasing much more slowly or not at all in control cells  (We previously reported that no differences were detected in the oxygen consumption rate (OCR) between patient derived cells and controls at rest or after Caalactose  A. Furthalactose  B. This < 0.001)  C. This P < 0.0001) as well as pre-treating cells for an hour with 10 mM 2-deoxyglucose, a glycolysis inhibitor (***P < 0.0001) . It seems plausible that a futile Ca2+ cycle undermines ATP generated by oxidative phosphorylation in ΔMICU1 cells, but that this is compensated at a steady state by increased glycolytic ATP generation. We therefore measured ATP content of the cells before and after inhibition of the NCLX with CGP-37157. In ΔMICU1 cells, CGP-37157 caused a small but significant increase in ATP (108% ± 1.9) while in control cells, the inhibitor caused no significant change in ATP (95.6% ± 4.9) (P = 0.04)   D. These3.32+ cycle might to some extent be balanced by Ca2+ dependent activation of the TCA cycle due to a high resting matrix [Ca2+], and so the net bioenergetic effect is difficult to predict and may also vary between cell types, depending on the capacity of the TCA cycle. Increased [Ca2+]m is expected to result in activation of the three mitochondrial dehydrogenases in the TCA cycle; isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase 2+]m and enhanced Ca2+ activated PDP activity, the pPDH/PDH ratio was significantly reduced in ΔMICU1 cells (0.48 ± 0.097) compared to controls (1.00 ± 0.13) (P = 0.0064) (P < 0.05) and 5 mM  compared to the absence of drug (0.79 ± 0.07), but had no significant effect in ΔMICU1 cells. Furthermore, the pPDH/PDH ratios in the ΔMICU1 cells for no drug treatment were comparable to phosphorylated state in the treated controls, implying that PDH is already maximally active in cells lacking MICU1   A & B. Tng MICU1  D. This 3.4The stability of the MCU complex has been linked to interactions between the different gatekeepers such as MICU1, MICU2 and EMRE, most notably in the direct correlation between MICU1 and MICU2 protein expression where loss of MICU1 leads to a downregulation in MICU2 levels. Previously, we have shown that loss of MICU1 expression did not influence the levels of MCU protein 3.5P < 0.0001) (We have previously reported that mitochondria were fragmented in the ΔMICU1 patient cells compared to the controls  0.0001)  A & B. T+ ATPase which therefore prevents fusion of autophagosomes with lysosomes and thus prevents their degradation, significantly increased the level of LC3-II in both control (P = 0.001) and ΔMICU1 cells (P < 0.0001). There was no significant difference (P = 0.827) in basal LC3-II levels between control (0.17 ± 0.02) and ΔMICU1 cells (0.26 ± 0.01), nor was there a significant difference between control (0.76 ± 0.05) and ΔMICU1 cells (1.05 ± 0.03) (P = 0.051) in LC3-II levels following exposure to bafilomycin (Fragmentation of the mitochondrial network could result from impaired removal of fragmented mitochondria by autophagy ilomycin  C&D.42+ uptake on [Ca2+]c was established over 50 years ago, but only very recently have we begun to understand how this relationship is defined through the regulation of MCU opening by MICU1 and MICU2, while the disease phenotype caused by its failure in children with MICU1 mutations highlights its functional importance. Our own and other recently published data suggest that the MICU1/MICU2 complex prevent mitochondrial Ca2 + uptake at resting cytosolic [Ca2 +], acts to discriminate ‘significant’ Ca2+ signals from noise and protects mitochondria from unwanted consequences of increased mitochondrial Ca2+ uptake at rest 2+ dependent upregulation of the TCA cycle that accompanies a rise in matrix [Ca2+], that should increase oxidative phosphorylation, the observation that a futile Ca2+ cycle consequent on loss of MICU1 function is sufficient to undermine oxidative phosphorylation highlights the energetic costs of mitochondrial Ca2+ signalling, an aspect that has been somewhat neglected. It seems plausible that the relative contributions of these two opposing mechanisms may differ between cells and tissues depending on the capacity of the TCA cycle, the capacity of the respiratory chain to respond, and the activity of the NCLX may all differ between cell types and so define the tissues affected in the patients with MICU1 mutations.The sigmoid dependence of mitochondrial Ca2 + cycle was confirmed by the simple experiment of blocking the efflux pathway, using CGP37157 to inhibit the NCLX. This revealed the continuous constitutive Ca2+ influx into the mitochondria in patient derived cells, as the matrix Ca2+ concentration rose immediately after inhibition of the extrusion pathway. To our knowledge, this is the first time that a futile mitochondrial Ca2+ cycle has been directly demonstrated in a human disease, although the mechanism has been widely considered 2+ in the patient derived cells, and has been described in the other cohort of patients lacking MICU1The presence of a futile Ca2+ cycle was confirmed by the small but significant increase in ATP seen in the patient cells following inhibition of the cycle using CGP-37157, while in the control cells, if anything, the drug caused a small decrease in ATP. It seems likely that the loss of the gatekeeping function in mitochondrial Ca2+ uptake would compromise the metabolic response to increased energy demand in the ΔMICU1 cells, with a particular impact on neurons and muscle, which show a very big dynamic range of metabolic activity.Understanding the impact of MICU1 loss on ATP homeostasis in the fibroblasts is difficult as the cells show a strong dependence on glycolysis for ATP production. Growing the cells in galactose, forced the cells to become more reliant on oxidative phosphorylation and so aids the detection of defects in mitochondrial respiration The stoichiometry and stability of the MCU complex is currently still being investigated. Increased EMRE levels in the cells lacking MICU1 might indicate a relationship between MICU1 and EMRE expression. Our data is consistent with the MICU1 KO mouse, where EMRE expression was directly correlated with severity of symptoms 2+ has been known to play a role in mitochondrial fragmentation in some studies 2+ homeostasis has been described in response to inhibition of the MCU, which caused downregulation of DRP1 expression 2+]c and dephosphorylates DRP1, linking mitochondrial fragmentation with [Ca2+] signalling, but it isn't entirely clear how this relates to changes in matrix Ca2+ handling. If anything, the increased rate of Ca2+ uptake into mitochondria associated with MICU1 mutations are accompanied by a decrease in cytosolic Ca2+, and so at present we cannot readily explain the mitochondrial morphological phenotype in the fibroblasts Perturbations in mitochondrial fusion-fission dynamics have been associated with disease, most notably in neurodegenerative diseases such as Charcot–Marie–Tooth Disease and Dominant Optic Atrophy MICU1 patients present with, and illuminates further avenues of investigation in order to delineate the underlying pathways that might explain tissue specific responses to this loss-of-function mutation.In conclusion, this study highlights a mechanism leading to a bioenergetics deficit, which could explain the pathological phenotype that the Transparency documentImage 1


Our data showed that SHL markedly dampened C48/80- or ST-induced MC degranulation in vitro and in vivo. Mechanistic study indicated that cytosolic Ca2+ (Ca2+[c]) level decreased rapidly and sustainably after SHL treatment, and then returned to homeostasis when SHL was withdrawn. Moreover, SHL decreases Ca2+[c] levels mainly through enhancing the mitochondrial Ca2+ (Ca2+[m]) uptake. After genetically silencing or pharmacologic inhibiting mitochondrial calcium uniporter (MCU), the effect of SHL on the Ca2+[c] level and MC degranulation was significantly weakened. Simultaneously, the activation of SHL on Ca2+[m] uptake was completely lost. Collectively, by activating MCU, SHL decreases Ca2+[c] level to stabilize MCs, thus exerting a remarkable anti-allergic activity, which could have considerable influences on clinical practice and research.Mast cells (MCs) are key effector cells of IgE-FcεRI- or MrgprX2-mediated signaling event. Shuang-Huang-Lian (SHL), a herbal formula from Chinese Pharmacopoeia, has been clinically used in type I hypersensitivity. Our previous study demonstrated that SHL exerted a non-negligible effect on MC stabilization. Herein, we sought to elucidate the molecular mechanisms of the prominent anti-allergic ability of SHL. MrgprX2- and IgE-FcεRI-mediated MC activation  The influx of Ca2+ is amplified by short transient potential Ca2+ channel 1. The increase in intracellular Ca2+ levels and the activation of PKC trigger MC degranulation10Mast cells (MCs) originate from the haematopoietic progenitor cells that enter nearly all vascularized tissue, where they complete their maturation and, under some circumstances, can then migrate into epithelia125792+ pools are the determining factors of MC degranulation2+ (Ca2+[m]) uptake is considered to buffer local or bulk cytosolic Ca2+ (Ca2+[c]) rises2+ channel, with the Ca2+ pore formed by mitochondrial calcium uniporter (MCU) protein142+[c] rises by accumulating Ca2+ into their matrix raises the question whether the activating MCU may dampen MC degranulation for the treatment of allergy, anaphylaxis and asthma, etc.Thus, calcium mobilization is a critical event to the activation of MCs and intracellular CaLonicerae Japonicae Flos, Scutellariae Radix and Fructus Forsythiae, is consistently prepared by stringent manufacturing procedure from Chinese Pharmacopoeia232527282+[c] level by activating MCU independent of Ca2+[c] rise, which is different from the conventional MC stabilizers .Shuang-Huang-Lian (SHL), a formula containing in vitro. As shown in P < 0.01), while SHL potently inhibited the β-hexosaminidase release concentration-dependently (P < 0.01) without cytotoxicity . Next, the effect of SHL on passive systemic anaphylaxis (PSA) was shown in We next determined the effect of SHL on ST-induced active systemic anaphylaxis (ASA) in mice. As shown in 2+[c] concentration. Thus, we next investigated whether SHL could affect the Ca2+[c] level. As expected, ST challenge markedly elevated Ca2+[c] level in the sensitized RBL-2H3 cells, while pretreatment with SHL significantly reduced Ca2+[c] level in a concentration-dependent manner  had been significantly reduced in response to pretreatment with SHL compared with the control  and mouse (P815) MCs (data not shown). In contrast, cromolyn sodium and ketotifen did not affect Ca2+[c] in the resting cells (data not shown) at their effective concentrations on MC degranulation  and plasma membrane Ca2+-ATPase (PMCA), and (or) clearance of Ca2+ by resequestration into the ER and mitochondria2+-ATPase (SERCA) by thapsigargin2+[c] was not affected . The overall permeability of the OMM for Ca2+ is relatively high, while the IMM presents a tight barrier for Ca2+ 2+ channel in lipid bilayer in the IMM, forms the basis of the primary mechanism for Ca2+[m] transport14152+[m] uptake can be completely blocked by ruthenium red, highly implicating that SHL enhanced Ca2+[m] uptake might through activating MCU. To verify whether MCU is indeed involved in the effect of SHL on Ca2+[m], we silenced MCU in mice using a Entranster TM in vivo transfection reagent. The resulting mice, termed MCU−/− mice, lack MCU protein in peritoneal MCs and liver mitochondria compared with the negative control mice (MCUF/F). The fluorescence intensity for the Ca2+[c] of FcεRI+ cells, namely MCs, was analyzed by a flow cytometer . It was found that SHL potently reduced Ca2+[c] levels of peritoneal MCs in MCUF/F mouse with a decreased percentage of 29%, while this effect was notably weakened in the MCU−/− cells with only a decreased percentage of 3.3%  and the extract of Lonicerae Japonicae Flos and Fructus Forsythiae (ELF). Thus, to identify the active constituents in SHL, we first evaluated the effects of ES and ELF at the equivalent concentrations in 2% SHL on Ca2+[c] in the resting RBL-2H3 cells. As shown in 2+[c] levels compared with untreated control in a nontoxic manner (data not shown). Next, we tested the effects of 26 constituents from ELF -quinic acid and methyl salicylate at 10 μg/ml in a nontoxic manner (data not shown), and the fore 3 constituents could be detected by the HPLC-UV according to the Chinese PharmacopoeiaAccording to the Chinese Pharmacopoeiafrom ELF  on the Cin vitro and in vivo  and PSA (in vivo), SHL exerted markedly anti-anaphylactic effects  to mimic type I hypersensitivity4851525456 effects . In ASA  effects . In part effects , which m2+[c] rises via accumulating Ca2+[c] into mitochondrial matrix. However, difficulties lie in the experimental practices. To our knowledge, MCU mediates Ca2+ uptake into the matrix to regulate metabolism, cytoplasmic Ca2+ signaling and cell death212+[m] is critical for the regulation of mitochondrial functions such as fission, fusion and ATP production2+[m] rise is the stimulation of Ca2+-sensitive dehydrogenases of the Krebs cycle, tuning ATP synthesis to the increased needs of a cell; on the other hand, uncontrolled Ca2+[m] overload can lead to the opening of the mitochondrial permeability transition pore with disruption of mitochondrial membrane potential (MMP)2+ entry in mitochondria has been associated with apoptosis and necrosis in many pathological states2+ depletion and overload by a unique complex involving Ca2+ sensors on both sides of the IMM, coupled through EMRE2+[m] is a highly sophisticated process. Thus, as a MC stabilizer through enhancing Ca2+[m] uptake, how to strike a better balance between the effectivity and toxicity is a serious challenge. Our findings indicate that it is through activating MCU that SHL, which has been using for the allergic diseases clinically, decreases Ca2+[c] level to stabilize MCs. Both the effectivity and safety (non-toxic) of SHL are compatible in vitro and in vivo, indicating that the Ca2+[m] increase induced by SHL through activating MCU is sustainable to a certain degree. Of course, the pharmacological reversibility of SHL is also an essential factor. Moreover, excess Ca2+ entry in mitochondria (Ca2+[m] overload) causes more reactive oxygen species (ROS) generation, a by-product of Krebs cycle, whose elevation is a key event that leads to further organelle depolarization and loss of MMP, thus resulting in a vicious cycle64Theoretically, it is feasible for a drug to stabilize MCs through buffering the Ca2+-activated Ca2+ channel whose activation depends on the increase of Ca2+[c] concentration2+[c] rise. Thus, we were able to observe that SHL rapidly reduced Ca2+[c] levels in the resting cells . Balb/c mice  and SD rats  were from Vital River Experimental Animal Services .SHL injection and its 2 intermediate fractions (ES and ELF), which were prepared according to the Chinese PharmacopoeiaThe MCU siRNA and their negative controls were synthesized by GenePharma Co., Ltd. . The plasmid pcDNA3.1-mito-GCaMP2 was a kind gift from Dr. Xianhua Wang . In this plasmid, the GCaMP2 calcium indicator was ligated with a mitochondrial targeting sequenceRat basophilic leukemia cell line (RBL-2H3) and mouse mastocytoma cell line (P815) were purchased from the cell bank of Chinese Academy of Sciences . Human LAD2 cell line  was presented as a gift from Prof. Renshan Sun . Peritoneal MCs were isolated from SD rats.Metapenaeus ensis was extracted and purified by an isoelectric precipitation method as previously describedST from the The preparation of antibody was similar to our previously described method5 cells/well and sensitized with anti-ST monoclonal IgE (25 μg/ml) at 37 °C overnight. The cells were washed by Hank’s balanced salt solution (HBSS) supplemented with 0.1% (w/v) BSA and pre-incubated with SHL in 120 μl of HBSS at 37 °C for 30 min followed by adding 20 ng/ml of ST for further 1.5 h incubation. 30 μl of the supernatant was transferred to a 96-well black flat-bottom plate accompany with 50 μl of substrate solution . The reaction proceeded at 37 °C for 1.5 h and was stopped by adding stop buffer . Fluorescence was determined with a fluorescence microplate reader at λex 355 nm and λem 460 nm.The β-hexosaminidase release assay was performed as previously described with some modifications4 cells/well) or the peritoneal MCs (1.0 × 106 cells/well) were seeded in the 96-well plates and pretreated with SHL at 37 °C for 30 min followed by adding C48/80 (10 μg/ml) for further 1.5 h incubation. β-hexosaminidase in the supernatant was determined.For the measurements of C48/80-induced β-hexosaminidase release, the LAD2 cells  with 40 μg/mouse of anti-ST monoclonal IgE, while the control group were given the equal volume of physiologic saline. 24 h later, the mice were challenged (i.v.) with 20 μg/mouse of ST after pretreatment with SHL or physiologic saline (i.p.) for 20 min. The rectal temperature was measured by a thermal probe  for 30 min using a polygraph .Mice received an i.p. injection of 100 μl of Imject Alum containing 60 μg/mouse ST and were immunized again 7 days later. 2 days after the second immunization, the mice were pretreated with SHL (2.5 ml/kg or 5 ml/kg) or physiologic saline (Control group and ST model group) for 30 min and then challenged by a rapid intravenous infusion  of 5 μg/mouse of ST. The mice in the control group were received the same solution without ST. To monitor changes in body temperature associated with anaphylaxis, rectal temperature was measured 30 min after ST challenge.2+[c] level was performed using the calcium-reactive fluorescence probe Fluo-3/AM as previously described with slight modifications6 cells/ml) and incubated for 30 min in the dark at 30 °C with Fluo-3/AM (4 μM) in the presence of 0.04% (w/v) Pluonic F-127 in HEPES buffer . 4 mM probenecid was added to avoid leakage of fluo-3. After removing the dye, the cells were treated with SHL or normal saline (the control group) and the fluorescent intensity was immediately determined at λex 485 nm and λem 538 nm using a spectrofluorimeter .Measurement of the Ca2+[c] levels in mouse peritoneal MCs, the cells in the mouse abdominal cavity were isolated and loaded with Fluo-3/AM (4 μM) at 30 °C for 30 min. MCs could be recognized and analyzed by a FACSCalibur flow cytometer after staining with PerCP-eFluor 710 labeled anti-mouse FcεR1 antibody at 25 °C for 15 min.To assay the Ca2+[m] level in RBL-2H3 cells was performed using the mitochondrially localizing Ca2+-reactive fluorescence probe, rhod-2/AM, as previously describedex 535 nm and λem 590 nm.Measurement of Ca2+[m] uptake was measured using Calcium Green-5N according to previously describedMouse liver mitochondria were isolated and further purifiedThe recombinant adenovirus (Ad.m-GCaMP2) based on the pcDNA3.1-mito-GCaMP2 was produced by Hanbio Biotechnology Co., Ltd. . Cells were infected with Ad.m-GCaMP2. 