However, the relationship between cytosolic and nucleosolic calcium signaling remains obscure. Here, we generated transgenic plants containing a fusion protein, comprising rat parvalbumin (PV) with either a nuclear export sequence (PV-NES) or a nuclear localization sequence (NLS-PV), to selectively buffer the cytosolic or nucleosolic calcium. Firstly, we found that the osmotic stress-induced cytosolic [Ca2+] increase (OICIcyt) and the salt stress-induced cytosolic [Ca2+] increase (SICIcyt) were impaired in the PV-NES lines compared with the Arabidopsis wildtype (WT). Similarly, the osmotic stress-induced nucleosolic [Ca2+] increase (OICInuc) and salt stress-induced nucleosolic [Ca2+] increase (SICInuc) were also disrupted in the NLS-PV lines. These results indicate that PV can effectively buffer the increase of [Ca2+] in response to various stimuli in Arabidopsis. However, the OICIcyt and SICIcyt in the NLS-PV plants were similar to those in the WT, and the OICInuc and SICInuc in the PV-NES plants were also same as those in the WT, suggesting that the cytosolic and nucleosolic calcium dynamics are mutually independent. Furthermore, we found that osmotic stress- and salt stress-inhibited root growth was reduced dramatically in the PV-NES and NLS-PV lines, while the osmotic stress-induced increase of the lateral root primordia was higher in the PV-NES plants than either the WT or NLS-PV plants. In addition, several stress-responsive genes, namely CML37, DREB2A, MYB2, RD29A, and RD29B, displayed diverse expression patterns in response to osmotic and salt stress in the PV-NES and NLS-PV lines when compared with the WT. Together, these results imply that the cytosolic and nucleosolic calcium signaling coexist to play the pivotal roles in the growth and development of plants and their responses to environment stresses.Calcium acts as a universal second messenger in both developmental processes and responses to environmental stresses. Previous research has shown that a number of stimuli can induce [Ca Althougchannels , have be systems , which a2+ signals, when viewed in terms of the spatial and temporal dynamics of the stimulus-induced changes in [Ca2+]cyt, have been called the ‘Ca2+ signature’ which is characterized by the duration, frequency, amplitude, and spatial location of [Ca2+]  in order to facilitate long-distance systemic responses in each plant, which may provide the feed-forward mechanisms to amplify and transmit the stimuli signals  is as important as [Ca2+]cyt to be involved in gene expression. Analyzing gene transcription in the hippocampal neurons, 2+]nuc activates gene transcription by a mechanism distinct from gene regulation as driven by [Ca2+]cyt. Previous study showed that EGF triggered the [Ca2+] increases in both the nucleus and cytosol. However, EGF-induced transactivation of the ternary complex factor, Elk-1, required [Ca2+]nuc but not [Ca2+]cyt in HepG2 or HEK293 cells  or nucleus (NLS-PV). Then we crossed the PV-NES and NLS-PV lines with those of NES-YC and NLS-YC, respectively, to yield four double-transgenic lines, NES-YC/PV-NES, NLS-YC/NLS-PV, NES-YC/NLS-PV, and NLS-YC/PV-NES, for measuring [Ca2+]cyt and [Ca2+]nuc in the plant response to hyperosmolarity and salt stresses. Using this toolkit, we deciphered the temporal and spatial characteristics of Ca2+ signatures in the nucleus and the cytosol in Arabidopsis root cells.Using tagged versions of Cameleon YC 3.6  that werp region , and funp region . Prior sG2 cells  or vasopG2 cells . In the Arabidopsis thaliana (Col-0 ecotype) plants were grown under 16 h light (120 μmol m-2 s-1)/8 h dark at 22°C and 60% relative humidity. Seeds of Arabidopsis were surface-sterilized by 75% ethanol and plated on Murashige and Skoog (MS) salts, 1% sucrose, 0.8% (w/v) agar, pH 5.8. After stratification at 4°C in the dark for 3 days, the dishes were transferred to a growth chamber for germination and seedling growth of 7 days. About 15–20 seedlings were planted into pots for continued growth and monitored during the experiment. Transgenic plants were generated using the floral-dip method  by separately crossing three transgenic plants which had nuclear-localized PV (NLS-PV) or cytosolic-localized PV (NES-PV) with either NES-YC or NLS-YC transgenic Arabidopsis  gene . The NES was fused to the C-terminal of PV, digested by BamHI and NotI, and cloned into pE2n and pE6n. The primers used to clone these gene fragments are listed in Supplementary Table The CDS of the 50) gene  and the 50) gene . The NLS1 with the Gateway LR II kit  to generate all of the plant expression vectors. These were introduced into the Agrobacterium tumefaciens strain, GV3101, for the Arabidopsis transformation. Construct maps containing both PV-NES and NLS-PV are shown in Supplementary Figure S1. All vectors were confirmed by sequencing.The pE2n, pE2c, pE6n, and pE6c constructs containing different PV fragments were used in combination with the destination vector pMDC32eYFP-PV-NES or NLS-PV-eYFP and the nuclear marker gene were co-transformed and transiently expressed in the Arabidopsis mesophyll protoplasts, as described before , and then centrifuged at 80 000 g, at 4°C, for 30 min. The ensuing supernatants were centrifuged again under the same conditions and collected as the cytosolic compartment. The pellet was re-suspended with an extraction buffer II that contained 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, 0.1% NP-40, 2 mM MgCl2, 5 mM DTT, and 1x protease inhibitor cocktail, and centrifuged again under same conditions as above. The nuclear proteins are in the pellet and were re-suspended with the buffer II in equal volume. In addition, 6-day-old Arabidopsis seedlings were collected, ground in liquid nitrogen, and used for the total protein extraction with a buffer .Four-week-old transgenic Protein extracts were resolved on 12%-SDS-polyacrylamide gels and electro-blotted onto Bio-Rad Immun-Blot PVDF membranes . After this transfer, the PVDF membranes were blocked in Tris-buffered saline-Tween 20 , which contained 5% bovine serum albumin , for 1 h at room temperature and then incubated overnight with a primary antibody at 4°C. The membranes were washed of 10 min for three times in the TBST and incubated with the secondary antibody—anti-rabbit immunoglobulin G, dilution 1:2000 or anti-mouse immunoglobulin G, dilution 1:3000—for 30 min. Next, the membranes were washed of 10 min for three times in the TBST. To examine the alkaline phosphatase activity, the Chemiluminescent Substrate (Roche) was used according to the manufacturer’s protocol. The primary anti-PV antibody  was used to detect the expression of PV in the transgenic plants. The non-specific band after blotted with anti-PV antibody was used as the loading control of the cytosolic proteins and the anti-histone antibody  was used as the loading control of the nuclear proteins.Arabidopsis seedlings were grown vertically on the half-strength MS medium for 5–7 days and their roots were prepared for Ca2+ imaging following ®Cell Chambers (Invitrogen). After digging a small tunnel in the agarose to expose the root, we gently applied 200 μl bathing solution buffer -KOH, pH 5.8] to the chamber. The shoot was not submerged in the solution. High concentrations of NaCl and sorbitol in the same solution were separately perfused as the stimulus into the chamber. The mature zone of the Arabidopsis roots was selected for the subsequent Ca2+ measurements.After germination, the To examine the FRET signals of the transgenic plants, we used an inverted fluorescence microscope  equipped with an iXon3 EMCCD camera , a Lambda DG4 fluorescent light source , and Bright Line filter sets . Images captured with the CFP (438Ex/483Em), CpVenus (500Ex/542Em), and FRET filters , respectively, were collected every 3 s at room temperature by using a 40× oil objective  and processed in Slidebook v.5.0 software .∗ FCFP–Fa/Da ∗ FCpVenus, where FRETc represents the corrected energy transfer, Fd/Dd represents the measured bleed-through of CFP through the FRET filter (0.826), and Fa/Da is the measured bleed-through of CpVenus through the FRET filter (0.048). To reduce the variation caused by different expression levels in the transgenic plants, the FRETc values were normalized against donor fluorescence (FCFP) to generate an N-FRET  signal.The FRET signal was calculated using a previously described formula : FRETc =t-test in the GraphPad Prism 5.0 software. The results are presented as means ± SD.To eliminate the influences of instrument-dependent factors, the apparent FRET efficiency, or Eapp, was calculated using the following equation: Eapp = N-FRET/(N-FRET ± G) , where GArabidopsis seeds of the wildtype (WT), NES-PV, and NLS-PV lines were sowed onto a sterile plate containing 1/2 MS salts, 1% sucrose, 0.8% (w/v) agar, and pH 5.8, and stratified at 4°C in the dark for 3 days. Then, the dishes were transferred to the growth chamber for the germination over 6 days. The seedlings were transferred onto 1/2 MS plates or 1/2 MS medium supplemented with either 150 mM of NaCl or 250 mM of sorbitol, and cultivated for 11 days to observe their root growth or for 6 h to perform qRT-PCR.