In vitro, PEITC decreased reduced glutathione (GSH), which resulted in decreased GSH/oxidized glutathione (GSSG) ratio and increased glutathionylation of Mcl-1, leading to rapid proteasomal degradation of Mcl-1. Furthermore, we identified Cys16 and Cys286 as Mcl-1 glutathionylation sites, and mutating them resulted in PEITC-mediated degradation resistant Mcl-1 protein. In conclusion, we demonstrate for the first time that CDDP resistance is partially associated with Mcl-1 in BTC cells and we identify a novel mechanism that PEITC can enhance CDDP-induced apoptosis via glutathionylation-dependent degradation of Mcl-1. Hence, our results provide support that dietary intake of watercress may help reverse CDDP resistance in BTC patients.Biliary tract cancer (BTC) is a highly malignant cancer. BTC exhibits a low response rate to cisplatin (CDDP) treatment, and therefore, an understanding of the mechanism of CDDP resistance is urgently needed. Here, we show that BTC cells develop CDDP resistance due, in part, to upregulation of myeloid cell leukemia 1 (Mcl-1). Phenylethyl isothiocyanate (PEITC), a natural compound found in watercress, could enhance the efficacy of CDDP by degrading Mcl-1. PEITC-CDDP co-treatment also increased the rate of apoptosis of cancer stem-like side population (SP) cells and inhibited xenograft tumor growth without obvious toxic effects.  Biliary tract cancer (BTC) refers to a group of cancers of the biliary tract, including gallbladder cancer, cholangiocarcinoma of intrahepatic and extrahepatic bile ducts, and cancers of the ampulla and papilla of Vater . BTC is Various naturally occurring compounds are being tested for their anti-tumor activity. Such compounds when used in combination with known chemotherapeutic agents may help in overcoming chemoresistance, and may provide new strategies and ideas for treatment in clinic. Phenylethyl isothiocyanate (PEITC) is present in high concentrations as its precursor gluconasturtiin in cruciferous vegetables, such as watercress. Upon chewing or chopping, PEITC is released as a product of hydrolysis mediated by myrosinase . AccumulThough recent data has shown that PEITC can sensitize some tumor cells to CDDP , 10–12, To examine the effect of PEITC-CDDP co-treatment on cell viability, human gallbladder cancer GBC-SD cells were treated with PEITC, CDDP, or a PEITC-CDDP combination, and cell metabolic activity was measured using the 3--2, 5-diphenyl-tetrazolium bromide (MTT) assay. Notably, PEITC-CDDP co-treatment led to a significant reduction in cell viability Figure . To quanTo determine whether PEITC in combination with CDDP decreases cell viability via an increase in apoptosis, annexin V-fluorescein isothiocyarate (Annexin V-FITC)/propidium iodide (PI) double labeling flow cytometry was used to determine the percentage of cells entering apoptosis. Flow cytometry analysis showed that CDDP caused about 10% of the cells to enter apoptosis, but co-treatment with PEITC dramatically enhanced CDDP-induced apoptosis to 40% Figure . A similRecent studies have shown that SP cells isolated from various cancer cell lines and primary tumors possess cancer stem-like properties –16. SP cin vivo, GBC-SD cells were transplanted into nude mice. When the tumor size reached approximately 50 mm3, mice were randomly sorted into four equal groups. The tumor-bearing mice were intra-peritoneally injected with physiological saline as a control, PEITC, CDDP or PEITC-CDDP combination for 10 days. Treatment of mice with CDDP alone moderately inhibited tumor growth, but PEITC-CDDP combination treatment resulted in a striking reduction in the average tumor weight by about 50% , B-cell lymphoma-extra large (Bcl-xl) and Mcl-1 are known to play key roles in cancer cell apoptosis. Therefore, we investigated the relationship between these anti-apoptotic proteins and CDDP resistance in GBC-SD cells. Interestingly, CDDP treatment increased Mcl-1 protein level in a time- and dose-dependent manner, but did not increase Bcl-2 or Bcl-xl protein amounts Figure . To detein vivo, tumor tissues harvested from two mice of each group were examined by immunoblotting. PEITC and/or CDDP-mediated changes in Mcl-1 protein level in the tumor tissue were generally in agreement with the molecular alteration observed in cultured cells  and human cholangiocarcinoma (RBE) cell lines were obtained from Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. GBC-SD and RBE cells were maintained in RPMI 1640 (Hyclone) supplemented with 10% fetal bovine serum (Hyclone). Cells were cultured in a humidified atmosphere of 5% COPEITC, NAC, Hoechst 33342, verapamil, DTT and cycloheximide were purchased from Sigma. MG132 was purchased from Calbiochem and CDDP was obtained from Qilu Pharmaceutical Co., Ltd . PEITC was dissolved in dimethyl sulfoxide (DMSO) and was freshly diluted in culture media before use in experiments.SP analysis was performed as previously described . BrieflyCell viability was assayed using the MTT assay (Sigma) as previously described . Cell apCI and DRI values of PEITC-CDDP co-treatment were determined using the CalcuSyn software using non-constant ratio combination design as previously described , 38.Mcl-1 forward 5′-TCCAAGGCATGCTTCGGA-3′ and reverse 5′-GGCACCAAAAGAAATGAGAGTCAC-3′; GAPDH forward 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse 5′-GAAGATGGTGATGGGATTTC-3′. Human GADPH mRNA served as an endogenous control for normalization.Reverse transcription and quantitative real-time PCR were carried out as previously described . PrimersGSH is a tripeptide with a free thiol group and function as a major antioxidant in cells. GSH/GSSG ratio reflects the cellular redox state. The total GSH and GSSG were determined by colorimetric microplate assay kit  as previously described , 41. GSHMcl-1 mRNA sequence were transiently transfected into GBC-SD cells using Lipofectamine 2000 (Invitrogen) as previously described [Mc1–1 mRNA were 5′ GUAUCACAGACGUUCUCGUdTdT 3′ and 3′ dTdTCAU AGUGUCUGCAAGAGCA 5′. The siRNA-2 sequences complimentary for Mcl-1 mRNA were 5′ GGACUUUU AGAUUUAGUGAdTdT 3′ and 3′ dTdTCCUGAAAAUC UAAAUCACU 5′.Two specific siRNA oligonucleotides complementary to the escribed , 43. A nMcl-1 gene was performed using the Quick Change Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) using the pcDNA3.1-Flag-Mcl-1 plasmid as the template. Cells were transiently transfected with Flag-Mcl-1 WT and mutants using the above-mentioned Lipofectamine method. Nonrelevant plasmid pcDNA3.1 was used as the transfection control. After 48 hrs, GBC-SD cells were lysed for immunoprecipitation (IP) or treated with PEITC for an additional 24 hrs and analysed by western blot. The anti-Flag monoclonal antibody was purchased from Sigma.Site-directed mutagenesis of the Mcl-1, Bcl-xl and Bcl-2 protein levels were determined by western blot as previously described . Cellulan = 28 mice, purchased from Shanghai Experimental Animal Center). Tumor volumes were measured twice a week using a calliper, and were calculated using the formula V = π/6 × length × width2 [3, the mice were sorted into four equal groups (n = 7 mice per group). The tumor-bearing mice were intra-peritoneally administered with physiological saline as a control, PEITC (25 mg/kg), CDDP (2.5 mg/kg) or PEITC/CDDP twice a week. Tumor volumes were measured twice a week with a calliper and body weights were also recorded. The mice were sacrificed after 10 days, and body weight and tumor weight were measured.All animal experiments were done in accordance with institutional guidelines for animal welfare. GBC-SD cells were harvested, washed, and resuspended in serum-free RPMI 1640 and then injected subcutaneously into 6-week old BALB/c-nu/nu mice (× width2 , 45. WheP < 0.05 was considered significant.Data were shown as mean value ± SD. SPSS17.0 software was used for statistical analysis. Analysis of variance was applied for comparison of the means of two or multiple groups. A value of


We conclude that serial expansion, genetic engineering and imaging represent valuable tools to improve the individualized xenograft mouse model of AML. Prospectively, these advancements enable repetitive, clinically relevant studies on AML biology and preclinical treatment trials on genetically defined and heterogeneous subgroups.Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor outcome. Adequate model systems are required for preclinical studies to improve understanding of AML biology and to develop novel, rational treatment approaches. Xenografts in immunodeficient mice allow performing functional studies on patient-derived AML cells. We have established an improved model system that integrates serial retransplantation of patient-derived xenograft (PDX) cells in mice, genetic manipulation by lentiviral transduction, and essential quality controls by immunophenotyping and targeted resequencing of driver genes. 17/29 samples showed primary engraftment, 10/17 samples could be retransplanted and some of them allowed virtually indefinite serial transplantation. 5/6 samples were successfully transduced using lentiviruses. Neither serial transplantation nor genetic engineering markedly altered sample characteristics analyzed. Transgene expression was stable in PDX AML cells. Example given, recombinant luciferase enabled bioluminescence  There is a strong demand for preclinical models to develop novel, targeted therapies based on a better understanding of the complex biology of AML . Althoug systems .in vivo studies on AML, engraftment of patient-derived cells in immuno-compromised mice has been established in the early 1990s using severe combined immunodeficiency (scid) mice [To allow id) mice –7. Engraid) mice –12 and lid) mice –19.in vitro and in vivo, but this approach has not yet been systematically explored. Second, genetic engineering was never reported in established PDX AML cells. Molecular studies were mainly restricted to AML cell lines, which were genetically altered both for in vitro and in vivo studies, and both for knockdown strategies and transgene overexpression, including in vivo imaging [in vivo is an important readout for preclinical studies, yet this remains challenging as PDX cells are detected in mouse peripheral blood (PB) only at late disease stages using flow cytometry or polymerase chain reaction [Using the individualized xenograft mouse model of AML has resulted in seminal insights, e.g., in stem cell biology ,21; neve imaging –24. Nevereaction ,17,19, areaction ,26. Analreaction ,17,19, wreaction .in vivo studies [Serial passaging and genetic engineering have already been established in studies using primary tumor cells from patients with acute lymphoblastic leukemia (ALL) by others and us and have proven to be valuable tools to facilitate preclinical  studies –31.in vitro and in vivo studies; (ii) introducing genetic engineering of PDX cells to express transgenes using lentiviral transduction; (iii) introducing repetitive and sensitive disease monitoring in vivo by bioluminescence imaging (BLI) and; (iv) establishing a stringent set of quality controls to monitor the effect of retransplantation and transgene expression on molecular, phenotypic and functional sample characteristics. Due to these advances, our model system will facilitate future studies on AML biology and novel treatment approaches in vivo.The aim of the present work was to develop an improved preclinical mouse model of AML, broadening and increasing the use and quality of studies performed in the model, by: (i) performing serial retransplantation of primary AML cells to repetitively provide PDX cells for Fresh BM or PB samples from adult AML patients were obtained from the Department of Internal Medicine III, Ludwig-Maximilians-Universität, Munich, Germany, during the years 2012 and 2013. Specimens were collected for diagnostic purposes before start of treatment. Written informed consent was obtained from all patients or legal guardians in the cases where patients were minors. The study was performed in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000. Pediatric AML samples were described previously [PrkdcscidIL2rgtm1Wjl/SzJ  were maintained under specific pathogen-free conditions in the research animal facility of the Helmholtz Zentrum München, Munich, Germany. Animals had free access to food and water, and were housed with a 12-hour light–dark cycle and constant temperature. All animal trials were performed in accordance with the current ethical standards of the official committee on animal experimentation . When clinical signs of illness became apparent , mice were sacrificed equally in all passages. If leukemia became not apparent, mice were killed and analyzed 25 weeks after cell injection by latest.NOD.Cg-6–1x107 total BM cells were reinjected into next recipient NSG mice for reexpansion (secondary transplantation).From first generation mice harboring primary AML cells see , PDX celGenetic characterization of patient specimens and PDX cells was performed using a targeted, multiplexed