24 h later, the fluorescence signal in the mitochondria was captured by confocal microscopy  using a 100× oil objective.in vivo transfection reagent was used to deliver the siRNA according to the manufacturer′s recommendations. On day 5, the mice were sacrificed after anesthetization. Fresh liver and the peritoneal MCs were immediately isolated for the assay.The mice were injected via tail vein with MCU siRNA  or the negative control siRNA (sense 5′-UUC UCC GAA CGU GUC ACG UTT-3′ and antisense 5′-ACG UGA CAC GUU CGG AGA ATT-3′) on days 1 and 4 (3 OD/mouse). Entranster TM-The rat MCU siRNA  or the negative control (sense 5′-UUC UCC GAA CGU GUC ACG UTT-3′ and antisense 5′-ACG UGA CAC GUU CGG AGA ATT-3′) was transfected into RBL-2H3 cells using Entranster TM-H4000 as described in the manufacturer’s protocol.t test was used when only two groups were compared. The difference was considered to be statistically significant when P < 0.05.Data represent the mean ± SD of at least three independent experiments. Statistical analysis was performed by one-way ANOVA. A student’s How to cite this article: Gao, Y. et al. The Three-Herb Formula Shuang-Huang-Lian stabilizes mast cells through activation of mitochondrial calcium uniporter. Sci. Rep.7, 38736; doi: 10.1038/srep38736 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The mechanisms driving these Ca2+ events remain incompletely understood. In this study, we integrate Ca2+ imaging, quantitative data analysis, and mechanistic computational modeling to study the spatial and temporal heterogeneity of cortical astrocyte Ca2+ transients evoked by focal application of ATP in mouse brain slices. Based on experimental results, we tune a single-compartment mathematical model of IP3-dependent Ca2+ responses in astrocytes and use that model to study response heterogeneity. Using information from the experimental data and the underlying bifurcation structure of our mathematical model, we categorize all astrocyte Ca2+ responses into four general types based on their temporal characteristics: Single-Peak, Multi-Peak, Plateau, and Long-Lasting responses. We find that the distribution of experimentally-recorded response types depends on the location within an astrocyte, with somatic responses dominated by Single-Peak (SP) responses and large and small processes generating more Multi-Peak responses. On the other hand, response kinetics differ more between cells and trials than with location within a given astrocyte. We use the computational model to elucidate possible sources of Ca2+ response variability: (1) temporal dynamics of IP3, and (2) relative flux rates through Ca2+ channels and pumps. Our model also predicts the effects of blocking Ca2+ channels/pumps; for example, blocking store-operated Ca2+ (SOC) channels in the model eliminates Plateau and Long-Lasting responses . Finally, we propose that observed differences in response type distributions between astrocyte somas and processes can be attributed to systematic differences in IP3 rise durations and Ca2+ flux rates.Astrocytes are a major cell type in the mammalian brain. They are not electrically excitable, but generate prominent Ca Activation of GPCRs leads to increases in intracellular Ca Haydon, . Ca2+ in Haydon, , enablin3-mediated Ca2+ responses in astrocytes have been used to investigate Ca2+ oscillations , to fit the evoked responses. This approach is more realistic . Coronal slices (400 μm-thick) were cut using a VT1200 Vibratome  and transferred to oxygenated artificial cerebrospinal fluid (aCSF) that contained 124 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgSO4, 26 mM NaHCO3, 1.25 mM NaH2PO4, 0.004 mM Na-Ascorbate, and 10 mM glucose . Slices were allowed to recover in oxygenated aCSF at room temperature for 1 h before experiments. During the recordings, the slices were placed in a perfusion chamber and superfused with aCSF gassed with 95% O2 and 5% CO2 at room temperature for the duration of the experiment. To evoke Ca2+ responses, 500 μM ATP  crossed with the GFAP-CreER mouse line, and thus express the GCaMP5G genetically-encoded Ca2+ images . Astrocyte Ca2+ signaling was recorded at a frame rate of 1 Hz. All imaging was done on cortical astrocytes in the primary somatosensory cortex.Two-photon imaging was performed using a Prairie two-photon microscope with a mode-locked Ti:Sapphire laser source emitting 140 fs pulses at an 80 MHz repetition rate with a wavelength adjustable from 690 to 1,040 nm . We used laser emission wavelengths of 920 nm to excite GCaMP5G or 1,040 nm to excite tdTomato. A 20 × 0.95 NA water-immersion objective was used for all the Camedfilt2 command). For each pixel, the median fluorescence of 20 frames preceding the agonist application was used to calculate the baseline fluorescence (F0). Using this baseline, the percent change in fluorescence (100 * ΔF/F0) of each pixel was calculated throughout the time-lapse image. Upon displaying the maximum ΔF/F0 projections, ROIs were manually selected for the soma, large processes, and small processes. Large processes . The Ca2+ response peaks were found using the findpeaks command in MATLAB and were defined as a ΔF/F0 exceeding n standard deviations above the baseline value (where n was 7 for the soma and 6 for the processes) and having a value of at least 40%. Only those ROI traces were included in our analyses that responded to the agonist and that had minimal or no spontaneous activity before the agonist application, in order to avoid mistaking spontaneous Ca2+ activity for evoked activity.The two-photon images were processed and analyzed using custom-written MATLAB  scripts. Each time-lapse image was first processed with a 3 × 3 median filter  the first response peak and after (Trough2) the last peak were automatically detected. Trough1 (or Trough2) was defined as the last (or first) data point, before (or after) the peak that had a value <3.3 standard deviations above the baseline. The time between the two troughs determined the duration of the experimental Ca2+ response. Due to the absence of noise in the mathematical model , simulated responses were determined as the first or last points where the Ca2+ concentration reached a value >40% of the baseline. In Figure 2+ responses.To calculate Ca2+ trace  and were from ROIs which had not responded to an agonist application for at least 3 min. The ΔF/F0 was then normalized such that the maximum peak value for each trace was 1 and the peaks of all traces were aligned . Finally, the average and standard deviation of the traces were calculated. Similar to ΔF/F0 traces, the model Ca2+ trace .For this comparison, we model astrocyte Ca2+ activity components in our model: activation of a metabotropic G-protein-coupled receptor  leads to IP3 production . IP3 then binds to IP3 receptors (IP3Rs) on the endoplasmic reticulum (ER) membrane, which consequently opens IP3R channels and allows Ca2+ to exit the ER and enter the cytosol. When there is significant depletion of Ca2+ from the ER, store-operated Ca2+ (SOC) channels are activated, and allow for additional Ca2+ to flow from the ECS into the cytosol. Sustained, elevated levels of cytosolic Ca2+ inactivate IP3Rs, and activate sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps and plasma membrane Ca2+ ATPase (PMCA) pumps, which then transfer the cytosolic Ca2+ back to the ER and ECS, respectively. After the degradation of IP3, these pumps and channels return the system to steady state.Figure 2+ dynamics .Unlike many previous studies on astrocyte Ca2+ concentration, c, and total intracellular Ca2+ concentration, ctot, as follows:Our resulting model consists of a system of three differential equations. Two of those equations model the changes in cytosolic CacER = (ctot – c)γ. We will also denote IP3 concentration as p. The Ji's in these equations represent fluxes through the pumps and channels that are shown schematically in Figure JIP3R represents the flux from the ER to the cytosol through the IP3R channel, JER_leak is the leak between the ER and the cytosol, JSERCA captures the flux due to the SERCA pump, JECS_add is the leak between the ECS and the cytosol as well as additional plasma membrane fluxes not explicitly modeled, JPMCA represents the PMCA pump, and JSOC is the flux through the SOC channels. Further, γ is ratio of the volume of the cytosol to the volume of the ER, and δ is the ratio of membrane transport to ER transport. The remaining differential equation tracks the deactivation rate of the IP3R,and the concentration in the ER is given by h∞ is the equilibrium binding probability and τh is the time constant. We define these quantities more specifically in the following subsections.where 3 receptor (IP3R) model to capture the Ca2+ dynamics through the IP3R channel , deactivating Ca2+, h, and IP3, m(p). They take the fast variables, the binding of activating Ca2+ and IP3, to be in quasi-steady state, and they model the slow variable, the binding of deactivating Ca2+, explicitly. When open, the flux through the channel is determined by the concentration gradient of Ca2+ between the cytosol and the ER. In total, the equations governing this model areWe use the Li-Rinzel IPwhereand the dynamics of h are governed by Equation (3).3 receptor found in astrocytes is most likely very similar to ones found here, the rate constants determining the open probability of receptor are likely to be different. However, little experimental data is available for comparison, and current astrocyte models use constants provided by Li and Rinzel  and the dissociation constant (kSERCA) were used in De Pittà et al.  of the rising and decaying phases, respectively.3 parameters A, rrise, ddec, and drise for use in our simulations, we examined and compared data from previous experimental  Ca2+ responses of COS-7 and HSY-EA1 cells. While they found differences in peak IP3 concentrations within one cell type, between the two cell types, and with different ATP concentrations . We compared these results with results from local, brief (<100 ms) glutamate applications in cultured astrocytes by Pasti et al. , rrise = , ddec = , and drise =  . All 600 IP3 traces were used throughout this paper, unless otherwise noted in the figure captions and text.To determine a reasonable range of the IPà et al.  to generi et al.  and acco2+ fluxes, and hence the mathematical form of these fluxes, are similar between cell types, it is known that the specific dynamics and time scales of these channels can vary. Further, many