Tulane virus is one of many enteric caliciviruses that cause acute gastroenteritis and diarrheal disease. Globally, enteric caliciviruses affect both humans and animals and amass >65 billion dollars per year in treatment and health care-associated costs, thus imposing an enormous economic burden. Recent progress has resulted in several cultivation systems  to study human noroviruses, but mechanistic insights into the viral factors and host pathways important for enteric calicivirus replication and infection are still largely lacking. Here, we used Tulane virus, a calicivirus that is biologically similar to human noroviruses and can be cultivated by conventional cell culture, to identify and functionally validate NS1-2 as an enteric calicivirus viroporin. Viroporin-mediated calcium signaling may be a broadly utilized pathway for enteric virus replication, and its existence within caliciviruses provides a novel approach to developing antivirals and comprehensive therapeutics for enteric calicivirus diarrheal disease outbreaks. Caliciviridae family cause acute gastroenteritis in humans and animals, but the cellular processes needed for virus replication and disease remain unknown. A common strategy among enteric viruses, including rotaviruses and enteroviruses, is to encode a viral ion channel  that is targeted to the endoplasmic reticulum (ER) and disrupts host calcium (Ca2+) homeostasis. Previous reports have demonstrated genetic and functional similarities between the nonstructural proteins of caliciviruses and enteroviruses, including the calicivirus NS1-2 protein and the 2B viroporin of enteroviruses. However, it is unknown whether caliciviruses alter Ca2+ homeostasis for virus replication or whether the NS1-2 protein has viroporin activity like its enterovirus counterpart. To address these questions, we used Tulane virus (TV), a rhesus enteric calicivirus, to examine Ca2+ signaling during infection and determine whether NS1-2 has viroporin activity that disrupts Ca2+ homeostasis. We found that TV increases Ca2+ signaling during infection and that increased cytoplasmic Ca2+ levels are important for efficient replication. Further, TV NS1-2 localizes to the endoplasmic reticulum, the predominant intracellular Ca2+ store, and the NS2 region has characteristics of a viroporin domain (VPD). NS1-2 had viroporin activity in a classic bacterial functional assay and caused aberrant Ca2+ signaling when expressed in mammalian cells, but truncation of the VPD abrogated these activities. Together, our data provide new mechanistic insights into the function of the NS2 region of NS1-2 and support the premise that enteric viruses, including those within Caliciviridae, exploit host Ca2+ signaling to facilitate their replication.Enteric viruses in the IMPORTANCE Tulane virus is one of many enteric caliciviruses that cause acute gastroenteritis and diarrheal disease. Globally, enteric caliciviruses affect both humans and animals and amass >65 billion dollars per year in treatment and health care-associated costs, thus imposing an enormous economic burden. Recent progress has resulted in several cultivation systems  to study human noroviruses, but mechanistic insights into the viral factors and host pathways important for enteric calicivirus replication and infection are still largely lacking. Here, we used Tulane virus, a calicivirus that is biologically similar to human noroviruses and can be cultivated by conventional cell culture, to identify and functionally validate NS1-2 as an enteric calicivirus viroporin. Viroporin-mediated calcium signaling may be a broadly utilized pathway for enteric virus replication, and its existence within caliciviruses provides a novel approach to developing antivirals and comprehensive therapeutics for enteric calicivirus diarrheal disease outbreaks. Caliciviridae family consists of small, nonenveloped single-stranded RNA viruses with 11 recognized genera (–Recovirus). Recoviruses are members of a newly approved genus of CVs initially identified in stool samples from rhesus macaques, of which Tulane virus (TV) is the prototype strain , with ORF1 encoding the nonstructural proteins (NS1-7), and ORFs 2 and 3 encoding the capsid proteins VP1 (ORF2) and VP2 (ORF3) , 14, 15.14–17–20–2+) storage organelles, with the ER Ca2+ concentration as high as 1 mM  cytoplasmic Ca2+ concentrations at cellular rest  to dysregulate Ca2+ homeostasis in order to usurp Ca2+-dependent host processes  from rotavirus (RV) and the 2B nonstructural protein of enteroviruses (EVs) and some other picornaviruses  require elevated cytosolic Ca2+ to facilitate replication  with different infectious doses  or γ-irradiated inactivated TV and performed live-cell fluorescence microscopy during the infection. GCaMP6s is a green fluorescent protein (GFP)-based genetically encoded Ca2+ indicator that reports changes in cytosolic Ca2+ as an increase in fluorescence . We. We2+ isignaling . TogetheMOI of 5 . To visuver time . Heatmapncreases . The heaed cells , consist+ spikes . Taken t10.1128/mSphere.00506-19.5MOVIE S12+ signaling but that replication-competent virus does. Quantification of this imaging run can be seen in Movie S1, MPG file, 7.8 MB.Representative imaging run of LLC-MK2 GCaMP6s cells infected at a wide infectious dose  compared to irradiated TV. The movies qualitatively show that irradiated virus does not induce aberrant CaCopyright © 2019 Strtak et al.2019Strtak et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00506-19.6MOVIE S22+. Changes manifest as discrete Ca2+ spikes which can be observed as large changes in cytosolic fluorescence. Download Movie S2, MPG file, 9.6 MB.IF staining for TV nonstructural protein anti-Vpg overlaid onto short, continuous imaging run of TV-infected cells (MOI of 5) at 12 HPI. TV-infected cells (red) exhibit dynamic changes in cytosolic CaCopyright © 2019 Strtak et al.2019Strtak et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2+ signaling during TV infection, we sought to determine whether Ca2+ was involved in TV replication. To test this, we manipulated extracellular and intracellular Ca2+ levels and determined the effects on TV yield. Doubling the extracellular Ca2+ concentration (∼4 mM) did not affect TV yield  was reduced up to 4 log units  on TV replication. TG is an inhibitor of sarco/endoplasmic reticulum (SERCA) Ca2+ ATPase, which pumps cytosolic Ca2+ into the ER to help maintain ER Ca2+ stores. We treated TV-infected cells with TG and measured TV yield as described in Materials and Methods and found that TV replication is ∼3 log units lower in TG-treated cells than in dimethyl sulfoxide (DMSO)-treated cells   . These s2+ had on the TV-induced Ca2+ signaling exhibited in 2+ concentrations as described in Materials and Methods and performed live Ca2+ imaging of mock-infected and TV-infected MK2-G6s cells. TV-infected cells in 2 mM Ca2+  exhibited increased Ca2+ signaling, as observed above  did not further increase the Ca2+ spikes, but removing extracellular Ca2+ abolished the TV-induced Ca2+ spikes . BAPTA treatment during TV infection abrogates TV-induced Ca2+ signaling and results in a Ca2+ signaling profile that mirrors mock LLC-MK2 cells (left). Quantification of this imaging run can be seen in Movie S3, MPG file, 12.0 MB.Representative imaging run of LLC-MK2 GCaMP6s cells infected with TV (MOI of 10) (middle) compared to TV-infected cells treated with 0CaCopyright © 2019 Strtak et al.2019Strtak et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2+ signaling involving the ER Ca2+ store, much like the dysregulation of Ca2+ homeostasis by other enteric viruses observed in RV and EV infections. Both RV and EV encode a viroporin, or viral ion channel, that targets the ER Ca2+ store to activate aberrant Ca2+ signaling pathways that are critical for virus replication  Multiple-sequence alignment results of NS1-2 sequences from various calicivirus strains. Sequence alignment results through T-Coffee show that the C-terminal domain (CTD) of NS1-2 is well conserved, particularly in the putative viroporin domain. Download FIG S1, PDF file, 2.84 MB.(A) Aligning the nonstructural regions of both the TV and EV genome reveals that TV NS1-2 is the positional homolog of enterovirus (EV) 2B, a Copyright © 2019 Strtak et al.2019Strtak et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00506-19.2FIG S2FIG S2, PDF file, 0.3 MB.Confidence of prediction values of secondary structure for NS1-2 amino acid residues. Computational prediction for the viroporin domain (VPD) of NS1-2 places it between aa195-215, which is predominantly helical, with ≥75% confidence of prediction throughout most of the C-terminal domain (aa160-232) (dark blue bars = conf). Download Copyright © 2019 Strtak et al.2019Strtak et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the First, we tested whether TV NS1-2 was an integral membrane protein and whether it localized to the ER similar to NS1-2 from other caliciviruses. To do so, we generated bacterial and mammalian expression vectors of full-length NS1-2. For mammalian expression vectors, we N-terminally fused full-length NS1-2 to mRuby3 (henceforth referred to as RFP-NS1-2 [RFP stands for red fluorescent protein]). From these constructs, we generated two truncation mutants of wild-type NS1-2 in both mammalian and bacterial expression vectors: the first, NS1-2 Δ176, was predicted to have TMD1 but lack the viroporin domain, and the second, NS1-2 Δ157, was predicted to lack both TMD1 and the VPD. We then transfected wild-type, full-length (WT) RFP-NS1-2, RFP-NS1-2 Δ157, and RFP-NS1-2 Δ176 into HEK 293FT cells and harvested cell suspensions next day. Samples after cell lysis, sonication, and fractionation were collected for SDS-PAGE Western blots. We found both Δ176 and WT TV NS1-2 in the total fraction (T) and membrane pellets (M), but not in the supernatant (S), suggesting that TMD1 mediates membrane association . Additio10.1128/mSphere.00506-19.3FIG S3FIG S3, PDF file, 1.46 MB.Immunoblot analysis of cell fractionation studies of bacterially expressed full-length WT NS1-2 and the Δ176 and Δ157 truncation mutants. Samples of total cell lysate (T), the soluble fraction (S), or the membrane fraction (M) were resolved by SDS-PAGE and detected by immunoblot using an anti-6xHis antibody. Oligomers of NS1-2 and the truncations are indicated by arrowheads. Download Copyright © 2019 Strtak et al.2019Strtak et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00506-19.4FIG S4E. coli BL21(DE3)pLysS cells transformed with the indicated expression vector were induced for 3 h with 1 mM IPTG or left uninduced (UI), cells were collected and lysed in PBS supplemented with 1% SDS. Lysates were resolved by SDS-PAGE, and proteins were detected by the anti-Vpg antisera (left) or anti-6xHis antibody (right). The