However, the translocation rate and activity of AddAB in vivo is not known, and how AddAB is regulated to prevent excessive DNA degradation around a break site is unclear. To examine the functions and mechanistic regulation of AddAB inside bacterial cells, we developed a next-generation sequencing-based approach to assay DNA processing after a site-specific DSB was introduced on the chromosome of Caulobacter crescentus. Using this assay we determined the in vivo rates of DSB processing by AddAB and found that putative chi sites attenuate processing in a RecA-dependent manner. This RecA-mediated regulation of AddAB prevents the excessive loss of DNA around a break site, limiting the effects of DSB processing on transcription. In sum, our results, taken together with prior studies, support a mechanism for regulating AddAB that couples two key events of DSB repair–the attenuation of DNA-end processing and the initiation of homology search by RecA–thereby helping to ensure that genomic integrity is maintained during DSB repair.In bacteria, double-strand break (DSB) repair via homologous recombination is thought to be initiated through the bi-directional degradation and resection of DNA ends by a helicase-nuclease complex such as AddAB. The activity of AddAB has been well-studied  in vitro, but is less well characterized in vivo. We describe a deep sequencing-based assay for assessing the early steps of DSB processing in bacterial cells by the helicase-nuclease complex AddAB. We find that a combination of chi site recognition and RecA loading is required to attenuate AddAB activity. In the absence of RecA, the chromosome is excessively degraded with a concomitant loss in transcription. Our results, along with prior studies, support a model for how chi recognition and RecA together regulate AddAB to maintain genome integrity and facilitate recombination.Double-strand breaks (DSBs) are a threat to genome integrity and are faithfully repaired via homologous recombination. The initial processing of DSB ends that prepares them for recombination has been well-studied  Double-strand breaks (DSBs) are a potentially lethal form of DNA damage as incorrectly repaired or unrepaired breaks can lead to the loss of genetic information, chromosomal rearrangements, mutations, or cell death. Cells have evolved the ability to faithfully repair DSBs via homologous recombination using a sister chromatid or sister chromosome as a template –3. In alchi (crossover hotspot instigator) that triggers a switch to a state that drives resection of only one strand, thereby producing the necessary ssDNA overhang needed to initiate homologous recombination [E. coli RecBCD, which has two independent helicase domains and a nuclease domain, and B. subtilis AddAB, which contains a helicase and a nuclease domain in AddA along with a nuclease domain in AddB  [B. subtilis show that, like RecBCD, recognition of chi sequences on the 3’-terminated strand during degradation converts AddAB from a double-stranded nuclease to a single-stranded DNA nuclease and slows its effective rate of translocation [chi recognition by AddB [chi-dependent manner, via a direct interaction with RecB [E. coli, that RecBCD initially unwinds a DSB end but without any degradation [chi site RecBCD then nicks one strand with subsequent helicase activity separating the two strands to create a single-stranded overhang that can load RecA and initiate homologous recombination.Precisely how DSB end processing occurs and how it is regulated to ensure the generation of single-stranded DNA required for recombination without an excessive loss of genomic information is not fully understood. Biochemical studies have led to two general models. In one model, the nuclease-helicase complex initially degrades both strands of DNA until it encounters a specific DNA element called bination –13. This domain) ,5,14. Bilocation –18. Stru by AddB . The sinith RecB ,21. How radation . Upon acin vivo to AddAB is not fully resolved. As noted, the molecular events underlying the initial processing of DSBs have been extensively studied in vitro, both in bulk and in single-molecule experiments [in vivo have provided important insights, but direct measurements of DNA processing by these helicase-nuclease complexes has been limited. The assays used often involve measuring the retention of radioactively labeled nucleotides in chromosomes subjected to UV damage [in vivo, but cannot easily be used to examine DNA processing on a global level. Advances in whole genome DNA sequencing in combination with the development of systems for the controlled introduction of single DSBs [in vivo activity of helicase-nuclease complexes like AddAB with higher resolution and more precision.Which of these two models applies eriments ,19,22–24eriments ,26, and eriments –30 activV damage , which pV damage ,33 have gle DSBs ,34–36 inCaulobacter crescentus is a useful organism for probing the in vivo dynamics and mechanisms underlying DSB repair. Unlike many bacteria, Caulobacter exhibits once-and-only-once replication of its chromosome under all growth conditions, and large populations of cells are easily synchronized, enabling the isolation and study of cells with a single chromosome. DSB repair has been previously visualized at the single-cell level in Caulobacter [in vivo processing of site-specific DSBs introduced in the Caulobacter chromosome, which is thought to require AddAB [chi sites, to resecting a single strand. We show that putative chi sites in Caulobacter attenuate the rate of AddAB-mediated DNA processing in vivo, but only with ~20% efficiency, similar to in vitro estimates for B. subtilis AddAB [in vivo are comparable to the previously measured in vitro rate of ~400 bp/s for B. subtilis AddAB [chi recognition is reduced ~4-fold. Successful attenuation of degradation requires the formation of a RecA filament, but not the SOS response or recombination. Collectively, our results indicate that RecA likely downregulates the translocation rate of AddAB after chi, possibly through a direct protein-protein interaction. This regulation of AddAB by RecA helps to limit DNA degradation around a break site, thus constraining the impact on transcription to a more limited region of the genome.lobacter . Here, wre AddAB ,38, usinre AddAB ,37. Usinis AddAB . We findis AddAB ,22,23,39Caulobacter [van promoter and induces gene expression [C. crescentus population. To isolate these swarmer cells and to prevent subsequent rounds of replication, cells were grown without IPTG for 1.5 h to deplete DnaA and arrest cells in a G1 state. These G1-arrested cells were then isolated using Percoll density gradient centrifugation [To assess DNA processing around a DSB induced on the chromosome, we used the I-SceI system previously developed in lobacter  Fig 1A)Caulobactlobacter ,40. Thisfugation ; flow cyIn cells with a single chromosome, induction of a DSB with 500 μM vanillate at +780 kb from the origin resulted in bidirectional loss of DNA around the break site, with an ~30% and ~40% drop in reads at the DSB site after 0.5 h and 1 h respectively . After 2ΔaddAB cells. These cells did not show any significant drop in reads near the DSB site, or elsewhere in the genome , which can report on total DNA, including any single-stranded DNA that may be missed in our sequencing assay. If AddAB were only unwinding the DNA flanking a DSB, then qPCR using primer pairs at loci adjacent to a DSB should yield significantly more product than a primer pair that spans the DSB site itself. However, the qPCR values for loci immediately adjacent to a DSB site at +780 kb were comparable to the value for the DSB site itself, and to the values measured by our sequencing-based profiling, supporting the notion that AddAB initially degrades both strands of DNA (chi sites (see below), switches to resecting a single strand. There is also a formal possibility that AddAB does initially just unwind the DSB end and that other nucleases in the cell degrade each strand, but such a model is less parsimonious as it invokes additional components that are not necessary in vitro.The library preparation procedure used should, in principle, only result in the sequencing of double-stranded DNA. However, DSB resection could, in principle, result in the production of some single-stranded DNA. To ensure that we were not sequencing single-stranded DNA, we treated genomic DNA extracted from a DSB-induced sample with Mung bean nuclease, which degrades single-stranded DNA. The resulting degradation profile was indistinguishable from that of a sample not treated with the nuclease , indicats of DNA . The qPCin vivo can be, at most, ~100 bp/s to the left and ~200 bp/s to the right. However, because the profiles are not step functions and instead feature a gradual decrease from +450 and +1300 kb toward the DSB site, it implies that most cells degrade DNA more slowly than 100–200 bp/s or initiate DSB processing at different times. To test this latter possibility, we measured cell viability as a function of time after adding vanillate to induce I-SceI and a DSB. Because we induce DSBs in swarmer cells containing a single chromosome, recombination-based repair cannot occur and a DSB is lethal. Thus, if all cells experienced a DSB immediately after the addition of vanillate, we would expect a precipitous drop in viability post-induction. Instead, we found a gradual decrease in viability suggesting that cells likely experience a DSB at variable times chi sequeing Figs  and S2A ing Figs  and S2B e origin  suggestiCaulobacter chi sequence and whether the presence of this sequence explains the asymmetry of our profiles, we inserted an array of 15 such sequences on the chromosome either +30 kb or +100 kb from the DSB site at +780 kb. This chi array was inserted either in the correct orientation for recognition by AddAB (chifor) or in the opposite orientation as a control (chirev). After inducing a DSB, the chirev construct had no effect on degradation  [in vivo  , which i[in vivo , but sencA cells . In asyncA cells . In cellcA cells . These rin vivo, both before and after chi recognition. These rates have been measured previously in vitro for B. subtilis AddAB, although only in the absence of RecA [B. subtilis AddAB translocation rate before chi recognition was reported to be between 400 and 2000 bp/s, with a chi recognition probability of ~0.25, and a post-chi translocation rate only ~15% slower than the pre-chi rate. To estimate the in vivo rates and chi recognition probability, we ran a simulation of DNA degradation/processing with four parameters: (i) the rate of DSB formation after adding inducer, which produces a distribution of times post-induction when DSB processing begins, (ii) the degradation rate before chi recognition, (iii) the probability of recognizing each chi site, and (iv) the DNA processing rate after chi recognition. Rates of DSB formation were estimated via simulations  where DnaA is not expressed, and DSBs were induced for indicated times by the addition of 500 μM vanillate. Note that the I-SceI construct used harbors an ssrA tag at the C-terminus which likely renders it unstable [ssrA tag is recognized by the ClpXP protease, which then degrades the I-SceI enzyme.Strains and plasmids used are listed in unstable ; this alE. coli BTH101. Co-transformants were grown until saturation in M63 media with maltose, IPTG and appropriate antibiotics and 5 μL of culture was spotted on MacConkey agar (40 g/L) plates with maltose, IPTG and appropriate antibiotics. Plates were incubated at 30°C for 2–3 days.Bacterial two hybrid experiments were performed as described in . BrieflyCells were pelleted and then resuspended in 1xSDS sample buffer and heated to 95°C for 5 min. Equal amounts of total protein were run on 10% Tris-HCl gels (Bio-Rad) at 150V for separation. Resolved proteins were transferred to polyvinylidene fluoride membranes and probed with 1:5000 dilution of primary antibodies