®Super  according to the protocols. Approximately 2 μg of total RNA was reverse transcribed into first-strand cDNA by using the First-Strand cDNA Synthesis SuperMix . The qRT-PCR was performed on the 7500 Fast Real-Time PCR System  which used a Power SYBR®Green PCR Master Mix . The Arabidopsis Actin2 served as an internal control. The stress-responsive genes selected for detecting their expression are listed in Supplementary Table Total RNA was isolated from seedling samples by using the Eastep2+-binding protein PV to either the nucleus or cytoplasm, by fusing it with a NLS (NLS-PV) or a NES (PV-NES), and transducing the vectors into Arabidopsis WT via Agrobacterium. Meanwhile, to verify the localization signal, two vectors with the indicated PV fused with yellow fluorescent protein—eYFP-PV-NES and eYFP-NLS-PV —were generated and transiently co-transformed with the mCherry-labeled nuclear marker into the Arabidopsis mesophyll protoplasts. We found that eYFP-PV-NES was restricted to the cytoplasm, whereas eYFP-NLS-PV was co-localized with the nuclear marker . Through resistance screening and the reverse transcription (RT)-PCR assay (data not shown), we firstly discarded the transgenic lines which has different growth phenotype with WT in whole life cycle, then obtained the single-insertion, T3 homozygous NLS-PV and PV-NES transgenic lines. So, the proteins extracted from the cytoplasm and nucleus of the three independent lines were isolated and detected by immunoblotting, revealing that the PV was distributed only in the nuclear compartment in the NLS-PV lines and in the cytoplasm compartment in the PV-NES lines . These results proved that the subcellular localization of PV-NES or NLS-PV is specific to the cytoplasm or nucleus in the transgenic plants, which lay the foundation for its selective buffering of [Ca2+]cyt or [Ca2+]nuc.To study the relationship between cytosolic and nucleosolic calcium signaling in plants, we constructed binary vectors targeting the Ca2+] elevations, we crossed the PV-NES lines with those Arabidopsis plants containing the cytoplasm-localized Yellow Cameleon 3.6 Indicator (NES-YC), and thus obtained the homozygous NES-YC/PV-NES lines with resistance screening. Then, the [Ca2+]cyt was monitored in response to different stimuli in the root cells of Arabidopsis plants either expressing NES-YC or co-expressing NES-YC/PV-NES. Firstly, we found that 250 mM of sorbitol, when used as an osmotic stress stimulus, can trigger a rapid increase of [Ca2+]cyt in the root cells of the NES-YC plants; the value of ΔEapp/Eapprest reached 0.16, which is similar to that of a previous study . Similarly, 125 of mM NaCl was also able to induce a single peak of [Ca2+]cyt increase in the root cells of the NES-YC plants, but it was impaired in the NES-YC/PV-NES lines . The increased FRET fluorescence and decreased CFP fluorescence after adding the 250 mM of sorbitol or 125 mM of NaCl to the root cells of the NES-YC plants are shown in . Together, these results demonstrated that the cytosolic-localized PV can effectively buffer the change of [Ca2+]cyt and thereby inhibit OICIcyt and SICIcyt in the Arabidopsis root cells.To explore the effect of PV on the cellular cyt and [Ca2+]nuc, we intercrossed NEC-YC with NLS-PV and NLS-YC with PV-NES plants. Doing so gave us the homozygous NEC-YC/NLS-PV and NLS-YC/PV-NES double-transgenic lines for detecting the OICI and SICI in the cytoplasm and nucleus, respectively. Firstly, the responses, in terms of the pattern and ΔEapp/Eapprest, for the cytoplasm calcium dynamics in the root cells of the NEC-YC/NLS-PV plants were similar to those seen in the NES-YC plants when they received 250 mM of sorbitol or 125 mM of NaCl . This shows that blocking nuclear calcium has no effect on OICIcyt and SICIcyt. Likewise, we separately measured the change of [Ca2+]nuc in response to the 250-mM sorbitol or 125-mM NaCl treatments in the root cells of the NLS-YC and NLS-YC/PV-NES lines and found that blocking the cytosolic calcium with PV-NES did not affect OICInuc and SICInuc . Interestingly, we want to show that the increase of [Ca2+]nuc in response to osmotic or salt stresses showed two kinds of patterns in the different cells of the root mature zone: one is a rapid increase following by oscillations , while the other is just a single peak . Each pattern accounted for approximately 50%. In addition, blocking the cytosolic calcium signal with PV-NES had no effect on these two patterns of [Ca2+]nuc. Via the Western blot, we also confirmed that PV is expressed in all these intercrossed lines . In order to rule out any bias about the increases of [Ca2+]cyt and [Ca2+]nuc due to stimulus application in this study, we perform the control experiment where only MS medium is added to the seedlings and show that the medium cannot trigger any change of ΔEapp/Eapprest . These results proved that cytosolic and nucleosolic calcium dynamics are independent of each other in Arabidopsis.To further reveal the inter-relationship between cyt and [Ca2+]nuc both participate in the various transcription of stress-related plant genes.Previous studies revealed that several genes, such as CML37 , DREB2A   acts as a Ca2+ store that releases Ca2+ for stimulus-induced [Ca2+]cyt increases; however, 2+ elevations in ER are followed by various stimuli-induced [Ca2+]cyt increases in Arabidopsis root-tip cells, with distinct dynamics, which does not support a significant role of ER [Ca2+] as a source of Ca2+ release that contributes to the formation of cytosolic Ca2+ signatures. In this study, we investigated the triggering of OICIcyt by 250 mM of sorbitol and that of SICIcyt by 125 mM of NaCl in the root cells of the NES-YC transgenic lines. We found a similar pattern of OICIcyt and SICIcyt, featuring a single peak of [Ca2+] elevation—consistent with findings for the root cells of aequorin-transgenic Arabidopsis seedlings . More interestingly, OICInuc and SICInuc in the root cells of the NLS-YC lines showed two distinct patterns: the transient increases and oscillation of [Ca2+], with each pattern about approximately 50% in the different cells of the mature root zone . In addition, we found that the targeted PV in the nucleus can block OICInuc and SICInuc in the root cells of NLS-YC/NLS-PV lines. However, 2+ transients in the nucleoplasm are kinetically similar to those in the cytoplasm of Arabidopsis guard cells. These results indicated that the temporal and spatial characteristics of calcium signatures present in different plant organs in response to various stimuli.Using the organelle-targeted Can planta . For exaeedlings —and a su2+]nuc and [Ca2+]cyt yet this has diverse patterns in different plants. In addition, the outer nuclear membrane is bordered by the endoplasmic reticulum, so they share a common Ca2+ pool, Ca2+-permeable channels, and Ca2+-ATPase carriers to produce Ca2+ signaling in plants. In this study, we first used the PV-NES fusion protein to buffer the [Ca2+]cyt increases to demonstrate a stimulus-induced [Ca2+]nuc that was independent of [Ca2+]cyt with distinct dynamics. Similarly, blocking [Ca2+]nuc with NLS-PV has no effect on the stimulus-induced [Ca2+]cyt increases in the Arabidopsis root cells. These results show that cytosolic and nucleosolic calcium signaling are independent of each other in Arabidopsis, which raises two interesting questions: (i) how do plants sense and transduce extracellular stimuli to simultaneously induce the [Ca2+] increases in the cytoplasm and nucleoplasm, and (ii) what is the physiological extent and function of organelle-specific calcium signaling?Extensive studies show that the same stimuli—such as osmotic shocks triggered by high concentration of sorbitol or NaCl , jasmoniPV-NES and NLS-PV plants exhibit a reduced salt stress-mediated inhibition of root growth, but not of osmotic stress-mediated inhibition of root growth; this suggests that salt stress-induced [Ca2+] increases in both the cytoplasm and nucleoplasm mediate the salt stress-induced growth inhibition in Arabidopsis. Another interesting result we found is the lateral root primordia density of PV-NES plants exceeding those of WT and NLS-PV plants, but only when treated with 250 mM of sorbitol; this indicates that the OICIcyt is somehow involved in the osmotic-stress mediated development of lateral roots. Prior studies have foundthat calcium signaling is crucial for plant adaptation to various stresses and that it participates in rapid changes in gene expression . These results further implied that cytosolic calcium signaling and nuclear calcium signaling function independently in the stress response pathways of plants. We also found that the expression of some genes, for instance RD22, is related neither to cytosolic or nucleosolic calcium in response to osmotic or salt stresses in Arabidopsis. This indicates that one or more calcium-independent signaling pathway(s) participated in the expression regulation of RD22 in Arabidopsis plants. It is also possible that overexpression of PV-NES or NLS-PV themselves could affect the gene expression in a [Ca2+]-independent pathway in the transgenic Arabidopsis plants. Further experiments are needed to address this issue. For example, we can generate transgenic plants overexpressing Ca2+-binding deficient mutants of PV (PV-NES and NLS-PV plants. In sum, our study is the first to show that cytosolic and nucleosolic calcium dynamics are mutually independent in plants, yet play coexisting roles critical in regulating gene expression for plant adaptation to various environmental stresses.Compared with the up-regulated expression of key stress-responsive genes, CML37 , DREB2A  (DREB2A , MYB2 (A2A (MYB2 , RD29A (2 (RD29A , RD29B (A (RD29B  in the Wts of PV , and demConceived and designed the experiments: SH, FH, and JL. Performed the experiments: FH, JL, TN, WC, and XJ. Analyzed the data: FH, JL, HZ, YW, and SH. Contributed reagents/materials/analysis tools: YW and SH. Wrote the paper: FH, HZ, YW, and SH.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.