positions of His-Vpg monomer and oligomer are indicated by arrowheads. (B) Validation of the specificity of the anti-TV antisera using mock-inoculated or TV-infected LLC-MK2 cell lysates. Blots were detected by the anti-TV antisera (left) or anti-GAPDH antibody (right). (C) Table of primers used to construct the bacterial and mammalian expression vectors. Download FIG S4, PDF file, 1.82 MB.Validation of antisera against TV Vpg (A) and purified TV particles (B). (A) Bacterial expression vectors for the indicated TV nonstructural proteins were generated using the pET46 vector, which includes a N-terminal 6xHis tag. Copyright © 2019 Strtak et al.2019Strtak et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2+ imaging data exhibited large changes in cytosolic Ca2+ during TV infection, we tested whether NS1-2 has viroporin activity. We performed the Escherichia coli lysis assay, which is a classical viroporin functional assay, wherein viroporin expression by E. coli BL21(DE3)pLysS results in permeabilization of the inner membrane, resulting in T7 lysozyme-mediated cell lysis  induces aberrant Ca2+ signaling in cells with similar activity to RV NSP4 (middle) and EV 2B (right). Quantification of this imaging run can be seen in Movie S4, MPG file, 14.1 MB.Representative signaling from viroporin transfection experiments in LLC-MK2 GCaMP6s cells. The movies show that cells exhibit aberrant CaCopyright © 2019 Strtak et al.2019Strtak et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Our computational studies above identified a putative TV NS1-2 VPD from aa195-212. To determine whether the NS1-2 viroporin activity maps to this putative VPD, we generated C-terminal truncation mutants in bacterial expression vectors with deletions after aa212 (A212-Δ), after aa194 (W194-Δ), or after aa176 (D176-Δ) and characterized them in the lysis assay . We foun2+ signaling in MK2-G6s cells. Since recombinant expression of full-length RFP-NS1-2 induced aberrant Ca2+ signaling  or infected with MNV-1 at an MOI of 5 (right). Infected cells show increased basal GCaMP6s fluorescence and an increased number of CaCopyright © 2019 Strtak et al.2019Strtak et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2+ signaling similar to TV NS1-2, we generated a mammalian expression vector of GII.3 NS1-2 N-terminally tagged with RFP and expressed it in MK2-G6s cells by transient transfection to perform Ca2+ imaging. Compared to the RFP control, expression of recombinant GII.3 NS1-2 causes aberrant Ca2+ signaling and significantly increases the number Ca2+ spikes similar to TV expression of RFP-NS1-2  of NS1-2 remains highly conserved . For MNV63–FP-NS1-2 . Represepression . Thus, t10.1128/mSphere.00506-19.10MOVIE S62+ signaling upon expression of RFP-tagged TV NS1-2 (top) or GII.3 HuNoV NS1-2 (bottom). Quantification of this imaging run can be seen in Movie S6, MPG file, 4.7 MB.Representative signaling from viroporin transfection experiments in LLC-MK2 GCaMP6s cells. The movies show that cells exhibit aberrant CaCopyright © 2019 Strtak et al.2019Strtak et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2+ signaling because Ca2+ signals are used by all cells to regulate a vast array of cellular functions. Therefore, this represents a powerful strategy to reconfigure host cell physiology via targeted disruption of host Ca2+ homeostasis. The overarching goal of this study was to determine whether dysregulation of Ca2+ signaling is a characteristic of caliciviruses and whether this is due to the production of a viroporin protein similar to picornaviruses. To address these questions, we studied TV, as a model calicivirus, using a combination of live-cell Ca2+ imaging and other classical techniques. The major new findings of this study are as follows. (i) TV infection causes aberrant Ca2+ signaling that coincides with viral protein synthesis and replication. (ii) Cellular Ca2+ is critical for TV replication, and buffering of cytosolic Ca2+ severely reduced viral yield. (iii) TV NS1-2 has viroporin activity and dysregulates Ca2+ signaling in mammalian cells similar to TV infection. (iv) NS1-2 viroporin activity maps to a C-terminal integral membrane viroporin domain, and truncation of this domain abrogates the NS1-2-induced activation of Ca2+ signaling. Finally, we extended these observations to show that both MNV-1 infection and expression of recombinant HuNoV NS1-2 induces aberrant Ca2+ signaling. To our knowledge, these results are the first to show exploitation of Ca2+ signaling by a calicivirus and identification of NS1-2 as a Ca2+-disrupting viroporin. These findings further extend the functional homology between the calicivirus nonstructural proteins and their picornavirus positional homologs.As obligate intracellular pathogens, viruses are adept at exploiting host pathways to facilitate replication. Viruses from many different taxonomic families activate aberrant Ca2+ signaling to facilitate virus replication is a common feature of many viruses  or block refilling of ER Ca2+ stores (TG). Further, as we recently reported for RV infection, the TV-induced increase in cytosolic Ca2+ manifests as many discrete Ca2+ signals rather than a monophasic increase in Ca2+ over the infection  How do they benefit TV replication? Both RV and EV have been shown to exploit Ca2+ signaling to activate the biosynthetic early stages of autophagy, which facilitates virus replication through rearrangement of cellular membranes to form replication complexes   and the P2-P3 nonstructural proteins of picornaviruses , 46, 47.iroporin , 45. We iroporin , 45. Thigomeric) , 39, 40.eficient . Analogo2+ signaling observed in TV-infected cells is phenotypically similar to that induced by recombinant expression of full-length NS1-2, but the Ca2+ signaling is abrogated by truncation of the viroporin domain. Further, NS1-2 primarily localized to the ER, which is a major intracellular Ca2+ storage organelle. Thus, our model predicts that NS1-2 directly releases Ca2+ from the ER; however, it is likely that both NS1-2 and activation of host Ca2+ signaling pathways contribute to the observed Ca2+ signals. Ca2+ signals from NS1-2 require it to directly conduct Ca2+ and have a high enough conductance that the ER Ca2+ release event can be detected by a fluorescent Ca2+ indicator, yet these unitary events are challenging to detect even for large channels like the inositol trisphosphate receptor (IP3R)  pathway, wherein decreased ER Ca2+ levels activate the ER Ca2+ sensing protein stromal interaction molecule 1 (STIM1). Activated STIM1 translocates to ER microdomains adjacent to the plasma membrane and opens Ca2+ influx channels, like Orai1, to elevate cytosolic Ca2+  . Future  channel . Second,lic Ca2+ , 33. Thi2+ signaling by TV may provide insights into the pathophysiology of enteric caliciviruses. Both IP3-mediated ER Ca2+ release and SOCE have been shown to activate chloride secretion from epithelial cells  and are a major cause of foodborne illnesses. However, the molecular mechanisms of how these caliciviruses cause vomiting and diarrhea, the chief symptoms of AGE, have not been characterized. The dysregulation of Caal cells , 74. In ntegrity . HyperacTV NS1-2 . This su2+ singling during infection, and cellular Ca2+ is critical for robust TV replication. Further, we found that the NS2 domain of the NS1-2 nonstructural protein is a viroporin that alone induces Ca2+ signaling similar to TV infection. Together, these results indicate that NS1-2 is functionally analogous to EV 2B and RV NSP4. While little is known about the function(s) of NS1-2, and particularly the NS2 domain of NS1-2, the similarity with other Ca2+-conducting viroporins may provide a broader insight for understanding NS1-2 functions. Finally, antiviral drugs against viroporins have been developed for influenza virus M2 and HIV Vpu . Cell lines were grown in high-glucose Dulbecco modified Eagle medium (DMEM)  containing 10% fetal bovine serum (FBS) (Corning lot no. 35010167) and antibiotic/antimycotic (Invitrogen), and maintained at 37°C with 5% CO2+ conditions , intracellular Ca2+ chelator 50 μM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM), or the sarco/endoplasmic reticulum calcium ATPase (SERCA) blocker thapsigargin (TG). Ca2+-free DMEM was purchased from Gibco . Standard high-glucose DMEM (Sigma) has 1.8 mM CaCl2, which we refer to as “2 mM Ca2+,” and media with 4 mM Ca2+ was made by adding 2 mM CaCl2 to the standard high-glucose DMEM (Sigma). We maintained TV-infected cells under these conditions until the positive control  had ∼90% CPE. Progeny virus was harvested by three freeze/thaw cycles, and the virus yield was determined by plaque assay. For plaque assays, cells are seeded at 75,000 cells/well in 24-well plates, and 2 days after seeding, the cells were inoculated for 1 h with 10-fold serial dilutions of the sample. Then, we removed the inoculum and added the overlay. Overlays for plaque assays were made by mixing equal parts of 1.2% Avicel (FMC Corporation) and 2× DMEM (Gibco). Plaque assays were harvested at 72 h and fixed and stained with crystal violet (3% solution) to visualize plaques. Titer is represented as plaque-forming units per milliliter (PFU/ml). To compare plaque size, images of wells were analyzed using Nikon Elements software to measure the longest diameter, and the resulting data were graphed using GraphPad Prism software.LLC-MK2 cells were seeded at 125,000 cells/well in 24-well plates  and inoculated the next day with TV at an MOI of 1 for 1 h. Inoculum was removed, and cell medium was replaced containing different extracellular CaOne-step growth curves for TV were performed using a modified protocol from previous reports , 15. Bri2. Time-lapse live-cell Ca2+ imaging experiments were conducted from ∼2 HPI until ∼18 to 24 HPI on a Nikon TiE epifluorescence microscope using a Spectrax LED light source (Lumencor) and a 20× Plan Apo  objective. Images were acquired at 1 or 2 images/well point/minute. Images were acquired and analyzed using the NIS elements advanced research software package (Nikon). Prior to image analysis, background camera noise was subtracted from the images using an averaged file of 10 no-light camera images. Cells that underwent division during the imaging run were excluded from analysis. Intracellular Ca2+ signaling over time was quantified by calculating the number of Ca2+ spikes per cell. This was determined as follows: raw fluorescence intensity values were measured from individual cells using Nikon software, then exported to Microsoft Excel to normalize the fluorescence to the first image (F/F0). The Ca2+ spikes were calculated by subtracting each normalized fluorescence measurement from the previous measurement to determine the change in GCaMP6s fluorescence (ΔF) between each time