amplicon resequencing approach . Mutational hotspots or the entire coding sequences, as appropriate, of 43 genes known to be recurrently mutated in myeloid malignancies  were amphttp://bioinf.wehi.edu.au/software/elda).Mean and standard deviation of the mean were calculated using the Microsoft Excel 2010 software . LIC frequencies were calculated according to Poisson statistics using the ELDA software application (in vivo bioluminescence imaging (BLI); quantification of BLI pictures; and Limiting dilution transplantation assay (LDTA).Please refer to in vivo studies on AML biology or treatment efficiency of novel therapeutic strategies.The aim of the present work was to develop the individualized mouse model of AML further and increase its capabilities for future in vivo growth characteristics , we detected a second, additional FLT3-ITD (96 bp length) in PDX cells obtained from three mice injected in parallel (VAF 2–4%). This second mutation was not detectable in the primary specimen (AML-373). Since the same, additional FLT3-ITD emerged in all three mice that were inoculated with the patient’s cells in parallel, we conclude that a small subclone carrying this variant was present in the patient sample, albeit below our limit of detection. After xenotransplantation, the subclone carrying the 96bp FLT3-ITD expanded relative to the 60bp FLT3-ITD-mutated clone, and thus had an engraftment or growth advantage in our model. Our data are in line with published results describing the challenge of reliably mimicking rare subclones upon xenotransplantation [AML is a genetically heterogeneous group of diseases, and various gene mutations have been shown to be associated with treatment response and patient outcome . Furtherrecently . Using arecently . In eachrecently ,42. Mostantation .These results illustrate several important characteristics of our model: (i) driver mutations present in the founding clone of primary AML specimens are preserved in xenografts; (ii) certain subclones present within the patients’ primary cell population may have an engraftment or growth advantage or disadvantage in mice, as indicated by the increase or decrease, respectively, of the VAF in PDX cells compared to the patient sample; (iii) in the three samples that were xeno-transplanted in multiple mice in parallel, engraftment characteristics of subclones are reproducible; (iv) PDX cells do not gain mutations in the studied AML driver genes, if they were unmutated in the patient's specimen.Next, capacity of initially engrafted cells to reengraft in next generation recipient mice was analyzed. 10/17 (59%) of established PDX samples, corresponding to 10/29 (34%) of all transplanted samples, reengrafted after first retransplantation. As positive predictive markers for successful repassaging, we identified the following growth characteristics of primary cells in the first transplantation: (i) overt leukemia in mice within 20 weeks after cell injection, accompanied by (ii) a substantial percentage of human cells in mouse BM and (iii) more than 5% leukemic cells in the blood at time of sacrifice . A thresFurthermore, serial transplantation of PDX cells was evaluated, to enable repetitive and reproducible experiments. Samples were retransplanted in up to six consecutive generations of mice. Until now, 6 samples could be analyzed for capacity to reengraft serially for more than three passages. 5/6 (83%) samples engrafted serially in all retransplantation experiments performed . Only on6, maximum 7x108) . In contrast, other characteristics varied widely between samples, including passaging time , percentage of leukemic cells in PB , or amount of leukemic cells in the spleen (minimum 1x10m 7x108) . Splenice spleen , but cone spleen .Serial transplantation might affect clonal composition of PDX cells. Targeted resequencing of serially transplanted cells revealed that they still contained the same leukemia-associated mutations as primary patient specimens and PDX cells after initial engraftment . In addiin vitro culture. The use of PDX AML cells will allow repetitive molecular studies on cells highly related to the patients, but these cells have never been used for genetic engineering so far.Until now, preclinical studies in AML were restricted to established AML cell lines –24, althHere, we established lentiviral transduction of PDX AML cells, which we and others had successfully used to genetically modify PDX ALL cells –31 or prNRAS mutation (VAF 5%) in t-PDX of AML-372  were included into further studies together with two pediatric PDX samples  that had been engrafted and serially transplanted before  S4 Tabl. After t AML-372 . Transge AML-372 . In contTaken together, lentiviral transduction was feasible in 5/6 (83%) PDX samples; both lentiviral constructs allowed successful transduction indicating a more general applicability of lentiviral constructs. Cell enrichment allowed generation of t-PDX cells without markedly altering sample characteristics. Successful genetic engineering in PDX AML cells now opens a broad variety of possible molecular studies.in vivo bioluminescence imaging (BLI) for sensitive and repetitive disease monitoring in single living mice over time [We next wanted to evaluate transgene expression in PDX cells and used the expression of luciferase as both a convenient example system and an attractive target protein. Expression of luciferase enables ver time . BLI wouin vivo [A codon-optimized form of firefly luciferase (effluc) was used which enables intense light emission for sensitive monitoring . BLI is in vivo –50 or toin vivo . We and in vivo –31, whil5 t-PDX cells of AML-372 were injected into mice and repetitively monitored using BLI  and 3x104 (2/3 mice) t-PDX AML cells resulted in a positive BLI signal at both femurs, whereas 1x104 cells yielded no detectable signal  and that (ii) within 18 hours from injection 100% of injected t-PDX cells had successfully homed to the BM, but (iii) cells had not yet started to proliferate, BLI was able to visualize less than 30000 leukemia cells per mouse, hence one human cell among 10000 murine BM cells (0.01%). As homing efficiency is unlikely to reach 100%, BLI probably is even more sensitive.To determine the sensitivity of BLI, different amounts of t-PDX AML cells were injected and mice were imaged as early as 18 hours after cell injection. Application of 1x10e signal . Thus, Pin vivo.Thus, expression of luciferase in PDX AML cells enables repetitive BLI for highly sensitive visualization of t-PDX cells in mice at tumor burdens which range within the different definitions of minimal residual disease in AML patients , to analAML represents a stem cell disease, and various scientific questions require quantification of leukemia initiating cells (LIC) as stem cell surrogates . The gol4 cells were positive in BLI one day after cell injection (4 cells) became positive in BLI; at day 29 after cell injection, all mice that would ever engraft were positive in BLI (3 (AML-372) and 1 in 2.8x103 (AML-346), consistent with data obtained from sorted primary AML samples [Limiting numbers of t-PDX AML-372 cells were injected into groups of mice and repetitive BLI was performed in all animals . Reprodunjection . Seven de in BLI . Calculae in BLI . Similare in BLI . LIC fre samples . Thus, Bin vivo trials on AML biology and novel treatment strategies.In the present work, we developed the individualized xenograft mouse model of AML further by evaluating serial transplantation and introducing lentiviral transduction and bioluminescence imaging. As these technical advances did not markedly alter important sample characteristics such as AML-specific mutations and growth behavior, the advanced model now represents a highly attractive tool for future The individualized xenograft mouse model of AML has been used in the past with a focus on primary transplantation and analysis of stem cell surrogates ,38,57–60The value of using the individualized mouse model as preclinical model depends on the capability of PDX cells to faithfully mimic the heterogeneity of the disease. Published results showed that PDX cells resemble the primary samples concerning gene expression profiles ,11,15,19in vitro and in vivo applications. However, three of the six samples allowing serial transplantation were from patients with adverse-risk genetic features who had suffered relapse after allogeneic transplantation, while the other three were from patients with newly diagnosed, ELN “Intermediate-I” risk group AML. All but one patient died within twelve months after sample collection. Therefore and as a limitation of the model, aggressive AMLs derived from adverse-risk patients might be especially suitable for allowing serial transplantation in NSG mice, consistent with data concerning initial engraftment capacity [Serially transplantable PDX cells offer important advantages compared to existing traditional AML cell lines, as cell lines may not be representative for the heterogeneity of AML. PDX cells might resemble the wide variety of genetic subgroups within AML and thus serve as clinically relevant model for drug testing. Furthermore, serial transplantation of PDX cells allows repetitive and reproducible analyses with stable and defined patient samples, both for capacity ,19.So far, molecular studies in AML were restricted to AML cell lines, as PDX AML cells were not systematically generated and genetic engineering was never performed in PDX AML cells. Our protocol for lentiviral transduction of PDX AML cells will allow multiple molecular studies, to gain a deeper and clinically more relevant understanding of AML biology.in vivo readout parameter for PDX AML cells. BLI enabled sensitive, reliable and repetitive disease monitoring and reduces required mouse numbers. BLI represents a convenient, simple and fast method to quantify stem cell frequencies.So far, using the individualized xenograft mouse model of AML for preclinical treatment trials was hampered by the inability to sensitively and repetitively monitor disease progression. Drug effects were determined post mortem , or at sin vivo imaging. As characterized patient cells are available repetitively and can be monitored in vivo in a highly sensitive way, the improved model will enable detailed reproducible in vivo studies on AML biology and therapy in the future.In conclusion, we advanced the xenograft mouse model of patient-derived AML by introducing and controlling serial transplantation, genetic engineering and S1 Fig7 cells were re-injected into next generation recipients and percentage of hCD45+ hCD33+ cells within BM within 20 weeks was measured. Each mark visualizes data obtained from one patient sample.(A) Exemplary FACS plots for staining of hCD33 hCD45 are presented for sample AML-372 PB (d61), BM and spleen (both d66). (B) 17 of 29 samples engrafted in NSG mice, defined by more than 0.5% hCD45+ hCD33+ cells within BM within 20 weeks after transplantation. 10(PDF)Click here for additional data file.S2 Fig(A) PDX AML cells which successfully engrafted in first recipients were re-isolated and serially transplanted into further recipient mice. Waved closure: in progress at time of manuscript preparation; black line: no re-engraftment could be observed after first re-transplantation in AML-373. (B) Serial transplantation of PDX samples was analyzed regarding passaging time  and percentage or absolute number of cells positive for both hCD45 and hCD33 at time of sacrifice within mouse PB, BM, or spleen, respectively. Each mark visualizes data obtained from a single mouse. Cross: Not determined.(PDF)Click here for additional data file.S3 Fig(A) Immunophenotype of primary cells and PDX cells after four retransplantation cycles was analyzed by multicolour flow cytometry; specific fluorescence intensity of six AML associated antigens and of the aberrantly expressed antigen CD7 (AML-361) is depicted. Raw data is depicted in (PDF)Click here for additional data file.S4 Fig(A) t-PDX AML cells show similar growth behavior as non-transgenic PDX cells. t-PDX cells (white bars) were compared to PDX cells (black bars) regarding passaging time and percentage or absolute number of cells positive for both hCD45 and hCD33 at time of sacrifice within mouse PB or spleen, respectively; shown are means +/- standard deviation. (B) Transgene expression remains stable over passaging. Expression of mCherry was analyzed by flow cytometry directly after cell enrichment by flow cytometry and cell amplification in mice, and after additional one, two or four retransplantation cycles.(PDF)Click here for additional data file.S5 Fig4 cells (B).Additional time points and reduced color scale to visualize lower BLI signals for the growth kinetics shown in (PDF)Click here for additional data file.S6 FigLDTA was performed in AML-346 and monitored by BLI as depicted in (PDF)Click here for additional data file.S1 Methods(PDF)Click here for additional data file.S1 Results(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.