against RecA (Sigma) and secondary horseradish-peroxidase-conjugated antibody (1:5000). Blots were visualized using a FluorChem M imager (ProteinSimple).Caulobacter cells were depleted of DnaA for 1.5 h and G1-arrested cells were then isolated by Percoll density gradient centrifugation. Swarmer cells were then released into DnaA depleting conditions (without IPTG) and DSBs were induced for 1 h by the addition of 500 μM vanillate. Cells were pelleted and genomic DNA was isolated using the DNeasy Blood and Tissue kit from Qiagen. For Mung bean treatment, isolated genomic DNA was incubated at 30°C for 15 min with Mung bean nuclease [nuclease  (NEB) anCaulobacter cells were depleted of DnaA for 1.5 h and G1-arrested cells were then isolated by Percoll density gradient centrifugation. Swarmer cells were then released into DnaA depleting conditions (without IPTG) and DSBs were induced for 1 h by the addition of 500 μM vanillate. Cells were pelleted and frozen in liquid nitrogen for RNA extraction. Cells (in pellets) were lysed by treatment with 400 μL of 65°C-preheated Trizol (Thermoscientific) for 10 min on a thermomixer at 200 rpm. They were frozen at -80°C for 30 min and then centrifuged at 4°C at maximum speed for 5 min. Supernatant was aspirated and added directly to 400 μL of 100% ethanol. The mixture was applied to an RNA-extraction spin column (Zymo Research). The column was then spun at 10000 rpm for 30 s and the spin column was washed with 400 μL of RNA Prewash solution twice and finally with 700 μL of RNA Wash buffer. Residual RNA Wash buffer was removed by an additional centrifugation step. RNA was eluted out with 90 μL of DEPC-treated water. DNase I treatment was carried out to remove any genomic DNA and the RNA was purified using acidic phenol-chloroform extraction. The integrity of the RNA was checked via agarose gel and submitted for Illumina sequencing .Caulobacter NA1000 reference genome (4.01 Mbp) (NCBI Reference Sequence: NC-011916.1) using Bowtie 1 [For analysis of DNA sequencing data, Hiseq 2500 Illumina short reads (40 bp) were mapped to the Bowtie 1  using th-2014-bowtie --sam *.fastqbowtie -m 1 -n 1 --best --strata -p 4 --chunkmbs 512 NA1000Caulobacter NA1000 genome was divided into 4000 bins and mapped Illumina reads were allocated to their corresponding bins to quantify the number of reads in each genomic bin. For samples where the DSB was introduced at 780 kb, datasets were normalized so that the experimental and the control dataset have the same number of Illumina reads between genomic position 2800 kb and 3400 kb. This genomic region was chosen since it is far from the DSB and was not affected by AddAB-induced DNA degradation. For samples where DSB was introduced at 3042 kb, datasets were normalized to have the same number of Illumina reads between genomic position 600 kb and 1200 kb instead. The enrichment between experimental datasets (DSBs were induced) and control dataset (no DSBs) is represented as the ratio of read counts in each bin between the experiment and the control, smoothed using the Lowess function in R with the smoothing bandwidth set to 0.01, and plotted against the genomic positions.The Caulobacter NA1000 reference genome (NCBI Reference Sequence: NC-011916.1) using Bowtie 1 using the following command:For analysis of RNA-seq data, Hiseq 2500 Illumina short reads (40 bp) were mapped back to the -2014-bowtie --sam *.fastqbowtie -m 1 -n 1 --best --strata -p 4 --chunkmbs 512 NA1000Caulobacter NA1000 was downloaded from NCBI (ftp://ftp.ncbi.nih.gov/genomes/archive/old_genbank/Bacteria/Caulobacter_crescentus_NA1000_uid32027/). The normalized value of reads per kb per million mapped reads (RPKPM) was calculated for each gene by a custom R script to enable comparison of gene expression within and between RNA-seq datasets. The enrichment between experimental datasets (DSBs were induced) and control dataset (no DSBs) is represented as the ratio of RPKPM of each gene between the experiment and the control and plotted against the genomic positions.The sequencing coverage was computed using BEDTools . The genaddAB cells treated with 2 or 500 μM vanillate to induce a DSB +780 kb from the origin. This was normalized to qPCR using probes across a control region  where no DSB is induced. As a control, qPCR across the DSB site was performed on chromosomal DNA isolated from swarmer or asynchronous cells with no DSB induction. Samples were taken 0, 5, 10, 15, 30, 60, 120 and 240 min after DSB induction. The rate of DSB induction was estimated by calculating the slope of the curve (excluding the 240 min time point). To measure total DNA at a DSB and at flanking loci, we performed qPCR using primer pairs as indicated in rpoA. These normalized values were then divided by similarly normalized values, but from cells in which a DSB was not induced. The resulting ratios (+DSB / -DSB) for each locus were are reported in recA) . The Ct values were converted to amounts using the following formula: 2^/slope).To estimate rates of DSB formation, qPCR was performed using one set of probes across the DSB site on chromosomal DNA isolated from swarmer or asynchronous Δ°C and a definite focus system for automatic maintenance of focus. Images were acquired via the metamorph imaging system and data analyzed on ImageJ. Swarmer cells were isolated as described above and then grown on PYE + 1.5% low-melting agarose pads with xylose and vanillate and imaged in a glass-bottomed petri dish. Images were acquired every 4 min for ML2402 and every 8 min for ML2401. Rate of degradation was calculated as the number of frames it took to go from the loss of the MipZ marker until the loss of the ParBpMT1 marker. Scale bars in figure = 1 μm.Fluorescence microscopy was performed on the Zeiss observer Z1 microscope with the LED-Collibri illumination system, 100x oil-immersion objective, Zeiss Temp module to maintain temperature at 30Sequencing data are available in GEO, GSE86913.S1 Fig(A) Flow cytometry profiles of swarmer cells before and after treatment with 500 μM vanillate for 1 h. (B) Representative DSB processing profiles for DSBs induced at +780 kb for 0.5 h (grey) or 1 h (black). 1 h profile is overlaid from (TIF)Click here for additional data file.S2 Figchi sequences on the leading strand (A) or lagging strand (B) with respect to a DSB site at +780 kb are overlaid on the profile. (C-D) Positions of chi sequences on the leading strand (C) or lagging strand (D) with respect to a DSB site at +3042 kb are overlaid on the DSB processing profile. (E-F) Genome-wide profiles from chi sequences (chifor) was inserted +30 kb (E) or +100 kb (F) from the DSB site at +780 kb and the profile is shown in green. As a control, the chi orientation is flipped (chirev) at the same location and the profile is shown in orange. Degradation profile of a DSB induced at +780 kb is shown from (A-B) Genome-wide profiles from (TIF)Click here for additional data file.S3 FigrecA cells. The location of putative chi sites on the top and bottom strands are indicated with red and blue, respectively, tick marks  was also performed. qPCR values at each locus normalized to rpoA are plotted for two independent repeats (a and b). DSB processing profile values for the same loci are also shown. (C) Representative DSB processing profile of ΔrecA swarmer cells when a DSB is induced at +780 kb for 1 h. Normalized data are shown in pink, with a Lowess smoothed curve overlaid in red. Location of the DSB site is indicated with a dashed line. Profile of wild type swarmer cells from ΔrecA cells. Each spot is a 10-fold dilution.(A-B) qPCR was performed using the primer pairs at the genomic positions indicated in Δalso see . Samples(TIF)Click here for additional data file.S4 FigrpoA). Error bars represent SD between two biological replicates. (D) Rates of DSB formation were calculated from panel C.(A) Histogram showing distribution of number of DSB events per cell in 100 s intervals expected over time  from the time of I-SceI induction to DSB onset. Dashed line indicates the value 1 h after DSB induction (B) Graph showing fraction of cells expected to have DSB over time from the time of I-SceI induction to DSB onset. Dashed line indicates the value 1 h after DSB induction. See (TIF)Click here for additional data file.S5 Figchi AddAB degradation rates are estimated. Pre-chi AddAB degradation rate is 400 bp/s and chi recognition probability is found to be ~0.22. DSB is induced at +3042 kb for 1, 2 or 4 h. Experimental profiles are overlaid in black, grey and brown respectively. Simulation profiles are in green (B-C) Simulation profiles obtained for Model 1 (red) and 2 (blue) that best fit the in vivo DNA processing profiles for a DSB induced at +3042 kb for 1 h and 2 h samples respectively. Experimental profiles from (A) Simulation profiles obtained for a model where post-(TIF)Click here for additional data file.S6 Figchi recognition probability (pchi) for models considered to best fit the in vivo wild-type DSB processing profiles. In all cases, dashed red lines represent the best fits and 95% confidence intervals. (B) Graph showing RMS deviation as a function of post-chi AddAB degradation rate (kslow) for Model 2. Post-chi AddAB degradation rate (kslow) for Model 1 is fixed at 340 bp/s. (C-D) Graphs showing RMS deviations as a function of post-chi AddAB dissociation rate (koff) for Models 1 and 2.(A) Graph showing RMS deviation for (TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


NFκB1 mRNA expression inversely correlated  with tri-methylation of lysine 27 on histone 3 near its promoter region in a time-dependent manner. These results indicate that PEITC may slow down the development of colon carcinogenesis in an inflammatory intestinal setting which is potentially associated with epigenetic modulation of NFκB1 signaling.We have previously reported alleviation of dextran sodium sulfate (DSS)-induced ulcerative colitis signs in phenethyl isothiocyanate (PEITC)-treated mice. Here we investigated chemoprotective activities of PEITC in mice with Azoxymethane-DSS induced colitis associated colon carcinogenesis. We also examined the molecular mediators associated with the PEITC effects using relevant cell lines. A 0.12% PEITC-enriched mouse-diet reduced mucosal and submucosal inflammation as well as glandular atypia by 12% and the frequency of adenocarcinoma by 17% with a concomitant improvement in overall disease activity indices compared to the diseased control group. Lipopolysaccharide-induced in vitro up-regulation of key mediators of inflammation, immune response, apoptosis, and cell proliferation were attenuated by 10 μM PEITC. Three of these mediators showed concentration-dependent reduction in respective mRNAs. Furthermore, PEITC inhibited Nuclear factor kappa B1 (NFκB1) proteins in a concentration-dependent manner. The  Colorectal cancer (CRC) is second among various cancers in terms of causing deaths in the United States, and it resulted in an estimated healthcare expenditure of $14 billion in 2014. Over 90% of localized CRC, when diagnosed early, would have a 5-year survival rate. However, early detection is less common due to lack of compliance to screening regimens. For late diagnosed metastatic disease, survival rate drops to 8–12%. Cytotoxic chemotherapy is the only available treatment option for unresectable metastatic disease, to which the initial response frequently tapers off due to ensuing resistance within six months [https://www.cdc.gov/ibd/data-statistics.htm). Currently, there is no medical cure for IBD and therefore, patients require a lifetime of care.The risk of developing CRC increases in patients with inflammatory bowel disease (IBD) . IBD is Phenethyl isothiocyanate (PEITC) is—derived from common cruciferous vegetables. Our team and other laboratories have reported earlier that PEITC has anti-inflammatory and chemopreventive effects against various cancers ,7,8,9,10p = 0.00015 and 0.026 respectively) compared to