point. Ca2+ signals with a ΔF magnitude of >5% were counted as Ca2+ spikes. For each condition tested, Ca2+ spikes in ≥30 cells were determined.Calcium imaging experiments were set up by adapting a protocol detailed in previous reports . For TV 2+ imaging experiments, we used the TidyR  (http://heliquest.ipmc.cnrs.fr/)  . Helicalnrs.fr/) .E. coli expression constructs for the lysis assay were generated via ligation-independent cloning (LIC) using the pET46-Ek/LIC kit . The pET46-Ek/LIC constructs all have an N-terminal six-histidine tag. Mammalian expression vectors were generated by inserting c-myc tag and mRuby3 red fluorescent protein (RFP) upstream of full-length NS1-2 and then subcloning this into the pTagRFP-N vector in place of TagRFP . This construct is referred to as RFP-NS1-2. The NS1-2(Δ176) and NS1-2(Δ157) truncation mutations in both bacterial and mammalian expression vectors were generated using the NEB Q5 site-directed mutagenesis kit . Primer sequences used for the bacterial and mammalian expression vectors are listed in 2+ imaging was performed beginning 8 h posttransfection to capture expression kinetics and up to 24 h posttransfection to measure changes in Ca2+ signaling during expression of the RFP-tagged proteins.MK2-G6s cells were seeded in 15 μ-slide 8-well chambers  and at 85% confluence transfected with mammalian expression constructs in Opti-MEM (ThermoFisher) and Lipofectamine 2000 (Invitrogen). Transfection was optimized so cells received 400 ng of plasmid DNA and 0.5 μl of Lipofectamine 2000 per well. Trichostatin A (TSA) (10 μM) was added from 1 to 3 h posttransfection. TSA is a histone deacetylase (HDAC) inhibitor used to increase expression from the vectors –81. Time–LLC-MK2 cells were seeded in 15 μ-slide 8-well chambers  and transfected 1 day prior to imaging. Cells were transfected with intracellular markers for the plasma membrane , endoplasmic reticulum , Golgi apparatus , and mitochondria . Control wells received TagRFP (Evrogen), while experimental wells received either full-length RFP-NS1-2, RFP-NS1-2(Δ157), or RFP-NS1-2(Δ176). Cells were imaged 24 h posttransfection on the DeltaVision LIVE high-resolution deconvolution microscope  using the 60×/1.4 Plan-Apo NA oil objective (Olympus), and acquired using a pco.edge sCMOS_5.5 camera. Images were acquired and deconvolved in SoftWoRx software. After the images were deconvolved, they were further processed in FIJI (ImageJ) to adjust for brightness/contrast and pseudocoloring .E. coli lysis assays were performed as previously described (E. coli BL21(DE3)pLysS cells. Transformations were plated on LB containing 1% glucose, 100 μg/ml ampicillin, and 35 μg/ml chloramphenicol and grown at 37°C overnight. Isolated colonies were picked the next day and cultured overnight in liquid LB containing 1% glucose, 100 μg/ml ampicillin, and 35 μg/ml chloramphenicol at 37°C in an orbital shaker at 250 rpm. The next day, overnight cultures were subcultured by 1:100 dilution into 200 ml LB containing 1% glucose, 100 μg/ml ampicillin, and 35 μg/ml chloramphenicol. Subcultures were grown at 37°C in an orbital shaker at 250 rpm for ∼3 h to an optical density at 630 nm (OD630) between 0.3 and 0.5 and then induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Absorbance measurements at 630 nm (OD630) were taken every 10 min for 90 min and normalized to the induction OD630 to determine the percent growth or lysis over time after induction. Each experiment was performed ≥3 times. Protein expression was determined by SDS-PAGE using a 4 to 20% Tris-glycine gel  and Western blotting for the six-histidine tag. An uninduced culture served as the negative control for viroporin activity and NS1-2 synthesis.escribed . Brieflyg for 20 min, and supernatants were decanted. Pellets were resuspended in phosphate-buffered saline (PBS) and sonicated three times for 1 min on ice. Total lysate was collected after sonication. The membranes were then pelleted by ultracentrifugation at 49,000 × g for 1 h using a TLA-100.3 rotor in an Optima TL ultracentrifuge , and the supernatant was collected for the soluble fraction. Finally, the membrane fraction pellet was resuspended in PBS containing 1% SDS to solubilize membrane proteins. Samples from the total lysate, soluble fraction, and membrane fractions were analyzed by Western blotting.Membrane association experiments were performed using a modified protocol from previously reported experiments , 42. For2 gradient-purified TV at 10 μg/dose in AddaVax adjuvant (InvivoGen). Immunizations were given at 3-week intervals. For the anti-Vpg antisera, adult BALB/c were immunized three times with 10 to 20 μg of purified Vpg expressed in E. coli per dose. The priming dose was given in Freund’s complete adjuvant, and the subsequent boosts were given in Freund’s incomplete adjuvant. Guide for the Care and Use of Laboratory Animals  were immunized five times with CsCl Animals .Samples were prepared using procedures adapted from reference t test for data sets with a parametric distribution or a Mann-Whitney test for data sets with a nonparametric distribution. Differences were determined statistically significant if the P value was <0.05. Authors had access to the data for this article, and all authors approved the final article.Statistical analyses were completed using GraphPad Prism (version 7.03). Data in this article are presented as means ± standard deviations. Unless otherwise noted, all experiments in this article were performed in biological triplicate, with at least two technical duplicates per biological replicate, when applicable. We performed column statistics to collect descriptive statistics and to determine the normality of the data sets. We then used the unpaired Student’s RConsole code for the heatmaps generated in this paper is available upon request.


Second harmonic generation (SHG) is forbidden for materials with inversion symmetry, such as bulk metals. Symmetry can be broken by morphological or dielectric discontinuities, yet SHG from a smooth continuous metallic surface is negligible. Using non-linear microscopy, we experimentally demonstrate enhanced SHG within an area of smooth silver film surrounded by nanocavities. Nanocavity-assisted SHG is locally enhanced by more than one order of magnitude compared to a neighboring silver surface area. Linear optical measurements and cathodoluminescence (CL) imaging substantiate these observations. We suggest that plasmonic modes launched from the edges of the nanocavities propagate onto the smooth silver film and annihilate, locally generating SHG. In addition, we show that these hotspots can be dynamically controlled in intensity and location by altering the polarization of the incoming field. Our results show that switchable nonlinear hotspots can be generated on smooth metallic films, with important applications in photocatalysis, single-molecule spectroscopy and non-linear surface imaging. Scientists have used nano-sized cavities etched onto a sliver surface for combining two photons to produce light with twice the frequency, opening the door for new applications in photo-catalysis, and sensing and imaging technologies. Second harmonic generation (SHG) is a process in which two photons with the same frequency combine to generate a photon with double the frequency. However, SHG cannot be produced in smooth continuous metallic surfaces unless the symmetry can be broken by abrupt changes in the material’s geometry or through the application of an electromagnetic field. Now, Adi Salomon and colleagues from Bar-Ilan University in Israel, in collaboration with Swiss researchers, have produced SHG from the flat area between pairs of triangular nanocavities etched onto a smooth silver surface, and could see use in photo-catalysis, non-linear surface imaging and single-molecule spectroscopy. SHG is forbidden in materials with inversion symmetry such as bulk metals, yet symmetry can be broken by material discontinuity3 or by electromagnetic (EM) field properties5. Significant SHG responses were observed from metallic surfaces and were attributed to nonlinear polarizability of free and bound electrons at the metal interface6.Second harmonic generation (SHG) is a process in which two photons at the fundamental frequency combine to generate a photon with a double frequency through nonlinear scattering15. Nonlinear responses of both nanoparticles and nanocavities with various sizes and shapes, including metasurfaces, were studied both in the visible and near-infrared regimes22. Additionally, metallic nanoparticles have been shown to increase the SHG responses of dielectric particles24. Typically, SHG emanating from nanostructures is enhanced when the plasmonic structure resonates with the electric field, E, either at the fundamental (ω) or harmonic frequency (2ω) or both29, since the SHG intensity is proportional to (|E(ω)|2|E(2ω)|)232. While most studies have focused on metallic structures with no inversion symmetry, SHG is also observed from symmetric plasmonic structures33. The reason for such an enhanced nonlinear conversion process has remained elusive.Plasmonic structures, whether particles or cavities, increase the nonlinear response dramatically due to their enhancement of the EM fields at the interface36. The localized modes of the nanocavities may lead to the excitation of surface plasmon polaritons (SPPs), and long-range coupling between cavities can occur38. In this respect, nanocavities differ from their so-called “complementary” nanoparticle structures and they exhibit dissimilar optical behavior. Elementary SPPs excited from nanocavities can combine and emit a more energetic photon39. In this case, SPPs do not boost local fields but are rather annihilated to form a photon at double the frequency. This implies that in practice, a sub-wavelength aperture is not necessarily needed to generate enhanced SHG. This was experimentally shown by Woggon and co-workers when measuring SHG emission from silver films using a Kretschmann configuration40 and theoretically supported by Finazzi and Ciccacci39.Nanocavities milled in a metallic film have emerged as a promising class of optical materialsHere, we experimentally demonstrate enhanced SHG emission from a smooth silver film area surrounded by several nanocavities. We used triangular nanocavities milled into a silver thin film in a configuration with a base-to-base distance of 400 nm. We show that SHG emission results solely from the strong interaction between the SPP modes propagating from each cavity edge. Those SPPs, at the fundamental frequency, propagate and annihilate to form a spatially confined SHG “hotspot” on the flat surface Fig. . In thisOur sample is composed of two pairs of triangular cavities. The triangular nanocavities have a typical side length