of these channels and pumps have not been investigated in astrocytes with sufficient detail to capture specific parameter values. As a result, we fitted several parameters  of our model in order to match the kinetics of an average, short-duration (<25 s) experimental Ca2+ transient  and the ER (~200 μM) , and examined the distribution of Ca2+ response types in each subspace. We also examined these distributions while limiting the ranges of IP3drise parameters, which were divided into increasingly larger ranges: drise-values from 1 to 11, 1 to 21, 1 to 31, and 1 to 41 s , Plateau (PL), Multi-Peak (MP), and Long-Lasting (LL). A general definition for each response type is as follows , with at least one trough reaching <50% of the maximum adjacent peak height. Only those peaks are considered that have heights >5% of the adjacent peak height.A signal that stays elevated continuously, without returning close to baseline  or having additional peaks (with troughs reaching <50% of the maximum adjacent height) for more than 70 s. When there are multiple peaks, if the signal remains elevated for >70 s between any two adjacent troughs, it will be considered a LL response; otherwise, it will be a MP response.A signal with one clear peak, without any subsequent major oscillations or bumps. A major oscillation/bump is one with a sufficiently large height (>5% of the adjacent peak height) or sufficiently long duration (lasting >50% of the main peak's duration).A signal with one main peak and subsequent bump or oscillation that either has a sufficiently long duration (>50% of the main peak's duration) or is elevated with its troughs >50% of the peak heights.3 time course, the simulated Ca2+ responses transition between response types as a continuum. As a result, to automatically determine the cytosolic Ca2+ response types of the mathematical model simulations, we developed an extensive MATLAB script to implement the classification procedure described above . It is also worth noting that Ca2+ responses that had amplitudes too small to be detected experimentally (<0.4 μM) or had amplitudes too high to be biologically reasonable (>3.5 μM) were not included in our analyses.In our mathematical model, when changing the IP2+ response types were determined using a similar algorithm; however, due to inherent noise in experimental signals, the algorithm was manually implemented rather than using the MATLAB script. Moreover, the following step was modified to avoid mistaking inherent experimental noise for additional peaks in the Ca2+ traces: in the case of a second peak or oscillation, to determine if the peak was large enough to be a MP response, we checked if its height was >15% of the adjacent peak height (rather than >5% as for the model traces).For experimental traces, Cat-test was used to compare SP kinetics between the soma and large processes. Pearson's chi-square test was used to compare the distributions of experimental Ca2+ response types recorded from the soma, large processes, and small processes. Fisher's exact 2-tail test was used to compare the frequencies of specific Ca2+ response types recorded in these subcompartments. The Kolmogorov-Smirnov test was used to compare the distribution of Ca2+ durations observed experimentally and generated in the model using different subsets of IP3 transient parameters.The paired sample 2+ responses in three subcompartments of each imaged astrocyte: the soma, large processes, and small processes . In Figure 2+ traces vs. time for the three cells, with cell 1 being the astrocyte from the time-lapse image in Figure 2+ responses , and to use the model to examine the sources of these forms of response variability (Table We applied brief 16–250 ms) local pulses of ATP and examined Ca50 ms loc2+ signaling, we divided all Ca2+ responses into four main categories according to their shape and duration: Single-Peak (SP), Plateau (PL), Multi-Peak (MP), and Long-Lasting (LL). This response type categorization is based on our observed experimental data, the astrocyte literature  were categorized into three classes based only on the response shape. We also categorized responses based on duration, particularly separating those responses that lasted 70 s or more . Second, IP3 uncaging experiments provide direct evidence that IP3 is the sole source of trial to trial variability. Fiacco and McCarthy  can respond differently to identical agonist pulses in different trials . We explore this idea in the simulations described later.Interestingly, Fiacco and McCarthy  did obsea Figure  and in ra Figure . We spec2+ kinetics between the astrocyte soma and processes in response to neural stimulation. Using ATP application, we investigated whether the kinetics of SP responses  varied between the soma and large processes of individual astrocytes. To control for trial to trial and cell to cell variability, we examined pairs of somas and large processes of the same astrocyte that responded to the same trial of ATP application with a SP response (N = 9). While we found that the Ca2+ amplitude is greater in large processes than in somas , we found no significant differences between the SP durations (p = 0.059), rise times (p = 0.586), or decay times (p = 0.367)  Figure . The difp = 0.026, and of the somas and small processes p = 0.0016). Further, PL and LL responses were observed at a lower rate in all three subcompartments. The following are the percentages of observed response types  in each astrocyte subcompartment: 63.64, 18.18, 0.0, 18.18% (somas); 47.06, 13.73, 33.33, 5.88% (large processes); 31.67, 10.0, 51.67, 6.67% . In comparing the overall response type distributions, we found the largest differences to be between the distributions of the somas and small processes .While we did not find significant differences between the paired SP response kinetics of the somas and large processes, we did note differences in the likelihood of observing certain types of responses in each of these subcompartments. In Figure 3 dynamics and the functional properties of Ca2+ channels and pumps. As above, the differences in IP3 dynamics within different astrocyte subcompartments could arise from differences in GPCR expression levels or functional properties, differences in agonist binding probability or IP3 diffusion within or between cells (due to differences in subcompartment size and shape), or differences in regulatory mechanisms downstream of GPCR activation. Moreover, differences in functional properties of Ca2+ channels and pumps in different astrocyte subcompartments could also arise from differences in subcompartment size and shape, differences in expression levels and spatial distributions of these Ca2+ components, or simply different activity levels of these components.The variability in response type distributions between subcompartments could be the result of differences in both the IP3 dynamics, (2) Ca2+ fluxes through various Ca2+ handling mechanisms . We will next examine the plausibility and consequences of each of these mechanisms in our mathematical model .To summarize, we suggest that the mechanisms behind the observed variability in all three cases Table  stem mai2+ handling mechanisms shown in Figure 3 (rather than the GPCR agonist) as the direct input. It consists of three ordinary differential equations describing changes in cytosolic Ca2+ levels, total intracellular Ca2+ levels, and the inactivation variable of the IP3 receptor (IP3R). See section Materials and Methods for details and Handy et al. , in agreement with the results discussed in Figure To verify our model of astrocyte Cae Figure . We exam2+ responses, we used an IP3 input within the lower end of the parameter range in Table 3 dynamics and Ca2+ fluxes driving this response, is shown in Figures 3 stimulus, the simulated system is at steady-state, with the Ca2+ fluxes governed by the ER leak and SERCA pump balanced. When IP3 is released into the cytosol, the IP3R becomes activated and Ca2+ flows from the ER into the cytosol. Depletion of ER Ca2+ causes SOC channel activation, though this flux remains small for the duration of this response. The increase in cytosolic Ca2+ also quickly activates the SERCA pumps, allowing for Ca2+ to be pumped back into the ER. As the cytosolic Ca2+ concentration continues to rise, the PMCA pumps also become activated and some Ca2+ is lost to the ECS. Additional Ca2+ is also released into the ECS due to the ECS_add term. Moreover, before cytosolic IP3 begins to degrade, the Ca2+ flux through the IP3R decreases as a result of negative feedback from elevated levels of cytosolic Ca2+. Together, the SERCA pump, PMCA pump, ECS_add, and the deactivation of the IP3R are able to slow the net change of Ca2+ in the cytosol. As IP3 is degraded and removed from the system, the IP3R begins to deactivate more rapidly, and the pumps are able to return the system to equilibrium. Even when the Ca2+ response measured in the cytosol has returned to baseline levels, the pumps and channels continue to work at a low level to restore the system back to pre-stimulus equilibrium. In the model, the process of refilling the ER to pre-stimulus levels is on the order of ~10 min.Because short-duration SP responses are the briefest observed astrocyte Ca2+ Response Variability and their Sources, IP3 stochasticity is likely a major source of all three forms of variability in evoked Ca2+ responses considered in this manuscript  over a biologically plausible range of values .As discussed in section Forms of Ca2+ response curve . For comparison, in Figure N = 71). Model and experimental responses are qualitatively similar, dominated by SP responses for durations <22 s; MP or LL responses for durations >70 s; and a mix of SP, MP, and PL responses for intermediate durations. For both the model and experiments, duration and total area under