In this study, we found that NCX1 expression was significantly higher in ESCC primary tissues compared to the noncancerous tissues and was overexpressed in tumor samples from the smoking patients. The expression of NCX1 proteins was also significantly higher in human ESCC cell lines compared to normal esophageal epithelial cell line. Moreover, NNK potentiated the [Ca2+]cyt signaling induced by removal of extracellular Na+, which was abolished by KB-R7943 or SN-6. NNK dose-dependently promoted proliferation and migration of human ESCC cells induced by NCX1 activation. Therefore, NCX1 expression correlates with the smoking status of ESCC patients, and NNK activates the Ca2+ entry mode of NCX1 in ESCC cells, leading to cell proliferation and migration. Our findings suggest NCX1 protein is a novel potential target for ESCC therapy.Tobacco-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a major environmental risk factor for the pathogenesis of human esophageal squamous cell carcinoma (ESCC). However, the molecular mechanisms by which tobacco induces ESCC are not well understood. Na+/Ca2+ exchanger 1 (NCX1) is a plasma membrane transporter protein that plays an essential role in maintaining cytosolic Ca Esophageal cancer is the sixth leading cause of cancer-related mortality in the world –3, incurCigarette smoking is a serious public health issue and is responsible for millions of deaths worldwide. Cigarette smoking is a major environmental risk factor for mortality of many cancers, including lung cancer, esophageal cancer, liver cancer, gastric cancer and so on . The ris2+ ([Ca2+]cyt) is a ubiquitous cell messenger, and Ca2+ entry is a critical component of Ca2+ signaling in human physiology. However, dysregulation of Ca2+ signaling is involved in tumorigenesis and tumor progression, such as metastasis, invasion and angiogenesis [2+ regulators plays an important role in the process of tumorigenesis [2+]cyt is elevated in a variety of human cancer cells [2+]cytvia blockade of Ca2+ entry suppress cancer cell growth, suggesting that remodeling of [Ca2+]cyt homeostasis might be valuable in cancer therapy [2+]cyt homeostasis is altered in cancer cells.Cytosolic free Caogenesis , 13. An igenesis , 15. A ner cells , 17, but therapy . Therefo2+]cyt homeostasis is precisely controlled by multiple proteins, including the plasma membrane Na+/Ca2+ exchanger (NCX). NCX is a family of membrane transporter that operates in either a forward mode (3 Na+ entry and 1 Ca2+ exit) or a reverse mode (3 Na+ exit and 1 Ca2+ entry), depending on the electrochemical gradient of Na+ and Ca2+ and membrane potential cyt. We analyzed both peaks and rising slopes of Ca2+ entry to quantitatively describe the NCX1 activity in the Ca2+ entry mode in cells. Since DMSO  was used as the solvent of all drugs, its effect was first tested, but it did not affect 0 Na+-induced [Ca2+]cyt signaling  were treated with different concentrations of NNK  for 16 h, and then the expression levels of NCX1 were analyzed by qPCR and Western blotting. As shown in Figure 2+ entry mode of NCX1 in human esophageal cells. Moreover, KB-R7943 and SN-6 significantly prevented NNK-induced Ca2+ influx in these cells  than in normal esophageal cells (NE-1)  Figure . Therefo2+ signaling that is implicated in tumorigenesis cyt homeostasis in various types of human cells. It can exchange Na+ and Ca2+ in either direction depending on transmembrane electrochemical gradients cyt under some pathological status [2+]cyt homeostasis induced by abnormal [Ca2+]cyt regulators, especially enhanced Ca2+ entry mode of NCX1, has been detected in some cancers, such as pancreatic cancer cyt under the restricted plasma membrane space and induce membrane depolarization [+]cyt and depolarization can switch on NCX1 to allow sustained Ca2+ entry cyt homeostasis in ESCC cells. Moreover, we found that NNK dose-dependently stimulated not only NCX1 expression in ESCC but its function as well. These findings indicate that tobacco smoking could cause Ca2+ entry through enhanced expression and function of NCX1 to finally result in the pathogenesis of ESCC.It has been reported that under pathological status such as during tumorigenesis, excessive cell metabolism could reduce intracellular pH to activate Narization , 33. A s2+ entry , 35. Ind2+]cyt is a pivotal messenger that regulates many biological functions including cell proliferation and migration cyt through enhanced expression and function of NCX1 in ESCC cells, finally leading to excessive proliferation and migration. A proposed schematic of NNK-evoked ESCC through the Ca2+ entry mode of NCX1 is shown in Figure 2+ entry induces human ESCC, our study has provided a novel perspective that targeting NCX1 may be a potential therapeutic strategy for ESCC.In conclusion, we demonstrate that NCX1 overexpression is correlated with the smoking status of ESCC patients. NNK may induce an increase in . After being washed with TBS with 1% Tween (TBST) for 3 times, the secondary antibody  was applied to the membrane. After being washed with PBST, the membrane was treated with a chemiluminescent solution (Millipore Corporation) according to manufacturer's instructions. Immunoreactive bands were visualized by ECL Western blotting analysis system . Densitometric analysis of the blots was conducted using ImageJ software and β-actin protein levels were used as a control for equal protein loading.2+]cyt was measured by fura-2/AM fluorescence ratio digital imaging. Briefly, cells grown on coverslips were loaded with 5 μM fura-2/AM dissolved in 0.02% Pluronic F-127 plus 0.1% DMSO in physiological salt solution  in the dark at room temperature (22°C) for 1 hour and then washed in PSS for 30 min. Thereafter, the coverslips with cells were mounted in a perfusion chamber on a Nikon microscope stage. The ratio of fura-2/AM fluorescence with excitation at 340 or 380 nm (F340/380) was followed over time and used the PTI RatioMaster fluorescence system . The corresponding ratios F(340/380) were used to obtain relative intracellular Ca2+ concentrations. As a measure for the increase of cytosolic Ca2+ activity, the rising slope and peak of the changes in the F(340/380) were determined for each experiment. Average fluorescence values of 5–10 attached cells were recorded. These cells were mostly grown in separate regions with no apparent connection. The experiments were performed more than 8 times. The PSS solution used in digital Ca2+ measurement contained the following (in mmol/L): 140 Na+, 5 K+, 2 Ca2+, 149 Cl−, 10 HEPES, and 10 glucose. In Na+-free PSS, NaCl was replaced by LiCl.[Ca3 cells were reseeded into 96-well culture plates, then treated with NNK at the concentrations of 0.2, 1, 5 μM for 24 hours. After incubation, the medium was removed and 20 μl MTT  was added to each well for 4 hours. The formazan precipitate was dissolved in 200 μl of DMSO and the optical density of each well was read on the plate reader at 492 nm. At least 4 independentents were performed, and the averages were calculated.For cell proliferation measurement, cells were either pretreated with KB-R7943 (10 μM) or SN-6 (10 μM) for 4 h or no drug treatment, 5 × 10Cells were propagated with complete media in 6-well plates, respectively, and allowed to grow to 90% confluence over-night. For scratch assay, cells were pre-treated on the following day with mitomycin C at 10 μg/mL (30 μM) and/or KB-R7943 (10 μM) or SN-6 (10 μM) for 4 h. By mechanically scratching the cell substrate with a pipette tip, characteristically sized wounds were performed in all wells. Remaining adherent cells were washed with PBS, respectively replenished with 1% serum medium, and allowed to stabilize for 1 hour. The cells were treated with 0.1% DMSO with 30 μM mitomycin C for vehicle control, 5 μM NNK with 30 μM mitomycin C, 10 μM NNK with 30 μM mitomycin C, 10 μM KB-R7943 with 30 μM mitomycin C, 10 μM NNK and 10 μM KB-R7943 with 30 μM mitomycin C for 16 hours. Gap closures of marked areas were recorded at 50 magnification using a Leica DMI3000B microscope  and Leica Application suite V4 capture software . Gap closures were measured over a time course to calculate the migration rate according to the following formula: migration rate = ((gap length at 0 h) − (gap length at 16 h) / (gap length at 0 h)) × 100%. The experiments were performed more than 3 times.t-tests for unpaired samples with GraphPad Prism 3.0 . The results were presented as mean ± SD unless otherwise stated. A difference with a P value of less than 0.05 was considered statistically significant.SPSS 13.0 software  was used for statistical analysis. The chi-square test was used to determine relationship exists between NCX1 expression and smoking status in ESCC patients. Data were analyzed by Student's


Circular RNAs (circRNAs) represent a widespread class of non-coding RNAs, which drew little attention in the past. Recently, limited data showed their promising future to act as biomarkers in human cancer, but the characteristics and functions remain largely unknown in hematopoietic malignancies, especially in leukemia. In this study, with the help of circRNA microarray, we demonstrated the expression profile of circRNAs in acute myeloid leukemia (AML) patients, and identified a large number of circRNAs possibly expressed in a leukemia specific manner. We also described a circRNA signature related to AML risk-status based on the bioinformatics prediction. In particular, a downregulated circRNA, hsa_circ_0004277, was characterized and functionally evaluated in a cohort of 115 human samples, thus offering a potential diagnostic marker and treatment target in AML. Interestingly, we found chemotherapy could significantly restore the expression of hsa_circ_0004277, indicating the increasing level of hsa_circ_0004277 was associated with successful treatment. Furthermore, a detailed circRNA–miRNA–mRNA interaction network was presented for hsa_circ_0004277, allowing us to better understand its underlying mechanisms for function in AML. Acute myeloid leukemia (AML) represents a group of myeloid malignancies characterized by acquired genetic abnormalities in hematopoietic progenitors. It shows remarkably complex biological and clinical heterogeneity in patients. Advances in molecular genetics promote the recognition of AML prognostic biomarkers, among which cytogenetic results and molecular abnormalities are considered as the most important factors. Nearly half of AML patients are found without cytogenetic abnormality, and constitutes the main part of intermediate-risk AML category . Howevercis-elements and/or trans-factors [Circular RNAs (circRNAs), a big class of noncoding RNAs, were discovered from a few of transcribed genes over twenty years ago  and were-factors . circRNA-factors . This st-factors . The exp-factors ,11 and a-factors ,12,13. F-factors ,15,16. HTo demonstrate the expression profile of circRNAs in AML, we performed a circRNA microarray in AML patients and healthy controls for the first time, and presented an AML-specific circRNA feature, as well as a unique signature related to AML risk-status. In particular, we identified one novel circRNA, hsa_circ_0004277, as a potential diagnostic marker and treatment target in AML. Moreover, with the help of bioinformatics analysis, a detailed circRNA-miRNA-mRNA interaction network was described for hsa_circ_0004277, allowing us to better understand its underlying mechanisms for function in AML.Firstly, we characterized circRNA transcripts using a microarray platform in 10 human samples, four healthy controls vs. six newly diagnosed CN-AML patients without prior treatment. The detailed clinical information of patients is available in We further confirmed the