This hypothesis was examined using Vicia faba cotyledons. On transferring cotyledons to culture, their adaxial epidermal cells synchronously trans-differentiate to epidermal transfer cells. A polarized and persistent Ca2+ signal, generated during epidermal cell trans-differentiation, was found to co-localize with the site of ingrowth wall formation. Dampening Ca2+ signal intensity, by withdrawing extracellular Ca2+ or blocking Ca2+ channel activity, inhibited formation of wall ingrowth papillae. Maintenance of Ca2+ signal polarity and persistence depended upon a rapid turnover (minutes) of cytosolic Ca2+ by co-operative functioning of plasma membrane Ca2+-permeable channels and Ca2+-ATPases. Viewed paradermally, and proximal to the cytosol–plasma membrane interface, the Ca2+ signal was organized into discrete patches that aligned spatially with clusters of Ca2+-permeable channels. Mathematical modelling demonstrated that these patches of cytosolic Ca2+ were consistent with inward-directed plumes of elevated [Ca2+]cyt. Plume formation depended upon an alternating distribution of Ca2+-permeable channels and Ca2+-ATPase clusters. On further inward diffusion, the Ca2+ plumes coalesced into a uniform Ca2+ signal. Blocking or dispersing the Ca2+ plumes inhibited deposition of wall ingrowth papillae, while uniform wall formation remained unaltered. A working model envisages that cytosolic Ca2+ plumes define the loci at which wall ingrowth papillae are deposited.Transfer cell morphology is characterized by a polarized ingrowth wall comprising a uniform wall upon which wall ingrowth papillae develop at right angles into the cytoplasm. The hypothesis that positional information directing construction of wall ingrowth papillae is mediated by Ca Cotyledons were surgically removed from their seed coats and prepared for aseptic culture on a Murashige and Skoog (MS) medium (Developing seeds were harvested from ) medium  as previ2+-sensitive fluorescent probe, Oregon Green 488 BAPTA-1-acetoxymethyl (AM) ester  following a protocol adapted from Supplementary Fig. S1 available at JXB online). To visualize the cellular distribution of Ca2+-permeable channels, cotyledons were stained with 600nM DM-BODIPY(–)-dihydropyridine  in MS medium for 2h at 20 °C . Calcofluor White was excited with a 405nm UV laser  and emitted fluorescence collected at 440–490nm, while Oregon Green, fl-DHP, and HPTS were excited with a 473nm diode laser  and their emitted fluorescence captured at 510–550nm. Gain of the photomultiplier tube was set to 500V for Calcofluor White and to 700V for Oregon Green, fl-DHP, or HPTS. Cotyledon sections were observed with a ×60 oil-immersion lens. Real-time intensity changes in Oregon Green fluorescence were recorded using a Hamamatsu™ spinning disc system coupled to a Zeiss confocal microscope  with a ×20 air objective, a 488nm argon laser (20 mW laser power set to 40%), and 488/515nm emission filters.2+ signal and Ca2+-permeable channels, Oregon Green-loaded or fl-DHP-stained cotyledons were counterstained with 20 μM RH-414, a plasma membrane marker (Molecular Probes), during the last 30min of cleaving Oregon Green ester or fl-DHP staining. Thereafter, cotyledon hand sections were floated for 20min on MS medium containing 0.1% (w/v) tetrazolium blue (cell viability) with their osmolalities adjusted to 300 mOsmol Kg–1 (turgid cells) or 500 mOsmol Kg–1 (plasmolysed cells) using betaine. Cell walls were stained with 0.1% (w/v) Calcofluor White. A 559nm diode laser  with a 625–725nm emission filter was used to visualize RH-414 fluorescence (gain of the photomultiplier tube was set to 500V). Spectrum settings for Calcofluor White, Oregon Green, and fl-DHP were as indicated previously.To identify the subcellular localization of the Ca2+]cyt were obtained by constructing a calibration curve from pixel intensities of Oregon Green fluorescence in epidermal cells of cotyledons equilibrated in a 10–1000nM clamped range of extracellular Ca2+ concentrations using CALBUF-2 buffer . Extra-/intracellular equilibration of Ca2+ was imposed 10min prior to confocal observation by permeabilizing and depolarizing membrane potentials of the epidermal cells by incubating the tissue sections in MS medium containing 10 μM A23187 and 10 μM CCCP, respectively. Thereafter, Oregon Green fluorescence of epidermal cells was captured by confocal microscopy as previously described.Relative estimates of cyt are reported as arbitrary units derived from a fitted calibration curve . Thus the algorithm provided a methodical non-biased detection of circular bright spots against noise in the images.To detect bright spots of fl-DHP or Oregon Green fluorescence in paradermal confocal images, the raw images were filtered and intensity peaks detected. A computerized algorithm was run to fit a two-dimensional Gaussian around each detected peak, given that bright punctate fluorescent spots are well represented by a point spread function. Fits with adjusted 2 nm–1=nm) using ImageJ software. Cytoplasmic volumes of inner and outer periclinal regions of epidermal cells were estimated as the product of their cytoplasmic widths using the same protocol as for wall widths (see above) and cell surface areas determined from scanning electron micrographs of epidermal peels.The percentages of cells with wall ingrowth papillae were obtained by scoring the presence/absence of wall ingrowth papillae in scanning electron microscopy images of epidermal peels . In the absence of a stable or transient transformation system for V. faba to introduce Ca2+ reporters  loaded with AM ester of the dye under low temperature .Compared with epifluorescence microscopy, optical sectioning by confocal microscopy of thick (100 μm) hand sections of cotyledons was found to capture clear fluorescence images of 2+ signal dynamics from Oregon Green fluorescence depended on there being no differences in intracellular dye concentrations and optical path lengths as well as no subcellular localization and cellular compartmentation of the dye  and in cells treated with Eosin Yellow to block cytosolic Ca2+ efflux by inhibiting plasma membrane Ca2+-ATPases (Supplementary Table S1). A corresponding even distribution of a non-Ca2+-sensitive and membrane-impermeant fluorophore, HPTS (Supplementary Table S1), confirmed an absence of any localized intracellular dye accumulation consistent with no detectable differences in subcellular cytoplasmic volumes within the epidermal cells (Supplementary Table S1). Organelle compartmentation of Oregon Green was considered unlikely as: (i) fluorescence was absent from anticlinal and inner periclinal cytoplasmic regions (2+ influx into cells was blocked (Supplementary Table S1). Finally, Oregon Green fluorescence was not detectable in epidermal cells of freshly harvested cotyledons . This suggests that the cytosolic Ca2+ signal was induced developmentally rather than from wounding on cutting hand sections. Collectively these findings indicate that the epidermal-cell-specific and polarized Oregon Green fluorescent band (Supplementary S1F), induced during cotyledon culture , resulted from a polarized intracellular elevation in [Ca2+]cyt detected by a uniform dye distribution throughout the cytosol of each epidermal cell.The cellular location of the Oregon Green fluorescent band was determined by co-staining hand sections with Calcofluor White . It is not known at this stage whether these channels belong to the cyclic nucleotide-gated channel and glutamate receptor-like channel families active in contributing to tip-high Ca2+ signals in elongating pollen tubes . Image overlays of turgid and plasmolysed epidermal cells indicated that fl-DHP fluorescence localized to the outer perimeter of their protoplasts . That fl-DHP bound to Ca2+-permeable channels in this location was supported by competition with non-labelled nifedipine substantially reducing the fluorescence intensity of, and hence binding by, fl-DHP .Whether the polarized cytosolic Ca  times more intense along the outer periclinal region of each epidermal cell compared with the remaining cell perimeter and on the inner cytoplasmic edge of each epidermal cell nucleus . These data suggest that the outer periclinal portion of the plasma membrane is enriched in nifedipine-sensitive Ca2+-permeable channels and that these channels are essentially absent from the plasma membrane lining anticlinal and inner periclinal walls of each epidermal cell.The reproducible presence of fl-DHP fluorescence circumscribing each epidermal cell nucleus  suggests2+-permeable channels were induced upon cotyledon culture , generate the polarized cytosolic Ca2+ signal , emitted from the outer periclinal cytosol  and Oregon Green  fluorescence appeared to be of uniform intensity across the outer periclinal interface of each epidermal cell . This analysis detected clumps of bright fl-DHP fluorescence at the outer periclinal cell wall–cytoplasm interface  blocking Ca2+ efflux from the epidermal cells . Under these conditions, deposition of wall ingrowth papillae was abolished whilst uniform wall formation of the ingrowth wall was unaltered .Similar diameters and separation distances  between ells see  by inhibn Yellow  versus Afedipine , wall infedipine . Togethe+ signal  selectiv+ signal ; Fig. 6A+ signal ; Fig. 6.-ATPases  versus Aiporters  versus A2+ signal that is temporally invariant but spatially complex in fully expanded cotyledon epidermal cells trans-differentiating to a transfer cell morphology. The cell-specific Ca2+ signal selectively functions to direct deposition of cell wall material to discrete loci, located on the outer periclinal walls of the trans-differentiating epidermal cells, for the construction of wall ingrowth papillae.The present work has identified a polarized cytosolic Ca2+ signals, which are known to regulate plant development, establish symbiotic partnerships and orchestrate responses to biotic or abiotic stresses, invariably are structured as single or oscillating spikes with periodicities ranging from seconds to minutes , the cytosolic Ca2+ signal exhibited temporal invariance for up to 1h . As found for tip growth systems  plays a major role in establishing the polarized Ca2+ signal , it is likely that the inner boundary of the cytosolic Ca2+ signal is constrained by the tonoplast of each epidermal cell. Restriction of the cytosolic Ca2+ signal to the outer periclinal region of each epidermal cell cytosol . Mathematical modelling confirmed this scenario as well as highlighting the co-operative role of plasma membrane Ca2+-ATPases in depleting the elevated [Ca2+]cyt between the Ca2+-permeable channel clusters to create the discrete plumes of cytosolic Ca2+ . Further evidence consistent with this assertion includes the finding that densities  to loci at which wall ingrowths are constructed. An additional role for the Ca2+ plumes could be to regulate the catalytic activity of plasma membrane-localized cell wall biosynthetic enzymes located at these loci. For instance, within sieve elements, putative Ca2+ hot spots have been shown to regulate the localized synthesis of callose deposits  in, together with cytoplasmic volumes of outer and inner periclinal regions of, epidermal cells of cultured cotyledons.Table S2. Competitive effect of nifedipine on intracellular distribution of fl-DHP, RH-414 fluorescence in epidermal cells of cultured cotyledons.Table S3. Effect of blockers of endomembrane Ca2+-ATPases  and Ca2+/proton antiporters (bafilomycin A1) on wall ingrowth papillae formation.