the curve  are positively correlated. Additionally, the range of Ca2+ response durations between simulations and experiments are similar. These similarities suggest that the range of our model parameters and selected IP3 kinetics are biologically plausible for evoked, IP3-dependent astrocyte Ca2+ activity. However, experimental Ca2+ responses are more sparsely and non-uniformly distributed than model Ca2+ responses, which we explore next.Furthermore, Figure 2+ response durations is similar in both model and data , with longer PL and LL responses overrepresented in simulations. Hypothesizing that IP3 kinetics may influence the distributions of model response durations, we explored the effects of restricting the IP3 waveform, with the goal of matching the distribution of experimental response durations without eliminating altogether the long-duration responses of Figure 3 total duration, decay duration, rise duration, and amplitude), we only succeeded in this goal by limiting the IP3 rise duration (drise) from Equation (4) to <22 s  and for restricted values of rise durations . As expected, the short-drise histogram exhibits many more SP responses and substantially fewer longer, more complex responses. This effect is compared with experimental data in section Variability between Astrocyte Subcompartments: Model.Because response durations and types are clearly correlated Figure , we migh2+ amount vs. the total IP3 amount . Data points are color-coded by response type, and the symbol type denotes the IP3 rise durations . We draw several conclusions from Figure 3 and Ca2+ are strongly positively correlated. In contrast, we found no correlation between other features of the IP3 and Ca2+ waveforms: IP3 rise duration, total duration, decay duration, amplitude, or the ratio of amplitude over duration did not correlate with Ca2+ total amount, duration, or amplitude. Second, although there is some correlation between total IP3 amount and Ca2+ response type, no two features or parameters of the IP3 waveform strictly predict the response type. Third, for intermediate values of total IP3 and Ca2+ amounts, the response type is particularly sensitive to small changes in the IP3 waveform  involved in Ca2+ responses (section Forms of Ca2+ Response Variability and their Sources). Therefore, we examined the individual contribution of each of these channels and pumps to Ca2+ responses , and found no major effects  change when SOC channels are fully blocked. As seen, the durations and amplitudes of the Ca2+ responses to the same IP3 input decrease when SOC channels are blocked. This was true for nearly 99% of the full set of 600 IP3 traces . In other words, our model predicts that blocking SOC channels decreases the likelihood that the astrocyte will respond to a brief application of agonist.The green traces in Figure 2+ responses is particularly surprising given that the SOC flux rates are low in our model . We then selected the subspace, or subspaces, that best matched each of the three experimentally-recorded distributions in Figure Thus, far, we have examined the separate effects of IPge Table , in ordege Table . To do t2+ response variability between astrocyte subcompartments stems from variability in both Ca2+ channel/pump properties and the underlying IP3 kinetics (see sections Variability between Astrocyte Subcompartments and Contribution of IP3 to Ca2+ Response Variability). It is noteworthy that the simulations for a given astrocyte subcompartment also include a range of Ca2+ channel/pump parameters and IP3 parameters. Since in the experiments multiple cells were used to generate the data for each astrocyte subcompartment, having a range of parameters in simulations reflects the variability in channel/pump properties and IP3 kinetics inherent to each cell . The range of IP3 parameters in simulations for each subcompartment also reflects experimental IP3 stochasticity stemming from trial to trial variability  as well as experimental variability .The rationale behind this parameter search is that Ca3drise values (≤11 s) and the following parameter ranges: high vSOC (1.83–2.36), medium vPMCA (8.33–11.67), and low vSERCA (0.45–0.75). We did not find any one subspace that matched the distributions of the large and small processes. By looking at combinations of two adjacent subspaces, we found that IP3drise values ≤21 s and the following parameter ranges provided a distribution similar to that of the large processes: high vSOC (1.83–2.36), low vPMCA (5.0–8.33), and medium and high vSERCA (0.75–1.35). Using this same parameter subspace of the large processes, but with the full range of IP3drise values in Table 2+ response type distributions from the mathematical model are shown in Figure Using this parameter search, we found that the only subspace with a distribution that closely resembled the somatic distribution consisted of very short IP values ≤1 s and t2+ response kinetics generated from these parameter subspaces are reasonable and comparable to the kinetics in the experimental data, we examined the ranges of Ca2+ kinetics for the soma and large processes of all response types in experiments  the Ca2+ flux rates through the PMCA and SERCA pumps are, respectively, lower and higher in the large processes. Response type distributions associated with small processes can be generated by increasing IP3 rise durations even further, but using the same Ca2+ flux parameters as for large processes. This gradient in IP3 rise durations from somas to fine processes could be related to the influx of IP3 through spatial diffusion from other subcompartments, the constricted volume of the smaller subcompartments compared to the larger ones, and different probabilities of the agonist binding to receptors (due to the different subcompartment sizes and GPCR densities or expression levels).In summary, our results predict that the main differences between the astrocyte somas and large processes that are responsible for different response type distributions are as follows: (1) large processes include IP2+ responses to brief focal ATP applications and, based on our results, developed a single-compartment model of astrocyte Ca2+ activity to investigate mechanisms of Ca2+ response variability. We categorized both experimentally-recorded and model Ca2+ responses into four types: Single-Peak (SP), Multi-Peak (MP), Plateau (PL), and Long-Lasting (LL). We found that, experimentally, SP response kinetics do not differ consistently between the soma, and processes, but we do see variability between cells and trials. However, the distributions of Ca2+ response types vary between different astrocyte subcompartments, with the likelihood of SP responses decreasing, and of MP responses increasing, in the large and small processes relative to the soma.We analyzed astrocyte Ca3 transients to the model with a range of biologically plausible kinetics, and found that this range of IP3 parameters was sufficient to reproduce all four Ca2+ response types in the model, without the need for feedback-induced oscillations in the IP3 waveform  astrocyte somas have higher PMCA flux rates and lower SERCA flux rates than the processes, and (2) when moving from the somas to the large and small processes, the underlying IP3 transients tend to include those with longer rise durations.Lastly, we propose that the experimentally observed response variability between astrocyte subcompartments can be explained by the differences in their channel/pump flux rates and IP2+ transients evoked in astrocytes, it is potentially very useful in determining which mechanisms are altered in disease states, reactive astrocytosis, or other cases in which Ca2+ events are potentially altered.Our modeling results highlight two major advantages of relatively simple, biophysically based, mathematical models. First, they are experimentally falsifiable, and thus can be straightforwardly improved, or if necessary, discarded. Second, verified models of this type can be used as diagnostic tools. For example, because our model makes strong predictions about how specific biophysical mechanisms determine the types of Ca3 time course variability may be a key contributor to all forms of response variability. For a given agonist application, variability between cells, or subcompartments within a cell, may arise from the profile of agonist reaching the GPCRs ; the local expression level or properties of GPCRs in a given cell or subcompartment; and differences in the diffusion and degradation of IP3. Our experimental and computational results suggest that the net effect of such differences between the soma, large processes, and small processes is to increase the effective IP3 rise duration (drise) in the periphery relative to the soma  are necessary to explain differences between the distributions of responses between somas and processes.We have presented three contexts in which astrocyte response variability has been observed Table . Our resa Figure . In our 3 uncaging experiments  channels, which are activated by GPCR agonists rather than by the depleted ER. ROC channels have been found in some astrocytes . Consistent with the suggestion that oscillatory IP3 events underlie growing Ca2+ oscillations, increases in Ca2+ peaks have been observed in HSY-EA1 cells during IP3 oscillations , which varies more smoothly with small differences in the upstream triggering events (Figure Much of our analysis has focused on dividing Cas Figure . Insteads Figure  is via ts Figure .MT collected and analyzed the experimental data under the guidance of JW. MT and GH built the mathematical model, ran the simulations, and analyzed the results under the guidance of AB. AB and JW oversaw the design and execution of project. MT, GH, AB, and JW wrote and reviewed the manuscript.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.