of 200 nm, and they are fabricated using a focused ion beam , in a 200-nm-thick smooth silver film deposited on a clean fused silica substrate Fig.. The sil41. A triangular cavity supports several plasmonic modes, among them a broadband side-length mode, which can launch SPP modes onto the surface43. Based on these earlier findings, we designed a plasmonic structure in which the “bases” of two adjacent triangular cavities are face-to-face, a configuration that leads to a confinement of the EM field onto the flat surface between the cavities and the frequency of which can be adjusted by the distance between the cavities16. The SHG response from such structures was investigated using custom-fabricated two-photon microscopes ijk. Figure 30. Next, for the pair of two equally aligned oriented triangular cavities, the off-axis shows an octupolar polarization behavior, the signature of a three-fold symmetric object45, while the in-axis reveals moderate coupling between the two triangular cavities. A highly polarized SHG emission is observed for the full structure composed of two oppositely oriented sub-units . The results are shown in Fig. its Fig. . The lin48 . Hence, the radiative plasmonic response of the metallic system can be probed, and high-resolution spectral imaging can be achieved51. In contrast, the  excite mechanism in CL is different from that of SHG in that both probe the plasmonic response and the optical properties of the plasmonic modes. Figure 20. The wavelength of interest in the CL measurements is 370 nm, correlated with the 470 nm in the SHG measurements due to differences in the refractive indices, i.e., PVA vs. air. Figure To further support this claim and to map these modes with increased spatial resolution, we also performed cathodoluminescence (CL) measurements on a similar plasmonic structure with identical geometrical parameters. In CL, the plasmonic modes are locally excited by an electron beam, and spatial mapping of the emitted photons at a given wavelengths directly maps the plasmonic modes of the systemx- and the y-axes.In this study, we demonstrate an enhanced SHG response from a centrosymmetric nanostructured silver film. We observed a very strong SHG signal from the smooth surface area between the triangular cavities, where the amplitude of the SHG signal is highly dependent on the polarization state of the driving fundamental field relative to the orientation of the triangular cavities. Based on several observations, we suggest that this strong SHG response arises from SPP modes launched from adjacent sub-units and subsequent plasmon–plasmon annihilation on the flat surface area: (i) in a base-to-base configuration and for in-axis polarization of the incoming field, the SHG emission emanates from the flat surface between the cavities, whereas the signal from the subunits is dramatically reduced. Individually, those sub-units enhance SHG Fig. . This ob54, nanocavities were used to boost the EM fields resulting in an enhanced nonlinear response. In stark contrast, the symmetric plasmonic systems introduced herein, with inversion symmetry, enhance SHG emission through what we believe to be a plasmon–plasmon annihilation processes.In previously studied systemsOne might consider the assembly of the cavities in the framework of the extended hybridization model. That is, the cavities are in analogous to the atoms and the SHG emanating we observed from the middle could be the probability function. However, the mode is too localized to support this explanation.55.From an application point of view, our findings may further be used to produce SHG hotspots at any given location on a given metallic surface. The ability to form such a strongly confined SHG spot in the optical regime may provide a useful tool to study physical-chemical transformations occurring on surfaces, such as photocatalysis processes. Moreover, it may be useful for molecular, label-free biological sensing and imaging near surfacesSecond Harmonic Generation Hot-Spot on a Centrosymmetric Smooth Silver Surface


While the role of the B cell receptor (BCR) Ca2+ pathway has been associated with disease progression, the importance of the newly described constitutive Ca2+ entry (CE) pathway is less clear. In addition, we hypothesized that these differences reflect modifications of the CE pathway and Ca2+ actors such as Orai1, transient receptor potential canonical (TRPC) 1, and stromal interaction molecule 1 (STIM1), the latter being the focus of this study.Dysregulation in calcium (Ca2+ entry (CE) pathway in CLL B cells was performed including constitutive Ca2+ entry, basal Ca2+ levels, and store operated Ca2+ entry (SOCE) activated following B cell receptor engagement or using Thapsigargin. The molecular characterization of the calcium channels Orai1 and TRPC1 and to their partner STIM1 was performed by flow cytometry and/or Western blotting. Specific siRNAs for Orai1, TRPC1 and STIM1 plus the Orai1 channel blocker Synta66 were used. CLL B cell viability was tested in the presence of an anti-STIM1 monoclonal antibody  coupled or not with an anti-CD20 mAb, rituximab. The Cox regression model was used to determine the optimal threshold and to stratify patients.An extensive analysis of the Ca2+ concentrations; (iii) independent from the BCR-PLCγ2-InsP3R (SOCE) Ca2+ signaling pathway; (iv) supported by Orai1 and TRPC1 channels; (v) regulated by the pool of STIM1 located in the plasma membrane (STIM1PM); and (vi) blocked when using a mAb targeting STIM1PM. Next, we further established an association between an elevated expression of STIM1PM and clinical outcome. In addition, combining an anti-STIM1 mAb with rituximab significantly reduced in vitro CLL B cell viability within the high STIM1PM CLL subgroup.Seeking to explore the CE pathway, we found in untreated CLL patients that an abnormal CE pathway was (i) highly associated with the disease outcome; (ii) positively correlated with basal Ca2+ entry in CLL evolution, provide new insights into CLL pathophysiology, and support innovative therapeutic perspectives such as targeting STIM1 located at the plasma membrane.These data establish the critical role of a newly discovered BCR independent CaThe online version of this article (10.1186/s40425-019-0591-3) contains supplementary material, which is available to authorized users. In addigression . Inductiphocytes . Finallypoptosis –12.2+ after BCR cross-linking, thus resembling B cells anergized in vivo after chronic antigenic stimulation [2+ was related to mutated IgHV patients, a reduced level of cell surface (s) IgM, and a defective signalosome. In contrast, CLL cases with a worse clinical outcome show an elevated basal Ca2+ level that can be enhanced upon sIgM triggering. The elevated Ca2+ signaling in the CLL group with progressive disease was associated with an unmutated IgHV status and an elevated level of CD38, but was not linked to any specific cytogenetic markers [2+ dysregulation in B-CLL cells such as a BCR autonomous signaling capacity due to an internal epitope present in the second framework of stereotyped IgHV that can be abrogated by using a BCR signaling inhibitor [2+ due to an inhibitory interaction between Bcl-2 (overexpressed in B-CLL cells) and the endoplasmic InsP3R [2+ pathway recently described in B-CLL cells [2+ deregulations in B-CLL cells and their correlation with disease evolution and severity are far from being fully understood. Reversing specific changes in deregulated Ca2+ fluxes may also represent new therapeutic opportunities to answer unmet needs in CLL treatment.At first glance, CLL cases with indolent and stable disease present B cells that are ineffective at mobilizing Camulation . For the markers . Howevernhibitor , an incac InsP3R , and lasLL cells , 18. Ca22+ entry deregulation in B-CLL cells and tested whether BCR-dependent or BCR-independent Ca2+ entry would be relevant in CLL outcome. The latter was critical for disease progression, and we therefore analyzed and characterized a novel Ca2+ signaling pathway, referred to as constitutive Ca2+ entry (CE), which is triggered by STIM1 located at the plasma-membrane (STIM1PM). Interestingly, we demonstrated that blocking CE with an anti-STIM1 monoclonal antibody (mAb) presents innovative therapeutic perspectives in CLL.In this study we deciphered CaIgHV mutational status, which were performed as previously described [Clinical information was retrospectively obtained from 74 untreated patients diagnosed with CLL according to the World Health Organization (WHO) classification , and 13 escribed . ConsentPeripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll-Hypaque density gradient centrifugation  and B cells were further enriched using the Pan B-cell Isolation Kit . Cell purity was assessed by fluorescence-activated cell sorting (FACS) analysis and was over 95% for B cells (CD19+).All monoclonal antibodies (mAb) were from Beckman-Coulter  unless specified: FITC-conjugated anti-IgM (clone: SA-D4), cyanin (Cy)5.5-conjugated anti-CD38 (LS198.4.3), electron coupled dye (ECD)-labelled anti-CD5 (BL1a), allophycocyanin (APC)-conjugated anti-IgD , APC-Alexa Fluor 700 (AF700)-conjugated anti-CD19 (J4–119), Pacific blue (PB)-conjugated anti-CD21 (1A4CD27) mAbs with saturating concentrations were incubated for 10 min at RT with 10 μl of PBS-washed blood. Versalyse solution (Beckman- Coulter) was added for 10 min in order to lyse red blood cells. The determination of the mean fluorescence intensity (MFI) of all markers required a minimum of 5000 events. The results were standardized to those obtained with isotype controls. Data were analysed using Kaluza 1.5 software (Beckman-Coulter).PM with the omission of the permeabilization step. In the transfection experiments a polyclonal rabbit anti-human Orai1 Ab  or anti-TRPC1 Ab  were used after the permeabilization step, and mAb staining was assessed with a FITC-conjugated goat anti-rabbit IgG . To assess PLCγ2 phosphorylation, 5 × 105 cells were fixed and permeabilized with cold 80% methanol solution during 30 min. After 2 washes in PBS-BSA 0.05%, APC-conjugated anti-pPLCγ2 mAb  was added.For intracellular staining, preliminary fixation and permeabilization were performed with the cytofix/cytoperm kit (BD Biosciences) followed by a 30 min incubation at 4 °C with Phyco-Erythrin (PE)-conjugated anti-STIM1 , PC7-conjugated anti-CD19 and APC-AF700-conjugated anti-CD5 mAbs. The same protocol was applied for plasma membrane STIM15 B cells/mL in RPMI-1640 (Sigma-Aldrich) supplemented with 2 mM L-glutamine, antibiotics, and 20% of decomplemented normal human serum AB . B cells were recovered, washed and stained for 15 min with FITC-conjugated annexin-V (AV) / propidium iodide (PI) and AF700-conjugated anti-CD19 antibody according to the Beckman-Coulter