mice that remained healthy throughout the experiment. The slowdown in weight gain was, however, partially recovered when mice received 0.12% PEITC enrichment in diet  and disease control (DC), and one PEITC-diet test group a. Body wp = 0.11) lower histological Disease Activity Index (hDAI) in the PEITC-diet group comparing with the DC group. The criteria included for scoring visible Disease Activity Index (vDAI) included rear end inflammation, rectal bleeding, and stool consistency and was 54% lower in the PEITC-diet group compared to DC mice  and colon tumor derived epithelial cells (SW480). The concentration range of PEITC was selected based on published studies ,16. At 6(NFκB) signaling network , chemokine (C–C motif) ligand 2 , and chemokine (C–X–C motif) ligand 10  as well as transcription factor NFκB1  mRNAs compared with positive control cells (lipopolysaccharide induced and vehicle treated) in colon epithelial and macrophage cells, respectively  with a time-dependent changes in NFκB1 mRNA expression  has remained elusive. However, growing evidence points to the NFκB-signaling pathway that regulates cell proliferation, apoptosis, and inflammatory responses. NFκB activation was reported in 40% of colon tumors and in the majority of CRC cell lines. The most commonly studied erodimer ,30,31. Herodimer ,33. Furterodimer . Since tresponse . The CCLgression . Finallypatients . At non-NFκB1 and associated expression of interacting genes were down regulated after PEITC exposure. The PEITC associated reduced expression of the NFκB1, correlated with changes in a gene-suppressing histone modification mark. This indicates at least partially that PEITC activity leads to a potential epigenetic regulation of NFκB1 that relates to its anti-inflammatory effects. However, since the epigenetic repression appeared to be reversible with time, further investigation is warranted in the future to understand if there are parallel inflammation-regulatory mechanisms induced by PEITC. Together, the results from this otherwise small scale pilot study provide a basis for further investigation of PEITC as a chemoprotective agent for CAC.In summary, using an AOM-initiated and DSS-promoted CAC model, we show that a PEITC-enriched diet may slow down colon cancer development in experimental mice. Follow-up molecular investigations in CAC-relevant cells showed inhibition of cancer cell proliferation by PEITC at higher concentrations. At non-cytotoxic concentrations, a key pro-inflammatory transcription factor Escherichia coli, O55:B5), penicillin/streptomycin, and phenethyl isothiocyanate (PEITC) were purchased from Sigma-Aldrich . The β-actin antibody was purchased from Santa Cruz Biotechnology , the NFκB1antibody was from Millipore , and the Dylight 800 anti-rabbit secondary antibody came from Li-Cor Biosciences . Also, for chromatin immuno-precipitation (ChIP) assay, the enzyme micrococcal nuclease (MNase), as well as anti-trimethyl-Histone H3 Lys27 and rabbit IgG for negative control samples were supplied by Cell Signaling , Upstate Biotechnology , respectively. IDT DNA Inc.,  synthesized all oligonucleotides. Dextran Sulfate Sodium  was obtained from MP Biomedicals .For cell culture, Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from HyClone , fetal bovine serum (FBS), and TrypLE were purchased from Invitrogen Gibco . Human interferon-γ (IFNγ) was purchased from R&D Systems . Dimethyl sulphoxide (DMSO), lipo-polysaccharide  for the protocol titled: An alternate therapy for treatment of IBD and prevention of colon cancer) and followed the guidelines set forth by the Animal Welfare Act and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Eight weeks old C57BL/6J male mice  were used. The housing for the mice maintained a constant temperature of 24–26 °C with 12 h light/dark cycle with free access to food and drinking water. Carbon dioxide inhalation was used for euthanasia after 16 weeks treatment. After terminal collection of the colons, they were flushed with sterile PBS, measured, and subsequently stored in 10% neutral buffered formalin for further processing. Hematoxylin and Eosin (H&E) stained cross-sections (~6 μm) were subjected to histological evaluation (×100 magnification) in a blinded manner.The mice were randomized into three groups, which are described in 2-humidified atmosphere at 37 °C. Briefly, RAW264.7 cells were treated with PEITC or DMSO (as a negative control) at a pre-determined dose for 6 h before elicitation with 1 μg/mL of LPS without IFNγ priming. SW480 cells were pretreated with IFNγ 10 ng/mL or control medium (as a negative control) for 12 h, and treated with PEITC or DMSO for 5 h and then stimulated with LPS 10 ng/mL for 4 h. PEITC treatments were performed at 5, 10 and 15 μM concentration. Relative number of viable cells were measured using Cell Proliferation Assay kit -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; Promega, Madison, WI, USA) following the manufacturer’s instructions.The mouse monocyte/macrophage cell line, RAW 264.7 (ATCC TIB-71), and human colon cancer cell line, SW480 (ATCC CCL-228), were purchased from ATCC  and cultured and treated as previously described by Dey et al.  and Liu Ct−ΔΔ method [RNA extraction, purification, cDNA synthesis and real-time PCR were performed as previously described by our group ,26. All t method . The mRNFor immunoblot analyses, IFNγ-primed PEITC-treated SW480 cells were activated with LPS for 4 h and harvested using RIPA lysis buffer. The protein concentrations were determined using Pierce BCA Protein Assay kit . Proteins (35–50 μg/lane) were separated by 12% SDS-PAGE prior to electrotransfer on polyvinyldene difluoride (PVDF) membranes . Blocking was acheived with 5% skim milk for 1 h. Incubation with primary antibodies occurred at 4 °C overnight, after which incubation with Dylight 800 anti-rabbit secondary antibody occurred for 1 h, followed by washing 3 times in PBS/T (0.1% Tween20 in PBS) at room temperature. Blots were imaged after rinsing in PBS containing 0.1% Tween20 followed by quantitative analyses using an Odyssey infrared imaging system (Li-Cor).ChIP assay was performed as described by Liu et al. . Brieflyt-test. All in vitro experiments were repeated 3 times. A probability (p) value of 0.05 or less was considered to be the criterion for a significant difference. For correlation analysis, Person’s r method was used.Data are expressed as mean ± SEM. We performed one-way analysis of variance (ANOVA) to determine the significance between groups followed by the post-hoc Dunnett test. Intergroup comparisons in western blot and ChIP assay in human cells were determined using


Roots are subjected to a range of abiotic stresses as they forage for water and nutrients. Cytosolic free calcium is a common second messenger in the signaling of abiotic stress. In addition, roots take up calcium both as a nutrient and to stimulate exocytosis in growth. For calcium to fulfill its multiple roles must require strict spatio-temporal regulation of its uptake and efflux across the plasma membrane, its buffering in the cytosol and its sequestration or release from internal stores. This prompts the question of how specificity of signaling output can be achieved against the background of calcium’s other uses. Threats to agriculture such as salinity, water availability and hypoxia are signaled through calcium. Nutrient deficiency is also emerging as a stress that is signaled through cytosolic free calcium, with progress in potassium, nitrate and boron deficiency signaling now being made. Heavy metals have the capacity to trigger or modulate root calcium signaling depending on their dose and their capacity to catalyze production of hydroxyl radicals. Mechanical stress and cold stress can both trigger an increase in root cytosolic free calcium, with the possibility of membrane deformation playing a part in initiating the calcium signal. This review addresses progress in identifying the calcium transporting proteins (particularly channels such as annexins and cyclic nucleotide-gated channels) that effect stress-induced calcium increases in roots and explores links to reactive oxygen species, lipid signaling, and the unfolded protein response. They lack kinase domains, suggesting these proteins must target others with enzymatic activity. CaMs modulate transcription by binding to Calmodulin-binding Transcription Activators (CAMTAs) (2+-Dependent Protein Kinases (CPKs) and Calcineurin-B Like proteins (CBLs). The latter target CBL-Interacting Protein Kinases (CIPKs) to effect cellular responses  while Phospholipase D catalyses production of phosphatidic acid, thus [Ca2+]cyt would have the capacity to trigger distinct lipid signals depending on the location and Ca2+-sensitivity of the phospholipases. Targets of lipid signals have been reviewed recently by Plant roots are exposed to a variety of abiotic stresses as they navigate the soil, foraging for nutrients and water. Cytosolic free calcium  . Other mesponses . Changesfamilies . Phospho2+]cyt measurements are from Arabidopsis thaliana seedlings and guard cells, achieved using the luminescent Ca2+-interacting aequorin protein. Far fewer studies have focused specifically on roots or utilized the greater sensitivity and spatial resolution of ratiometric fluorescent dyes. The genetically encoded YC3.6 Ca2+ reporter is now being used for both Arabidopsis and rice roots  or depolarize (render it less negative). Manipulating PM voltage elicits distinct [Ca2+]cyt signatures and resultant transcriptional responses (Hyperpolarization-Activated Ca2+ Channels (HACCs); Depolarization-Activated Ca2+Channels (DACCS); Voltage-Independent Ca2+Channels (VICCs) (2+ influx routes is afforded by reactive oxygen species (ROS) that are produced during development and stress responses . This regulation depends on the specific ROS, its position, the cell type and the cell’s developmental state. In Arabidopsis roots, sensitivity of PM Ca2+ channel activation by extracellular H2O2 decreases as epidermal cells mature but is still greater than that of the cortex  have advanced our understanding of the Caesponses . Studies (VICCs) . Thus che cortex . A similericycle . In epidlar H2O2 , 2007. T2+]cyt elevation in roots remains poorly understood in terms of the genes encoding the PM or endo membrane Ca2+ channels involved. Plants have multi-gene families of Glutamate Receptor-Like channels  and Cyclic Nucleotide-Gated channels , with each gene encoding a potential subunit of a potentially tetrameric channel. Some members have been characterized as having Ca2+ channel forming ability  thus leading to the threat of hypoxic challenge from waterlogged soil . Salinit+ influx into the root are not fully known but include the PM cyclic nucleotide-gated channels CNGC3 . Both channels types are present in Arabidopsis root epidermal PM  was sensitive to inhibition of soluble guanylyl cyclase activity and the signal evoked by an equivalent osmotic stress was insensitive . This channel mediates the [Ca2+]cyt response to hyperosmotic stress , with [Ca2+]cyt activating these enzymes through their EF hands . In accordance with this, Arabidopsis root cortical cells lacking RBOHD and F have much lower PM hyperpolarization-activated Ca2+ activity in response to NaCl challenge than wild type  acts to limit NaCl-induced [Ca2+]cyt increase, partly by limiting Ca2+ release from mitochondria. In the arp2/3 mutant, the [Ca2+]cyt increase is greater than wild type and so is the extent of mitochondrial-driven cell death  and inositol trisphosphate (InsP3) suggested involvement of the InsP3 pathway in Arabidopsis roots  are firmly implicated in salt stress responses  in Arabidopsis leaves  would bind Ca2+, and open to release K+ to the cytosol to maintain a favorable Na+/K+ ratio  acts to promote Na+ efflux  would restore [Ca2+]cyt to pre-stimulus levels  which can then react with the serine/threonine protein kinase CIPK24 (SOS2) (+ antiporter SOS1 