Inositol 1,4,5-trisphosphate receptors  are assumed to regulate SOCE solely by mediating ER Ca2+ release. We show that in Drosophila neurons, mutant IP3R attenuates SOCE evoked by depleting Ca2+ stores with thapsigargin. In normal neurons, store depletion caused STIM and the IP3R to accumulate near the plasma membrane, association of STIM with Orai, clustering of STIM and Orai at ER–plasma-membrane junctions and activation of SOCE. These responses were attenuated in neurons with mutant IP3Rs and were rescued by overexpression of STIM with Orai. We conclude that, after depletion of Ca2+ stores in Drosophila, translocation of the IP3R to ER–plasma-membrane junctions facilitates the coupling of STIM to Orai that leads to activation of SOCE.Store-operated Ca Summary: In Drosophila neurons, mutant IP3 receptors disrupt store-operated Ca2+ entry by destabilizing interaction of STIM with the Ca2+ channel, Orai. The interactions could coordinate store emptying with Ca2+ entry. The latter is usually mediated by store-operated Ca2+ entry (SOCE), an almost ubiquitously present pathway through which empty Ca2+ stores stimulate Ca2+ entry across the plasma membrane (Olf186-F in flies) -enriched membrane domains  . Orai fomembrane  and STIMembranes . Ca2+ di to Orai . That in to Orai . Additio to Orai  or by fa to Orai  or the o domains .2+ stores by inhibiting the ER Ca2+ pump, but for SOCE evoked by physiological stimuli, the Ca2+ stores are depleted by activation of IP3Rs. In the present study, we use genetic manipulations in Drosophila neurons to ask whether IP3Rs regulate SOCE solely by mediating Ca2+ release from the ER or whether they can also play additional roles downstream of store depletion. Drosophila is well suited to this analysis because single genes encode IP3R, STIM and Orai, whereas vertebrates have several genes for each of these proteins. Our results demonstrate that in Drosophila, its IP3R contributes to assembly of the STIM–Orai complex. Comparison of results from Drosophila and vertebrates suggests that the STIM–Orai complex might assemble in plasma-membrane–ER regions equipped to allow local depletion of Ca2+ stores.SOCE can be activated by thapsigargin, which depletes Ca2+ stores with thapsigargin in Drosophila neurons was abolished by RNA interference (RNAi) treatment for STIM or Orai  in Drosophila, in regulating SOCE. To characterize SOCE in Drosophila neurons with mutant itpr, we examined five hetero-allelic combinations of a 15-residue C-terminal deletion and three point mutations located in different parts of the IP3R  were measured in primary neuronal cultures for each genotype  to report cytosolic free Ca2+ concentration ([Ca2+]c) is vindicated by evidence that [Ca2+]c in unstimulated cells was unaffected by mutant IP3R  and the peak fluorescence changes evoked by SOCE in wild-type neurons were only 32±14%  of those evoked by saturating the indicator with Ca2+.SOCE evoked by depleting ER CaOrai see . This isthe IP3R  . However, the resting [Ca2+]c and thapsigargin-evoked release of Ca2+ from intracellular stores were unaffected, confirming that mutant IP3R selectively perturbed SOCE . The reduced SOCE in itpr mutant neurons was not, therefore, restricted to a single combination of mutant alleles. Mutant combinations in the ligand-binding domain (S224F), modulatory domain (G1891S and G2117E) and C-terminus (Δ2814–2828) of the IP3R all inhibited SOCE. SOCE was not significantly affected in neurons heterozygous for the individual mutants (Table S1). In adult flies, these heterozygous itpr mutants also have no significant effect on viability or flight phenotype .SOCE was significantly reduced in all five henotype . The resked SOCE D. It migenotypes , and resenotypes . The oriitpr were observed in cultured haemocytes from Drosophila larvae, where itpr mutants attenuated thapsigargin-evoked SOCE without affecting basal [Ca2+]c or Ca2+ release from intracellular stores .Similar functional consequences of mutant 3R or mutations within IP3R attenuate SOCE without affecting the Ca2+ content of the intracellular stores. The effects are not due to loss of STIM or Orai.The results so far establish that loss of IPitpr combination itprS224F/G1891S to examine the effects of overexpressing STIM and Orai on SOCE in cultured neurons. We chose this combination because it has been the most extensively studied of the heteroallelic mutant itpr combinations    . A simil (siRNA) . Hence, and Orai D. The re3R mutations affect interactions between STIM and Orai using an ectopically expressed +dOrai-CFP transgene with a pan-neuronal driver (C155Elav). This allowed immunoprecipitation of Orai with an anti-GFP antibody. Expression of Orai–CFP did not restore SOCE in itpr mutant neurons . Treatment with thapsigargin enhanced the pulldown of STIM with anti-GFP antibody from lysates of wild-type brain, consistent with enhanced interaction between STIM and Orai after store depletion. However, the pulldown of STIM from thapsigargin-treated brains with mutant IP3R was much reduced (3R in this immunoprecipitate (data not shown), suggesting that any interaction between IP3R and STIM or Orai, whether direct or through other proteins, might be too weak to survive immunoprecipitation. It was impracticable to assess the effects of mutant IP3R in these immunoprecipitation experiments because STIM–YFP rescued the mutant IP3R phenotypes  or total fluorescence within a plane that included mostly plasma membrane (3R (itprS224F/G1891S) (3R immunostaining near the plasma membrane (3R in thapsigargin-treated neurons expressing mutant IP3R (Drosophila neurons with mutant IP3Rs (itprS224F/G1891S); and siRNA for the IP3R appeared to have a similar effect  revealed that only STIM redistributed after thapsigargin treatment . These results demonstrate that IP3R and STIM accumulate in peripheral ER near the plasma membrane after store depletion, and loss of IP3R or mutations within it inhibits the translocation of STIM.To avoid reversal of attenuated SOCE in neurons with mutant IP of STIM D, we usemembrane C,D (see /G1891S) C,D. Stormembrane A–D. Therant IP3R A,B. Thapant IP3R E, althour effect E,F. The 2+ stores with thapsigargin stimulated formation of Orai puncta in neurons expressing wild-type IP3R, but not in neurons expressing mutant IP3Rs (S224F/G1891Sitpr) . These results suggest that overexpression of STIM with Orai can override the requirement of IP3R for formation of Orai puncta or SOCE after store depletion. However, when STIM and Orai are expressed at native levels in Drosophila, the interaction between them, the formation of Orai puncta and the activation of SOCE are enhanced by IP3R.Using an antibody to endogenous Orai, we observed that depletion of Ca/G1891S) A. Furthepe IP3Rs B–D. Overpe IP3Rs D. Howeve3Rs , was originally suggested to reflect IP3R coupling to a SOCE channel  and examined SOCE after graded depletion of intracellular Ca2+ stores to assess whether partially depleted stores might more effectively activate SOCE in the presence of IP3R. However, the relationship between store depletion and SOCE was unaffected by expression of IP3R1 , and RNAi lines for itpr , STIM (no. 47073) and Orai (no. 12221) were procured from the Vienna Drosophila Resource Centre, Austria. The Canton-S strain was used as the wild-type control.Single-point mutants of the eviously . UAS traDrosophila third-instar larvae were dissociated by incubation for 20 min at 25°C in Schneider's medium containing collagenase (0.75 µg/µl) and dispase . After centrifugation (600 g for 5 min), cells were plated onto poly-l-lysine-coated coverslips in HEPES-buffered medium  or (for most experiments) Dulbecco's modified Eagle's medium (DMEM) with F12 and Glutamax-I, NaHCO3 and sodium pyruvate, and supplemented with 20 mM HEPES (pH 7.2) and 10% fetal bovine serum. This enriched medium substantially reduced the heterogeneity of the Ca2+ signals between neurons. All culture media contained 50 units/ml penicillin, 50 µg/ml streptomycin and 10 µg/ml amphotericin B. Cells were incubated at 25°C in humidified air with 5% CO2. After 14–16 h, cells were loaded with fluo 4 by incubation at 25°C for 30 min with fluo 4-AM (2.5 µM) and Pluronic F-127 (0.02%) in HBM. Medium was then replaced with HBM, and after a further 10–30 min, with Ca2+-free HBM containing 0.5 mM EGTA. Cells were immediately imaged at 15-s intervals with excitation at 488 nm and emission at 520 nm using a Nikon TE2000 microscope with a 60×1.4 NA objective, Evolution QEi CCD