microarray results by quantitative Real-Time PCR (qRT-PCR) using the same cohort of patient and control samples as in microarrays. Five abundant circRNAs  were chosen based on the significant difference and raw signal intensity of expression. As shown in p-value cut-off: 0.05). By following those patterns, two upregulated circRNAs  and three downregulated circRNAs  were found within the whole profile  and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to investigate the functions of their target genes. The results revealed that the target genes related to this circRNA signature participated in various biological processes, such as developmental process, regulation of cellular process and cell junction A,B. Somep < 0.0001). Meanwhile, patients achieved complete remission (CR group) after treatment restored the expression of hsa_circ_0004277, showing no difference compared to the control group. However, in the relapsed-refractory patients (RE group) after CR stage, downregulated hsa_circ_0004277 expression was observed again  curve analysis to examine its potential diagnostic value in distinguishing individuals with AML from healthy individuals. Area under the ROC curve (AUC) was 0.957 B, indicaWDR37, located at chr10:1125950-1126416. Little is known about this gene in general, and barely anything in specific regards to AML; therefore, we started with checking its expression in AML in the same cohort of samples as in n = 5) had a wide range, not valid enough to show the differences with other groups. Furthermore, a positive correlation between hsa_circ_0004277 and WDR37 was observed in all the AML patients at different stages  samples acquired from those seven AML patients at both ND and CR stage, we demonstrated a significant increase of hsa_circ_0004277 expression in CR stage compared with ND stage E, suggesPPARGC1A, PIP4K2C, SH2B3, ZNF275, and ATP1B4, which could be our main focus for further study.Considering what we have seen above, hsa_circ_0004277 is very likely to be a special AML-related circRNA. We assumed that hsa_circ_0004277 could also act as a miRNA sponge and regulate its own circRNA-miRNA-mRNA network. The molecular interactions between hsa_circ_0004277 and its top five miRNA targets are displayed in Recently, circRNAs have been a hotspot in the field of RNA, although the understanding of its nature and extent involved in biological events is still very limited. The formation of circRNAs is comparatively clearer than other aspects. To date, two mechanisms are proposed to form mammalian exonic circRNA: lariat-driven circularization and intron-pairing-driven circularization ,21,22. TIt is reported that circRNAs are widely expressed in human cells, and their expression can be higher by 10-fold or more than their linear counterparts . Due to As to circRNA function, one of the most novel discoveries would be circRNAs acting as miRNA sponges. Certain circRNAs can bind and negatively influence miRNAs, which are substantially involved in the competing endogenous RNA (ceRNA) network, thereby regulating linear RNA transcription and protein production. Thomas et al. reported that circRNA ciRS-7 could strongly suppress miR-7 activity, resulting in increased levels of miR-7 targets ,12. AnotNFIX and hsa_circ_0049657 could be worth exploring in our future research projects.Besides profiling circRNAs between AML patients and healthy controls, our microarray designs also offer comparisons between better-risk and poor-risk AML subgroups. Five circRNAs were found to be dysregulated especially in the poor-risk subgroup, which can potentially contribute to AML risk-status evaluation. Combined with the sponge theory above, we further predicted their potential target miRNAs and performed GO and KEGG pathway analysis. The result revealed that these circRNAs contribute to an aggressive leukemia phenotype through certain biological mechanisms and pathways controlled by MAPK, PI3K-Akt, and so on. In addition to the most popular “sponge” theory, other possible mechanisms for those five circRNAs linked with AML risk-status remain comparatively unclear. The complicated interaction between the circular and the linear isoforms could be a possibility. It is reported one circRNA, cirZKSCAN1, varied in hepatocellular carcinoma patients with different tumor numbers, cirrhosis, vascular invasion, or the tumor grade, and furthermore, ZKSCAN1 mRNA and circZKSCAN1 cooperated closely with one another . It becaSH3GL2 and PPARGC1A were reported to be involved in several solid tumors, and SH2B3 especially as “a new leukemia predisposition gene” [Based on the findings of Denzler et al., low levels of circRNAs may not be sufficient to affect the target miRNAs . Therefoon gene” . These eBM samples from 113 AML patients and 12 healthy controls were obtained following informed consent at Qilu Hospital, Shandong University. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Qilu Hospital . Ten samples were used for the circRNA microarray, and 115 samples were used for further investigation. Mononuclear cells from BM aspirates were separated by Ficoll-Paque Plus  density gradient centrifugation. Two million cells from each sample were collected for further procedure.Total RNA was isolated from 10 samples using TRIzol reagent  according to the manufacturer’s protocol, and then treated with RNase R  to remove linear RNAs and enrich circRNAs. The enriched circRNAs were amplified and transcribed into fluorescent complementary RNA (cRNA) utilizing a random priming method . Purification of the labeled RNAs was done by RNeasy Mini Kit , and next the concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000. Five microliter 10× Blocking Agent and 1 μL of 25× Fragmentation Buffer were used to fragment 1 μg of each labeled cRNA, followed by heat at 60 °C for 30 min. Finally, 25 μL 2× Hybridization buffer was added to dilute the labeled cRNA and 50 μL of hybridization solution was dispensed into the gasket slide and assembled to the circRNA expression microarray slide, incubated for 17 h at 65 °C in an Agilent Hybridization Oven .After incubation, the hybridized arrays were washed, fixed, and scanned using the Agilent Scanner G2505C. Scanned images were imported into Agilent Feature Extraction software  for raw data extraction. Quantile normalization and subsequent data processing were performed using the R software package. Next, low intensity filtering was performed and circRNAs with at least 4 out of 10 samples flagged in “P” or “M”  were kept for further analysis. The Gene Expression Omnibus (GEO) accession number is GSE94591.Ct) method was used to analyze the gene expression level. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. Primer sequences were presented in Total RNA isolated from 115 samples was reversely transcribed into cDNA using MLV RTase cDNA Synthesis Kit . Quantitative RT-PCR was conducted in Roche Applied Science LightCycler 480II Real-time PCR systems  in accordance to the manufacturer’s instructions. A comparative cycle threshold (http: //www.cytoscape.org/) was applied to build a circRNA–miRNA–mRNA/gene interaction network. The predicted gene functions in the networks were annotated using Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.The circRNA-miRNA interaction was predicted with Arraystar’s home-made miRNA target prediction software based on TargetScan and miRanda. Cytoscape . Significant differences were determined by Student t test and Spearman correlation test.Significant differences between different groups from the microarray were estimated by In conclusion, our study provided a unique circRNA profile in AML patients, based on which a substantial circRNA signature was demonstrated to indicate possible involvement in AML risk-status. Furthermore, we characterized and functionally evaluated one abundant circRNA, hsa_circ_0004277, thus offering a potential diagnostic marker and treatment target in AML.


Whether Doc2B functions as a calcium sensor akin to synaptotagmins, or in other calcium-independent or calcium-dependent capacities is debated. We here show by mutation and overexpression that Doc2B plays distinct roles in two sequential priming steps in mouse adrenal chromaffin cells. Mutating Ca2+-coordinating aspartates in the C2A-domain localizes Doc2B permanently at the plasma membrane, and renders an upstream priming step Ca2+-independent, whereas a separate function in downstream priming depends on SNARE-binding, Ca2+-binding to the C2B-domain of Doc2B, interaction with ubMunc13-2 and the presence of synaptotagmin-1. Another function of Doc2B – inhibition of release during sustained calcium elevations – depends on an overlapping protein domain (the MID-domain), but is separate from its Ca2+-dependent priming function. We conclude that Doc2B acts as a vesicle priming protein.Doc2B is a cytosolic protein with binding sites for Munc13 and Tctex-1 (dynein light chain), and two C2-domains that bind to phospholipids, Ca The calcium signal triggering fast synchronous secretion in chemical synapses and neuroendocrine cells is confined to nanodomains or microdomains around calcium channels ; it is o2+-coordinating aspartates created a mutant (denoted ‘6A’), which could not bind to Ca2+, but still supported spontaneous release  and the RRP, respectively  of vesicles was decreased – especially during repetitive stimulation – and the sustained component of release was potentiated . Consistectively . The eff2+]i before stimulation increases the size of the exocytotic burst . The results (To demonstrate the role of Doc2B in chromaffin cells we first compared secretion from Doc2B knockout (KO) adrenal chromaffin cells with Doc2B KO cells overexpressing Doc2B . Doc2B wic burst . In the  results , while i results . Because results . We conc results . Thus, eWe performed kinetic analysis to quantify the changes induced by Doc2B expression in fast and slow burst secretion, which originate from the Readily Releasable vesicle Pool (RRP) and the Slowly Releasable Pool (SRP), respectively . These aA promoting effect on RRP size implies that Doc2B stimulates RRP filling (priming) during period of rest (i.e. before stimulation). We next wanted to understand whether Doc2B requires the presence of other exocytotic priming proteins in the cell in order to stimulate priming, or whether it acts independently. Synaptotagmin-1 (syt-1) knockout (KO) chromaffin cells display a smaller-than-normal fast burst (RRP), whereas the slow burst is preserved ; this slMunc13-proteins are the canonical priming proteins in neurons  and chro2+-dependent, with higher Ca2+ concentrations in the sub-μM range leading to a larger burst of release  causes Doc2B to reside permanently at the plasma membrane  to uncage some of the Ca2+-cage ~20 s before stronger stimulation using a flash UV-lamp to probe secretion itself , Doc2B WT will be fully Ca2+-saturated and membrane localized . Therefore, the DN-mutant is a gain-of-function mutant in chromaffin cells when studied from a low basal [Ca2+]. Kinetic analysis indicated that the DN-mutant decreased the time constant for fast fusion  blocks Ca2+ binding to both C2-domains  depolarizations fuse the entire RRP , and calcium-dependent priming can be occluded by mutations at the Ca2 P2 , and the2 . The Dociposomes . We receiposomes . We theriposomes . Using eiposomes . The resiposomes , whereas2 interactions could drive this function. In syt-1, lysines can interact alternatively with SNAREs or PIP2 P2 , and rec,5)P2 . The ΔTCT-mutant was expressed similarly to WT Doc2B , which is located near the N-terminal end of Doc2B, encompassing amino acids 14 to 38  of the MID-domain , which wof Doc2b . Expressof Doc2b  showed tof Doc2b , indicatof Doc2b , and theof