at its auto-inhibitory C-terminus to achieve activation of Na+ efflux (Arabidopsis by Mitogen Activated Protein Kinase6 (MPK6) and loss of MPK6 function impairs root growth under salt stress  pathway lies downstream of the root cyt increase activates the root PM Phospholipase C2  as a diagnostic of a UPR response  cells containing the Fura-2 Ca2+-reporting dye lead to the identification of Calcium-permeable Stress-gated cation Channel1   . PERK4’songation . This imlevation . Anotherlevation .. GLRs abidopsis . The [Cass genes . Droughtss genes . In maizss genes .2+]cyt and is relevant to waterlogged soils. In Arabidopsis, root PM harbors two mechanosensitive Ca2+-permeable channels, MCA1 and MCA2  PM voltage as H+ efflux is curtailed. The extent and duration of membrane depolarization varies with sensitivity to O2 deprivation and cell type. Values of -70 to -80 mV have been reported for O2-deprived barley root cells  are activated  or absence (anoxia) causes transient increases in root cyt with clearance to the apoplast by ACA10-13 and sequestration to the vacuole by CAX3. How they are regulated remains to be determined, as does the involvement of the structurally compromised wall and the consequence of this higher level of apical [Ca2+]cyt. The area of higher [Ca2+]cyt reported appears to correspond with the zone of inhibition of primary root elongation and the induction of cell death , as were three CPK genes  all suggesting a distinct change in intracellular Ca2+ signaling (WRKY transcription factors (TF) , two three MYB family TF  and downregulation of two BZIP family TFs   . B depriroot tip . It has  nucleus  and this nucleus , suggest3 and above). Although some of these metals  are required as micronutrients they can be damaging in excess whilst others (such as Cd) have no physiological role and can be deleterious even at low concentrations, often impairing mineral nutrition. The consequences of heavy metal exposure have been reviewed recently by 2+), intersects with hormonal responses and detoxification.At the opposite end of nutritional deprivation is heavy metal stress. Industrial activity, mining and modern agricultural practices can lead to soil contamination by heavy metals (defined here as 7 g/cm2+-based processes because of its similar size. A recent review by 2+ a key question is whether Cd (as a Ca2+ “substitute”) generates a signal in its own right or whether its impairment of Ca2+ homeostasis will be, in effect, the signal. Certainly Cd depolarises the rice root epidermal PM, which would impair Ca2+ influx and results in inhibition of root elongation  and activate a possible DACC in wheat root PM  were identified in Arabidopsis due to their sequence similarity to the bacterial Mechanosensitive channels of small conductance (MscS)  . These aArabidopsis mechano-stimulated [Ca2+]cyt increase and downstream transcriptional regulation  may be influenced by mechano-sensing. At this point, it is unclear whether FERONIA also plays a role in the mechanical induction of lateral root formation   have been reviewed by es e.g., . By usinn leaves . Using ae CRLK1; . This woe CRLK1; . This pae CRLK1; . [Ca2+]cignaling . Part ofFactor2) .Triana bogotensis  and elevation of root [Ca2+]cyt (aequorin and cameleon determinations). This indicates that heterotrimeric G proteins lie upstream of PM Ca2+ channels in the cold response. In common with other stress responses, NADPH oxidase activity also appears in cold stress and needs to be placed in relation to Ca2+ channels. Arabidopsis roots exposed to cold stress use AtSRC2 (Soybean gene Regulated by Cold2) to enhance Ca2+ activation of the AtRBOHF NADPH Oxidase (2+ channel activity. Curtailing cold-induced [Ca2+]cyt elevation could involve vacuolar CAX1 activity in Arabidopsis (Cold stress has been found to depolarise the PM of root cells from cucumber and gotensis , consistof roots . Cold haof roots  but this PM DACC , this cotoplasts . Actin don cells . This inon cells . It woulolerance . Converseedlings . The posubunit α . It is r Oxidase . This subidopsis .2+]cyt increases, and by extension those of organelles. Many members of channel gene families still await characterization. The identification of new families of channels is challenging and will require different approaches linking forward and reverse genetics to electrophysiology. Targets of Ca2+-binding and interacting proteins also require further study. Components common to different abiotic stresses are emerging such as Arabidopsis CIPK23 in K+ and nitrate deprivation. These common regulatory components are likely to represent critical steps where complex stress signals encountered in the soil are integrated in unified responses. Receptor like kinases such as FERONIA or PERK4 have emerged as new components in [Ca2+]cyt signaling and perhaps other related proteins will be found to have a role in abiotic stress signaling. Remodeling of calcium signaling machinery after stress is also apparent with the possibility of components common to different stresses. For example, Arabidopsis CNGC19 is upregulated under B limitation and salinity stress (2+]cyt-dependent transcriptional responses can be delineated and future work could include the impact of stress-induced calcium signaling on epigenetic inheritance (A repeated message from this review is how incomplete our knowledge is of the channels mediating stress-induced [Cay stress . Finallyeritance .All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication. KW, EM, SS, and JD wrote the review. KW produced the figures.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.


It is now known that Stromal Interaction Molecule (STIM), the Ca2+ sensor of the intracellular compartments, together with Orai and Transient Receptor Potential Canonical (TRPC), the subunits of Ca2+ permeable channels on the plasma membrane, cooperate in regulating multiple cellular functions as diverse as proliferation, differentiation, migration, gene expression, and many others, depending on the cell type. In particular, a growing body of evidences suggests that a tight control of SOCE expression and function is achieved by megakaryocytes along their route from hematopoietic stem cells to platelet production. This review attempts to provide an overview about the SOCE dynamics in megakaryocyte development, with a focus on most recent findings related to its involvement in physiological and pathological thrombopoiesis.Store-Operated Calcium Entry (SOCE) is a universal calcium (Ca In resting states, the sarco/endoplasmic reticulum (SR/ER) Ca2+-ATPase (SERCA) pump, along with plasma membrane Ca2+-ATPase (PMCA) and the Na+/Ca2+ exchanger (NCX), determine the basal [Ca2+]i; while, upon stimulation by extracellular signals, [Ca2+]i increases by Ca2+ release from intracellular stores or extracellular Ca2+ influx into the cell ER) ranges between 400 and 600 µM, STIM2 is activated by significantly smaller depletion of the luminal Ca2+ pool as compared to STIM1 i resulting in impaired platelet activation i upon collagen stimulation i is achieved by megakaryocytes along their route to platelet production by different stimuli that integrate in order to ensure the fine balance between cell proliferation and differentiation.All together, these data suggest that a tight control of [CaSTIM1Sax/+), resulting in the constitutive activation of SOCE and consequent high cytoplasmic Ca2+ levels [2+ levels in circulating platelets responsible for a pre-activation state and consequent increased platelet consumption [STIM1Sax/+ mice was accompanied, at the age of 6 months, by the appearance of bone marrow fibrosis and severe splenomegaly [A pronounced megakaryocyte hyperplasia has been shown in heterozygous mice expressing an activating EF hand mutant of STIM1 (+ levels . Macrothsumption , furthernomegaly , all symnomegaly ,184. It nomegaly . Interesnomegaly ,186,187.nomegaly . ImportaMPL gene, leading to a constitutive activation of c-Mpl, the TPO receptor, and in other ~60% by mutations in JAK2 gene, resulting in the constitutive activation of its signaling pathway which is normally activated downstream of c-Mpl upon TPO stimulation [CALR, the gene encoding for the ER chaperone calreticulin, which was found in ~25% of patients with MPL or JAK2 unmutated disease [MPL and JAK2 gene mutations, megakaryocytes expressing the mutated calreticulin present constitutive stimulation of c-Mpl downstream signaling, due to an unexpected activating interaction between the receptor and the mutated protein [2+ storage and buffering [2+ homeostasis in megakaryocytes harboring the different mutations. To this regard, we demonstrated that the type-1 mutation enhanced both ER-dependent intracellular Ca2+ release and SOCE with respect to healthy controls, and JAK2 or type-2 mutated megakaryocytes [2+ mobilization or to the end tail modifications of calreticulin mutants still remains to be elucidated. The higher intraluminal Ca2+ discharge is in line with the notion that, in type-1 mutation, the negatively charged amminoacids responsible for Ca2+ binding at the C-terminal are almost entirely replaced by either positively charged or neutral residues, while type-2 mutation does not result in the loss of stretch II and III [2+ entry may have a major impact in promoting cell proliferation and migration, thus resulting in increased platelet release, justifying the augmented platelet count observed in these patients (Both PMF end ET are caused in ~10% of cases by mutations of mulation . Recent  disease ,190. As  protein ,192. Caluffering . All theuffering . In partryocytes . Whether and III . Intrigu and III ,196,197, and III . Based opatients . Further2+ entry, many researches have been devoted to identify molecular and mechanistic process related to SOCE. In this review, we have summarized the whole machinery of SOCE, which involve STIMs, Orais and TRPCs in an elegant choreography that connects intracellular Ca2+ release to plasma membrane channels promoting extracellular Ca2+ entry. In megakaryocytes SOCE regulates fundamental cellular functions, such as proliferation, migration and sensing of extracellular environment. However, little is still known about the specific molecular and biochemical signals that are targeted by Ca2+ in order to drive thrombopoiesis. Understanding in details the exact mechanisms by which SOCE control megakaryocyte functions would be instrumental for the identification of novel pathways that may be involved in the pathogenesis of megakaryocyte-related diseases.After 30 years form the first description of capacitative Ca