camera and QED imaging software . Background fluorescence (measured from an area without cells) was subtracted from all measurements before calculation of ΔF/F0, where F0 is the initial fluorescence and ΔF is the difference between basal and peak fluorescence.Materials, unless stated otherwise, were from ThermoFisher Scientific . Methods for primary cultures were adapted from 3R genes are disrupted (DT40-KO cells) (3R1 (DT40-IP3R1) were used to determine the contribution of IP3R to SOCE in cells from vertebrates. DT40 cells (107 cells/ml) were loaded with fluo-4 by incubation at 20°C with fluo-4 AM (2 µM) in HBS containing BSA (1 mg/ml) and Pluronic F-127 (0.02% w/v) . After 60 min, cells were centrifuged , re-suspended in HBS (5×106 cells/ml) and distributed (50 µl/well) into poly-l-lysine-coated half-area 96-well plates. After centrifugation  fluorescence  was recorded at 1.44-s intervals at 20°C in a FlexStation 3 plate-reader. Fluorescence signals (F) were calibrated to [Ca2+]c from:min and Fmax are the fluorescence values determined in parallel wells by addition of Triton X-100 (0.1% w/v) and either BAPTA (10 mM) for Fmin, or CaCl2 (10 mM) for Fmax, and KDCa=345 nM.Avian DT40 cells in which endogenous IPO cells)  or the sg, 20 min), the supernatant (0.5 µg protein/µl in PBS) was incubated with Dynabeads (2 mg) bound to anti-GFP antibody . After 18–20 h at 4°C, the beads were washed and lysed according to the manufacturer's protocol, and used for western blotting. The antibodies used were: Drosophila STIM  (Drosophila IP3R (1:300) (Drosophila STIM (1:10) (Drosophila Orai (1:1000)  containing 1% NP-40 and 5 mM EDTA, Roche protease inhibitor tablet and 10 µM MG-132. The lysate was homogenized by passage through a 26 G needle, mixed  and after centrifugation  , GFP (1: (1:300) , mouse fM (1:10)  and rat (1:1000) ]. Cells 3R in each neuron, most of which have a near-circular profile  as described previously . To quan profile A, an aut3R, we used Orai immunostaining to manually identify the confocal section within which most plasma membrane apposed the coverslip . These data are presented as box and whisker plots showing medians, 25–75th percentiles (boxes), 10–90th percentiles (whiskers) and points for values beyond the 10th and 90th percentiles. Student's rn blots B and Ca240 cells A.


RKs) are important for plant growth and stress responses. This family has three clades: SnRK1, SnRK2 and SnRK3. Although plant SnRKs are thought to be activated by upstream kinases, the overall mechanism remains obscure. Geminivirus Rep‐Interacting Kinase (GRIK)1 and GRIK2 phosphorylate SnRK1s, which are involved in sugar/energy sensing, and the grik1‐1 grik2‐1 double mutant shows growth retardation under regular growth conditions. In this study, we established another Arabidopsis mutant line harbouring a different allele of gene GRIK1 (grik1‐2 grik2‐1) that grows similarly to the wild‐type, enabling us to evaluate the function of GRIKs under stress conditions. In the grik1‐2 grik2‐1 double mutant, phosphorylation of SnRK1.1 was reduced, but not eliminated, suggesting that the grik1‐2 mutation is a weak allele. In addition to high sensitivity to glucose, the grik1‐2 grik2‐1 mutant was sensitive to high salt, indicating that GRIKs are also involved in salinity signalling pathways. Salt Overly Sensitive (SOS)2, a member of the SnRK3 subfamily, is a critical mediator of the response to salinity. GRIK1 phosphorylated SOS2 in vitro, resulting in elevated kinase activity of SOS2. The salt tolerance of sos2 was restored to normal levels by wild‐type SOS2, but not by a mutated form of SOS2 lacking the T168 residue phosphorylated by GRIK1. Activation of SOS2 by GRIK1 was also demonstrated in a reconstituted system in yeast. Our results indicate that GRIKs phosphorylate and activate SnRK1 and other members of the SnRK3 family, and that they play important roles in multiple signalling pathways in vivo.Sucrose non‐fermenting 1‐related protein kinases (Sn Since plants face diverse, often coincidental stresses throughout their lifetime, they have to integrate multiple signals to organize growth, development and stress responses. This work shows that GRIKs, which are essential players in sugar signalling pathway as upstream kinases of SnRK1s, also work in salt tolerance of Arabidopsis and identified SOS2, a SnRK3, as a potential substrate of GRIKs in the salinity signalling pathway. As expected based on homology, SnRK1s play important roles in energy signalling /H+ exchangers NHXs /AMP‐activated protein kinases (AMPKs) are involved in the responses to energy depletion in yeasts and mammals  is an important regulatory mechanism of SnRK proteins  that no full‐length mRNA for either GRIK1 or GRIK2 is expressed in the double mutant  plates with 1% sucrose, although phosphorylation was not entirely eliminated , in which the ATP‐binding residue Lys137 was changed to Arg, was also produced as a negative control. In vitro kinase assays revealed that GRIK1 had autophosphorylation activity, whereas GRIK1‐KR did not ‐fused GRIK1 recombinant proteins in in vitro , because they are fully conserved in all the members of the Arabidopsis SnRK3 subfamily, and replacement of these residues with Asp to mimic phosphorylation significantly increased the activity of SOS2 in vivo. For this purpose, phosphorylation site‐mutated forms of SOS2 were expressed in the sos2‐2 background under the control of the 35S promoter of Cauliflower Mosaic Virus (35S promoter). Transgenic plants expressing wild‐type SOS2 in the sos2‐2 background could grow well under high‐salt conditions  removes the autoinhibitory domain of SOS2, yielding a SOS3‐independent form of the kinase  experiments in Nicotiana benthamiana. The results demonstrated that SOS2 and GRIK1 interact at least in the cytosol , which lacks all three upstream regulatory kinases of SNF1  failed to complement the salt‐sensitive phenotype , the residual salt tolerance imparted by the full complement of SOS proteins was identical to that conferred by SOS1 alone, indicating that the SOS2–SOS3 regulatory module was essentially inactive in Δ3K4E  is identical to that of previous reports, the mutant lines harbouring T‐DNA insertions in GRIK1 were different. Previous studies used the GABI line 713C09 with the T‐DNA insertion in the 8th intron, and we have used line Salk_142938 bearing the T‐DNA insertion at the 1st intron  contributing to the full activation of SOS2, perhaps by phosphorylating residues S159 and/or Y175. SOS2 is also activated by SOS3 and SCaBP8 in a Ca2+‐dependent manner  and reverse‐transcribed with the Superscript II RT (Invitrogen). GRIK1 cDNA was amplified by PCR with KOD polymerase (Takara) under the following conditions: 1 × 95°C for 5 min; 30 × . Primers used to obtain a 1207‐bp fragment encoding the GRIK1 are listed in Table EcoRI and XhoI sites. Inserted fragments of all constructs were sequenced.Total RNA prepared from 10 days seedling of GRIK1 cDNA was inserted into the vector p425GPD . For expression in A. thaliana, fragments were cloned into a binary vector (pCAMBIA1200) under the control of the CaMV 35S promoter derived from pRT105. For expression in yeast, the full‐length mutant allele SOS2‐AAA and the truncated version SOS2‐AAA∆308 were subcloned as BamHI/EcoRI fragments in p414GPD . The following conditions were used: 1 × 95°C for 5 min; 35 ×  with primers described in Table The seeds of T‐DNA insertion lines  were obtained from Arabidopsis Biological Resource Center  with primers as listed in Table cDNAs purified from 10‐day‐old seedlings of Col‐0 plants and T‐DNA insertion lines as mentioned above were used as templates for RT‐PCR. The following conditions were used: 1 × 95°C for 5 min; 35 ×  was used as a probe. The mRNA of grik1‐2 was amplified by RT‐PCR with primers given in Table Northern blot analysis was performed as described in Fujii and Zhu . For GRIet al.  was used for hydroponic cultures. This medium was designed to maximize the toxicity of Na+ ions, while minimizing the osmotic effects of supplemental NaCl. The final composition of the LAK base solution was as follows: 1 mm KH2PO4, 2 mm Ca(NO3)2, 1 mm MgSO4, 30 mm H3BO3, 10 mm MnSO4, 1 mm ZnSO4, 1 mm CuSO4, 0.03 mm (NH4)6Mo7O24 and 100 mm Fe2+ as Sequestrene 138‐ Fe, pH 5.3. Seedlings were grown in LAK medium for 1 week followed by salt treatment at the indicated NaCl concentrations.For salt treatment in hydroponic culture, seeds were directly grown on hydroponics as described by Barragan et al. . A