Doc2b . Therefo+ . Theref+ . This fu+ , which r+ . Althoug+ , which i+ , this sh2+-elevations, leading to a shallower sustained release component. Thus, Doc2B joins the ranks of a number of exocytotic proteins – including complexin, syts and Munc18s – that have positive and negative effects on different phases of release. Here, we have dissected the involvement of the different Doc2B effectors in this phenotype. Cross-rescue showed that syt-1 and ubMunc13-2 are required for the overall stimulating effect on priming, whereas syt-7 is dispensable. Even in the absence of syt-1 or syt-7, an inhibitory function of Doc2B was seen, and inhibition dominated in the absence of Munc13-2. Mutagenesis showed that interactions with SNAREs, Munc13, and Ca2+ are involved in the positive effects of Doc2B on priming, whereas they were not required for the inhibitory function. Thus, the stimulatory and inhibitory effects of Doc2B can be separated by mutation. This rules out that the stimulatory effect on RRP size could be a secondary effect of the inhibition of sustained release, which in principle could lead to a larger RRP by preventing loss of vesicles.In chromaffin cells, Doc2B has dual effects: it stimulates the size of the RRP from rest by boosting priming, but it inhibits priming/fusion during sustained Ca2+]i. With the DN mutation, the primed vesicle pools attained their maximal size irrespective of basal [Ca2+]i, occluding Ca2+-dependent priming (2+-channels. Other factors (for instance the number or availability of Ca2+-channels) might therefore limit the size of the IRP.Strikingly, both the DN and 6A mutations rendered the exocytotic burst size independent of basal i. The extracellular solution was composed of (in mM): 145 NaCl, 2.8 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES and 11 glucose, pH 7.2 (osmolarity adjusted to ~305 mOsm).Intracellular calcium concentrations were determined using a mixture of two fluorescent dyes with different Caitrogen; ] which, Amperometric recordings were performed using carbon fiber electrodes with 5 µm in diameter , insulated using the polyethylene method . AmperomPool sizes and fusion rates were determined by fitting individual capacitance traces with a sum of three exponential functions, each of the form A(1-exp(-t/τ)), representing the fast burst , the slow burst  and the sustained component , respectively, using procedures written in IGOR Pro software. Since the time constant of the sustained component often approached or exceeded the measurement time (5 s), only the mean linear rate of sustained release within 5 s was reported.priming 1 and priming 2 , which yields0 and SRP0 are the steady-state values of the two pool sizes. We estimated the RRP0 and SRP0 from the response to Ca2+-uncaging; the underlying assumptions are that pools are at equilibrium, that the product of 1k and the Depot size is a constant and that there was no ongoing fusion before stimulation.where RRPFor immunostaining against Doc2B and VAMP2, chromaffin cells were prepared from Doc2A/B double-knockout pups at postnatal day P1. Doc2A/B double knockouts were used for these experiments due to availability, but note that Doc2A is not expressed in adrenal chromaffin cells . Eight hFor immunostaining against syntaxin-1, cells were first prefixed for 5 min in isosmotic fixative containing 0.9% PFA in 0.05 M 1,4-piperazinediethanesulfonic acid (PIPES)/NaOH  2.8 mM KCl and 1 mM MgCl. This was followed by 8 min of incubation in 3.7% PFA in 0.05 M PIPES/NaOH (pH 7.4). Afterward, the cells were washed three times with PBS (pH 7.4) and permeabilized for 10 min in PBS containing 0.1% Triton X-100 (Sigma-Aldrich), followed by 30 min of blocking in PBS containing 3% BSA. The blocking solution was used for diluting antibodies. The chromaffin cells were incubated overnight with a cocktail of anti-syntaxin 1 and anti-GFP antibodies  at room temperature, washed three times with PBS, and incubated with secondary goat anti-mouse Alexa 546 and goat anti-chicken Alexa 488 antibodies (1:1000: Life Technologies) overnight at 4°C. After extensive washings with PBS, cells were rinsed with water and mounted on microscopic slides with Prolong Gold (Invitrogen). For this series of experiments, confocal images were acquired with a HCX PL APO CS lens  with frame size of 512 × 512 and a zoom factor 10 on a LEICA SP5-X Confocal microscope (Leica Microsystems). Images were corrected for background fluorescence by subtracting an average intensity value of an area not containing any cells. Fluorescence levels of the plasma membrane were quantified by measuring the integrated intensity of a ROI1, defined as the outer edge of the plasma membrane, and subtracting the integrated density of a ROI 2, defined as the inner edge of the membrane. The increase in syntaxin-1 staining by Doc2B expression is more modest using this isosmotic fixation method than with standard PFA-fixation .Adrenal glands were collected from P0-2 Syt-7 oe E18 Syt-1 mice and lysed in RIPA buffer (Invitrogen) supplemented with Protease Inhibitor Cocktail (Invitrogen). The protein concentrations were determined by use of a BCA Protein Assay Kit . 20 μg  of protein were resolved by SDS-PAGE, transferred to nitrocellulose and blotted using anti Doc2B , Synaptotagmin-1 , Synaptotagmin-7 , or VCP  antibodies. The blots were developed by chemiluminescence using the ECL plus western blotting substrate system (Pierce). Immunoreactive bands were detected using the FluorChemE image acquisition system (ProteinSimple) and quantified with ImageJ 1.47q .n indicating the number of cells, and statistical comparisons were made in Graphpad Prism. For every mutant or overexpression condition, we performed control experiments (non-expressing or Doc2B WT expressing) and overexpression/mutant experiments in parallel using cells isolated from the same animals. When testing two conditions, we used a F-test to test for differences in variance. In case the F-test was significant, we performed a Mann-Whitney test, otherwise we used an unpaired two-tailed Student’s t-test. In case of three conditions measured in parallel, we tested deviations from homoscedasticity using a Bartlett’s test; if the test was significant, we used a Kruskal-Wallis test and Dunn’s test for multiple comparison, otherwise we used an ANOVA and Tukey’s post-hoc test. For live imaging experiments  and asynchronous neurotransmitter release . In addition, Do2B binds to Munc13-proteins and the SNAREs. Most experiments assess exocytosis by capacitance measurements and amperometry. Exocytosis is stimulated in these experiments by uncaging intracellular Ca2+ in the patch clamp whole cell configuration. The experiments are well designed, and the overall quality of the data is excellent.The manuscript by Houy et al. addresses the role of the Ca2+ binding and associated translocation to the plasma membrane and that mutations that do not bind Ca2+ and are permanently associated with the plasma membrane are constitutively active, independent of Ca2+ binding. In contrast, no changes in Ca2+-dependent priming could be detected in Doc2B null mutants  they find that Doc2B action on priming relies on the presence of sytI, Munc13-2 and SNARE binding activity.The results show that Doc2B increases the exocytotic burst and thus the readily releasable pool but it also inhibits the sustained component, which is generally thought to reflect the recruitment followed by the release of unprimed vesicles. They conclude that the action of Doc2B involves CaEssential revisions:Reviewer 1:While the manuscript contains very interesting data leading to novel conclusions some data are less clear and/or not clearly presented, which need to be addressed. Also, the Discussion section is rather confusing. The discussion should specifically address points 2, 4, and 9, listed below.1) In 2) 3) Subsection “Doc2B requires synaptotagmin-1 and Munc13-2 to stimulate priming”, Figure 1Bii: The authors state that in absence of Syt-1, Doc2B has no effect on slow burst. However, the slow burst in syt1 KO is almost 50% larger than that in syt1 KO + Doc2B WT although the error bars are too large to achieve apparently p<0.05 significance. Is this due to the smaller sample size? In Figure 1Ai, similar amount of difference between Doc2B KO and Doc2B KO+Doc2B WT is statistically significant, and the sample size is also larger than that in Figure 1Bii. As it stands, it remains unclear whether Doc2B has no effect on slow burst.4) In the Discussion section and elsewhere in the manuscript: The increase in burst amplitude in the presence of Doc2B is interpreted as promotion of a step towards priming upstream of fusion. It is not obvious why this could not as well be a consequence of Doc2B inhibiting release, which could also lead to a larger RRP and could at the same time explain the reduction of the sustained phase. While the data may contain evidence for rejecting such an alternative hypothesis it should at least be explicitly discussed in the Discussion section.5. The authors state that chromaffin cells do not express Doc2A (Subsection “Doc2B requires synaptotagmin-1 and Munc13-2 to stimulate priming”). However, 6) 7) In 8) What are the Doc2B expression levels for the different KO cells as used in 2+-dependent priming as indicated by relative burst amplitude change is even higher in Doc2B KO than in Doc2B overexpressing cells. How does this fit with the author's conclusion that Doc2B is the Ca2+ sensor for priming?9) 10) In the long pulse stimulations of 11) In 12) In Reviewer 2:2+-sensor for vesicle priming, a notion that is largely based on the observation that the DN mutant and the 6A mutant abolishes any Ca2+-dependency of the vesicle priming reaction  and others suggesting a function as a Ca2+-sensor for asynchronous transmitter release . Furthermore, the authors themselves have previously reported, otherwise . By analyzing the exocytotic burst size over a wide range of preflash [Ca2+]I in wild type and Doc2B-KO cells, they could show that Doc2B has no obligatory role in driving the Ca2+-dependent priming reaction upstream of the slowly releasable pool. Also in the present manuscript the authors find the Ca2+-dependency of the priming reaction of chromaffin vesicles to be unperturbed in cells of Doc2B null mutants when compared with controls (see 1) My major concerns deals with the central conclusion of the manuscript implicating now Doc2B as a Careaction  and 4M. rols see .2+-binding) resulted in an anomalous increase in constitutive membrane binding and thereby provide Doc2B with novel functional properties . Thus, I'm not convinced that these experiments with the mutant proteins (DN and 6A mutation) provide clear evidence that Doc2B acts as a Ca2+-sensor in vesicle priming. The apparent gain-of-function mutation may rather result from anomalous membrane binding activity. The latter is only displayed as exemplary results  Previously, the authors have shown that overexpressing Doc2b increased significantly the total number of vesicles per cell as well as the number of membrane-proximal vesicles . This raises the question, to what extent apparent changes in priming with the DN and 6A mutation are accompanied by alterations in vesicle number and whether a similar phenotype can be observed with the mutant proteins. In this context, I do not understand the authors' statement in subsection “Doc2B supports vesicle priming as a calcium sensor”: "The DN-mutant was expressed at similar levels as WT Doc2B, and VAMP2/synaptobrevin-2 staining also showed similar levels, consistent with an unperturbed granule density in the presence of Doc2B expression ." The au2+]i. This action of Doc2B is dependent on its MID-domain (3) The most