Tridacna squamosa, lives in symbiosis with zooxanthellae which reside extracellularly inside a tubular system. Zooxanthellae fix inorganic carbon (Ci) during insolation and donate photosynthate to the host. Carbonic anhydrases catalyze the interconversion of CO2 and HCO3−, of which carbonic anhydrase 2 (CA2) is the most ubiquitous and involved in many biological processes. This study aimed to clone a CA2 homolog (CA2‐like) from the fleshy and colorful outer mantle as well as the thin and whitish inner mantle of T. squamosa, to determine its cellular and subcellular localization, and to examine the effects of light exposure on its gene and protein expression levels. The cDNA coding sequence of CA2‐like from T. squamosa comprised 789 bp, encoding 263 amino acids with an estimated molecular mass of 29.6 kDa. A phenogramic analysis of the deduced CA2‐like sequence denoted an animal origin. CA2‐like was not detectable in the shell‐facing epithelium of the inner mantle adjacent to the extrapallial fluid. Hence, CA2‐like is unlikely to participate directly in light‐enhanced calcification. By contrast, the outer mantle, which contains the highest density of tertiary tubules and zooxanthellae, displayed high level of CA2‐like expression, and CA2‐like was localized to the tubule epithelial cells. More importantly, exposure to light induced significant increases in the protein abundance of CA2‐like in the outer mantle. Hence, CA2‐like could probably take part in the increased supply of inorganic carbon (Ci) from the host clam to the symbiotic zooxanthellae when the latter conduct photosynthesis to fix Ci during light exposure.The fluted giant clam,  Tridacna squamosa, belongs to Phylum: Mollusca, Class: Bivalvia, Order: Veneroida, Family: Cardiidae, Subfamily: Tridacninae. It inhabits the shallow tropical water of the Indo‐Pacific coral reefs   Trench .i), nitrogen, and phosphorus to the symbionts in support of their metabolism and growth , according to the equation Ca2+ + HCO3− ⇌ CaCO3 + H+, from the extrapallial fluid onto the inner surfaces of the shell valves. The calcification process requires the participation of the thin and mostly nonpigmented inner mantle tissue delineated by the shell's pallial line. The inner mantle secretes Ca2+, HCO3−, and an organic matrix to the extrapallial fluid where calcification occurs, and CaCO3 is deposited onto the inner surface of the shell valve  which are zinc‐containing enzymes catalyzing these reactions with dramatic acceleration of CO2 hydration , with α‐CA being the largest and the most ubiquitous family  from the outer and inner mantle of T. squamosa. As described previously . Furthermore, the effects of 3, 6, or 12 h of light exposure on the mRNA expression levels of CA2‐like in the outer and inner mantle of T. squamosa, as compared with the control kept in darkness for 12 h, were determined. Based on the deduced CA2‐like sequence, an anti‐CA2‐like antibody was custom‐made to examine the effects of light exposure on its protein abundance in the outer and inner mantle of T. squamosa. It was hypothesized that the gene and protein expression levels of CA2‐like/CA2‐like in the outer and inner mantle, like those of Na+/H+ exchanger 3‐like transporter  approval is required for invertebrates including giant clams at the time the laboratory experiments were performed. The animals were anesthetized with 0.2% phenoxyethanol before killing to minimize any stress and suffering.T. squamosa  were obtained from Xanh Tuoi Tropical Fish Co., Ltd. (Vietnam), and kept in an indoor aquarium. They were maintained in 350 L of recirculating seawater in a glass tank (L 90 cm × W 62 cm × H 60 cm) under a 12‐h light:12‐h dark regime, with seawater conditions and light intensity as described previously  were sampled at the end of the 12‐h dark period of the 12‐h light:12‐h dark regime. In addition, four individuals (N = 4 for each time point) were sampled each at 3, 6, or 12 h after exposure to light . Parallel controls were not used in order to accurately simulate the dark/light hours experienced by the clams in their natural habitat. After being anesthetized in 0.2% phenoxyethanol, the giant clam was forced open, and the adductor muscle was cut. The pigmented outer mantle was dissected, and the nonpigmented inner mantle was excised. Samples of the ctenidia, hepatopancreas, foot muscle, byssal retractor muscle, adductor muscle, kidney, and heart were also collected. Collected tissue samples were blotted dry, immediately freeze clamped with aluminum tongs precooled by liquid nitrogen, and stored at −80°C until analysis. Tissues for immunofluorescence microscopy were sampled from T. squamosa which had been exposed to darkness or light for 12 h (N = 4 each) and anesthetized in 0.2% phenoxyethanol.For molecular work, four individuals of T. squamosa were performed using TRI Reagent™  and RNeasy Plus Mini Kit , respectively. The purified RNA was quantified by a Shimadzu BioSpec‐nano spectrophotometer , and its integrity was determined by electrophoresis. Then cDNA was synthesized from 4 μg of purified RNA using a RevertAid™ first‐strand cDNA synthesis kit .The extraction and purification of total RNA from the tissues of CA2‐like sequence from the outer and inner mantle of T. squamosa, degenerate primers (forward: 5′‐GGGWTACGAKRAKCATAATGG‐3′ and reverse: 5′‐CCCAGRTAGGTCCAGTAKTY‐3′) were designed based on the conserved regions of Crassostrea gigas CA2 (XM_011449590.2), Mytilus galloprovincialis CA2 (KT818923.1), Octopus bimaculoides CA2‐like (XM_014929272.1), and Danio rerio CA2 (NM_199215.1). PCR and cloning experiments were performed as described previously in Hiong et al.  were performed using the following primers: 5′‐ACCTGTGTTCGCAATTTCAAGACCTGAC‐3′ and 5′‐GGATTATCGCCAATATCAGACGGAGCCC‐3′.In order to obtain the partial g et al. . AnalysiCA2‐like has been deposited into GenBank (accession number MF042362).Samples were processed for sequencing using BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) with subsequent ethanol/sodium acetate precipitation. Purified products were automatically sequenced using the 3130XL Genetic Analyzer (Thermo Fisher Scientific). Sequence data were analyzed with BioEdit version 7.2.5 Hall . The cDNCA2‐like in the cDNAs of outer mantle, inner mantle, ctenidia, hepatopancreas, foot muscle, byssal retractor muscle, adductor muscle, kidney, and heart of T. squamosa by PCR using a set of gene‐specific primers (forward: 5′‐TCCATTACAACGAGAAGTACGG‐3′ and reverse: 5′‐GGCGATAATCCATCATACTGCT‐3′). Each PCR was performed in 10 μl reaction volume with thermal cycling conditions of 94°C for 3 min, followed by 24 cycles of 94°C for 30 sec, 50°C for 30 sec, 72°C for 1 min, and 1 cycle of final extension at 72°C for 10 min. The products were then separated on a 1% agarose gel by gel electrophoresis.The gene expression of CA2‐like sequence obtained from T. squamosa was translated using the ExPASy Proteomic server (http://web.expasy.org/translate/)  was extracted from the inner and outer mantle of T. squamosa as mentioned above and reverse‐transcribed using random hexamer primers with RevertAid™ first strand cDNA synthesis kit. qPCR was performed in triplicates using a StepOnePlus™ Real‐Time PCR System (Applied Biosystems). The mRNA expression level of CA2‐like was determined using specific forward (5′‐TCCATTACAACGAGAAGTACGG‐3′) and reverse (5′‐GGCGATAATCCATCATACTGCT‐3′) qPCR primers. The qPCR reactions contained 5 μL of 2X Fast SYBR® Green Master Mix (Applied Biosystems), 0.3 μmol L−1 of forward and reverse primers each, and various quantities of standard (to construct the standard curve) or 1 ng of sample cDNA in a total volume of 10 μL. The cycling conditions, melt curve analysis, and construction of a standard curve were performed according to the method of Hiong et al.  or anti‐α‐tubulin antibodies  for 1 h at 25°C. The primary antibodies were diluted in TTBS prior to use. Subsequently, the membranes were incubated in alkaline phosphatase‐conjugated secondary antibodies  for 1 h at 25°C. Bands were visualized using a BCIP/NBT Substrate Kit (Life Technologies). The blots were scanned using a CanoScan 9000F Mark II flatbed scanner in TIFF format at 600 dpi resolution. The optical density of the band was quantified using ImageJ , calibrated with a 37‐step reflection scanner scale . Results were presented as relative protein abundance of CA2‐like normalized with α‐tubulin. In order to validate the specificity of the anti‐CA2‐like antibody, the primary antibody (25 μg) was incubated with the immunizing peptide (125 μg) for 1 h at 25°C. The incubated mixture was diluted in TTBS and the aforementioned western blotting procedure was carried out as usual.A custom‐made anti‐CA2‐like polyclonal antibody (epitope sequence PLTKDDWGYEDHNG) was raised in rabbits by a commercial firm . Protein extraction and SDS‐PAGE were performed according to the methods of Hiong et al. ,b. Fiftyμg mL−1.Samples of outer mantle and inner mantle were excised and immersed in 3% paraformaldehyde diluted in seawater at 4°C overnight. Sample preparation and immunostaining were performed following the method of Hiong et al.  except tAfter immunostaining, sections were viewed under an Olympus BX60 epifluorescence microscope  mounted with an Olympus DP73 digital camera (Olympus Corporation) for image capturing. All images were captured under predetermined optimal exposure settings. Of note, zooxanthellae are known to contain pigments that autofluoresce  was used to perform statistical analyses. Levene's test was used to check homogeneity of variance. One‐way analyses of variance (ANOVA) followed by multiple comparisons of means by Dunnett's T3  or by Tukey's test  were used to evaluate differences among means and differences with CA2‐like was obtained from the outer and inner mantle of T. squamosa (GenBank accession number MF042362). No nucleotide polymorphism was observed between the CA2‐like cDNA sequences obtained from these two tissues. The open reading frame encoded for a 263‐amino acid polypeptide with an estimated molecular mass of 29.6 kDa.The complete cDNA coding sequence (789 bp) of T. squamosa was comparable to those of CA2 from other animal species. Phenogramic analyses showed that the CA2‐like of T. squamosa was grouped closely with the intracellular  forms of CAs of Homo sapiens, distinctly separated from the CAs of algae  and responsible for binding the substrate CO2 were fully conserved in the CAs of all the species analyzed  was conserved in the CA2‐like of T. squamosa. The gatekeeping residues Glu114 and Thr211 were also conserved . In both cases, the substituting residue was identical to either CA2 of mussel or teleost at the same location.An alignment of the deduced amino acid sequence of the CA2‐like of CA2‐like was detected in all the organs/tissues examined, except the kidney, with particularly strong expressions in the outer and inner mantle , the outer mantle, which contains the greatest density of zooxanthellae, had higher mRNA expression levels of CA2‐like  in the protein abundance of CA2‐like in the outer mantle of rol Fig. B and S1BT. squamosa. CA2‐like immunofluorescence was also detected in epithelial cells of the tertiary tubules located close to the seawater‐facing epithelium of the inner mantle  than azooxanthellate species  ⇌ ZnH2O + HCO3−. In the second stage, the Zn2+‐bound H2O molecule transfers one H+ to another residue within the active site of the enzyme, represented as X  are released toward the environment  which allows a proton to be transferred off the Zn2+‐bound water molecule to regenerate the active Zn2+‐hydroxide form of the enzyme  catalyzes a reaction essential to Ci from the host clam to the symbiotic zooxanthellae through the epithelium of the zooxanthellal tubules because of the following reasons. First, the outer mantle exhibited the highest level of host CA2‐like gene expression which corroborates the highest density of tertiary tubules and zooxanthellae therein. Second, CA2‐like was localized to the epithelial cells that form the zooxanthellal tubules. Third, exposure to light induced significant increases in the protein abundance of CA2‐like in the outer mantle. In light, CO2 is rapidly hydrated to HCO3− catalyzed by CA2‐like in the cytoplasm of the epithelial cells surrounding the zooxanthellal tubules. The HCO3− generated intracellularly can be transported into the lumen of the zooxanthellal tubule via some kind of apical HCO3− transporters  subunit A, ATP6V1A, from the outer mantle of T. squamosa, and confirmed that its gene and protein expression levels can be enhanced by light . Furthermore, ATP6V1A was localized to the apical (lumen‐facing) membrane of the epithelial cells of the zooxanthellal tubules , indicating that VHA is positioned to secret H+ into the lumen of the tubules during light exposure. When taken together, these results suggest that the epithelial cells of the zooxanthellal tubules in T. squamosa may possess a light‐enhanced and host‐mediated carbon‐concentrating mechanism (CCM) to facilitate the supply of Ci to the symbionts.Our results suggest for the first time that CA2‐like may participate in the increased supply of CPCO2 to enhance catalytic efficiency  in the outer mantle and inner mantle of Tridacna squamosa.Click here for additional data file. Click here for additional data file.