apoptosis kit protocol. The decrease in the percentage of live cells (CD19+ AV-PI-) was recorded.For each patient, B-CLL cells were incubated 48 h in 24-well plates at 37 °C with either (i) 10 μg/ml murine IgG2a  (ii) 10 μg/ml anti-STIM1 (Gok), (iii) 10 μg/ml RTX , or (iv) 2x10μg/ml anti-STIM1 + RTX. All cultures were set at 5 × 102, 10 mM HEPES, 10 mM Glucose with an 7,4-adjusted pH (Buffer A) supplemented with 5 mM CaCl2. Cells were washed and left to attach in the same buffer on 12 mm Cell-Tak  precoated coverslides for 20 min, allowing the de-esterification of the dye. Fura-2 was excited alternatively at 340 and 380 nm , and fluorescence emission was recorded at 510 nm using a fluorescence microscope  equipped with a dichroic mirror (415DCLP) and a 14-bit CCD camera . After the stabilization of basal fluorescence, the extracellular medium was replaced with Buffer A supplemented with 0.5 mM CaCl2 for 100 s and again with the original 5 mM CaCl2-containing Buffer A after curve stabilization. Excitation/emission ratio (F340nm/F380nm) was calculated for each time point and each cell with the Metafluor 6.3 Software . The amplitude of CE was calculated after normalization to the basal ratio (ΔF/F0), as the difference between average values of basal ratio measured in 5 mM external Ca2+ and the average ratio value in 0.5 mM Ca2+.For CE measurements, B cells were loaded with 2 μM Fura-2/AM dye  and 2 μM Pluronic acid  for 30 min at 37 °C in a medium containing: 135 mM NaCl, 5 mM KCl, 1 mM MgCl2 and 2 μM Fura-2/AM  for 1 h at 37 °C in Cell-Tak precoated 96-well plates, and fluorescence acquisition  was performed on the Flexstation 3 microplate reader with SoftMax Pro 5.4.5 software . For anti-IgM induced Ca2+ response, the extracellular medium was replaced with Buffer A supplemented with 10 mM CaCl2, before reading, and 10 μM of polyclonal goat anti-human IgM (Jackson Immunoresearch) were injected after 150 s. TG-induced ER Ca2+ release, extracellular medium was replaced with Buffer A supplemented with 100 μM EGTA just before starting the reading protocol. A stimulation with 2 μM of TG was performed after 100 s of recording, and 1.8 mM CaCl2 was added after 700 s in order to quantify SOCE entry. Ca2+ entry were quantified after value normalization (ΔF/F0) with the exception of basal Ca2+ concentrations estimated as the average of initial F340nm/F380nm values.For anti-IgM and Thapsigargin (TG)-induced calcium entry, B cells were loaded in Buffer A containing 1.8 mM CaClIn selected experiments, 1 μM Ibrutinib , 5 μM LY294002 (Sigma Aldrich), 2.5 μM Synta66 (Sigma Aldrich), DMSO, 10 μg/ml anti-STIM1 (clone GOK) or 10 μg/ml IgG2a mAb were added to the loading buffer.2+ analysis was performed.The small interfering RNAs (siRNA) targeting Orai1 , STIM1 , TRPC1 (s7311), and the control siRNA (4390843) were purchased from Ambion unless specified and transfected in B-CLL cells from 3 CE+ CLL patients by nucleofection  as previously described , 21. Aft7 B cells for 30 min on ice in a lysis buffer containing: 20 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X100, 2.5 mM Na+ pyrosodium tetraphosphate, 1 mM glycerophosphate, 1 mM Na+ orthovanadate, 1 μg/ml leupeptin and protease inhibitor cocktail (Roche). Protein extracts were sonicated and centrifuged for 12 min at 16,000 g. The protein concentration of the cell lysates was determined using the micro-bicinchoninic acid assay, and 50 μg were run on SDS-PAGE (10% polyacrylamide gels) in denaturing conditions, and transferred onto PVDF membrane sheets . After unbound sites were blocked, blots were probed overnight with 5% fat milk in PBS, 0.1% tween 20, and either mouse monoclonal anti-STIM1 , rabbit polyclonal anti-Orai1 , mouse monoclonal anti-TRPC1  or mouse monoclonal anti-GAPDH antibody . Washed blots were incubated with Horseradish Peroxidase (HRP)-conjugated goat anti-mouse or rabbit IgG Ab . Image acquisition was performed on a Chemi-Smart 5100 system (Vilber-Lourmat) with the Chemi-Capt 5000 software and analysed on Image J. All results were normalized upon GAPDH quantification.Protein extraction was performed by incubating 10P values under 0.05 were considered significant. Statistical analyses and the correlation matrix were performed using GraphPad Prism 7.0a .Continuous data are described as mean ± standard error of the mean (SEM). Following normality and equality of variance tests, nominal values were compared to controls using the student’s t test or alternatively by using a nonparametric test (Mann-Whitney rank sum test). Differences among groups were analyzed by one-way ANOVA in a non-parametric test and the Dunn’s test was used for post-hoc comparisons. For categorical data the Fisher’s exact test was used, and for correlation analysis the Pearson’s coefficient r was calculated. The profile likelihood method using a Cox regression model of PFS was used in univariate analysis to determine the optimal threshold and stratify patients into two groups as previously described . PFS, TF2+ signaling is an important hallmark of B-CLL cells, and suspected to vary during CLL disease progression [2+ entry in the absence of BCR engagement, designated as CE, was evaluated in resting B-CLL cells. To this end 30 untreated CLL patients were selected and, as reported in Fig. 0: 0.10 ± 0.01 in B-CLL cells versus 0.06 ± 0.01 in controls, P = 0.03). CLL patients were further dichotomized into CE+ (high levels) versus CE-  using the profile likelihood method in a Cox regression model of PFS for optimal cut-off identification (cut-off = 0.083).As deregulation in Can = 16), a significant difference with regards to parameters associated with disease outcome such as PFS  and lymphocytosis (P = 0.003) were associated with an elevated CE, which was not the case for the cytogenetic risk status, IgHV mutational status, and CD38 positivity , however no difference was observed when comparing the two CE subgroups within CLL patients. Interestingly, by conducting a bivariate analysis of PFS on both CE and IgM Ca2+ mobilization, we further observed that CLL patients with disease progression were restricted to CE+/IgM+ (n = 11) and CE+/IgM- (n = 5) CLL patients but not to CE−/IgM+ (n = 4) and CE−/IgM- (n = 9) CLL patients  the anti-IgM Ca2+ response; (iii) the ER Ca2+ release by thapsigargin (TG), an inhibitor of the ER Ca2+ ATPase pumps, that artificially and maximally deplete Ca2+ stores in the absence of extracellular Ca2+; and (iv) the TG SOCE response observed after Ca2+ reffiling.Next, 29 B-CLL cells  were selected and a correlation matrix was performed for all in order to compare CE with (i) the basal intracellular Ca2+ responses in B-CLL cells , but not CE with anti-IgM and Tg SOCE response (data not shown). Second, a proximal BCR-InsP3R signaling pathway as the anti-IgM Ca2+ response was correlated with both TG ER Ca2+ release and TG SOCE  but not with basal Ca2+ levels and CE.Results from the correlation matrix were effective to highlight two groups of Ca2+ channel influx in CE+ B-CLL cells, three strategies were developed using (1) the Orai1 channel blocker, Synta66 (S66); (2) specific siRNA for Orai1, TRPC1 and STIM1 to modulate CE amplitude; and (3) a quantitative analysis of Orai1, TRPC1 and STIM1 expression by Western-blot.Based on our previous work showing a role for Orai1, TRPC1 channels and STIM1 , 23 in CP = 0.03), the anti-IgM Ca2+ response (P = 0.01), and TG SOCE (P = 0.05) but not TG ER Ca2+ release in CE+/IgM+ B-CLL cells compared to control conditions  , and, although not significant, there is a trend for higher TRPC1 expression in CE+ B-CLL cells compared to CE- B-CLL cells. STIM1 analysis by WB reveals higher expression of both the 75 kDa non-glycosylated isoform and the 85 kDa glycosylated isoform that were overexpressed in CE+ B-CLL cells (P = 0.03).Third, Western blot (WB) was used to analyze the expression of Orai1, STIM1 and TRPC1 isoforms in B-CLL cells from 19 patients (11 CE+ and 8 CE-). When comparing CE+ and CE- patients Fig. , the twoPM) regulates store-independent Ca2+ influx [PM controls CE and contributes to its enhancement in CE+ B-CLL cells. To address this issue . In agreement with WB results, FACS analysis revealed that both total-STIM1 and STIM1PM were increased in CE+ B-CLL cells , and their levels correlated with CE amplitude  to inhibit CE. In contrast to the IgG2a isotype control mAb that had no effect on CE , while no effects were reported on the anti-IgM Ca2+ response, TG ER Ca2+ release and TG SOCE responses . Characteristics of STIM1PM high and low patients are presented in Table p = 0.05), but not the other parameters tested, was increased in the CLL STIM1PM high subgroup.As CE determination is difficult to manage in routine practice, we further compared the patient’s characteristics according to their plasma membrane STIM1 status in 74 untreated CLL that included those tested for Caves Fig. a, the CLPM were related to the control of cell survival [PM high n = 9; and STIM1PM low n = 8) when used alone or in combination with RTX, an anti-CD20 mAb , an effect which was not significant in the STIM1PM low subgroup (33.5 ± 6.5% with IgG2 versus 20.3 ± 4.7% with RTX + GOK).Finally and as the initial descriptions of STIM1survival , 28, we mAb Fig. b. Used a2+ signaling in CLL outcome, and describe for the first time a novel STIM1PM-dependant and constitutively active Ca2+ entry, independent from BCR signaling, and that constitutively active CE can be modulated and targeted by an anti-STIM1 mAb. We found that both CE and STIM1PM are clinically relevant in CLL and their determinations present important prognostic value.The overall data add new support to the critical role played by the Ca2+ signaling in CLL B cells and with the paradox that Ca2+ mobilization is altered in “anergic” CLL B cells from non-progressive patients, while a response is reported in CLL B cells from patients with disease progression as observed in our study [2+ concentrations in this study, we were able to extend the observation performed by Muggen and colleagues who have described elevated basal Ca2+ concentrations in B-CLL cells in contrast to normal B cells [2+ reported in B-CLL cells are, in fact, independent from the BCR-PLCγ2-InsP3R pathway and are instead related to an enhanced CE and are independent from store depletion. In contrast, Duhren-Von Minden and colleagues have associated the elevated basal Ca2+ signaling downstream Syk phosphorylation in CLL B cells to an antigen-independent recognition of the BCR framework domains (FR2 or FR3), or alternatively through an occupation of the BCR with repetitive motifs [2+ level (data not shown) which is in agreement with Muggen report who failed to associate the basal Ca2+ level in CLL B cells with the FR2/3 amino-acid sequence. Based on the report of Le Roy and