modifE. coli Rosetta cells (Novagen). Single colonies were grown overnight at 37°C, transferred to fresh 20 × volume of Luria‐Bertani media, and further cultured for 1 h. Recombinant protein expression was induced by 0.2 mm isopropyl beta‐d‐thiogalactopyranoside for 4 h at 37°C. The cells were harvested by centrifugation , and the pellets were resuspended in pre‐chilled lysis buffer , incubated on ice for 15 min. After dithiothreitol (50 mm), phenylmethanesulphonyl fluoride (1 mm) and Triton X‐100 (1.5%) were added, and the suspension was centrifuged at 30 000 g for 5 min at 4°C. Then, glutathione‐agarose beads (Sigma) were added to the supernatant, and the mixture was incubated with gentle agitation for at least 1 h at 4°C. The beads were washed six times with pre‐chilled buffer .The constructs encoding GST‐fused proteins were transformed into grik1‐2 grik2‐1 seedlings, grown on MS media with 1% sucrose, were collected and ground in liquid nitrogen. The tissue samples were added to Laemmli sample buffer . Signals from the blots were detected using Westernbright ECL , and band strengths were evaluated with LiCor Image Studio program . Experiments were replicated with five biological samples.Two‐week‐old Col‐0 and Laemmli,  and heatIn vitro phosphorylation assays were performed as described previously , 10 or 40 mm MgCl2, 10 μm ATP, 5 μCi [γ‐32P] ATP and 2 mm dithiothreitol. Reaction mixtures were incubated at 30°C for 40 min. The reaction was stopped by the addition of Laemmli's sample buffer, followed by SDS–PAGE.∆ena1::HIS3::ena4, nha1::LEU2, nhx1::KanMX, PGK1prom::AtSOS1::CYC1ter; Guo et al., sak1Δ::KanMX elm1Δ::KanMX tos3Δ::TRP1) lacking all three upstream regulatory kinases of SNF1 . To test growth in non‐fermentable carbon sources, glucose was substituted by 3% ethanol, 2% glycerol. The ability of yeast cells to grow in salt was tested on AP medium  vector 173 has been described before . The bacterial cultures were grown at 28°C overnight and centrifuged at 15 000 g for 10 min. Pellets were resuspended with infiltration buffer  and kept at room temperature for 5 h. Cell cultures were adjusted to a final OD600 nm of 0.2. Appropriate combinations of cultures were mixed with equal amounts of an Agrobacterium suspension carrying the p19 suppressor of post‐transcriptional gene silencing  for 3 days until analysis by confocal microscopy. Images were taken with a FluoView FV1000 Confocal Microscope (Olympus) using a 488‐nm Ar/ArKr laser and 60 × objective. The Olympus FluoView 4.2 software was used to analyse the images.For BiFC assays, the full‐length cDNA of t‐test and one‐way anova followed by Tukey's multiple comparison test were performed for single and multiple comparisons, respectively. For normalized values, the non‐parametric binomial test was adopted with 0.5 as a priori probability of > 1.Student's grik1‐2: Salk_142938; grik2‐1: Salk_015230.GRIK1: AT3G45240; GRIK2: AT5G60550; SOS2: AT5G35410; SOS1: AT2G01980; SOS3: AT5G24270; The authors thank the Arabidopsis Biological Resource Center for providing the T‐DNA insertion mutants. This work was supported by the Turku Collegium for Science and Medicine and by the Academy of Finland  to HF, by National Institutes of Health (Grant R01GM059138) to JZ, and by grants BFU2015‐64671 to JMP and BIO2015‐70946‐R to FJQ from the Spanish Ministry of Economy and Competitiveness, co‐financed by FEDER, and with additional support from the SSAC grant PJ01105105 from the Rural Development Administration, Republic of Korea.The authors declare that there is no conflict of interest.Figure S1. Other phenotypes of grik1‐2 grik2‐1 lines.Click here for additional data file.Figure S2. Control experiments for the in vitro kinase assays.Click here for additional data file.Figure S3. Transgenic lines with similar expression levels of SOS2‐S159A (SA), T168A (TA) and Y175A (YA) in the sos2‐2.Click here for additional data file.Figure S4. The complementation of the SNF1 upstream kinase by GRIK regarding carbon source use.Click here for additional data file.Table S1. Primer sequences.Click here for additional data file.


The advent of high throughput sequencing technologies has helped to reveal the tremendous genetic heterogeneity that underlies acute myeloid leukemia (AML), including pediatric AML. This level of complexity had already been suspected given the variability in patient outcomes associated with standard chemotherapy regimens, composed essentially of a cytarabine and anthracycline backbone, irrespective of disease subgroup. Currently in children, cure rates can vary dramatically from above 90% for acute promyelocytic leukemia (PML-RARA fusion) to below 30% for high risk subgroups, such as selected MLL- (Mixed Lineage Leukemia) rearranged AML . The dramatic improvements in treatment outcomes obtained with the introduction of retinoic acid (ATRA) and arsenic trioxide (ATO) for the treatment of acute promyelocytic leukemia highlights the importance of tailoring therapies according to specific genetic AML subgroups. In the emerging era following the large scale sequencing of tumor genomes, engineered human models of high fatality pediatric AMLs will be seminal to identify disease subtype specific functional dependencies and develop novel therapeutic strategies. It is these models that will be required to overcome the paucity of primary human samples, the genetic heterogeneity of the disease, and the clonal evolution of commercially available cell lines.in vitro and in vivo. In addition, a panel of leukemia activated genes was identified [In pediatric AML, translocations that generate chimeric fusion genes are critical transforming events and present in a high proportion of cases, including the highest risk categories. We have exploited the oncogenic potential of MLL fusions, considered hallmarks of infant and therapy-related AML, to generate human models of MLL-AF9 driven AML , using oentified , a signa+CD117+ AML, but not to the CD34-CD41+CD61+ leukemic blasts typically seen in pediatric AMKL, possibly indicating distinct cells of origin. In addition, the anti-tumor activity of novel pharmacological compounds will need to be assessed in models that more accurately reflect human disease, as well as potential cross toxicity on the normal hematopoietic system. Clearly, the recent development of HSPC self-renewal agonists has shown considerable variability in responses between model organisms. As reported, the aryl hydrocarbon receptor inhibitor StemRegenin 1 (or SR1) can expand human, monkey and dog cells, but had no impact on mouse HSPC [Human leukemia models will unmistakably become an indispensable complement to the currently developed model organisms used to gain further insights into relevant disease biomarkers and drug targets. For example, differences across species have been reported, especially in the cytological subtype of leukemia that is induced by a given oncogene, depending on the cell type that is used. Overexpression of MLL-AF4 in HSPC, for instance, gives rise to AML in mouse, but to acute lymphoblastic leukemia (ALL) when using adoptive transfer of engineered human cells into immunocompromised recipient mouse strains , giving in vitro genetic or chemical based screens. More restricted screens involving the most promising lead compounds (as single agents or in combination) can then be carried using AML subtype specific xenografts, to streamline pre-clinical testing, and only bring the most promising treatment modalities to clinical trials. A current inherent limitation of xenograft models, of course, remains the study of immune responses and bone marrow niche interactions, but improvements in this direction are actively being pursued [While human cells initially proved difficult to transform, the optimisation of human HSPC culture conditions and viral transduction protocols were critical milestones that paved the way to AML modeling. It is now possible to maintain engineered human AML cell lines in culture long-term , setting the stage for large-scale  pursued , 8.Overall, engineered human AML models constitute yet another weapon in our quest to conquer leukemia, that will enable us to tackle the heterogeneity of the disease with the identification of subtype specific functional susceptibilities, as well as improved biomarkers to gauge treatment responses. These models will be particularly critical for the rare and highly lethal AML subgroups, as seen in children, for which cure rates are stagnating and remain unacceptably low.