prominent phenotype in Doc2B KO seems to be the enhanced sustained release under prolonged elevation of I in wild type and Doc2B-KO cells, they could show that Doc2B has no obligatory role in driving the Ca2+-dependent priming reaction upstream of the slowly releasable pool. Also in the present manuscript the authors find the Ca2+-dependency of the priming reaction of chromaffin vesicles to be unperturbed in cells of Doc2B null mutants when compared with controls (see 1) My major concerns deals with the central conclusion of the manuscript implicating now Doc2B as a Careaction  and 4M. rols see .2+-binding) resulted in an anomalous increase in constitutive membrane binding and thereby provide Doc2B with novel functional properties . Thus, I'm not convinced that these experiments with the mutant proteins (DN and 6A mutation) provide clear evidence that Doc2B acts as a Ca2+-sensor in vesicle priming. The apparent gain-of-function mutation may rather result from anomalous membrane binding activity. The latter is only displayed as exemplary results , but it also displays a much higher membrane localization at rest than the WT; thus it is only a minor fraction of the DN mutant that cycles, since most resides at the membrane. The 6A-mutation does not cycle on and off the membrane, but remains at the plasma membrane. The reviewer raises the question whether the DN mutation has ‘anomalous increase in constitutive membrane binding and thereby provides Doc2B with novel functional properties’. The decisive experiments that we performed here was to study the effect of expressing Doc2B WT and DN when stimulated from a low and a high basal-[Ca2+]. The striking result is that the DN is a gain-of-function mutant when studied from a low basal-[Ca2+], but – when studied from a high basal [Ca2+] – it is indistinguishable from the WT. This latter fact indicates that the DN is not special or anomalous, except that it is already abundant at the membrane at very low [Ca2+]. The WT protein would not be at the membrane at low basal-[Ca2+], and thus the gain-of-function of the DN-mutant is entirely understandable from the trafficking behavior. The only exception is the fusion time constant of the RRP, which is significantly faster for the DN-mutant when studied from high basal-[Ca2+] . In addition, we see basically the same behavior by another mutant – the 6A-mutant – which is unable to bind to Ca2+ at all, but which is also constitutively at the membrane. Also, this mutation is gain-of-function when studied from low basal-[Ca2+], although it is slightly inferior to the WT at high basal [Ca2+]. This permanent membrane localization of the 6A-mutation sheds new light on this mutation, which was previously used to argue that Doc2B might have Ca2+-independent effect ; now we see that also this mutation bypasses Ca2+-binding by being permanently at the plasma membrane. We have not been able to extend the range of preflash [Ca2+] that we study, because of the huge amount of work that would be involved, in combination with the modest yield of cells from a single Doc2B embryo. As far as we can see, the current data provide very strong evidence for our conclusion that Ca2+-binding to Doc2B drives it to the membrane, which leads to membrane priming.We have now performed new experiments and included a quantification of the membrane binding activity of Doc2B WT, DN, 6A, and ΔMID in a new figure . This sh2+-dependent (due to the trafficking of Doc2B) SNARE-complex formation could very well in other systems lead to spontaneous, or even  asynchronous release.Our data here are consistent with our previous data , since we noted – and discussed carefully in the Discussion that calcium-dependent priming is still present in Doc2B KO cells, probably because of redundant processes/proteins (as the reviewer mentions), which can be overridden by overexpression of Doc2B DN. When comparing to data obtained in synapses, it is worth remembering that the vesicles we study here (large dense-core vesicles) are different from synaptic vesicles, and there is nothing surprising in having different functions of the protein in the two (very) different systems. Also, the groups studying synaptic vesicles cannot agree as to the function of Doc2B in asynchronous release. Nevertheless, we think the data might be reconciled, since priming in chromaffin cells is driven by SNARE-complex assembly, and Ca2) Previously, the authors have shown that overexpressing Doc2B increased significantly the total number of vesicles per cell as well as the number of membrane-proximal vesicles . This raises the question, to what extent apparent changes in priming with the DN and 6A mutation are accompanied by alterations in vesicle number and whether a similar phenotype can be observed with the mutant proteins. In this context, I do not understand the authors' statement in subsection “Doc2B supports vesicle priming as a calcium sensor”: "The DN-mutant was expressed at similar levels as WT Doc2B, and VAMP2/synaptobrevin-2 staining also showed similar levels, consistent with an unperturbed granule density in the presence of Doc2B expression ." The auWe have repeated the stainings, as explained above. In the process of doing this, we realized that quantification of Doc2 and Syb2 cannot be done accurately on samples stained for both proteins, because the expression and staining of Doc2 reduces the staining for Syb2, probably because of antibody interference. We have therefore performed separate stainings for Doc2 and syb2, in an attempt to solve this question. However, this didn’t change the conclusion. We can only speculate why there is a difference between the EM and the immunostaining. These are very different measures, and there are plenty of reasons these measures could turn out different. In the EM the levels of proteins were not quantified; In Immunostaining we did not quantify granules, only overall syb2-intensity. Thus, formally one possibility is that the total syb2 content is distributed between more granules after overexpression, although because these measurements were done several years apart by different people, we would not be comfortable making such a conclusion at the present time. We now pointed out this possible discrepancy in the Discussion section.2+]i. This action of Doc2B is dependent on its MID-domain (3) The most prominent phenotype in Doc2B KO seems to be the enhanced sustained release under prolonged elevation of [CaD-domain  but curiD-domain . The autWe agree, studying the Doc2B-CAPS interaction directly would certainly be helpful. However, repeating the experiments in CAPS DKO cells has not been possible, because we currently don’t have CAPS DKO mice available in the laboratory. We have tried to make it clearer in our text that we are discussing the possibilities; we are not drawing a conclusion.4) It remains unclear why specifically the RRP but not the IRP is altered with Doc2B DN mutant expression.2+-channels. Therefore, there are more factors involved in making an IRP-vesicle than a RRP-vesicle. Most likely another factor (number of Ca2+-channels?) is limiting for the IRP-vesicles.The IRP is larger on average, but it is far from significant. We now discuss this. The point is that the IRP is a subpool of the RRP, consisting of RRP-vesicles that are close to Ca


Mechanistically, the adverse effects of MD1 deletion on LV remodelling were related to hyperactivated CaMKII signalling and increased impairment of intracellular Ca2+ homeostasis, whereas the increased electrophysiological instability was partly attributed to exaggerated prolongation of cardiac repolarisation, decreased action potential duration alternans threshold, and increased diastolic SR Ca2+ leak. Therefore, our study on MD1 could provide new therapeutic strategies for preventing/treating heart failure.Myeloid differentiation protein 1 (MD1) has been implicated in numerous pathophysiological processes, including immune regulation, obesity, insulin resistance, and inflammation. However, the role of MD1 in cardiac remodelling remains incompletely understood. We used MD1-knockout (KO) mice and their wild-type littermates to determine the functional significance of MD1 in the regulation of aortic banding (AB)-induced left ventricular (LV) structural and electrical remodelling and its underlying mechanisms. After 4 weeks of AB, MD1-KO hearts showed substantial aggravation of LV hypertrophy, fibrosis, LV dilation and dysfunction, and electrical remodelling, which resulted in overt heart failure and increased electrophysiological instability. Moreover, MD1-KO-AB cardiomyocytes showed increased diastolic sarcoplasmic reticulum (SR) Ca High blood pressure is widely recognised to induce left ventricular (LV) hypertrophy and lead initially to ventricular wall thickening and stiffening, a process which is compensatory and adaptive in nature. However, sustained pressure overload contributes to maladaptive LV remodelling, progressive LV dilatation, and cardiac dysfunction4, and this results in arrhythmias and HF6, a major underlying cause of increased cardiovascular morbidity and mortality7. Despite being the focus of substantial research effort in recent decades, the precise pathogenesis of maladaptive LV remodelling and the mechanisms that determine how long-standing hypertrophy ultimately progresses to HF remain unclear8. Therefore, enhanced understanding of the factors and mechanisms that modulate pathological LV remodelling could lead to novel strategies for the treatment of HF.Hypertension is the single most critical risk factor for heart failure (HF)13. Upon stimulation, TLR4 signalling ultimately activates numerous signalling pathways15, including the MAPK pathway, NF-κB pathway, and PI3K/Akt pathway. Furthermore, a link between Ca2+/calmodulin-dependent kinase II (CaMKII) signalling and TLR4 signalling has been clearly demonstrated18. More importantly, therapies targeting against TLR4 have shown effectiveness in attenuating murine cardiac remodelling caused by pressure overload21. Furthermore, a recent study demonstrated that stimulation of TLR4 in rat ventricular cardiomyocytes in vitro promoted an electrical remodelling that led to action potential duration (APD) prolongation associated with delayed afterdepolarisation and triggered activity22.Evidence gathered over the past two decades has shown that Toll-like receptor 4 (TLR4) signalling is involved in several aspects of the cardiac pathological process, such as cardiac remodelling, ischaemia/reperfusion injury, hypertension, and atherosclerosis23. MD1 is expressed predominantly in B cells, macrophages, dendritic cells, and other immune cells24. MD1 forms a complex with radioprotective protein 105 (RP105), which is abundantly present in heart tissue. MD1-RP105 complex can directly interact with the MD2-TLR4 complex by a lateral binding, acting as physiological negative regulators of TLR4 signalling25. Furthermore, recent evidence suggests that MD1-RP105 complex is associated with several pathophysiological processes, including immune regulation, obesity, insulin resistance, and inflammation28. However, to the best of our knowledge, no previous study has reported whether MD1 regulates sustained pressure overload-induced LV structural and electrical remodelling.The aforementioned data led us to investigate whether a naturally occurring molecule can inhibit LV remodelling and reduce the susceptibility to ventricular tachycardia (VT) during chronic pressure overload by blocking TLR4 signalling. We speculated that a favourable candidate molecule might be myeloid differentiation protein 1 (MD1), an endogenous negative modulator of TLR4 signalling2+ homeostasis, the increased electrophysiological