Familial Alzheimer’s disease (AD) is caused by mutations in the genes that encode amyloid precursor protein (APP) and presenilins. Disturbances in calcium homeostasis have been observed in various cellular and animal models of AD and are proposed to underlie the pathogenesis of the disease. Furthermore, wildtype presenilins were shown to regulate endoplasmic reticulum (ER) calcium homeostasis, although their precise mechanism of action remains controversial. To investigate whether APP also affects ER calcium levels, we used RNA interference to target the APP gene in cultured T84 cells in combination with two types of ER calcium sensors. Using a genetically encoded calcium indicator, GEM-CEPIA1er, we found that APP-deficient cells exhibited elevated resting calcium levels in the ER and prolonged emptying of ER calcium stores upon the cyclopiazonic acid-induced inhibition of sarco-endoplasmic reticulum calcium-ATPase. These effects could be ascribed to lower ER calcium leakage rates. Consistent with these results, translocation of the endogenous ER calcium sensor STIM1 to its target channel Orai1 was delayed following ER calcium store depletion. Our data suggest a physiological function of APP in the regulation of ER calcium levels. It plays an important role in a multitude of cellular activities, ranging from gene transcription to neurotransmission. Inside the cell, Ca2+ ions are predominantly sequestered in the endoplasmic reticulum (ER). The steep gradient of Ca2+ concentrations between the cytosol and ER is maintained by sarco-endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA) pump1. In resting cells, the activity of SERCA is counteracted by poorly defined Ca2+-conducting passive leak channels2. Upon cell stimulation, Ca2+ that is stored in the ER is released into the cytosol through the activity of inositol triphosphate-3 (IP3) receptors and ryanodine receptors1. The resulting drop in ER Ca2+ concentrations ([Ca2+]ER) is sensed by stromal interaction molecule 1 (STIM1), an integral ER membrane protein3. The dissociation of Ca2+ from its EF-hand motif results in STIM1 oligomerisation and translocation toward ER-plasma membrane junctions where it binds and activates Orai Ca2+ channels3. The subsequent Ca2+ influx is referred to as store-operated calcium entry (SOCE), which both refills Ca2+ stores and sustains Ca2+ signalling6. Orai channels are composed of homologous Orai1-3 proteins, from which Orai1 contributes most to SOCE in different cell types7. Moreover, the interaction between the ER Ca2+ sensor STIM1 and Orai1-based Ca2+ channels has been demonstrated to be sufficient for SOCE8.Calcium 10. Most AD cases are sporadic and affect elderly people, but some cases (1–6%) have an early-onset and are caused by mutations in the genes that encode presenilin-1 (PS1), presenilin-2 (PS2), and amyloid precursor protein (APP)11. Although such familial AD (FAD) cases are relatively rare, the disease-linked proteins have been intensively studied to elucidate the pathogenesis of AD. Most FAD-causing mutations map to PS1, the enzymatic component of the γ-secretase proteolytic complex12. PS1 FAD mutations have been repeatedly shown to enhance ER Ca2+ signalling in patient cells and various cellular and animal disease models, supporting the “calcium hypothesis” of AD14. The expression of FAD-causing PS1 mutants also reduces SOCE, whereas the downregulation of PS1 or inhibition of γ-secretase activity enhances SOCE14. However, still debatable is whether PS1 affects SOCE machinery directly or only indirectly by altering ER Ca2+ content15. The precise effects of presenilins (PSs) and PS FAD mutations on ER Ca2+ levels are also disputed because measurements of [Ca2+]ER with the help of ER-targeted indicators have yielded contradictory results24. Consequently, several different mechanisms have been proposed to explain the role of PS FAD mutations in the observed enhancement of ER Ca2+ signalling25.The dysregulation of Ca2+ homeostasis. APP is a single-pass transmembrane protein that undergoes sequential proteolytic cleavage26. Amyloidogenic processing is performed by β- and γ-secretases, which liberate two short fragments from the APP molecule: β-amyloid and APP intracellular C-terminal domain (AICD). β-amyloid peptides may lead to an elevation of cytosolic Ca2+ levels by activating Ca2+ influx mechanisms or forming Ca2+-permeable pores themselves10. AICD was shown to be required for bradykinin-evoked ER Ca2+ release in fibroblasts27. However, the Ca2+-related functions of APP-derived fragments were inferred solely from changes in cytosolic Ca2+ levels. In contrast, using both cytosolic and ER-targeted Ca2+ indicators, Oules et al. recently reported that the overexpression of a FAD-causing APPSWE mutant reduced ER Ca2+ load capacity through higher activity of Ca2+ release mechanisms from this organelle28. In the present study, we used a different approach to shed light on the physiological function of APP and examined ER Ca2+ levels in cells with downregulated APP expression. For this purpose, we used both the ER-targeted genetically encoded Ca2+ indicator (GECI) GEM-CEPIA1er29 and the endogenous ER Ca2+ sensor STIM1. We found that APP-deficient cells had elevated resting levels of Ca2+ in the ER and exhibited delayed translocation of STIM1 to Orai1 upon ER Ca2+ store depletion. Our data suggest a regulatory role for APP in ER Ca2+ homeostasis.Even less is known about the role of APP in ER Ca2+ store depletion, STIM1 proteins oligomerise and translocate within ER membranes toward cell surface-localised Orai1 Ca2+ channels3. This process can be experimentally elicited by blocking SERCA with cyclopiazonic acid (CPA), which depletes Ca2+ from the ER through passive leakage. To investigate the potential effect of APP on the translocation of STIM1 to Orai1, we searched for a cell line that produces all three proteins at high levels. We confirmed substantial amounts of Orai1 in lysates from T84 and Jurkat cells as previously reported by others30. However, among the tested cell lines, only T84 cells satisfied our search criteria  using virally-delivered specific short-hairpin RNA (shRNA) sequences . Using this approach, a time-dependent increase in the fraction of STIM1 that was present with Orai1 was found, with half maximal co-localisation values reached 6.3 min after the addition of CPA  and YM-58483 33  did not prevent the translocation of STIM1 to Orai1 . Altogether, these results indicate that T84 cells can be used to analyse the kinetics of endogenous STIM1 translocation toward native Orai1 channels by quantitative co-localisation.During ER Caria Fig. . Thereforia Fig. . The speces Fig. . The kinCPA Fig. . Pretrealls Fig. . In cont2+ content in T84 cells was experimentally modulated to evaluate the effect of initial [Ca2+]ER on subsequent CPA-evoked STIM1 translocation to Orai1. T84 cells were first grown for 12 or 24 h in media that contained 10 mM Ca2+ (high Ca2+ medium) to increase intracellular [Ca2+], including [Ca2+]ER. The co-localisation of STIM1 with Orai1, measured 12 min after the addition of CPA in Ca2+-free buffer, was significantly reduced in these cells compared with control cells that were grown in normal media , the expression of SERCA2 was downregulated by RNAi using a specific shRNA sequence  when perfused with the CPA-containing Ca2+-free buffer  and APP KD i2 cells . Thus, the downregulation of APP expression increased resting [Ca2+]ER, which may be explained by the slower leakage of Ca2+ from the ER.After establishing the procedure to measure STIM1 translocation to Orai1, we knocked down NAs Fig. . The dowins Fig. . APP-defCPA Fig. . In contCPA Fig. , at whicCPA Fig. . To invefer Fig. . To deteell Fig. . Therefoell Fig.  and subj2+ sensors to estimate Ca2+ levels in the ER in APP-deficient cells: a stably expressed GECI (GEM-CEPIA1er) and an endogenous ER Ca2+ sensor (STIM1). Ratiometric GECIs, such as GEM-CEPIA1er and D1ER, are advantageous for measuring resting ER Ca2+ levels in intact cells because they can be calibrated to obtain absolute [Ca2+]ER values, and they do not require any cell manipulation prior to the measurements. The latter cannot be avoided when working with some other frequently used ER Ca2+ indicators, such as aequorin (which requires reconstitution with its cofactor) or the chemical probe MagFura-2 (which requires loading into cells and subsequent cell permeabilisation). Moreover, when using either of these two indicators, steady-state ER Ca2+ levels are recorded after store refilling and may not necessarily reflect physiological resting levels. As demonstrated in this work using GEM-CEPIA1er, T84 cells with downregulated APP expression had substantially elevated resting [Ca2+]ER compared with APP-containing cells. Furthermore, they exhibited delayed translocation of the endogenous ER Ca2+ sensor STIM1 to the Ca2+ channel Orai1 upon CPA-evoked Ca2+ store depletion, reflected by the STIM1/Orai1 co-localisation coefficient. Although we cannot exclude a direct effect of APP on the translocation of STIM1, these two lines of experimentation suggest that the lack of APP results in an elevation of resting [Ca2+]ER, which in turn causes a delay in reaching the threshold for STIM1 oligomerisation and translocation upon Ca2+ store depletion. The advantage of the latter experimental method is that it does not perturb resting [Ca2+]ER by the presence of exogenous indicators, which unavoidably act as Ca2+ buffers. The translocation of STIM1 to Orai1 in wildtype T84 cells was a relatively slow process, with half-maximal values reached at 6.3 min after the addition of CPA. The observed translocation kinetics was likely limited by the kinetics of store depletion. The inhibition of SERCA with CPA causes passive and slow Ca2+ release through poorly characterised “leak channels”2. This approach, however, was advantageous for our analysis because we sought to reveal differences in initial ER Ca2+ levels.In the present study, we utilised two different types of ER-localised Ca2+ store depletion. This is consistent with our previous findings that APP had no effect on SOCE in cells with fully depleted Ca2+ stores37. Thus, the APP-dependent effect on SOCE that was observed by some authors  may be merely a consequence of changes in ER Ca2+ content. A similar conclusion was reached regarding the observed effects of PS1 on SOCE38.Interestingly, we