Nicotiana benthamiana N–acetylglucosaminyltransferase I (GnTI) for its cis/medial-Golgi localization and for protein–protein interaction in the Golgi. The individual GnTI protein domains were replaced with those from the well-known trans-Golgi enzyme α2,6–sialyltransferase (ST) and transiently expressed in Nicotiana benthamiana. Using co-localization analysis and N–glycan profiling, we show that the transmembrane domain of GnTI is the major determinant for its cis/medial-Golgi localization. By contrast, the stem region of GnTI contributes predominately to homomeric and heteromeric protein complex formation. Importantly, in transgenic Arabidopsis thaliana, a chimeric GnTI variant with altered sub-Golgi localization was not able to complement the GnTI-dependent glycosylation defect. Our results suggest that sequence-specific features in the transmembrane domain of GnTI account for its steady-state distribution in the cis/medial-Golgi in plants, which is a prerequisite for efficient N–glycan processing in vivo.Golgi-resident type–II membrane proteins are asymmetrically distributed across the Golgi stack. The intrinsic features of the protein that determine its subcompartment-specific concentration are still largely unknown. Here, we used a series of chimeric proteins to investigate the contribution of the cytoplasmic, transmembrane and stem region of  N–glycans by Golgi-α–mannosidase II, for example, is blocked by prior action of β1,4–galactosyltransferase , subjects them to extensive processing in different subcompartments and transports the cargo to other destinations within the endomembrane system. The compartmentation of biosynthetic activities in different cisternae of a polarized Golgi stack is a major function of the Golgi. Many processing steps involve modifications of protein- or lipid-bound oligosaccharides that are carried out by a large number of Golgi-resident glycosyltransferases and glycosidases. The overlapping but non-uniform distribution of these glycosylation enzymes across the Golgi stack is well documented, and has been shown for plants by immunoelectron and confocal microscopy . GnTI is a cis/medial-Golgi-resident type–II membrane protein that plays a key role in N–glycan processing, because it initiates the formation of complex N–glycans in animals and plants , which is the most widely used trans-Golgi marker in plants  and ST (RRR). We chose the Golgi targeting domains  consisting of the IgG1 heavy chain fragment (Fc domain) and GFP , which confirms processing in the Golgi apparatus. Peaks representing incompletely processed or further elongated N–glycan structures were only found in low quantities  to their substrates is blocked, resulting in the formation of incompletely processed N–glycans . We hypothesized that the co-expression of chimeric CTS-GALT enzymes leads to alterations in N–glycosylation dependent on the sub-Golgi distribution of CTS-GALT. To test our approach we fused the CTS regions from GnTI and ST to the catalytic domain of Homo sapiens GALT, and analyzed the generated N–glycans of a co-expressed monoclonal antibody (mAb), which served as a glycoprotein reporter  were more abundant  in N. benthamiana leaves and purified GnTI-GFPglyc by binding to protein A. Immunoblot analysis revealed that the quantity of co-purified RNN-mRFP was similar to NNN-mRFP, whereas binding of NNR-mRFP, RNR-mRFP and NRR-mRFP was as low as RRR-mRFP , co-expressed RNR-GNTI-GFP with the control NNN-GNTI-mRFP  and performed co-immunoprecipitation (co–IP) followed by immunoblot detection. In agreement with our previous data, no marked interaction could be found between RNR-GNTI-GFP and NNN-GNTI-mRFP . To exclude any overexpression effect the chimeric AtGNTI proteins were expressed under the control of the endogenous GnTI promoter. The complementation of the N–glycan processing defect of gntI plants was analyzed by the immunoblotting of protein extracts with antibodies directed against complex N–glycans. As expected, AtNNN-AtGNTI complemented the N–glycan processing defect of gntI, and restored complex N–glycan formation , p31 (expression of mRFP tagged proteins), pF , p57 (complementation of gntI plants with the full-length GnTI protein) or p46 .The RNN expression construct was generated by ligation of two overlapping synthetic oligonucleotides  into BamHI/XhoI-digested PCR product was ligated into the BamHI/SalI sites of pPT2M. For the generation of the pF-GCSI construct the CTS region from A. thaliana α–glucosidase I  and the GnTI catalytic domain into the HindIII/BamHI site of vector p27GFP. For this purpose, the GnTI promoter region was amplified by PCR from genomic DNA using primers AthGnT_12F/13R, the CTS region was amplified with primers AthGnT_14F/16R and the catalytic domain was amplified using primers AthGnT_15F/9R. These DNA fragments were assembled using the Gibson Assembly Cloning Kit . For vector p46 the catalytic domain of N. tabacum GnTI was amplified by PCR from p20-GnTI , RRR-GFP (ST-GFP), NNN-GFPglyc (GnTI-CTS-GFPglyc), NNN-mRFP (GnTI-CTS-mRFP), MNS1-GFPglyc, GCSI-GFPglyc, GnTI-mRFP, MNS1-mRFP and AtCASP-mRFP were all available from previous studies . After a brief incubation on ice, the extract was cleared by centrifugation (9000 g for 20 min at 4°C) and incubated for 1.5 h at 4°C with 20 μL of rProteinA Sepharose™ Fast Flow . The sepharose was collected by centrifugation, washed three times with 1× PBS using Micro Bio-Spin™ Chromatography Columns  and the bound protein was eluted by incubation in Laemmli buffer for 5 min at 95°C. Approximately 1 μg of chimeric CTS-GFPglyc or mAb was separated by SDS–PAGE (10%) under reducing conditions and stained with Coomassie Brilliant Blue. The corresponding protein band was excised from the gel, destained, carbamidomethylated, in-gel trypsin digested and analyzed by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS), as described in detail previously   that bind to complex N–glycans carrying β–1,2-xylose and core α–1,3-fucose residues (N. benthamiana plants were infiltrated with agrobacterium suspensions carrying the protein(s) of interest with the following optical densities (OD600): NNN, RRR, NNR, RRN, NRN, RNR, RNN and NRR, 0.05; mRFP-AtCASP, 0.10; RRR-mRFP, 0.07. High-resolution images were acquired 2 and 3 days post infiltration (dpi) on an upright Leica SP5 II confocal microscope using the Leica las af software system (http://www.leica.com). GFP and mRFP were excited with 488- and 561–nm laser lines, respectively, and detected at 500–530 and 600–630 nm, respectively. Dual-color image acquisition of cells expressing both GFP and mRFP was performed simultaneously. Post-acquisition image processing was performed in Adobe photoshop cs5.Leaves of 5–week-old glyc-fused protein of interest together with the cis/medial-Golgi marker mRFP-AtCASP  were generated by drawing a line across dual-labelled Golgi stacks using the ‘Line Profile’ intensity tool of the Leica las af software. Statistical analyses were performed using the Student's t–test for the comparison of two samples, assuming equal variances .Images of cells expressing the GFPin jacop . As everm in dimethyl sulphoxide; Calbiochem, now EMD Millipore, https://www.emdmillipore.com) at a concentration of 25 μm to inhibit Golgi movement (http://www.gsig.com). Laser light at a wavelength of 920 ± 5 nm was obtained from a mode-locked titanium sapphire laser , producing 180–fs pulses at 75 MHz. Two-photon excitation at 920 nm was chosen to allow reduced autofluorescence emission from chloroplast and guard cells. The laser beam was focused to a diffraction-limited spot through a VC 60 × /1.2 NA water immersion objective . Fluorescence emission was collected without descanning, bypassing the scanning system, and passed through a BG39  filter to block the near infrared laser light. Line, frame, and pixel clock signals were generated and synchronized with an external fast microchannel plate photomultiplier tube  used as the detector. These were linked via a time-correlated single-photon-counting PC module SPC830  to generate the raw FLIM data. Prior to FLIM data collection, the GFP and mRFP expression levels in the plant specimens within the region of interest were confirmed using a Nikon eC1 confocal microscope with excitation at 488 and 543 nm, respectively. A 633–nm interference filter was used to further minimize the contaminating effect of chlorophyll autofluorescence emission, which would otherwise obscure the mRFP emission. FLIM images were analyzed by obtaining excited-state lifetime values of a single cell. Calculations and image processing was performed using SPCIMAGE (Becker and Hickl). Lifetime values were collected on a single pixel basis from the center of individual Golgi bodies. Decay curves of a single point highlight an optimal single exponential fit when chi-square (χ2) values are 1 (points with χ2 from 0.9 to 1.4 were taken). The collected data values were used to generate histograms depicting the distribution of lifetime values of all data points within the samples. Results are from two or three independent experiments .Infiltrated leaf samples were excised and, prior to image acquisition, treated for 45–60 min with the actin-depolymerizing agent latrunculin B (stock solution at 1 mE). The percentage efficiency (E%) can be calculated using equation DA and τD are the mean pixel-by-pixel excited-state lifetimes of the donor in the presence and absence of the acceptor determined for each pixel. We have previously shown that a reduction of as little as ∼200 ps or 8% in the excited state lifetime of the GFP-labelled protein represents quenching through a protein–protein interaction (An observed protein–protein interaction is described by the decrease of the donor fluorescence lifetime (quenching) as a result of energy transfer to the acceptor , and canN. benthamiana plants were co-infiltrated with agrobacteria (OD600 0.2), containing p20-GnTI-CTS-Fc-GFP (NNN-GFPglyc) and different p31 constructs expressing the chimeric CTS regions  or controls (NNN and RRR) fused to mRFP. NNN-GFPglyc was purified by binding to rProtein A–Sepharose™ Fast Flow, as described in detail by http://www.miltenyibiotec.com) and anti-mRFP  antibodies. Similarly, constructs for expression of chimeric CTS-GFPglyc were co-expressed with NNN-mRFP and analyzed in the same way. For analysis of MNS1-CTS and NNN interaction, MNS1-CTS-GFPglyc was co-expressed with constructs expressing chimeric CTS-regions fused to mRFP, and for monitoring of the interaction between full-length proteins NNN-GNTI-GFP or RNR-GNTI-GFP (in p46) were co-expressed with NNN-GNTI-mRFP (in p31), co-purified using GFP-Trap-A beads (ChromoTek) and analyzed by immunoblotting.Co-purification experiments were performed as previously described (