instability is at least partly due to an increased prolongation of cardiac repolarisation, enhanced reduction of the APD alternans threshold, and exaggerated increase of diastolic sarcoplasmic reticulum (SR) Ca2+ leak.Here, using loss-of-function approach, we discovered that pressure overload-induced LV remodelling was aggravated in MD1-knockout (KO) mice, which led to overt HF and increased electrophysiological instability. Whereas these adverse effects of MD1 deletion on LV remodelling are related to the hyperactivation of CaMKII signalling and an exaggerated impairment of intracellular CaTo explore the correlation between MD1 and LV remodelling, we first examined MD1 expression in the failing left ventricles. Western blot analysis revealed that MD1 protein levels were significantly lower in the heart samples of DCM patients than in those of normal donors Fig. . MoreoveBNP, β-MHC) were markedly higher in the LV tissues of MD1-KO mice than in those of WT mice  recordings were performed on mice under light anaesthesia. Mice from the groups indicated in Fig. 90, threshold interval for APD alternans, and VT inducibility rate. After 4 weeks of AB, APD90 was markedly prolonged in both WT-AB and MD1-KO-AB mice, but the prolongation was considerably greater in the KO mice than in the WT mice .Langendorff-perfused hearts and the monophasic action potential (MAP) recording technique were used to determine the changes in three electrophysiological parameters: APDice Fig. . Among tice Fig.  were simP < 0.05 vs. WT-AB; Fig. We applied Western blotting to investigate the effects of MD1 loss on TLR4 signalling and CaMKII signalling after 4 weeks of AB. Our results showed that a 4-week course of AB significantly decreased RP105 expression, and increased MD2 and TLR4 expression in both WT and MD1-KO LV tissues Fig. . However2+ transients in LV myocytes  values revealed that diastolic SR Ca2+ leak was markedly higher in MD1-KO-Sham myocytes than in WT-Sham myocytes  expression and SERCA2 phosphorylation were drastically decreased, whereas the expression of Na+/Ca2+ exchanger 1 (NCX1) and phospholamban (PLN) were significantly increased in both MD1-KO and WT hearts, although these changes were considerably greater in MD1-KO hearts than in WT hearts .Under baseline conditions, the expression and phosphorylation of major Cains Fig. , and theins Fig.  were simrts Fig. . Moreoverts Fig. . Phosphorts Fig.  and the rts Fig.  were marrts Fig.  was sign+ and Na+ channels are also associated with electrical remodelling during HF, so we investigated the expression of some K+ and Na+ channels in the mouse left-ventricle samples. Our western blotting results demonstrated that after 4 weeks of AB, both WT and MD1-KO mice showed a significant reduction in protein levels of KCNH2  density  and sudden cardiac death42, and interventions that eliminate TWA might prevent VT and sudden death42. TWA corresponds to APD alternans at the cellular level43. Therefore, any factor that decreases the APD alternans threshold might induce VT. Our results showed that MD1 loss decreased the APD alternans threshold in isolated pressure-overload hearts, which suggests that reduction of the APD alternans threshold is one of the mechanisms underlying the MD1 deletion-elicited arrhythmogenic potential in response to pressure overload. A recent literature review revealed that increased SR Ca2+ leak through RyR2 channels and/or decreased SERCA pump activity contributes to alternans43. Consistent with this review’s finding, our study has shown that SERCA2 expression and phosphorylation levels were lower but RyR2 phosphorylation was higher in MD1-KO-AB mice than in WT-AB mice. In addition, alternans can be caused by instabilities originating from voltage and is influenced by INa in electrically remodelled diseased hearts43. INa is affected by CaMKII activity41. Although INa might be reduced as Nav1.5 expression was demonstrated to be markedly decreased in MD1-KO-AB mice in this study, but the activation and kinetic properties of INa remain to be explored in future work.T-wave alternans (TWA) is a risk marker of cardiac repolarisation that has been associated with VAs2+ leak, which can cause Ca2+ waves and activate an arrhythmogenic inward Na+/Ca2+ exchange current, causing delayed afterdepolarisations (DADs) and triggering arrhythmias46. Here, MD1 loss enhanced the increase in CaMKII activity and CaMKII-mediated RyR2 phosphorylation in response to chronic pressure overload. Moreover, the LV myocytes isolated from MD1-KO-AB mice exhibited an increased arrhythmogenic potential in vitro as compared with WT-AB mice, including enhanced diastolic SR Ca2+ leak, reduced SERCA2 pump activity, and increased NCX1 expression. Growing evidence indicates that increased SR Ca2+ loading is necessary for enhanced RyR2 Ca2+ leak to produce arrhythmia47, but no direct evidence is available to demonstrate that spontaneous Ca2+ sparks arise only when a specific SR cistern becomes ‘overloaded’ and that a lower-than-physiological concentration of Ca2+ in the SR does not affect Ca2+ spark production48. Collectively, these data indicate that the enhanced diastolic SR Ca2+ leak is likely sufficient to contribute to the electrophysiological instability, although the occurrence of spontaneous Ca2+ waves, and DADs needs to be further investigated.RyR2 hyperphosphorylation due to CaMKII overactivity is widely accepted to lead to increases in diastolic SR Ca2+ handling. (1) The analytical results of ICa,L inactivation  The increased NCX1 expression as a factor for the decreased SR Ca2+ content in MD1-KO-AB mice was not confirmed. Although enhanced NCX activity could decrease SR Ca2+ content in HF53, and increased NCX expression was proved to be associated with the decreased SR Ca2+ content in right atrial myocytes isolated from the patients with chronic atrial fibrillation54, additional analysis are required to calculate the contribution of NCX to Ca2+ removal. The way to do that is to fit exponential decay both on the systolic and caffeine transients and calculate rate constants of decay, as previously described55. Due to technical problems and insufficient experience, we couldn’t completely solve these in a short time.This work has limitations in the study of cardiomyocyte Ca2+ homeostasis. The increased electrophysiological instability is partly due to aggravated electrical remodelling, including prolonged cardiac repolarisation, decreased APD alternans threshold, and increased diastolic SR Ca2+ leak. These findings suggest that endogenous MD1 protects against LV structural and electrical remodelling during long-standing pressure overload. Therefore, MD1 upregulation in the heart might serve as a new therapeutic strategy for the treatment of HF.In summary, the present study indicates that MD1 loss exacerbates LV structural and electrical remodelling in response to chronic pressure overload, resulting in overt HF and increased electrophysiological instability. Mechanistically, the adverse effects of MD1 deletion on LV remodelling are associated with hyperactivated CaMKII signalling and increased impairment of intracellular CaSamples of human failing hearts were collected from the left ventricles of DCM patients undergoing heart transplants. Control samples were obtained from the left ventricles of normal heart donors who had died as a result of accidents, and whose hearts were not used for transplantation due to non-cardiac reasons. After excision, all specimens were immediately flash-frozen in liquid nitrogen and stored at −80 °C for biochemical analysis. Written informed consent was obtained from the family of prospective heart donors. The study was approved by Renmin Hospital of Wuhan University Review Board and was conducted in accordance with the Declaration of Helsinki.Male MD1-KO mice  and their WT littermates aged 8–10 weeks were used for experiments. All animals were housed in a 12/12-h light/dark cycle and provided food and water ad libitum. Mice were genotyped by means of PCR performed using the protocol provided by RIKEN , and ratios of HW/BW (mg/g), HW/TL (mg/mm), and LW/TL (mg/mm) of the sacrificed mice from the different groups were assessed. The LV tissues from each heart were dissected, snap-frozen in liquid nitrogen, and stored at −80 °C for biochemical studies.All surgeries and subsequent analyses were performed in a blinded fashion for all groups. AB surgery and sham operation were performed as described previously57 to obtain these values: LVEF, LVFS, LVEDD, LVESD, LVESP, interventricular septum diameter in diastole and systole , LV posterior wall diameter in diastole and systole , and dp/dt max and dp/dt min.We performed pressure-volume relationship measurements and echocardiography as described previouslyHearts were excised and washed with saline solution, arrested in diastole with 10% KCl, fixed in 4% paraformaldehyde solution, and embedded in paraffin. The paraffin-embedded hearts were sectioned transversely at the level of the LV papillary muscles. Several slices (4–5 mm thick) of heart were prepared and stained with H&E for morphometric analysis or picrosirius red (PSR) for evaluation of myocardial fibrosis. All micrographs were acquired using a high-resolution optical microscope. Image-Pro Plus 6.0 software was used to determine the CSA of cardiomyocytes and the volume of LV collagen deposition.GAPDH, and data were analysed according to the 2−∆∆Ct method58. The method details are described in Supplementary Methods.Total RNA from human or mouse LV tissues was extracted and reverse transcribed to generate cDNAs; qRT-PCR was performed by using an ABI-PRISM 7900 Sequence Detection System with the SYBR Color qPCR Master Mix . Gene expression was normalised relative to the housekeeping gene Total proteins were extracted from frozen LV samples, and a BCA Protein Assay Kit  was used to determine protein concentrations. Proteins (40 μg) were separated using SDS-PAGE and transferred onto polyvinylidene difluoride membranes, which were then incubated with primary antibodies overnight at 4 °C and subsequently with secondary antibodies for 2 h at room temperature. Chemiluminescent detection was performed using the SuperSignal West Pico Chemiluminescent Substrate . Additional details are presented in Supplementary Methods.2). Surface electrodes were placed subcutaneously and the surface-lead ECG (lead II) was recorded. Data were analysed off-line using LabChart 7 Pro (AD Instruments). To correct for heart rate, the QTc interval was calculated using Bazett’s formula59: QTc = QT/(RR/100)1/2. Supplementary Methods contains additional details.Mice were lightly anaesthetised using inhaled isoflurane . The paired platinum stimulating electrode was positioned on the basal surface of the right ventricle, and it delivered regular pacing. Method details are described in Supplementary Methods.Langendorff-perfused hearts were prepared according to published methods90 and APD alternans threshold; pacing was performed with a series of pulse trains at a regular pacing cycle length (PCL) for 30 s, and then interrupted for at least 30 s to minimise pacing memory effects. The PCL was shortened from 150 to 100 ms in 10-ms steps and then to 50 ms in 5-ms steps. The APD90 at the PCL of 150 ms was defied as the average 90% repolarisation time for 6–8 successive MAPs. Alternans was judged to have occurred when alternate APDs differed by 5% over at least 10 beats61. The APDs were measured at 90% repolarisation and the longest PCL-induced APD90 alternans was defined as the APD alternans threshold interval. To test for VT inducibility, burst pacing at cycle lengths of 100–50 ms or 2:1 capture occurrence was used63. Unlike in previous studies, we applied