did not observe any differences between APP-depleted and control cells in the co-localisation of STIM1 with Orai1 12 min after the addition of CPA . This suggests that the same amount of STIM1 binds Orai1 when given sufficient time for Ca2+ homeostasis has most often been investigated using cytosolic Ca2+ indicators. As recently reported, Fura-2-loaded hippocampal neurons from APP knock-in mice (bearing two pathogenic mutations) exhibited enlarged ionomycin-sensitive intracellular Ca2+ stores39. This Ca2+ overload was proposed to result from persistent overactivation of mGluR5 receptors by Aβ42. In addition, wildtype APP was implicated in ER Ca2+ handling by Linde et. al. who demonstrated attenuation of CPA-evoked ER Ca2+ release in cortical astrocytes isolated from APP knockout (KO) mice compared with wildtype astrocytes40. In contrast, APP KO neurons had normal ER-releasable Ca2+ pools41 and knockdown of APP in neuroblastoma cells resulted in larger cytosolic Ca2+ transients upon SERCA inhibition42. It should be noted that such measurements with cytosolic indicators provide a net outcome of the activities of ER Ca2+ channels from one side and Ca2+ extrusion or buffering mechanisms from the other side. Therefore, the reported discrepancies may reflect not only differences in resting ER Ca2+ levels between different cell types but also differences in the activities of Ca2+ handling machineries. This emphasises the importance of using organelle-targeted Ca2+ probes to investigate organellar Ca2+ levels. However, evidence of a role for APP in ER Ca2+ handling that is supported by direct measurements of [Ca2+]ER is scarce. Using ER-targeted aequorin, the overexpression of a FAD-causing APPSWE mutant in neuroblastoma cells was found to reduce steady-state ER Ca2+ loads through accelerated ER Ca2+ release28. No data were provided for wildtype APP. In this work, we found that knocking down APP expression with either of two different APP-targeting shRNAs led to an elevation of resting ER Ca2+ levels in intact T84 cells  increases in the levels and function of ryanodine receptors45, (ii) augmented IP3 receptor activity17, (iii) SERCA activation46, and (iv) the loss of leak channel activity22. However, none of these mechanisms gained broad acceptance, and some were questioned21. These studies usually also investigated the Ca2+-related functions of wildtype PSs compared with PS1 mutants. When the measurements were made with the D1ER indicator, PS double knockout (PS DKO) fibroblasts had elevated resting Ca2+ levels compared with wildtype fibroblasts, and this phenotype was rescued by the expression of wildtype PS120. The same conclusions were reached when the measurements were made with MagFura-2-loaded and permeabilised PS DKO fibroblasts24. To explain these observations, wildtype PS1 was shown to act as a Ca2+-permeable ER leak channel itself22. This finding has recently gained further support by a study reporting the dual function of archeobacterial PS homologues as proteases and ion channels47. Furthermore, a screen for Ca2+ regulatory proteins identified PS2 as an important ER Ca2+ leak factor48. An alternative mechanistic explanation for the effects of PSs was provided by Brunello et al., who found that PS2 accelerated ER Ca2+ leakage through IP3- and ryanodine receptors and simultaneously inhibited SERCA216. In addition, other data did not support the inverse relationship between wildtype PS1 levels and ER Ca2+ levels or the proposed mechanisms of action of wildtype PS121. These discrepancies may be attributable to the different cell types and methodologies employed to measure ER Ca2+ dynamics.Compared with APP, Ca2+ levels. In particular, we show that T84 cells depleted of APP exhibited elevated resting [Ca2+]ER and slower passive ER Ca2+ leakage. We also demonstrate that differences in the resting levels of ER Ca2+ between cells can be estimated by measuring the translocation of endogenous STIM1 to Orai1 upon SERCA inhibition. Our results reveal a physiological function of APP in ER Ca2+ homeostasis, which may have important implications for AD research.In conclusion, the present findings indicate that wildtype APP, similar to PSs, may also be involved in regulating ER CaProLong Gold and Hoechst 33342 were purchased from Life Technologies. Ionomycin (free acid) was purchased from Calbiochem. CPA, ML-9, polybrene, poly-L-lysine (PLL), fetal bovine serum (FBS), puromycin, and penicillin/streptomycin were purchased from Sigma-Aldrich. Escin was purchased from Santa Cruz. YM-58483 and SKF-96365 were purchased from Abcam. The following antibodies (Abs) were used: anti-STIM1 (HPA012123), anti-SERCA2 (S1439), anti-β-actin (A5441) were from Sigma-Aldrich, anti-APP (Y188) was from Abcam, and anti-Orai1  was from Santa Cruz.49: APP KD i1 (GAAGGCAGTTATCCAGCAT), APP KD i2 (GGTGCAATCATTGGACTCA), STIM1 KD (GAAAGTGATGAGTTCCTGA), ORAI1 KD i1 (GCAACGTGCACAATCTCAA), ORAI1 KD i2 (GTGTGTGTGACACATAAAT), and SERCA2 KD (GCAACTCAGTCATTAAACA). Control non-targeting shRNA was CCTAAGGTTAAGTCGCCCT. The shRNAs were cloned between EcoRI and ClaI sites into modified pLVTH vectors (originating from Addgene plasmid no. 12262), in which GFP cDNA was replaced with the GEM-CEPIA1er sequence (originating from Addgene plasmid no. 58217), or both GFP cDNA and the EF1α promoter were replaced with a puromycin selection marker and PGK promoter. DsRed2-ER cDNA was cloned into a pQCXIP retroviral vector (Clontech).The following shRNAs were designed using the Whitehead Selection Web Server2+-phosphate transfection method. Supernatants were collected 48–72 h after transfection, filtered through 0.45-µm membranes and concentrated in Vivaspin 100-kDa units (Sartorius) in a swing-out rotor at 1,000 × g. The viral titers were ~106 transducing units per ml. T84 and HeLa cells were transduced in the presence of 8 µg/ml polybrene for 8 h. T84 cells transduced with viruses carrying the puromycin marker were selected with 5–10 µg/ml puromycin for 2 weeks. Experiments with these cells started at least one week after puromycin withdrawal. Experiments with cells expressing GEM-CEPIA1er were conducted between 1–2 weeks after transduction. The ER localisation of GEM-CEPIA1er in APP KD and control T84 cells is presented in Supplementary Fig. All cells used in this study were obtained from ATCC. HEK293T/17, HeLa, and U-87 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), Jurkat cells were grown in RPMI-1640, and T84 cells were grown in DMEM/F-12 supplemented with 15 mM HEPES. All media contained 10% FBS and 1% penicillin-streptomycin. All gene knockdowns (KD) were performed by transducing cells with lentiviruses that carried targeting shRNA sequences and the puromycin selection marker or the GEM-CEPIA1er sequence. Viruses were prepared in 293 T/17 cells using the Ca2, 10 mM glucose, and 10 mM HEPES, pH 7.4) supplemented with 1.8 mM CaCl2. To deplete ER Ca2+ stores, the cells were washed in Ringer solution supplemented with 0.5 mM EGTA, and incubated for defined periods of time in Ringer solution that contained 0.5 mM EGTA and 30 μM CPA. The incubations were carried out at room temperature. In some experiments, SOCE inhibitors  or DMSO (drug vehicle) were added to the CaCl2-containing Ringer solution (pretreatment of cells for 5 min) and also to the EGTA and EGTA/CPA solutions (CPA treatment). For experiments presented in Fig. 2 (as in high-Ca2+ medium) or 1.8 mM CaCl2  before starting ER Ca2+ store depletion. After treatments, the cells were fixed in 3.7% paraformaldehyde in PBS, permeabilised in 0.1% Triton X-100 and 0.05% SDS in PBS, and stained against endogenous STIM1 and Orai1 proteins. Nuclei were stained with Hoechst 33342. The cells were then mounted in ProLong Gold. Images were captured under Zeiss LSM5 Exciter or Zeiss LSM800 confocal microscope using a Plan-Apochromat 63×/1.4 oil objective. Acquired cells were chosen based exclusively on Hoechst staining. All of the images were batch processed using custom-written scripts in ImageJ software. Mander’s M1 colocalisation coefficient50 was calculated with the Manders Coefficients ImageJ plugin. To obtain the extent of specific co-localisation of STIM1 with Orai1 (referred to as “M1 STIM1/Orai1” in figures) for each image pair, co-localisation coefficients were first calculated for images that were displaced vertically or horizontally by 2.35 µm in each direction, and their mean value was subtracted from the co-localisation coefficient that was calculated for aligned images. The results were imported into Microsoft Excel software for statistical analysis.T84 cells were seeded on PLL-coated coverslips. The next day, they were transferred to Ringer solution  supplemented with 1.8 mM CaCl2 under a Zeiss LSM800 confocal microscope with an EC Plan-Neofluar 40×/1.3 oil objective at room temperature. GEM-CEPIA1er signals were excited with a 405 nm laser, and the fluorescence light at wavelengths of 410–470 nm and 510–700 nm emitted by the Ca2+-bound and Ca2+-free indicator, respectively, was passed through a partially opened pinhole (460 µm) and simultaneously collected using two GaAsP detectors. 4–6 images from one field of view were acquired at 10 s intervals. An example of fluorescence intensity and ratio traces together with a calibration procedure is shown in Supplementary Fig. 2+ leakage were performed in cells continuously perfused with Ringer solution that contained either 1.8 mM CaCl2 or 0.5 mM EGTA and 30 μM CPA. Perfusion was driven by syringe pump 33 (Harvard Apparatus) at 1–2 ml/min. Images were acquired at 5 s intervals. At the end of each measurement, the cells were permeabilised in 150 μM escin for 2 min, and Rmin and Rmax values were obtained in the presence of 10 μM ionomycin under Ca2+-free and Ca2+-saturating conditions, respectively. The signals were calibrated as described previously29. All of the images were processed using ImageJ or MetaFluor software. The Ca2+ leakage rates were calculated over a 60 s time-window from the start of [Ca2+]ER decline using the SLOPE function in Excel and plotted against initial [Ca2+]ER.T84 cells were seeded on PLL-coated coverslips. Measurements of resting ER Ca37. Proteins were separated on 7–12% SDS-PAGE using Tris-glycine buffer, and visualized by ECL reaction as described previously37. Uncropped blots are presented in Supplementary Fig. Lysates were prepared as described previously2+ leak rates were subjected to analysis of covariance as described51 using the StatTools web server (http://www.obg.cuhk.edu.hk). This analysis included the initial [Ca2+]ER as a covariate. In short, the regression slopes for all cell lines . The differences in the adjusted means are differences in leakage rates between the APP KD and control cells having the same initial [Ca2+]ER.ER Canes Fig.  were anat-tests. The co-localisation data (n = 3–6) with calculated mean values and standard errors are presented in aligned dot plots. One n for the co-localisation analyses is defined as the mean result of six image pairs collected from an independent experiment. The calculated mean values of the Ca2+ measurements (n ≥ 6) are shown as bars with standard errors. One n for the Ca2+ measurements is defined as the mean result of all cells within one field of view, with the exception of the data presented in Fig. n indicates one ER region. Values of p < 0.05 were considered statistically significant.Other data were analysed using Excel or GraphPad Prism software using two-tailed unpaired or one-sample All data generated or analysed during this study are included in this published article and its Supplementary Information files.Supplementary Information