Anoctamin‐1  is a principal CaCC subunit in many cell types, yet our understanding of the mechanisms of ANO1 activation and regulation are only beginning to emerge. Ca2+ sensitivity of ANO1 is rather low and at negative membrane potentials the channel requires several micromoles of intracellular Ca2+ for activation. However, global Ca2+ levels in cells rarely reach such levels and, therefore, there must be mechanisms that focus intracellular Ca2+ transients towards the ANO1 channels. Recent findings indeed indicate that ANO1 channels often co‐localize with sources of intracellular Ca2+ signals. Interestingly, it appears that in many cell types ANO1 is particularly tightly coupled to the Ca2+ release sites of the intracellular Ca2+ stores. Such preferential coupling may represent a general mechanism of ANO1 activation in native tissues.Ca ANOanoctaminAPaction potentialBAPTAN,N,N′,N′‐tetraacetic acid1,2‐bis(2‐aminophenoxy)ethane‐CaCC2+‐activated Cl− currentCaCRAC2+ release‐activated channelCaDIDS4,4′‐diisothio‐cyanostilbene‐2,2′‐disulfonic acidDRGdorsal root ganglionEGTAN,N,N′,N′‐tetraacetic acidethylene glycol‐bis(2‐aminoethylether)‐ERendoplasmic reticulumGPCRG protein‐coupled receptorHEK293human embryonic kidney 293 cell lineICCinterstitial cells of Cajal3RIPinositol 1,4,5‐trisphosphate receptorβMCDmethyl‐β‐cyclodextrinNFAniflumic acidNMDAN‐methyl‐d‐aspartic acidNPPB5‐nitro‐2‐(3‐phenylpropylamino) benzoic acidPAR‐2protease‐activated receptor 2PLAproximity ligation assayPLCphospholipase CP2Ypurinergic G protein‐coupled receptorRyRryanodine receptorSOCE2+ entrystore‐operated CaSTICspontaneous transient inward currentsSTOCspontaneous transient outward currentsTMEM16transmembrane protein 16TRPV1transient receptor potential cation channel subfamily V member 1VGCC2+ channelvoltage‐gated Ca2+‐activated Cl− channels (CaCCs) play crucial roles in numerous physiological processes including epithelial transport, smooth muscle contraction and sensory processing. In epithelia CaCCs are important for Ca2+‐activated Cl− secretion and mucus production  activate CaCCs and produce depolarization as most of these neurons accumulate high intracellular Cl− concentrations benzoic acid (NPPB), niflumic acid (NFA) and 4,4′‐diisothio‐cyanostilbene‐2,2′‐disulfonic acid (DIDS)  domains, cytosolic N‐ and C‐termini and a re‐entrant loop between TM5 and TM6 (probably pore‐lining) has been proposed  opening of sensory cation channels (such as TRP) or any ligand‐gated cation channels, (ii) activation of voltage‐gated Ca2+ channels (VGCCs), (iii) release of Ca2+ from intracellular stores, or (iv) store‐operated Ca2+ entry (SOCE) all result in intracellular Ca2+ transients . The question arises: would all these diverse processes uniformly activate CaCCs in a given cell type? Let us consider a pain‐sensing (nociceptive) sensory neuron as an example. Figure 2+]i, CaCC activation, membrane potential and activity of an excitable cell. Nociceptive sensory neurons are normally silent and respond only to strong adverse stimuli that can result in tissue damage. Action potentials (APs) that are triggered by such stimuli in the peripheral terminals of nociceptors  travel to the spinal cord where these neurons synapse. Second order neurons in the spinal cord then relay these peripheral nociceptive signals to the higher brain centres where these signals can be perceived as a sensation of pain. Since CaCC activation depolarizes and excites nociceptive neurons, an action that may result in pain sensation  is especially obvious in the case of possible relationships between CaCCs and VGCCs . Indeed, if Ca2+ influx through the VGCCs  did reach CaCCs and activate them, the depolarization produced by such activation might in turn further activate VGCCs  Ca2+ stores. Another current had linear current–voltage relationships, lacked slow activation kinetics and was activated by Ca2+ influx through SOCE  in order to be able to activate endogenous ANO1 in oocytes. It was further observed that a direct Ca2+ influx through STIM1–Orai1 complexes into the cytosol cannot activate ANO1, presumably due to the lack of close proximity between the ANO1 and the STIM1–Orai1 complexes. Thus, it appears that both types of oocyte CaCCs are indeed activated by the Ca2+ released from the ER via the IP3Rs, but when SOCE is engaged as well, the Ca2+ release is stronger, rendering ANO1‐mediated CaCCs less voltage dependent. Although this was not the first study showing preferential coupling of ANO1 channels to the IP3R‐mediated Ca2+ release (see below), it did complement earlier studies on native CaCCs in oocytes to point to such preferential coupling.There is a growing body of evidence indicating that in multiple cell types CaCCs mediated by ANO1 are particularly well coupled to the Ca2+ signals is important for ANO1 activation. Specifically, they proposed that functional proximity of ANO1 to the ER‐localized IP3Rs is important for the activation of ANO1 channels by the Gq/11‐ and PLC‐coupled purinergic P2Y receptors in HEK293 cells and oocytes . This observation could be explained by an assumption that the increase in P2Y receptor density results in stronger IP3 release upon ATP application. This, in turn, engages additional IP3Rs in the response. In such a scenario Ca2+ imaging with cytosolic Ca2+ indicators may not reveal any difference between the P2Y‐receptor‐overexpressing and control cells; yet additional IP3R Ca2+ release sites could activate more co‐localized ANO1 channels. An additional suggestion for close association between Ca2+ release sites and ANO1–CaCCs came from oocyte recordings: in oocytes overexpressing ANO1 and P2Y2 receptors, whole‐cell currents activated by the Ca2+ ionophore ionomycin displayed an outward rectification, while ATP‐activated currents had linear current–voltage relationships, suggesting higher local Ca2+ concentrations near ANO1 channels in the latter case  neurons express VGCC‐coupled CaCCs  such as bradykinin receptor 2 (B2) or protease‐activated receptor 2 (PAR‐2) activate CaCCs reliably  channels , a highly specific proteomics method that enables optical detection of closely associated proteins  and (ii) B2 receptors localize to plasma membrane lipid rafts in sympathetic and sensory neurons . In accord with these reports we found that B2 and PAR‐2 receptors co‐immunoprecipitated with the IP3R and with the lipid raft marker caveolin‐1 . In accord with this idea, membrane fractionation experiments revealed that in DRG neurons ANO1 localized to the same membrane fractions as caveolin‐1, B2 receptors and PAR‐2. However, this distribution was disrupted by cholesterol depletion with methyl‐β‐cyclodextrin (βMCD), a procedure known to disrupt lipid rafts  pull‐down experiments using GST‐fused cytosolic domains of ANO1 revealed that both the C‐terminus and the cytoplasmic TM2/3 loop but not the N‐terminus of ANO1 were able to precipitate endogenous IP3R1 in DRG preparation. We reasoned that if lipid raft disruption distorts ANO1 membrane localization and interrupts the interaction with IP3R1, this may also affect the ability of ANO1 to differentiate sources of intracellular Ca2+. Indeed, after the βMCD treatment, activation of neither PAR‐2 nor B2 receptor was able to activate CaCC currents in DRG neurones. In contrast, in 50% of such βMCD‐treated neurones we observed CaCC activation coupled to the VGCC activation. Thus, disassociation of the ANO1 signalling complex in a small DRG neuron resulted in loss of functional coupling between the GPCRs and the ANO1 but instead some coupling between ANO1 and VGCCs appeared. We hypothesize that the latter effect is due to the proximity between ANO1 and VGCCs acquired by chance after the raft disruption as both proteins reside in the plasma membrane . Consistent with a VGCC–CaCC–VGCC positive feedback loop idea , current clump experiments revealed that most small‐diameter DRG neurons fired single APs in response to strong depolarizing current injections in control conditions; however, lipid raft disruption resulted in overexcitable neurons that fired APs trains. Thus, we proposed a model outlined in Fig. 3Rs in order to confer efficient activation of the channel by the Ca2+ release. At the same time, such assembly may also protect ANO1 from the ambient Ca2+; this is probably why Ca2+ influx via the VGCCs is mostly insufficient to activate ANO1 in these neurons. In support of this hypothesis, in several DRG neurons we were unable to activate CaCCs by dialysis of as high as 10 μm free Ca2+ through the patch pipette, yet, bradykinin application still activated CaCCs .In our laboratory we investigated a phenomenon closely related to the one described above; we found a preferential coupling of ANO1 channels to IPs Mayer, . However3R regulating protein ERLIN1 has been identified as part of the ANO1 interactome  of the latter . The latter possibility was recently inferred by Courjaret & Machaca (3Rs but we do not know yet if such an interaction is constitutive.Recently in an unbiased SILAC (stable isotope labelling by amino acids in cell culture) proteomics screen of the proteins that physically interact with ANO1 channels in cells, an ER‐localized IP Machaca . Our datet al. et al. It is worth noting that during evolution the role of anoctamins in plasma membrane–ER interactions may even have preceded their ion channel function. Thus, in yeast an anoctamin orthologue, Ist2, is apparently a tethering protein that holds plasma and ER membranes together to maintain plasma membrane–ER junctions ‐mediated Ca2+ release  that are tightly coupled to RyR‐mediated Ca2+ sparks . Using a combination of biophysical methods the authors further determined that CaCCs in their smooth muscle preparation were densely clustered in specific spots of the plasma membrane that were less than 600 nm away from the RyR. This proximity established an environment in which CaCCs are exposed to a local [Ca2+]i of at least 2.4 μm during the Ca2+ spark event. In smooth muscle cells Ca2+ sparks activate not only STICs but also spontaneous transient outward currents (STOCs) carried by Ca2+‐activated K+ channels . A plausible explanation of this effect is that, similarly to nociceptive DRG neurons, lipid raft‐localized microdomains in smooth muscle cells may play a dual role: (i) providing a mechanism for coupling of ANO1 channels to intracellular stores and (ii) protecting these channels from ‘global’ cytosolic Ca2+ elevations. Therefore, lipid raft disruption in smooth muscle cells may have exposed ANO1 channels to global Ca2+, making them more easily activated by voltage.Another study has suggested that similar to the DRG neurons, ANO1 is enriched within the caveolin‐1‐containing plasma membrane lipid rafts in the murine portal vein myocytes  control secretion. Mechanisms of Cl−‐dependent secretion in epithelia are outside the scope of this brief review . ICCs are specialized cells of the gastrointestinal tract which control phasic contractions of gastrointestinal smooth muscles by generating waves of electrical activity (‘slow waves’). Release of Caet al. et al. et al. et al. 2+ source preference as compared to ANO1. Huang and colleagues  [Ca2+]i the relationships become linear. In turn, at negative membrane potentials the channel requires much higher [Ca2+]i for activation than at positive potentials  were reported to be in the range of 3–5 μm while at high, depolarizing voltages the EC50 is about tenfold lower . These complexes are likely to be further supported by scaffolding proteins and cytoskeleton. Indeed, interaction of ANO1 with the ezrin–radixin–moesin network has been identified  channels as these are needed for refilling the ER Ca2+ stores. Moreover, STIM1–Orai1 interactions that bring about CRAC activation  the indirect nature of most available methods for protein–protein interaction ; (ii) the resolution limits of most common microscopy techniques  and (iv) below); (iii) antibody specificity issues; (iv) limited suitability of expression systems since overexpressed proteins often ‘disobey’ endogenous localization rules. The latter point is also exemplified by the fact that overexpressed ANO1 channels are usually easily activated by ‘global’ Ca2+ sensitivity and therefore require close association with the source of intracellular Ca2+ for reliable activation in their native environment. Growing evidence suggests that ANO1 channels often preferentially couple to the Ca2+ release sites in the ER. Examples of such preferential coupling include ANO1‐containing signalling complexes in nociceptive sensory neurons and ANO1‐mediated spontaneous transient inward currents (STICs) in airways smooth muscle cells. In both cases plasma membrane ANO1 channels are found in close proximity to either IP3R (nociceptive neurons) or RyRs (smooth muscle cells). Such distinctive coupling may serve to ensure specificity and fidelity of Ca2+ signalling pathways in native cells. The mechanisms and physiological significance of specific coupling of ANO channels to the localized Ca2+ signals requires further investigation.Members of the anoctamin protein family ANO1 and ANO2 mediate CaCC currents in various cell types. These channels have low CaNone declared.The work that led up to this review was supported by Wellcome Trust, MRC and National Science Foundation of China.


Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2−/−), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca2+ responses as assessed by single-cell Ca2+ imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PIP2 were only partially dependent on TPCs. In Tpcn1/2−/− cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca2+-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca2+ release. High-affinity [32P]NAADP binding still occurs in Tpcn1/2−/− tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca2+-permeable channels indispensable for NAADP signalling.The second messenger NAADP triggers Ca In wild-type MEFs, NAADP/AM evoked robust Ca2+ signals which were inhibited by pre-treatment with bafilomycin A1, GPN, and nigericin, agents that deplete acidic Ca2+ stores, and by the NAADP antagonist trans-Ned-19  or ryanodine receptor (RyR), that is, Ca2+-induced Ca2+ release (CICR). We confirmed the co-involvement of the ER in several ways, first by depleting the ER with the Ca2+-ATPase inhibitor cyclopiazonic acid (CPA)  and ryanodine, respectively, abolished NAADP/AM-stimulated Ca2+ signals  Fig and F, w(CPA) Fig and H. G MEFs Fig, we testgnals Fig and L. Ntores Fig and B, t2+ signals. In WT MEFs, NAADP/AM evoked robust Ca2+ signals  within the endo-lysosomal system, either of which could potentially affect NAADP-induced Ca2+ release , cytosolic nanomolar concentrations of NAADP stimulated an inward current (lumen to cytoplasm) . Importantly, NAADP-induced currents were undetectable in similar preparations from Tpcn1/2−/− and Tpcn2−/− cells, while they were still present  in preparations from Tpcn1−/− cells  Fig and B wicells Fig and B. T+ . With cytosolic K+, the reversal potential was +76 ± 2 mV which equates to a PCa/PK permeability ratio of 268 ± 47 , which equates to a permeability ratio PCa/PNa of 0.86 ± 0.22  were stimulated by PIP2, whereas PIP2 was without effect  Pffect Fig and H. Iduced Fig and H, w2-induced currents can also be mediated by other endo-lysosomal channels as may be predicted for a permissive lipid endo-lysosomal channel modulator PTpcn1/2−/− MEFs was due to the specific lack of TPCs, we restored expression of TPCs and assessed NAADP-induced Ca2+ responses. Thus, Tpcn1/2−/− MEFs were transduced with lentiviruses for expression of either mouse TPC1 or TPC2 tagged with a C-terminal mCherry. Immunoblot analysis confirmed that transduction resulted in expression of TPC1 and TPC2  and in the number of responding cells (70–100% of transduced cells)  Fig–G.Tpcn1/2Dren mice used in the previous studies . This has allowed us to express various channels on a null background, record endogenous endo-lysosomal TPC currents, and image Ca2+ signals in the same cell type, permitting a direct comparison of results.In view of such contentions, we have investigated the role of TPCs in NAADP-dependent signalling in embryonic fibroblasts from 2+ signalling. We conclude this because: (i) NAADP-dependent Ca2+ responses were eliminated in Tpcn1/2−/− cells whereas Ca2+ storage, pHL, and PIP2 responsiveness were preserved; (ii) NAADP responses were selectively rescued by TPCs and not by another Ca2+-permeant endo-lysosomal channel, TRPML1 , even though TPC1 supports NAADP-induced Ca2+ release as we have shown in the rescue experiments; it is possible that TPC1-decorated endosomes are simply absent from the organelle preparation or its coupling to NAADP is less robust and lost upon purification.Although clearly essential, are TPCs actually activated by NAADP? Our electrophysiological recordings suggest that they are. NAADP-evoked currents were robust in planar patch-clamp recordings of single endo-lysosomes from WT but undetectable in et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, Our recordings differ from those of the recent papers in several key ways: first, we successfully observed NAADP-stimulated currents in endo-lysosomal preparations, which mirrors previous work  is present in MEFs from WT mice (but not in MEFs from our Tpcn1/2−/− mice).Moreover, the expression of truncated TPCs can indeed occur physiologically; at least for Tpcn1/2Dren mice  of these shorter functional TPC proteins in the 2+-permeable channels ; under bi-ionic conditions, the PCa/PK was quantified as ∼270. These indicate that, under these conditions, endogenous mouse TPCs are highly selective for Ca2+ over K+, in agreement with our previous results with mouse channels , which broadly agrees with other studies showing permeability of mammalian TPCs to various cations such as K+, Cs+, Ba2+, Ca2+, Na+ and H+ P2 response. The residual PIP2-stimulated current is attributable to other endogenous channels, a likely candidate being TRPML1 given the characteristic inward rectifying curve P2 activates multiple channel families such as TPCs, TRPML1 and RyR Pet al, et al, et al, et al, Tpcn1 or Tpcn2 knockout mice were used and the gene disruption strategy used to generate them meant that large portions of TPC proteins could potentially still be produced  mask any TPC contribution.Recent studies suggest that NAADP may not bind to TPCs directly but via a smaller molecular weight NAADP-binding protein(s) P2 as a non-selective modulator of endo-lysosomal channels. Expression of various channels in this TPC-null background reinforces this conclusion in demonstrating that only Ca2+-permeable TPCs can rescue NAADP signals. Our data contradict recent assertions that TPCs are NAADP-insensitive Na+-selective channels and establish TPCs as NAADP-regulated Ca2+-permeable channels.In conclusion, the use of the first demonstrable TPC double-knockout mice affirms TPCs as CaTpcn1T159  mice Byg) mice ; Tpcn2 . Reaction products were analysed by agarose gel electrophoresis. Mice with genotype corresponding to Tpcn1T159/Tpcn2YHD437 were born at the expected Mendelian proportion .Homozygote Tpcn1 ; Tpcn2 ; Actb . Reaction products were analysed by agarose gel electrophoresis.For analysis of gene expression, RNA was extracted following an RNeasy QiaRNA extraction procedure (Qiagen) with an in-column DNase I treatment. One-step RT–PCR was performed in a reaction containing extracted total RNA, SuperScript III RT/Platinum Taq High Fidelity Enzyme Mix (Invitrogen), and gene-specific primers: Tpcn1  and Tpcn2  with Universal Probes (#95 for Tpcn1 and #106 for Tpcn2) in a Light Cycler 480 System (Roche). cDNA copy numbers were determined against a standard curve using a custom-made double-stranded DNA fragment containing the amplicon sequences for Tpcn1 and Tpcn2 .For RT–qPCR, cDNA was synthesized from RNA using high-capacity cDNA Reverse Transcription kit (Applied Biosystems). cDNA was subjected to qPCR using gene-specific, intron-flanking primers for Cells were fixed in 4% paraformaldehyde in PBS and permeabilized/blocked with 0.1% saponin/5% goat serum in PBS (a methanol permeabilization step was included for anti-PDI labelling). Antibody incubations were performed in PBS/0.01% saponin/5% goat serum. The primary antibodies used were anti-RFP , anti-mCherry , anti-Lamp1 , anti-TfR , anti-EEA1 , and anti-PDI . The secondary antibodies used were derived from goat serum, cross-absorbed, and conjugates of Alexa 488 (for organelle markers) or Alexa 546 (for mCherry) (Invitrogen). Cells were viewed on a Zeiss 510 META confocal microscope, in multitrack mode, using the following excitation/emission parameters (nm): Alexa 488 (488/505–530) and Alexa 546 (543/>560).2+ indicator Fura 2-AM and where indicated pre-treated with pharmacological agents before addition of NAADP/AM, followed by ATP. The maximum amplitude and the mean [Ca2+] were calculated on a single-cell basis. Further details are given in Supplementary Materials and Methods.MEFs were loaded with the ratiometric CaLTAs isolated using differential centrifugation . Inward currents are defined as ion movement from the endo-lysosomal lumen to cytoplasm , 0.2 mM Ca-MSA, and 10 mM HEPES (pH adjusted with KOH to 7.2); luminal solution was 70 mM K-MSA, 60 mM Ca-MSA, 1 mM MgCl2, and 10 mM HEPES (pH adjusted with MSA to 4.6). Mannitol was used to adjust osmolarity.For experiments using mixed Ca2+/Na+ solutions, the cytoplasmic solution contained 60 mM NaF, 100 mM Na-MSA, 0.2 mM Ca-MSA, 5 mM Hepes, and 5 mM MES (pH adjusted with NaOH to 7.2). Luminal solution was 70 mM Na-MSA, 60 mM Ca-MSA, 1 mM CaCl2, 5 mM Hepes and 5 mM MES (pH 4.6).For experiments using mixed Ca2, 5 mM Hepes, and 5 mM MES (pH 4.6). For Na+ experiments, all K+ salts were replaced by their equimolar Na+ version.For the bi-ionic experiments, the cytoplasmic solution contained 60 mM KF, 100 mM K-MSA, 5 mM Hepes, and 5 mM MES (pH 7.2 with KOH), whereas the luminal solution was 105 mM Ca-MSA, 2 mM CaClet al, 2, PIP2, and PIP2 were from A.G. Scientific. NAADP was from Tocris Bioscience.Currents in the absence of NAADP (or phosphoinositides) were subtracted from the currents in the presence of these stimulators as previously described  where appropriate and significance determined as P < 0.05. Graphs were usually annotated using the following conventions: P > 0.05 (ns), P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***). The number of responding cells (FigP < 0.00625.Data are presented as mean ± SEM and analysed by Student's cells Fig was asse


Functional analysis in a set of different GIC lines regarding sensitivity to disturbed homeostasis using A23187 and Thapsigargin, revealed that NSC-proximal GICs were more sensitive, corroborating the transcriptome data. Furthermore, Ca2+ drug sensitivity was reduced in GICs after differentiation, with most potent effect in the NSC-proximal GIC, supporting a stemness-associated Ca2+ sensitivity. NSCs and the NSC-proximal GIC line expressed a larger number of ion channels permeable to potassium, sodium and Ca2+. Conversely, a higher number of and higher expression levels of Ca2+ binding genes that may buffer Ca2+, were expressed in NSC-distal GICs. In particular, expression of the AMPA glutamate receptor subunit GRIA1, was found to associate with Ca2+ sensitive NSC-proximal GICs, and decreased as GICs differentiated along with reduced Ca2+ drug sensitivity. The correlation between high expression of Ca2+ channels (such as GRIA1) and sensitivity to Ca2+ drugs was confirmed in an additional nine novel GIC lines. Calcium drug sensitivity also correlated with expression of the NSC markers nestin (NES) and FABP7  in this extended analysis. In summary, NSC-associated NES+/FABP7+/GRIA1+ GICs were selectively sensitive to disturbances in Ca2+ homeostasis, providing a potential target mechanism for eradication of an immature population of malignant cells.Tumor-initiating cells are a subpopulation in aggressive cancers that exhibit traits shared with stem cells, including the ability to self-renew and differentiate, commonly referred to as stemness. In addition, such cells are resistant to chemo- and radiation therapy posing a therapeutic challenge. To uncover stemness-associated functions in glioma-initiating cells (GICs), transcriptome profiles were compared to neural stem cells (NSCs) and gene ontology analysis identified an enrichment of Ca Glioblastoma multiforme (GBM) is a highly malignant form of brain cancer with poor prognosis for affected individuals. Despite the combination of surgery, chemotherapy and radiotherapy, more than 90% of the patients show recurrence Although several signaling pathways such as Notch, Hedgehog-Gli, RTK-Akt, BMP/TGF-β, WNT-β-catenin and STAT3 have been shown to support self-renewal of stem cells and immature cancer cells 2+ channel signaling control diverse functions as proliferation and migration in stem cells and cancer cell lines 2+ has also been implicated in cancer cell survival 2+ channel subunit was able to drive liver tumor-initiating cells into apoptosis Ion channels have long been assigned the role of governing basic cellular processes in addition to electrical excitability and for example potassium and Ca2+ disturbances compared to more mature cell types.In this study, we set to investigate mechanisms unique for the stemness-associated functions in glioma cells and conclude that stem-like cells are more sensitive to CaGliNS1, G179NS and G166NS GIC lines were grown in culture as previously described Novel human malignant glioblastoma initiating cell (GIC) cultures (U3NNN-MG series of GIC lines) used in this study are part of the Uppsala University Human Glioma Cell Culture (HGCC) collection, which comprises well-characterized GBM-derived cancer initiating cell cultures . This work was approved by the Uppsala ethical review board (2007/353). All GIC lines were used between passages 15 and 30.50 values.GliNS1, G179NS and G166NS GIC lines, both undifferentiated and differentiated, were seeded on day 1 at 20% density