colleagues who detected pSyk at a basal level in IgM+ responder patients, it could be proposed that blocking pSYK controls both CE and the IgM response in CE+/IgM+ responder patients, an hypothesis that needs to be tested as well as the capacity of Syk to phosphorylate STIM1 [Several reports have demonstrated altered Caur study . Moreove B cells . Our stue motifs . ImportaPM was associated with the regulation of a store independent Ca2+ entry pathway activated by arachidonic acid [PM and Orai1 as well as an enhanced expression of TRPC1. This is important because STIM1PM can interact with Orai1 or TRPC1, two Ca2+ channels activated in CE+ B-CLL cells as demonstrated by using specific siRNAs and in agreement with the Chen KT et al. report [STIM1 was initially identified as a plasma membrane protein , and mornic acid  and to Snic acid . Similar. report . STIM1 d. report , 32, and. report , 33, as . report .PM for CE, we demonstrated that pre-incubating cells with antibodies targeting STIM1PM reverses B-CLL cell capacity for CE and in turn impairs cell survival when associated with RTX. Therefore, we propose to use anti-STIM1 mAb targeting STIM1PM and CE as a new innovative therapeutic option for CLL. An additive/synergic effect of RTX or BCR inhibitors with CE inhibitors, such as anti-STIM1 mAb, should be addressed in future studies.The clinical success of RTX in monotherapy is limited in CLL and, in order to improve its efficacy, RTX is associated with chemotherapy (RFC) or with BCR inhibitors , however relapses and side-effects remain important suggesting a need to develop new therapeutical options and in particular to combine RTX with new drugs targeting a non BCR survival pathway , 36. Con2+ entry in CLL B cells; (ii) the use of samples from a cross-sectional and monocentric center; and (iii) a bias due to the selection of untreated patients. However and to reduce these limitations, a large panel of approaches  has been used in order to demonstrate that STIM1 and in particular STIM1PM controls CE in CLL B cells from patients with progressive disease. The selection of untreated patients for this study represents also an advantage as drug exposure may affect the analysis of Ca2+ entry, as observed in vitro with ibrutinib. Future studies are however mandatory in order to study whether variations in Ca2+ entry and Ca2+ actor variations vary following treatment introduction and in those patients who relapse.Relevant limitations of our study include the following: (i) a small sample size used to analyze Ca2+ signaling deregulation in cancer cell progression is well established, but the identification of mechanisms controlling Ca2+ entry are poorly understood. In the present work, an extensive analysis of the Ca2+ entry in CLL cells was performed, revealing, in patients with progressive disease, the implication of a constitutive and BCR-independent Ca2+ entry pathway. Next, it was further observed that a pool of STIM1 present in the plasma membrane characterizes tumor progression and controls constitutive Ca2+ entry. Finally, the capacity of an anti-STIM1 mAb to block constitutive Ca2+ entry and to reduce in vitro CLL cell viability, when associated with Rituximab, was reported within the high STIM1PM CLL subgroup. This supports the idea that targeting STIM1PM and therefore constitutive Ca2+ entry represents a new 1st in class therapeutic pathway in leukemia treatment. The potential use of mAb targeting STIM1PM in cancer therapy that can be used alone or in synergy with existing drugs needs to be further evaluated.In CLL the involvement of CaAdditional file 1:Figure S1. Two pathways control Ca2+ signaling in B cells from patients with chronic lymphocytic leukemia. In the BCR-induced store operated Ca2+ entry pathway, B cell receptor (BCR) interaction with the antigen results in the formation of the signalosome consisting of an active complex composed of the tyrosine kinases Lyn and Syk, B-cell linker protein (BLNK), Bruton-tyrosine-kinase (BTK), phospholipase C gamma 2 (PLCγ2), and phosphatidylinositol-4,5-bisphosphate 3-kinase δ (PI3Kδ) that phosphorylates CD19. Signalosome activation cleaves the membrane phospholipid phosphatidyl inositol 4.5-biphosphate (InsP2) into diacylglycerol (DAG) and inositol 1,4,5-triphosphate (InsP3), which subsequently, through binding to the endoplasmic reticulum (ER) IP3 receptor (InsP3R), mobilizes initially Ca2+ from stores and secondarily extracellular Ca2+ through the interaction between the multimerized reticular stromal interaction molecule 1 (STIM1ER) and the plasma-membrane Orai1 channel. In the constitutive Ca2+ pathway, Ca2+ entry is triggered by STIM1 located at the plasma-membrane (STIM1PM). (PPTX 90 kb)Additional file 2Table S1. B cell receptors (BCR) and co-receptors analysis in B-CLL cells (n = 30) according to the capacity of the cells to possess an elevated constitutive Ca2+ entry (CE+) and their capacity to mobilize Ca2+ in response to BCR engagement (IgM+). The phosphorylated phospholipase Cγ2 (pPLCγ2), an immediate downstream BCR effector, was added to the list. Figure S2. Representative kinetic plots of anti-IgM Ca2+ response (A), and thapsigargin (TG) Ca2+ response (B) in representative healthy donor (HD) control B cells (n = 8), CE- B-CLL (n = 13) and CE+ B-CLL (n = 16) samples. Cox regression model of progression free survival (PFS) was used to dichotomize CLL patients in IgM- and IgM+ (dash line). From the TG Ca2+ response analysis, the basal Ca2+ level was evaluated before normalization (1), the TG capacity to release Ca2+ from the endoplasmic reticulum (ER), and the TG capacity to release SOCE following extracellular medium supplementation with 1.8 mM Ca2+. P values are indicated when significant. Figure S3. Basal calcium (Ca2+) entry is related to constitutive calcium entry (CE) but not to store operated Ca2+ entry (SOCE), while the anti-IgM Ca2+ response correlated to thapsigargin (TG) capacity to induce endoplasmic reticulum (ER) Ca2+ release and SOCE. Figure S4. The pool of STIM1 in plasma membrane (STIM1PM) is correlated with basal Ca2+ levels but independent from anti-IgM Ca2+ response and thapsigargin (TG) capacity to release Ca2+ from the endoplasmic reticulum (ER) and to induce SOCE. Correlations between STIM1PM levels with basal Ca2+ (A), anti-IgM Ca2+ response (B), TG capacity to induce ER Ca2+ release (C), and TG SOCE (D). Values were obtained from 18 CLL, see material and methods for details. P and r2 values are indicated when significant. (DOCX 531 kb)


TMCO1 dysfunction in humans is associated with dysmorphism, mental retardation, glaucoma and the occurrence of cancer. Here we show an essential role of TMCO1 in osteogenesis mediated by local Ca2+/CaMKII signaling in osteoblasts. TMCO1 levels were significantly decreased in bone from both osteoporosis patients and bone-loss mouse models. Tmco1−/− mice exhibited loss of bone mass and altered microarchitecture characteristic of osteoporosis. In the absence of TMCO1, decreased HDAC4 phosphorylation resulted in nuclear enrichment of HADC4, which leads to deacetylation and degradation of RUNX2, the master regulator of osteogenesis. We further demonstrate that TMCO1-mediated Ca2+ leak provides local Ca2+ signals to activate the CaMKII-HDAC4-RUNX2 signaling axis. The establishment of TMCO1 as a pivotal player in osteogenesis uncovers a novel potential therapeutic target for ameliorating osteoporosis.Transmembrane and coiled-coil domains 1 (TMCO1) is a recently identified Ca 2+ channel. Here, the authors show it is important for osteoblast function and bone formation in mice, and identify a novel pathway linking local increases in Ca2+ at the ER surface with the posttranslational modification of RUNX2.TMCO1 is a recently described endoplasmic reticular Ca These transcription factors are regulated by a range of developmental signals and play important roles during various stages of cellular development of osteoblast lineages. Among them, RUNX2 is essential for differentiation of mesenchymal cells into osteoblasts and inhibition of osteoblast differentiation into adipocytes and chondrocytes. As the master regulator of osteoblast differentiation, RUNX2 is tightly regulated at both the transcriptional and post-translational levels during the bone formation processes6.Osteoporosis is a disease that develops with age and is characterized by systemic impairment of bone mass and microarchitecture. Bone metabolism disorders, with reductions in bone formation by osteoblasts, play an important role in this process. However, the molecular mechanisms that mediate impaired bone formation are poorly understood. Many transcription factors are known to be involved in the regulation of osteoblast function in response to different cellular stimuli, such as RUNX2, ATF4, Osterix, and AP12+ signaling is essential for bone remodeling. Release of calcium from the endoplasmic reticulum (ER) to trigger Ca2+ signaling plays a significant role in osteoblast proliferation and differentiation. The effects of Ca2+ are mediated by the Ca2+ binding protein calmodulin (CaM). Calmodulin-dependent kinase II (CaMKII) is the major target of Ca2+/CaM. After binding to Ca2+/CaM, CaMKII is phosphorylated and activates the CREB/ATF and ERK signaling pathways, which induce changes in osteoblast functions7. Calcineurin (Cn) is another protein complex that responds to Ca2+ signaling. Upon binding to Ca2+/CaM, Cn directly binds to and dephosphorylates the NFAT transcription factors that regulate osteoblast differentiation9. The mechanisms by which changes in Ca2+ signaling regulate osteoblastic-specific transcription factors remain unknown.Ca2+ from intracellular stores is facilitated by a series of ion channels in the ER. However, little is known about how intracellular Ca2+ signaling is regulated by these channels during osteoblast differentiation. Transmembrane and coiled-coil domain 1 (TMCO1) have been reported to be a highly conserved, transmembrane-spanning protein located in the ER10. Loss of TMCO1 function has been identified as the pathogenic cause of an autosomal-recessive syndrome characterized by craniofacial dysmorphism, skeletal anomalies, and intellectual disability13. Further research has demonstrated that TMCO1 acts as a Ca2+ channel, preventing intracellular Ca2+ stores from overfilling and maintaining calcium homeostasis in the ER10. Ca2+ homeostasis in the ER is important for the intracellular Ca2+ signaling and is involved in the regulation of a variety of cellular processes, such as proliferation, differentiation, and programmed cell death, in bone cells15. Disarrangement of ER Ca2+ homeostasis is related to many severe bone diseases19. The role of TMCO1 in bone formation and remodeling remains unclear.Release of Ca1, was found to mediate the effects of TMCO1 on