V1.2 channels (LTCCs), which trigger the release of calcium (Ca2+) from the sarcoplasmic reticulum (SR). This results in an increase in intracellular Ca2+ (Cai) that initiates contraction. During pulsus alternans, cardiac contraction is unstable, going from weak to strong in successive beats despite a constant heart rate. These cardiac alternans can be caused by the instability of membrane potential (Vm) due to steep AP duration (APD) restitution , instability of Cai cycling , or both, and may be modulated by functional coupling between clustered CaV1.2 (e.g. cooperative gating). Here, mathematical analysis and computational models were used to determine how changes in the strength of cooperative gating between LTCCs may impact membrane voltage and intracellular Ca2+ dynamics in the heart. We found that increasing the degree of coupling between LTCCs increases the amplitude of Ca2+ currents (ICaL) and prolongs AP duration (APD). Increased AP duration is known to promote cardiac alternans, a potentially arrhythmogenic substrate. In addition, our analysis shows that increasing the strength of cooperative activation of LTCCs makes the coupling of Ca2+ on the membrane voltage (Cai→Vm coupling) more positive and destabilizes the Vm-Cai dynamics for Vm-driven alternans and Cai-driven alternans, but not for quasiperiodic oscillation. These results suggest that cooperative gating of LTCCs may have a major impact on cardiac excitation-contraction coupling, not only by prolonging APD, but also by altering Cai→Vm coupling and potentially promoting cardiac arrhythmias.In ventricular myocytes, membrane depolarization during the action potential (AP) causes synchronous activation of multiple L-type Ca V1.2 channels (LTCCs) can open and close in unison  and that this gating modality may regulate excitation-contraction coupling in the heart. However, whether amplification of Ca2+ influx by cooperative gating of LTCCs promotes alternans is unknown. In this study, we developed a novel computational model of cooperative gating of LTCCs from experimental data. We incorporate the model into a physiologically detailed action potential (AP) model and investigated how changes in coupling strength of LTCCs may impact dynamics of AP and Ca2+ alternans. Our data suggest that increasing coupling strength of LTCCs prolongs AP duration and leads to Ca2+ overload. In addition, our theoretical and computational approaches elucidate that increasing coupling strength of LTCCs promotes positive Cai→Vm coupling, which could lead to Vm-driven and Ca2+-driven alternans.Recent experimental studies have suggested that clusters of L-type Ca V1.2 channels (LTCC) play a critical role in triggering cardiac muscle contraction during the action potential  coupling) Cleft) and junctional SR Ca2+ concentration ([Ca2+]JSR). SERCA pumps are distributed equally over the cell.The dimension of this model is 121 μm × 25 μm × 11 μm and there are 19,305 (65×27×11) Ca10 LTCCs , 25. Eac2+ cycling is governed by the following equations:vi is the local cytosolic volume, vs is the local submembrane space volume, vp is the local proximal space volume, vJSR is the local JSR volume, βi is the instantaneous buffer function for ci, βs is the instantaneous buffer function for cs, βp is the instantaneous buffer function for cp, βJSR is the instantaneous buffer function for cJSR, ITCi is time-dependent buffering to Troponin C for ci, ITCs is time-dependent buffering to Troponin C for cs, Idsi is the diffusive current between cs and ci, Iup is the uptake current, Ici is the nearest-neighbor diffusive current for ci, Ics is the nearest-neighbor diffusive current for cs, Idps is the diffusive current between cp and cs, INCX is the sodium-calcium exchange current, ICaBk is the background sarcolemmal membrane Ca flux, ISLCaP is the sarcolemmal membrane Ca pump, Ir is the release current, ICaL is the L-type Ca current, Itr is the JSR refilling current, IcNSR is the nearest-neighbor diffusive current for cNSR, superscript n shows the n-th compartment.Intracellular Caet al.  (cs), pox and csx are parameters, which control the sensitivity of the coupling, w is the coupling strength.To replicate the Markov model shown above, cooperative gating and its Ca2+ dependent inactivation are given byVoltage-dependent inactivation and the Caa = VmF/RT.The single channel current is given byτf) of the inactivation gate (f) of LTCCs to control the stability of the Vm system since it is known that the steepness of APD restitution is sensitive to it. In order to control the stability of the Ca2+ system, we varied the steepness of the slope (u) of the SR Ca2+ release function, which controls the sensitivity of release to SR load. The degree of Ca2+ dependent inactivation (γ) was varied to obtain positive Cai→Vm coupling (γ = 0.7) and negative Cai→Vm coupling (γ = 1.5).We varied the recovery time constant .We built a stochastic model of cooperative gating of LTCCs and incorporated this gating modality into the subcellular CaCaL and the activation curve. To measure these curves, we used the same protocol used in the experimental study by Dixon et al.  vs time. (C) APD vs the beat number. (D) peak cytosolic [Ca2+] vs the beat number. (E) APD alternans amplitude vs the beat number. (C) peak cytosolic [Ca2+] alternans amplitude vs the beat number.The initial conditions are the same in both cases. Alternans was developed within 100 beats in both cases. (TIFF)Click here for additional data file.S2 Fig2+ transient alternans was observed. This demonstrate Ca2+ cycling is unstable and contributes development of alternans.Ca(TIFF)Click here for additional data file.S3 FigKr was reduced by 50%. (A) positive Cai→Vm coupling (B) negative Cai→Vm coupling.G(TIFF)Click here for additional data file.S4 FigBlack: w = 0, Red: w = 1.(TIFF)Click here for additional data file.


Ion mobility experiments coupled with electrospray ionization (ESI) were conducted to evaluate the folding states of bovine carbonic anhydrase 2 (CA2) under three different pH conditions. Collision cross-section (CCS) of the CA2 ions generated by ESI revealed the presence of six discrete conformers in the gas phase under the conditions employed in this study. The CCS of the most extended conformer was three times larger than that of the most compact one. The charge state distribution of the CA2 ions was indicative of three conformers being present. Although there was consistency in conformer assignment conducted by CCS and charge state distribution, the CCS measurement was shown to be more effective because the information obtained provided more detailed knowledge of the conformation of the protein. Although the sample solution passes through an electrospray needle with a high electric field, it is thought that analytes remain in solution until the final ionization step. If a protein is in a folded conformation, ESI produces gaseous ions with a relatively small charge number, because compactly folded polypeptide chains only allow protonation at the basic side chains that are exposed at the protein surface during the ionization process. In contrast, for an unfolded protein, a significantly large number of protons on the polypeptide chain are accessible, and the ions are shifted to a lower m/z region. A multimodal charge state distribution suggests the coexistence of several conformers.One of the commonly accepted methods of analyzing protein folding states by mass spectrometry (MS) is to monitor the shift in charge state distribution of multiply-protonated protein molecules, n+ corresponding to apo-CA2  and 28+ (m/z 1037.5 for the apo-ion), as shown in m/z 2645.2, ESI mass spectra of CA2 obtained at three different pH conditions are shown in  apo-CA2 . In the 2+, and the enzyme has both hydration and dehydration activities at pH 6.5. The CA2 ions observed at pH 6.5 were holo-CA2 ions and belonged to component C. Therefore, component C reflects the folded conformation of CA2, while the ions comprising component A are all in the apo-form. Indicating that the component likely consists of inactive extended conformers. On the other hand, component B was observed at pH 2.6 and pH 3.8 but not at pH 6.5, indicating that ions belonging to this component do not represent physiologically active conformers. At pH 3.8, component B contains some holo-CA2 ions with lower charge numbers. It can be concluded that component B consists of apo- and holo-ions, suggesting that the component might be in a transition state to component A which is dominantly observed at pH 2.6.We also conducted a chemometric analysis to determine the number of independent conformers in the multimodal charge state distribution of ESI mass spectra . The res2+ on the conformation of the molecule.Ion mobility experiments were performed to obtain information concerning the gas-phase conformation of the CA2 ions at pH 2.6, 3.8, and 6.5. The driftgrams obtained are shown in At pH 6.5 , doubletThe peak top CCS of the driftgrams was obtained for the three pH conditions and the CCS was plotted against the charge number of the ions, as shown in 2  with CCS ranging from 20 to 120 nment A–C, , whereasent A–C,  and 5. A