onto laminin-coated 96 or 384 black well, flat bottom microplates (Corning). Compounds were added to the plates on day 2, followed by incubation for 48 hrs. FBS differentiated cells received serum-free media , supplemented with Glutamax, Hepes, B27, ¼ N2, no growth factors) during chemical compound treatment. DMSO was used as negative control. Viability assay was performed using the CellTiterGlo assay  according to the manufacturer's recommendations. Briefly, assay reaction buffer was added to the wells using an automated multipipette, followed by shaking the microplate for 30 seconds and 7 min incubation in the dark. Luciferase intensity reading was then taken using Victor2 (Perkin Elmer) with a setting of 1 s luminescence reading per well. Z-factor was calculated for each experiment. For every cell line , at least 3 replicates were analyzed. Statistical calculations were performed with GraphPadPrism . Dose-response data were processed using log-linear interpolation to obtain log ICDrug assays in novel GIC lines (U3NNN-MG series) were seeded in 384-well microplates  24 hours prior to treatment using a Multidrop 384 liquid dispenser . To ensure growth phase at end of the assay (∼70% confluency) cells were seeded at a density ranging between 2000–4000 cells/well. Drugs were transferred using the ECHO550 non-contact liquid dispenser  to a 384 V- bottom polypropylene plate. The drugs were then diluted in medium and transferred using the MDT 384 head on a Janus automated workstation  to the cell plates. Drugs were tested in 11-point dose dilution series and assayed for viability after 72 hours of treatment on an EnVision Multilabel reader  using resazurin , at the excitation/emission wave- length 560/590 nm http://david.abcc.ncifcrf.gov) and GeneVenn (http://genevenn.sourceforge.net).Two replicates were analyzed for the undifferentiated and differentiated cell lines GliNS1, G166NS and G179NS, while three replicates were analyzed for DMSO, A23187 and Thapsigargin treated GliNS1 and G166NS. Total RNA was extracted from cells grown to subconfluency using the RNeasy Mini Kit (Qiagen), following the manufacturer's instructions. Fluorometric quantitation of RNA concentration and quality was done using the Qubit RNA assay kit (Invitrogen). We used 300 ng of total RNA in the preparation of the TruSeq library, for which we used the Illumina Low-Throughput TruSeq RNA Sample Preparation Kit protocol resulting in barcoded cDNA. 50 ng of barcoded TruSeq products were used for Illumina RNA sequencing. Samples were sequenced on an Illumina HiSeq 2000 sequencer as single-end 51-nucleotide reads according to the manufactures protocol. Raw reads were mapped to the reference human genome and normalized data was generated for each genomic feature using STRT software The Uppsala U3NNN-MG cell lines were not analyzed in replicates. For each cell line total RNA was extracted from cultured cells using the RNeasy Mini kit (Qiagen) and was labeled and hybridized on Affymetrix GeneChip Human Exon 1.0 ST arrays following the instructions of the manufacturer. The expression values were RMAnormalized using the Affymetrix Expression Console software.Cells cultured attached on cover glass were fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature (RT) followed by antibody incubation at 4°C overnight. The following primary antibodies were used: rabbit anti-glial fibrillary acidic protein (GFAP) (DAKO Cytomation) and mouse anti-beta III tubulin (Tuj1) (Millipore). The cells were then incubated with fluorescence-labeled secondary antibodies anti-rabbit Alexa Fluor 555 and Alexa Fluor 488 (Invitrogen) for 1 hr at RT. Nuclei were counterstained with DAPI and mounted using Immumount (DAKO). Images of stained cells were acquired with an Olympus FV1000 confocal microscope and processed with Adobe Photoshop CS5.1 software.Cells were lysed in RIPA buffer and protein concentration was quantified using the Bicinchoninic Acid (BCA) Protein Assay Kit . Equal amounts of protein samples were separated by electrophoresis using 10% Mini-PROTEAN TGX gels (Bio-Rad) or 4–12% Bis-Tris polyacrylamide gradient gel  under reducing conditions and transferred onto a PVDF membrane (Bio-Rad) or nitrocellulose membrane . Membranes were blocked in 5% milk for 1 hour and incubated overnight at 4°C with the following primary antibodies: rabbit anti-GRIA1 antibody (Abcam), rabbit anti-S100 alpha 6 antibody (Abcam), mouse anti-BLBP antibody (Millipore) and mouse anti-beta actin antibody (Abcam). The membranes were then incubated with the appropriate horseradish peroxidase-labeled secondary antibody  before being revealed by chemiluminescence ). The band intensities were quantified using ImageJ .et alTo determine the relationship between human GIC lines and human fetal neural stem cells (NSC), we re-analyzed the microarray data from a previous study by Pollard et al study  and a NSC line (hf5205NS) showed that more genes were expressed in NSCs than GIC lines  on one hand, and Ca2+ binders that reduce free intracellular Ca2+ on the other (here termed Ca2+ buffers) 2+ provokers compared to the NSC-distal GIC line (G166NS). These ranged from Ca2+ permeable ion channels , voltage-gated Ca2+ ion channels (CACNA1C/-E/-H) and Ca2+-activated potassium channels (e.g. KCNN3) (2+ buffers (for example S100 family genes and CALB1).Cytosolic Ca. KCNN3) . In cont2+ gene expression between tested GIC lines and NSCs, expression of Ca2+ provokers and buffers was analyzed in more detail in all three GIC lines (2+ permeable ionotropic glutamate receptor), which showed the highest expression among glutamate receptors, ranked the GIC lines identically to the order GliNS1> G179NS> G166NS seen in the PCA analysis  belonging to the SLC8 family and the SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) pump localized in the endoplasmic reticulum (ER) membrane, actively remove cytosolic Ca2+ to maintain homeostasis 2+ ion channels and Ca2+ binding genes, we next performed a drug-mediated challenge of Ca2+ homeostasis and signaling, in the GIC lines. Cells were exposed to either to the target independent cation (Ca2+) ionophore A23187 or the SERCA pump inhibitor Thapsigargin . Validation of differentiation was done by transcriptome analysis of the GIC lines and their differentiated progeny using RNA sequencing  in differentiated GICs and compared to undifferentiated GICs revealing a clearly reduced effect on cell viability in all GIC lines upon differentiation and with the strongest effect in the drug sensitive NSC-proximal GIC line (GliNS1). These f2+ sensitivity, transcriptome data from nine novel GIC lines (derived in an independent laboratory) was compared to Ca2+ sensitivity data from exposure to Thapsigargin (72 h exposure). 7 out oexposure). Analysiexposure) mRNA expexposure). The corexposure), which wexposure). Furthersitivity .2+ (Ca2+ provokers), i.e. GRIA1 and the inward rectifier K+ channel KCNJ4, which may participate in maintaining a depolarized membrane potential required to activate voltage-gated Ca2+ channels and Ca2+ permeable glutamate receptors. In summary, the correlation between functional Ca2+ drug sensitivity and gene expression suggests participation towards sensitivity to drug-elicited Ca2+ overload, by a network of genes involved in maintaining Ca2+ homeostasis and membrane potential.To identify genes in this data set that also associated with a NSC-proximal stemness signature in GICs, the set was further filtered for genes, which also (1) had a higher expression in GliNS1 compared to G166NS and (2) were downregulated upon differentiation . This re2+ underlying the differential level of Ca2+ sensitivity in GICs, the NSC-proximal GliNS1 and NSC-distal G166NS were exposed to A23187 for 7 hours, followed by transcriptome analysis by RNA sequencing (“drug reactome mapping”) were analyzed by gene enrichment and gene ontology, which showed that cell cycle related genes were altered (To identify intracellular responses to Camapping”). In the  altered ., sugges altered .2+ signaling in sensitive GICs. This may be the consequence by an inability to effectively reduce cytosolic Ca2+ levels. Interestingly, a very similar set of genes were altered in both the NSC-proximal (GliNS1) and the NSC-distal (G166NS) GIC lines, including Ca2+-binding genes acting as buffers (e.g. HSPA5 and HSP90B1) and Ca2+ related ER stress response (e.g. HERPUD1 and CALR). Also Ca2+-activated transcription factors (e.g NFATC2) were induced in both lines  and BCL2 genes  to identify robustly altered genes with a potential biological significance. Strikingly, the GliNS1 line induced a clearly higher global transcriptome fold change than the less sensitive G166NS  suggestith lines , suggestL2 genes , providi2+ homeostasis and signaling. This finding was functionally validated by increasing cytosolic Ca2+ using the ionophore A23187, which causes uncontrolled uptake of Ca2+ over the plasma membrane, and Thapsigargin, which causes elevated cytosolic Ca2+ via depletion of Ca2+ stored in ER. GICs with a NSC-like transcriptome profile were more sensitive to disrupted Ca2+ homeostasis and undifferentiated GICs were more sensitive than differentiated GICs. The degree of similarity in the transcriptome profile of individual GIC lines and NSCs correlated with Ca2+ sensitivity. In contrast, NSC-distal GICs, expressing markers of reactive astrocytes and microglia, expressed higher levels of Ca2+ buffers and were comparatively less sensitive to Ca2+ drugs. Ca2+ signaling is well known to be critical for normal somatic cells and embryonic 2+ that we identified appears to correlate uniquely to stemness (e.g. expression of nestin and BLBP), suggesting that certain functions in NSC-like immature cancer cells, are affected differently by perturbing Ca2+ homeostasis than are more differentiated mature cells.Our study identified a stemness-associated sensitivity in GICs related to Ca2+-sensitivity appeared to be capacity to reduce cytosolic Ca2+, either via lower expression of Ca2+ channels or via expression of Ca2+ buffers, such as S100 proteins. In this context the AMPA receptor subunit GRIA1 appeared as an interesting marker predicting sensitivity and potentially involved mechanistically in increasing sensitivity by allowing Ca2+ influx in response to glutamate. GRIA1 is a marker of GICs, as previously published 2+ homeostasis and membrane potential in immature cells, may contribute to the observed differential sensitivity to Ca2+ overload.An important component in the observed Ca2+ drug reactome analysis identified upregulated expression of a set of Ca2+ binding proteins and Ca2+-activated transcription factors, suggesting a positive feedback loop of Ca2+-responsive transcription that may be expected to further increase a Ca2+ transcriptional response. This is somewhat surprising, unless Ca2+ transcriptional response also includes functions to reduce or abort an intracellular calcium surge. A comparison between GliNS1 and the slightly less Ca2+ sensitive GIC line G166NS was made to potentially identify genes that may correlate with Ca2+ overload and hence may protect the cells. The drug reactome profile unique for G166NS identified genes protecting from apoptosis, thus contributing to the relative lower sensitivity to Ca2+ overload and giving an alternative explanation than interplay between Ca2+ provokers and buffers.Interestingly, Ca2+ sensitivity may be a novel such target with ability to eradicate a potentially malignant subpopulation in brain tumors. In this context, nestin, BLBP and GRIA1 are potential biomarkers predicting sensitivity to this drug in brain tumor cells.We recently reported a novel target in GICs critical for cell volume homeostasis S1 Fig2+ provokers such as permeable glutamate receptor subunits (A) or Ca2+ buffers (B) in GliNS1, G179NS and G166NS.Analysis of expression of Ca(TIF)Click here for additional data file.S2 FigOverview of genes involved in cell cycle progression with direction of altered expression indicated in red (upregulation) or green (downregulation).(TIF)Click here for additional data file.S1 TableIllumina mRNA sequencing data in simple reads of undifferentiated and differentiated GICs.(XLSX)Click here for additional data file.S2 TableTop hits of pearson correlation analysis of genes and sensitivity to Thapsigargin in novel GIC cell lines.(XLSX)Click here for additional data file.S3 TableA list of the novel GIC cell lines, their tumor initiating property and the median survival in mice (n = 5–10 mice per line).(XLSX)Click here for additional data file.S4 TableDifferential gene expression analysis of vehicle control vs A23187 treated GliNS1 and vehicle control vs A23187 treated G166NS.(XLSX)Click here for additional data file.