a pacing train of 20 stimuli. VT was defined as the occurrence of at least 4 consecutive ventricular waveforms after the last paced beat.S1-S1 pacing was used for determining APDSingle LV myocytes were isolated from the mice by using collagenase type II digestion. Freshly isolated LV myocytes were stored at room temperature until use. Only rod-shaped myocytes showing clear striations were studied, and experiments were performed at room temperature (20 °C–25 °C) within 6 h after cell isolation. Experimental details are presented in Supplementary Methods.Whole-cell membrane currents were obtained and assessed by using an EPC-9 patch-clamp amplifier  in the whole-cell mode and Pulse/Pulsefit software (HEKA Electronik). Supplementary Methods contains the experimental details.2+ indicator Fluo-4 AM  in Tyrode’s solution  at 37 °C for 30 min; cells were suspended every 5 min during incubation. The loaded cells were washed for 15 min for de-esterification, superfused for 10 min with dye-free Tyrode’s solution, and then transferred to a chamber equipped with a pair of parallel platinum electrodes. The chamber was placed on a Leica AF6000 fluorescence microscope . Systolic Ca2+ transients were recorded in steady-state conditions under constant field stimulation . We evaluated the average time to peak Ca2+ transient amplitude and duration of Ca2+ transient decay of a tested cardiomyocyte by measuring 4 consecutive and well performed Ca2+ transients during field stimulation. To assess SR Ca2+ content, the amplitude of caffeine-induced Ca2+ transients was measured; at 4 s after the stimulation was stopped, 10 mmol/L caffeine was applied directly onto the cells, which led to an immediate and complete SR Ca2+ release. The fluorescence intensity of Fluo-4 (measured at 526 nm upon excitation at 488 nm) was recorded and the Ca2+ transient amplitude (F/F0) and duration were assessed, both by using the Leica AF6000 Modular system.Isolated cardiomyocytes were loaded with the Ca2+ sparks of the loaded cardiomyocytes were recorded at resting conditions after repetitive field stimulation . Ca2+ sparks were analysed using SparkMaster and Image J64. The mean CaSpF of each recorded cell was expressed as sparks/100 μm/s.Loaded cardiomyocytes were prepared as described above and transferred to the recording chamber, which was placed on a Leica TCS SP8 STED 3X laser-scanning confocal microscope (Leica Microsystems Inc.). Fluo-4 was excited using a 488-nm argon-ion laser and the emitted fluorescence was measured at 526 nm. Diastolic Cat test or two-way ANOVA followed by Bonferroni post hoc test. Categorical data are expressed as percentages and were analysed using Fisher exact test. P < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS or GraphPad Prism software. Continuous variables are shown as means ± SEM and were evaluated with Student’s 2-tailed unpaired Supplementary Materials


Because of the many different calcium‐binding proteins distributed throughout cells, signalling precision requires localized rises in the cytosolic Ca2+ concentration. In electrically non‐excitable cells, for example epithelial cells, this is achieved by primary release of Ca2+ from the endoplasmic reticulum via Ca2+ release channels placed close to the physiological target. Because any rise in the cytosolic Ca2+ concentration activates Ca2+ extrusion, and in order for cells not to run out of Ca2+, there is a need for compensatory Ca2+ uptake from the extracellular fluid. This Ca2+ uptake occurs through a process known as store‐operated Ca2+ entry. Ideally Ca2+ entering the cell should not diffuse to the target site through the cytosol, as this would potentially activate undesirable processes. Ca2+ tunnelling through the lumen of the endoplasmic reticulum is a mechanism for delivering Ca2+ entering via store‐operated Ca2+ channels to specific target sites, and this process has been described in considerable detail in pancreatic acinar cells and oocytes. Here we review the most important evidence and present a generalized concept.Ca AChacetylcholineANO1anoctamin‐1CaCC2+‐activated Cl− channelCaCCKcholecystokininCRAC channel2+ release‐activated Ca2+ channelCaERendoplasmic reticulum3IPinositol trisphosphate3RIP3 receptorIPMCU2+ uniportermitochondrial CaNAADPnicotinic acid adenine dinucleotide phosphateNFATnuclear factor of activated T‐cells2PIPphosphatidyl inositol bisphosphatePMplasma membranePMCA2+‐activated ATPaseplasma membrane CaRyRryanodine receptorSERCA2+ activated ATPasesarco/endoplasmic reticulum CaSOARSTIM1 Orai1‐activating regionSOCE2+ entrystore‐operated CaSTIMstromal interaction moleculeTMEM16Atransmembrane member 16ATRPtransient receptor potential2+ signalling depends on specific Ca2+ sensors and transport proteins expressed differentially on organelle and plasma membranes, as well as Ca2+ buffers with different affinities and kinetics in different cellular compartments   into the cytosol ‐evoked Ca2+ release into the nucleoplasm through the inner nuclear membrane  being tunnelled through the ER lumen directly into the nuclear envelope. In this article, we describe and review the most important evidence for Ca2+ tunnelling, primarily based on studies of pancreatic acinar cells and oocytes.Effective and precise intracellular Ca Südhof, . However Putney, . Diffusilem Fig. . The pro2+ signals is encoded in their spatial, temporal and amplitude features. These Ca2+ dynamics combine to activate a defined subset of Ca2+‐dependent downstream effectors to transduce the cellular response . These elementary events due to the opening of one or a few channels can either remain localized resulting in Ca2+ signals in the microdomain around the channel(s), or coalesce through complex mechanisms into more global Ca2+ events that often encompass the entire cell  activation in T‐cells in response to antigen stimulation  at the plasma membrane and results in the production of IP3 and diacylglycerol. IP3 diffuses and gates open IP3 receptors at the ER membrane, releasing store Ca2+ to mediate the first phase of the Ca2+ signal. Should the Ca2+ release phase result in significant store depletion, it leads to the activation of Ca2+ influx at the plasma membrane through store‐operated Ca2+ entry (SOCE). SOCE is mediated by members of the stromal interaction molecule (STIM) and Orai family  junctions that are 20 nm apart /CRAC‐activating domain, which interacts with Orai1, traps it within the STIM1‐defined ER–PM junctions, and gates it open, thus allowing Ca2+ flow into the cell. SOCE activation not only results in store refilling but also shapes Ca2+ signal dynamics. There is therefore a tight functional link between IP3‐dependent Ca2+ release in response to agonist stimulation and Ca2+ influx through SOCE.Ca2+, liberating messengers – IP3 in the case of ACh and cyclic ADP‐ribose and nicotinic acid adenine dinucleotide phosphate (NAADP) in the case of CCK stimulation – thereby releasing Ca2+ from intracellular stores  and/or the hormone cholecystokinin (CCK). Interaction with specific surface membrane receptors activates signal transduction mechanisms that generate intracellular Ca2+ concentration ([Ca2+]i) changes evoked by ACh or CCK consist of repetitive short‐lasting elevations mostly confined to the apical region /anoctamin‐1 (ANO1)  are found in the basolateral membrane  opening of Cl− channels and exocytosis  inevitably activates Ca2+ pumps in the plasma membrane  resulting in extrusion of Ca2+ , ACh stimulation was discontinued and a period of Ca2+ entry was enabled by switching the pipette potential from negative to positive. No change was observed in [Ca2+]i during this Ca2+ entry period, but after discontinuation of Ca2+ entry (switching the pipette voltage back to negative), a new period of ACh stimulation caused a local rise of [Ca2+]i in the apical pole near the apical membrane, exactly as under normal conditions. A rise in [Ca2+]i near the cell‐attached pipette during Ca2+ entry could only be observed when the ER Ca2+ pumps  were arrested by thapsigargin  and that the whole of the ER was re‐equilibrated with regard to [Ca2+]ER within a few seconds   apical Ca2+ release, occurred through the basolateral membrane after a delay of about 6–7 s  current previously discovered in immune cells  during individual apical Ca2+ spikes shows  , there is primary generation of NAADP  allowing for easy spatial resolution of Ca2+ dynamics, which becomes particularly important for studies focused on the generation and propagation of Ca2+ waves, and elementary Ca2+ release events because their large spatial footprint in the oocyte makes them more amenable to investigation. The size of the oocyte also favours biochemical analyses and importantly linking them directly to Ca2+ signalling and other cell physiological processes at the single cell level , which are critical for oocyte biology and fertilization as they contribute significantly to the maintenance and regulation of the membrane potential. The Ca2+ transient generated at fertilization is initially localized at the site of sperm entry but gradually sweeps across the entire egg in the form of a Ca2+ release wave, which activates CaCCs and depolarizes the cell to prevent polyspermy ‐ethylenediamine (TPEN) and thapsigargin . TPEN is a transition metal chelator with low Ca2+ affinity that is freely membrane permeant and chelates high lumenal ER Ca2+, thus simulating Ca2+ store depletion and inducing SOCE   . Various approaches to block the IP3R or SERCA were used to show that the high CaCC current in response to SOCE when IP3 is present requires active IP3Rs  and of the Ca2+ effector (CaCC), and the distance to be covered by Ca2+ ions in the cytoplasm from the mouth of the Orai channel to the CaCC is incompatible with a diffusion mechanism given the speed of activation of CaCCs and the measured size and distribution of SOCE puncta as compared to CaCC‐rich membrane domains  has been directly monitored. The rate of rise of [Ca2+]ER decreases, as expected for a diffusional process, with increasing distance from the uncaging site , Ca2+‐sensitive K+ channels are strongly activated, supporting the idea that SOCE ‘fuels’ the IP3 receptors when the stores are empty to provide an efficient activation of the K+ channel , the SERCA pump and the IP3R. Store depletion stabilizes the STIM1–Orai1 puncta at ER–PM junctions thus providing the source for Ca2+ entry from the extracellular space. Ca2+ flowing through SOCE channels is taken up by the SERCA pump into the ER lumen preventing its diffusion out of the SOCE microdomain. In turn ER Ca2+ is released through IP3Rs thus delivering it to the appropriate effectors  with high efficiency and specificity. The Ca2+ tunnelling machinery has been adapted to very different cell physiological needs in the oocyte as compared to the pancreatic acinar cell. In the oocyte it modulates resting membrane potential and the temporal features of Ca2+ signals, whereas in the acinar cell it drives enzyme secretion and fluid flow. Given that the molecular machinery underlying Ca2+ tunnelling is ubiquitous, it is likely that this pathway is involved in Ca2+ signalling in a plethora of other physiological functions. The remarkable functional link between SOCE, SERCA and IP3R conscripted to allow Ca2+ tunnelling results in the delivery of Ca2+ to effectors that could easily be missed experimentally and interpreted as signalling downstream of SOCE directly. It is therefore likely that Ca2+ tunnelling activates additional cell physiological events that remain to be defined.Interestingly, the molecular mechanisms underlying CaNone declared.All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.O.H.P. is a Medical Research Council Professor (G19/22/2).