osteoblast function. We further demonstrated that loss of TMCO1 in osteoblasts disrupted ER Ca2+ homeostasis and promoted CaMKII-HDAC4 axis-mediated RUNX2 degradation in a local Ca2+ signaling-dependent manner. These results define the critical functions of TMCO1 in osteoblasts and highlight the importance of Ca2+ signaling in RUNX2 protein stability regulation, which suggests a key role for TMCO1 in the pathophysiological process that leads to reduced bone formation in osteoporosis.In this study, we found that TMCO1 levels were significantly decreased in bone specimens from both osteoporosis patients and osteoporotic mice. TMCO1 deficiency inhibited osteoblast differentiation and bone formation in vivo and in vitro. RUNX2, one of the primary transcription factors required for osteoblast functionTMCO1 in femurs from patients with fractures (Supplementary Table TMCO1 mRNA and protein levels in osteoporosis patients (T ≤ −2.5) were obviously lower than those in control patients (T > −2.5)  and OCN  mRNA levels in these human samples   showed growth retardation and died before weaning. While those survived adult Tmco1−/− mice had no differences in body size or weight compared with the control mice  mice , Ocn, and Col1a1, was significantly decreased in femurs from Tmco1−/− mice compared with control femurs  Fig. . TMCO1 eles Fig. . We furtles Fig. , b. To iles Fig. . To deteion Fig. . Trabecuice Fig. . Meanwhisis Fig. . Quantiturs Fig. . Accordirum Fig. . In contice Fig. . Also, wTmco1−/− osteoblasts displayed markedly reduced ALP activity compared with control osteoblasts  influenced by TMCO1, we used synthetic DNA containing a concatenated tandem array of consensus TF response elements (catTFREs) for most known TF families to enrich TFs from control and Luc Fig. . Then, wice Fig. . Accordiued Fig. . AlthougTmco1-siRNA in the presence or absence of MG132, a proteasome inhibitor. Immunoblotting results demonstrated that the RUNX2 protein levels decreased in the absence of MG132 following Tmco1-siRNA knockdown, but remained constant in the presence of MG132  treatment. The results showed that the half-life of RUNX2 dramatically decreased in the absence of TMCO1 Fig. . We next132 Fig. . Ubiquitlls Fig. . Previouown Fig. . To idenrf1 Fig. . These rels Fig. . We tranion Fig. . As experol Fig. . These d21. HDAC−/− mice exhibit premature ossification of developing bones, and overexpression of HDAC4 in proliferating chondrocytes results in phenotypes similar to those observed in RUNX2−/− mice, such as chondrocyte hypertrophy deficiency and endochondral bone formation22. Based on the above-mentioned studies, we explored the role of HDAC4 in TMCO1 deficiency-induced RUNX2 degradation. We found that the inhibitory effect of TMCO1 on RUNX2 degradation was weakened when HDAC4 was knocked down in osteoblasts , an inhibitor of the ER-localized Ca2+ ATPase (SERCA). The results showed that Ca2+ release from the ER was much greater and long lasting in the absence of TMCO1 than in WT cells  that lacks the Ca2+ permeation function. We found that the increased Ca2+ release after TG treatment in Tmco1−/− osteoblasts was completely rescued by the expression of WT TMCO1, but not by the D140A mutant  calcium pumps and channels in the ER. However, little is known regarding the effect of these ER membrane proteins on osteoblast function. PANX3 is an ER Ca2+ channel that is required for normal progression of skeletal development and late-stage bone growth in vertebrates. PANX3 can regulate both chondrocyte and osteoblast differentiation through activation of intracellular Ca2+ signaling pathways32. Panx3-deficient osteoblasts exhibit decreased resting [Ca2+]i levels. In Tmco1−/− osteoblasts, we found that the ER Ca2+ store was significantly upregulated. However, the intracellular resting Ca2+ concentration remained unchanged. Surprisingly, we found that CaMKII phosphorylation was significantly reduced. BAPTA but not EGTA treatment can eliminate the differences between WT and Tmco1−/− osteoblasts. This result indicates that the changes in the Ca2+ concentration and CaMKII activation occur at the nanodomain scale near the ER surface. The simulation model of CaMKII activation and the distribution of Ca2+ also confirmed this result. The intracellular Ca2+ regulates CaMKII activation, which further phosphorylates Smad and c-jun to affect osteoblast function33. Here, we determined that the TMCO1-mediated CaMKII/HDAC4 pathway can enhance RUNX2 stability and promote bone formation.TMCO1 is an evolutionarily conserved ER transmembrane-spanning protein that has been reported to be an ER Ca2+ homeostasis in osteoblasts and plays a key role in bone formation in a manner that is dependent on the Ca2+ permeability of the TMCO1 channel. TMCO1 deficiency causes ER Ca2+ overload, and RUNX2 stability is regulated by the TMCO1–CaMKII–HDAC4 axis in a local Ca2+ signaling-dependent manner. Our study establishes TMCO1 as an important regulator in bone formation and provides a new therapeutic target for osteoporosis.In summary, we demonstrated that TMCO1 mediates ER CaPatients who had fracture caused by falling without obvious violence from the Second Affiliated Hospital of Soochow University from January 2016 to January 2017 were included in our study (inclusive criteria). Excluding criteria as below: (1) participants with fracture caused by high-energy injury and pathologic fracture due to other diseases were excluded; (2) participants with hyperparathyroidism, other congenital or acquired bone disease, history of malignancy, significant liver, or renal disease; (3) participants that had taken glucocorticoids or estrogen, or selective estrogen receptor modulators within 6 months; (4) participants that had ever taken parenteral bisphosphonates, teriparatide, calcitonin, or strontium ranelate within 12 months. Above all, we recruited 18 female patients aged 60 years or older with fracture caused by low-energy injury. The osteoporosis group was defined as a BMD T score of ≤−2.5 at the lumbar, and a T score of å −2.5 as the normal group. We obtained informed consent from the participants. All the clinical procedures were approved by the Committees of Clinical Ethics in the Second Affiliated Hospital of Soochow University .All the female C57BL/6N mice used were maintained under standard animal housing conditions . The mice were ovariectomized or sham-operated at 3 months of age. Twomonths after surgery (5 months of age), all the sham-operated and ovariectomized mice were euthanized for dissecting bilateral femurs. All the experimental procedures were approved by the Committees of Animal Ethics and Experimental Safety of China Astronaut Research and Training Center.The hindlimb-unloading procedure was achieved by tail suspension. Briefly, the 3-month-old male C57BL/6J mice were individually caged and suspended by the tail using a strip of adhesive surgical tape attached to a chain hanging from a pulley. The mice were suspended at a 30° angle to the floor, with only the forelimbs touching the floor, this allowed the mice to move and access food and water freely. The mice were subjected to hindlimb unloading through tail suspension for 28 days. After killing, bilateral femurs and tibiae were dissected and processed for microCT examination, bone histomorphometry analysis, and real-time PCR analysis. All the experimental procedures were approved by the Committees of Animal Ethics and Experimental Safety of China Astronaut Research and Training Center.Tmco1−/− mice were generated by CRISPR/Cas9. SgRNA targeting the sequence GTGCACCGCGCTGCTCGCCGAGG  on exon 1 of mouse Tmco1 was designed with the online tool (http://crispr.mit.edu/)34 and inserted into the BbsI site of the pX330 vector  to generate px330-sgTMCO1. T7 promoter was added to Cas9 and sgRNA coding region by PCR amplification. T7-Cas9 and T7-sgRNA PCR product were gel purified and used as the template for in vitro transcription (IVT) using RiboMAX Large Scale RNA Production Systems (Promega). Both the Cas9 mRNA and the sgRNAs were purified using MEGAclear kit (Life Technologies) and eluted in RNase-free water. B6D2F1 (C57BL/6 X DBA2) female mice strains were used as embryo donors, and RNA samples were used for one-cell embryo injection to produce the offspring at Tsinghua University Laboratory Animal Research Center. The primers employed for TMCO1 genotyping primers were 5′-GTCCCGCCACGTCTCT-3′ and 5′- AGTGAGGCTCCCGATC-3′. The gene disruption was ultimately validated by Sanger’s sequencing. All mice were bred and maintained under specific pathogen-free conditions. All the experimental procedures were approved by the Committees of Animal Ethics and Experimental Safety of China Astronaut Research and Training Center.We determined serum concentrations of OCN using the OCN ELISA kit (R&D) according to the instructions provided. The levels of serum CTX-1 were measured using the mouse CTX-1 ELISA kit (Sangon Biotech).For analysis of bone formation in vivo, 2-month-old mice were injected intraperitoneally with calcein (20 mg/kg body weight) in a time sequence of 10 and 3 days before being killed. Tibias were harvested, cleared of soft tissue, fixed in 70% ethanol. Undecalcified sections were cut at a thickness of 5 µm and examined using fluorescence microscopy.The total RNA from bone tissues or cells was extracted with TRIzol reagent (Invitrogen) as per the manufacturer’s instructions. The cell samples were washed with PBS before being treated with TRIzol. RNA (0.5 μg) was reverse transcribed with PrimeScript RT reagent Kit  according to the manufacturer’s instructions. Real-time PCR was performed using SYBR Premix Ex Taq II Kit  to detect expression of osteogenic genes. Gapdh was used as a normalization control for mRNA using the 2-△△CT method.Gapdh, forward: 5′-A A C A T C A A A T G G G G T G A G G C C-3′ and reverse: 5′-G T T G T C A T G G A T G A C C T T G G C-3′; Alp, forward: 5′-A T C T T T G G T C T G G C T C C C A T G-3′ and reverse: 5′-T T T C C C G T T C A C C G T C C A C-3′; Bglap, forward: 5′-C C A A G C A G G A G G G C A A T A-3′ and reverse: 5′-T C G T C A C A A G C A G G G T C A-3′; Col1α1, forward: 5′-G G G A C C A G G A G G A C C A G G A A G T-3′ and reverse: 5′-G G A G G G C G A G T G C T G T G C T T T-3′; Runx2, forward: 5′-C A A G A A G G C T C T G G C G T T T A-3′ and reverse: 5′-T G C A G C C T T A A A T G A C T C G G-3′; Tmco1, forward: 5′-T G A T G G T A G A G T G G T G G C A A A G-3′ and reverse: 5′-G G T G G T G G G C C A A G A A A T C C-3′.Mouse primers used in this study: GAPDH, forward: 5′-A C A A C T T T G G T A T C G T G G A A G G-3′ and reverse: 5′-G C C A T C A C G C C A C A G T T T C-3′; TMCO1, forward: 5′-T G T T T G C T A T T G G C T T T T G T-3′ and reverse: 5′-A G T C T G T G G T G T C A T C T C C C-3′.Human primers: High-resolution micro-CT analyses were performed on the distal femurs from each mouse using a model of μ40 Scanco . In the femurs, the trabecular bone proximal to the distal growth plate was selected for analyses within a conforming volume of interest  commencing at a distance of 840 μm from the growth plate and extending a further longitudinal distance of 1680 μm in the proximal direction. Cortical measurements were performed in the diaphyseal region of the femur starting at a distance of 3.57 mm from the growth plate and extending a further longitudinal distance of 210 μm in the proximal direction. All trabecular bone from each selected slice was segmented for three-dimensional reconstruction to calculate the following parameters: BMD, BV/TV, Tb.Th, and Tb.Sp.35. After mice were skinned, eviscerated and fixation with 95% ethanol for 3 days, the samples were transferred into acetone for an additional 48-h incubation. Skeletal preparations were stained for 3 days in Alcian blue solution. After staining, the samples were washed three times for 30 min in 95% EtOH followed by treatment of 2% KOH for 3–4 h. After stained with ARS solution for 3–4 h, skeletons were cleared in 1% KOH/20% glycerol and dehydrated in 95% EtOH overnight.Skeletal preparations were double stained with alcian blue and alizarin red S (ARS)The tibia of mice were fixed with 4% paraformaldehyde for 48 h followed by decalcification in 10% EDTA for 3–4 weeks, and 6 -μm sections were prepared on a rotation microtome. Paraffin-embedded sections were deparaffinized in xylene and rehydrated. The samples were stained with hematoxylin–eosin and TRAP (Sigma) according to the standard protocol.2O2 was used to block the activity of endogenous peroxidase. The sections were then incubated overnight at 4 °C with rabbit polyclonal antibody to osteocalcin  and Col1a . After three times washes in PBS, biotinylated secondary antibodies were then added and incubated for 1 h at room temperature, followed by color development with DAB kit (ZSGB-bio). Negative control experiments were done by omitting the primary antibodies. The sections were examined using a microscope .For immunohistochemical staining, sections were deparaffinized in xylene and rehydrated. Antigen retrieval was performed with protease K at 37 °C for 15 min. A solution of 3% H36. For osteoblast differentiation, primary osteoblasts were cultured in α-MEM containing 10% FBS and 10 nM dexamethasone (Sigma), 50 µg/ml of ascorbic acid, and 5 mM β-glycerophosphate.Human embryonic kidney cell line (HEK293A) were purchased from NICLR  and cultured with the high-glucose minimum essential medium (Dulbecco’s modified Eagle’s medium) containing 10% fetal bovine serum (FBS). Mouse MSC cell line C3H10T1/2 were purchased from NICLR  and cultured with the high-glucose minimum essential medium (Dulbecco’s modified Eagle’s medium) containing 10% fetal bovine serum (FBS). MC3T3-E1 cells were purchased from NICLR  and maintained in the minimum essential medium (MEM) alpha containing 10% FBS. For the culture of osteoblast lineage cells, calvariae from newborn mice were dissected aseptically and treated with 0.1% collagenase and 0.2% dispase. The isolated cells were maintained in the minimum essential medium (MEM) alpha containing 10% FBSTo generate the Myc-Runx2 construct, the full-length Runx2 open-reading frame was amplified from the total cDNA of mice and cloned into the pCMV-Myc vector containing Myc-tag. The point mutant construct  was generated by site-directed mutagenesis. Full-length and truncated of HDACs plasmids were provided by Dr. Eric Olson .Tmco1, Wwp1, Smurf1, Hdac4, and Camk2d siRNAs were purchased from GenePharma.The control, mouse-specific Tmco1 siRNA sequences: 5′-UUAAACAUUCCCAUUAAGGCA-3′mouse Wwp1 siRNA sequences: 5′-GCAGAGAAAUACUGUUUAU-3′mouse Smurf1 siRNA sequences: 5′-UUAAACAUUCCCAUUAAGGCA-3′mouse Hdac4 siRNA sequences: 5′-UUAAACAUUCCCAUUAAGGCA-3′mouse Camk2d siRNA sequences: 5′-UCUAGAAUCUGUUGUAUACAA-3′.mouse For adenoviral expression, the human TMCO1 and mouse RUNX2 CDS were cloned into pAd/CMV/V5-DEST vector (Life Technologies) following the manufacturer’s instructions. The TMCO1-D140A mutant was generated from the wild-type TMCO1 construct (Myc-tagged) by point mutation using site-directed mutagenesis kit (Takara). Ad-CaMKII-DN ) was a gift from Dr. Yan Zhang . The adenovirus were produced by transfecting adenoviral vector into HEK293A cells and purified using Vivapure AdenoPACK™ 20 RT kit (Sartorius).Alkaline phosphatase staining was monitored using a Vector Blue Substrate Kit . According to the protocol, MC3T3-E1 or primary osteoblast cells were incubated with the substrate working solution for 20–30 min. The whole procedure was protected from light. After 2 min of rinsing in deionized water, slides were treated with Mayer’s hematoxylin solution for 10 min.2O. Cells were stained with 40 mM Alizarin red S (Sigma), pH 4.0, for 15 min with gentle agitation. Cells were rinsed five times with double-distilled H2O and then rinsed for 15 min using 1 × PBS with gently agitating.Cells were fixed in 70% ice-cold ethanol for 15 min and rinsed with double-distilled H37. Then the samples from catTFRE in-gel digestion were analyzed by LC-MS/MS.Transfected cells were washed thrice with phosphate-buffered saline, then suspended in 600 μl of cytoplasmic extraction reagent I (CER I) buffer . Nuclear proteins were extracted in accordance with the manufacturer’s instructions. Protein concentrations were determined using the Bradford method . Two milligrams of control or KD NEs were used for catTFRE pull-down and trypsin digestiong at 4 °C for 30 min and then separated by 10% SDS–PAGE followed by western blotting according to a standard protocol. The membranes were blocked with 5% bovine serum albumin and incubated with specific antibodies overnight. A horseradish peroxidase-labeled secondary antibody was added and visualized using an Enhanced Chemiluminescence Kit (Millipore). Antibodies used were: TMCO1 , Wwp1 , RUNX2 , HDAC4 , p-HDAC4 , Myc-Tag , CaMKII , p-CaMKII , Smurf1 , GAPDH . Western blots were reproduced by three independent experiments.Cells were lysed in lysis buffer  on ice for 30 min. Protein fractions were collected by centrifugation at 15,000 g, 15 min). Lysates were subjected to IP with Anti-Ac-K-100  at 4 °C overnight, then protein A/G (Santa Cruz) was added into cell lysates for 2 h. Followed by washing with lysis buffer, cell lysates were subjected to SDS–PAGE electrophoresis and immunoblotting with the indicated antibody.For immunoprecipitations, cells were lysed in lysis buffer  with protease inhibitor cocktail (Roche Applied Science) at 4°C for 30 min and lysates were collected by centrifugation  for 6 h, and proteins were immunoprecipitated with the RUNX2 antibody , followed by immunoblotting with anti-ubiquitin antibody .4 cells/well into 24-well plate, allowed to settle overnight, then transfected using lipofectamine 3000 with a luciferase reporter plasmid (6X OSE2) and pRL-TK (Promega) along with various combinations of expression plasmids as indicated. Reporter plasmid with binding-region mutations was used as a control. After 48 h, the cells were lysed with passive lysis buffer (Promega), and lysates were used for the dual luciferase reporter assay (Promega) according to the manufacturer’s instructions. Luminescent signals were quantified by luminometer , and each value from the firefly luciferase construct was normalized by Renilla luciferase assay. All luciferase experiments were repeated at least three times.C3H10T1/2 cells were seeded at 2 × 10After transfected with plasmid and/or adenovirus, cells grown on confocal dish were fixed with 4% paraformaldehyde for 15 min, washed twice with PBST (PBS + 0.5% Tween 20), and permeated with 0.5% Triton X-100 for 25 min. Cells were blocked in 1% BSA, 0.1% Triton X-100 in PBS for 1 h at 37 °C. Cell samples were incubated with relevant primary antibodies against HDAC4  and Myc-Tag  overnight. After extensive washes with PBST, cells were incubated with TRITC or FITC-conjugated anti-mouse IgG or anti-rabbit IgG (ZSGB-Bio) antibodies. Cells were counterstained with 5 μg/ml 4, 6-diamidino-2-phenylindole (DAPI) in PBS and then mounted onto the slides in antifading solution containing 0.25% DABCO. Images were acquired using Nikon ECLIPSE Ci-S epifluorescence microscope.2+ measurement, the coverslip was transferred to chamber and cells were loaded with 5 μM fluo-4, AM (Molecular Probe) for 20 min at 37 °C in Tyrode solution: 137 mM NaCl, 20 mM HEPES (Sigma), 10 mM glucose (Sigma), 1.2 mM MgCl2•6H2O (Sigma), 1.2 mM NaH2PO4•2H2O (Sigma), and 5.4 mM KCl (Sigma), pH 7.35. Cells were then rinsed twice with Tyrode solution and mounted on the inverted stage of a confocal scope (Zeiss LSM 710). Fluorescence excitation was performed using 488 -nm laser, and detection filters were set at 530 nm. Images were acquired every 3 s and analyzed using Interactive Data Language  software. For testing of resting [Ca2+]i and [Ca2+]i released from the ER, cells were scanned for 20–30 s to obtain Fresting, then replaced the solution with 0 Ca2+ Tyrode solution including 4 mM EGTA (Invitrogen), 5 μM thapsigargin (Molecular probes), and 10 μM A23187 (Sigma). Stored calcium was released to the cytoplasm immediately. We defined the peak value as FER release. Added 100 μM BAPTA, AM (Molecular probes) into solution to obtain Fmin. Then replaced the solution with 10 mM Ca2+, 5 μM thapsigargin, 12 μM A23187 (Sigma), 3 μM FCCP (Sigma), and 20 mM 2-DG (Sigma) in Tyrode solution. The stable value was Fmax. Finally, [Ca2+]i was calibrated using the equation [Ca2+] = Kd × (F–Fmin)/(Fmax–F). The method is modified from Grzegorz Grynkiewicz38.Cells were seeded on 25-mm glass cover slips and cultured with the α-MEM medium. For Ca2+] and buffers near the tubule are depicted by the following ODES:2+] refers to the free calcium concentration, [CaEN], [CaEG], and [CaBA] refer to binding endogenous buffer, EGTA, and BAPTA concentration. JEN, JEG, and JBA refer to the reaction flux between buffers and calcium.Here, we bring up a simple simulation of CaMKII activation near the ER tubule surface to investigate the impact of calcium leak on local environment. Calcium from the ER tubule can freely diffuse, bind on the buffer and diffuse as the form of calcium buffer, or dissociate from buffer. The calcium leaks are uniformly distributed on the tubule surface.  and CaMKII activity along  can be gotten from two integrations  with a post hoc test to determine group differences in the study parameters. All statistical analyses were performed with Prism software . Statistical significance was evaluated using either unpaired Student’s Further information on experimental design is available in the Supplementary InformationReporting Summary