SOCE is activated by Ca2+ release from the endoplasmic reticulum (ER), whereupon stromal interacting molecule 1 (STIM1) forms oligomers, redistributes to ER–plasma-membrane junctions and opens plasma membrane Ca2+ channels. The mechanisms by which mitochondria interfere with the complex process of SOCE are insufficiently clarified. In this study, we used an shRNA approach to investigate the direct involvement of mitochondrial Ca2+ buffering in SOCE. We demonstrate that knockdown of either of two proteins that are essential for mitochondrial Ca2+ uptake, the mitochondrial calcium uniporter (MCU) or uncoupling protein 2 (UCP2), results in decelerated STIM1 oligomerization and impaired SOCE following cell stimulation with an inositol-1,4,5-trisphosphate (IP3)-generating agonist. Upon artificially augmented cytosolic Ca2+ buffering or ER Ca2+ depletion by sarcoplasmic or endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors, STIM1 oligomerization did not rely on intact mitochondrial Ca2+ uptake. However, MCU-dependent mitochondrial sequestration of Ca2+ entering through the SOCE pathway was essential to prevent slow deactivation of SOCE. Our findings show a stimulus-specific contribution of mitochondrial Ca2+ uptake to the SOCE machinery, likely through a role in shaping cytosolic Ca2+ micro-domains.Mitochondria contribute to cell signaling by controlling store-operated Ca Correlations between the dynamics of ER Ca2+ depletion and STIM1 oligomerization upon different modes of Ca2+ mobilization suggest that mitochondrial Ca2+ sequestration predominately shapes IP3-mediated cytosolic Ca2+ micro-domains, which facilitates STIM1 oligomerization under physiological conditions of cell stimulation. In addition, MCU-dependent mitochondrial buffering of entering Ca2+ is crucial for the maintenance of SERCA-inhibition-induced SOCE signals. In summary, we highlight a special and tight regulation of STIM1 activation and SOCE maintenance by mitochondrial Ca2+ uptake upon physiological and non-physiological stimuli.In this study, mitochondrial Cather MCU  (MCUKD)  or UCP2  (UCP2KD)+ uptake . MCU, thlization , whereas release . Accordisupplementary material Fig. S1A) strongly reduced mitochondrial sequestration of intracellularly released  and entering Ca2+ . By contrast, HeLa cells stably depleted of UCP2  showed an impaired mitochondrial Ca2+ signal upon intracellular Ca2+ mobilization, whereas mitochondrial uptake of entering Ca2+ was not affected . These results confirm our recent findings, which have revealed the exclusive involvement of UCP2 and UCP3 in mitochondrial Ca2+ sequestration upon IP3-mediated intracellular Ca2+ release , oxygen consumption rate  and cellular ATP content  in stable knockdown cells. All of these variables remained unaffected by the knockdown of MCU or UCP2. Moreover, the morphology of mitochondria , ER structure  and the contact sites between these organelles  were not altered by the stable knockdown of MCU or UCP2. In summary, these data demonstrate that the knockdown of MCU or UCP2 results in specific effects on distinct modes of mitochondrial Ca2+ uptake. Therefore, MCUKD and UCP2KD cells can serve as suitable models to specifically investigate the role of mitochondrial Ca2+ sequestration in SOCE regulation. In addition, due to the selective engagement of UCP2 in mitochondrial Ca2+ sequestration depending on the source of Ca2+   , these c2+]ER ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), which accumulates in the cytosol and prevents the formation of high-concentration cytosolic Ca2+ micro-domains ; thus indicating that Ca2+ entry is required to maintain elevated cytosolic Ca2+ levels during stimulation. By contrast, in MCU- or UCP2-depleted cells, the SOCE-dependent second phase of cytosolic Ca2+ elevation was strongly reduced , a dominant-negative form of the wild-type Orai1 protein (2+ responses upon stimulation of control cells expressing Orai1(E106Q) showed a strongly reduced plateau phase in the presence of extracellular Ca2+ . Notably, the already transient elevation of cytosolic Ca2+ in response to histamine in the MCUKD and UCP2KD cells remained unaffected by an additional expression of the dominant-negative Orai . This indicates that STIM1–Orai1-dependent SOCE is not activated in cells lacking the mitochondrial Ca2+ sequestration of intracellularly released Ca2+. Similar results were obtained in all cell models  upon incubation with FCCP and oligomycin A prior to histamine stimulation . Interestingly, the sustained phase of the cytosolic Ca2+ signal in MCU- and UCP2-silenced cells in the absence of FCCP and oligomycin A was reduced to the same level as in control cells pre-exposed to the drugs  in combination with fura-2 , the thapsigargin-induced SOCE signal was almost completely terminated within several minutes , whereas the lack of this protein did not affect mitochondrial uptake of entering Ca2+ . Although it has been hypothesized that UCP2 and UCP3 are preferentially located within sites of mitochondrial Ca2+ uptake that face areas of ER Ca2+ release , the proposed pore-forming subunit of a mitochondrial Ca2+ channel , the electrophysiological correlate of SOCE  of 60 µM . Prior to experiments, cells were washed and maintained for 20 minutes in a HEPES-buffered solution containing 138 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM HEPES, 2.6 mM NaHCO3, 0.44 mM KH2PO4, 0.34 mM Na2HPO4, 10 mM d-glucose, 0.1% vitamins, 0.2% essential amino acids and 1% penicillin-streptomycin, the pH of which was adjusted to 7.4 with NaOH. During the experiments, cells were perfused with a Ca2+-containing buffer, which consisted of 145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM d-glucose and 10 mM HEPES, the pH of which was adjusted to 7.4 with NaOH. During experiments in which a Ca2+-free solution was applied to the cells, the CaCl2 was replaced with 1 mM EGTA.Cell culture materials were obtained from PAA laboratories . FCCP, oligomycin A, histamine, BHQ and EGTA were purchased from Sigma Aldrich . Thapsigargin and BAPTA-AM were from Abcam® knockdown cell lines were purchased from Tebu-bio . We used HeLa cells stably expressing scrambled shRNA (control) or shRNA against MCU (MCUKD) (KD).HeLa SilenciX (MCUKD)  or the UTM transfection reagent at 3 µg/well  in 1 ml of serum- and antibiotic-free medium. Cells were maintained in a humidified incubator  for 16–20 hours prior to changing the culture medium. All experiments were performed either 24 hours or 48 hours after transfection.HeLa cells were grown in Dulbeccos's Modified Eagle Medium (Sigma Aldrich) containing 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin, and they were plated on 30-mm glass coverslips. At 60–80% confluence, cells were transfected with 1.5 µg (per 30-mm well) of plasmid DNA encoding the appropriate sensor/fluorophore using TransFastKD and UCP2KD HeLa cells using a Total RNA isolation kit . For reverse transcription, a High Capacity cDNA Reverse Transcription Kit from Applied Biosystems  was used. The analysis of the expression of MCU and UCP2 was performed by conventional PCR using GoTaq Green master mix (Promega) and by real-time (RT)-PCR using QuantiFast SYBR Green RT-PCR kit  on a LightCycler 480 . RNA polymerase II (RPOL2) was used as the housekeeping control. Primers for RPOL2 and MCU were obtained from Invitrogen , and their sequences were as follows: RPOL2 forward, 5′-CATTGACTTGCGTTTCCACC-3′; RPOL2 reverse, 5′-ACATTTTGTGCAGAGTTGGC-3′; MCU forward, 5′-TTCCTGGCAGAATTTGGGAG-3′; and MCU reverse, 5′-AGAGATAGGCTTGAGTGTGAAC-3′. For human UCP2, the QuantiTect®Primer Assay  was used.RNA was isolated from SilenceX control, MCUg for 5 minutes and resuspended in the Ca2+-containing buffer described above. JC-1 fluorescence was detected using a fluorescence spectrophotometer . JC-1 was excited at 490 nm and 540 nm and emission was collected at 540 nm and 590 nm, respectively. The basal fluorescence ratio was normalized to the ratio after dissipation of mitochondrial membrane by treatment with 10 µM FCCP.HeLa cells were loaded with 1 µM of the ratiometric dye JC-1 (Invitrogen) in culture medium at 37°C for 40 minutes, washed with PBS, dissociated by trypsinization, centrifuged at 262 d-glucose and 1 mM sodium pyruvate at 37°C in a non-CO2 environment. Oxygen consumption rates were subsequently measured using an XF96 extracellular flux analyzer.Cells were plated in XF96 polystyrene cell culture microplates (Seahorse Bioscience) at a density of 30,000 cells per well. After an overnight incubation, cells were washed and preincubated for 30 minutes in unbuffered XF assay medium (Seahorse Bioscience) supplemented with 5.5 mM Separation of adenine nucleotides  after cell lysis was performed on a Hypersil ODS column , using a L2200 autosampler, two L-2130 HTA pumps and a L2450 diode array detector , as described previously .2+]ER and [Ca2+]mito were followed in cells expressing the ER- or mitochondria-targeted cameleons D1ER (535/F480)/R0 , to correct for photobleaching and/or photochromism, as described previously , using a 40× objective (Zeiss). Fura-2 and the 4mtD1GO-Cam were alternately excited at 340 nm or 380 nm and at 477 nm, respectively, with an ultra-fast switching monochromator, the Polychrome V (Till Photonics), equipped with an excitation filter (E500spuv) and a dichroic filter . Emitted light was simultaneously collected at 510 nm (Fura-2 and GFP of GO-Cam) and at 560 nm (FRET-channel of GO-Cam) using a single beam splitter design  that was equipped with a dual band emission filter  and a second dichroic filter . Images were recorded with a CCD camera . The digital imaging system was controlled by the live-acquisition software v2.0.0.12 (Till Photonics), as described previously  and a 100× objective . Fluorophores were illuminated at 488 nm  and 515 nm . Emitted light was acquired with a CCD camera  using the emission filters ET535/30m and E570LPv2 (Chroma Technology) mounted on a computer-controlled fast-filter wheel (Ludl Electronic Products). All devices were controlled by VisiView Premier Acquisition software (Visitron Systems). The ER and mitochondria z-stacks were deconvoluted using the iterative quick maximum likelihood estimation algorithm (QMLE) of Huygens 2.4.1p3 . Subsequently, combined three-dimensional rendering of the organelles was performed with Imaris 3.3 software . Quantitative mitochondrial shape analysis and colocalization computations were performed with the integrated morphometric analysis plug-in of MetaMorph 7.7.0.0 software (Visitron). The intensity threshold values of shape analysis and colocalization computations did not significantly differ within all the samples analyzed and were determined over a range that completely eliminated background fluorescence but preserved organelle structures.High resolution n reflects the number of cells. Statistical analyses were performed by using the unpaired Student's t-test, and P<0.05 was considered to be significant.Data shown represent the mean±s.e.m., where