Schizosaccharomyces pombelacking the major double-stranded telomere DNA binding protein Taz1. Telomeric DNA repeats in cells lacking Taz1 are 10 times longer than normal and contain single-stranded DNA regions. DNA damage checkpoint proteins associate with these damaged telomeres, but the DNA damage checkpoint is not activated. This severing of the DNA damage checkpoint signaling pathway was reported to stem from exclusion of histone H4 lysine 20 dimethylation (H4K20me2) from telomeric nucleosomes in both wild type cells and cells lacking Taz1.  However, experiments to identify the mechanism of this exclusion failed, prompting our re-evaluation of H4K20me2 levels at telomeric chromatin.  In this short report, we used an extensive series of controls to identify an antibody specific for the H4K20me2 modification and show that the level of this modification is the same at telomeres and internal loci in both wild type cells and those lacking Taz1.  Consequently, telomeres must block activation of the DNA Damage Response by another mechanism that remains to be determined.DNA double-strand breaks (DSBs) activate the DNA damage checkpoint machinery to pause or halt the cell cycle.  Telomeres, the specific DNA-protein complexes at linear eukaryotic chromosome ends, are capped DSBs that do not activate DNA damage checkpoints.  This “checkpoint privileged” status of telomeres was previously investigated in the yeast  The link to dataset 2 in the version 1 paper was not provided due to an editorial error. Thus, we now include below the original dataset 2 for readers to see labelled, alongsi1 and recruit proteins to repair the DNA lesion3. Telomeres, the physical ends of linear eukaryotic chromosomes, are specialized DSBs that suppress DNA damage checkpoint activation by an unknown mechanism(s), even though telomeres are bound by many of the DNA damage checkpoint proteins that signal cell cycle arrest4. Carneiroet al. (Nature 467: 228-232) addressed this question usingSchizosaccharomyces pombe cells that lack Taz1, the protein that binds to double-stranded telomere repeats5. Telomeres intaz1∆ cells have single-stranded DNA regions that are bound by checkpoint and DNA repair proteins, but cells do not arrest6. Immunofluorescence co-localization results from Carneiroet al. indicated that the ortholog of the human DNA damage checkpoint protein 53BP1 (Crb2) found at DSBs was not recruited to telomeres5. Crb2 can bind to dimethylated lysine 20 of histone H4 (H4K20me2) in nucleosomes7. Carneiroet al. presented data that H4K20me2 was depleted near telomeres in wild type andtaz1∆ cells, suggesting a mechanism for checkpoint suppression5. Efforts to pursue this exciting result by ourselves and others failed. We therefore carefully re-evaluated the presence of H4K20me2 at different chromosomal loci, and found that H4K20me2 is not depleted near telomeres, indicating that checkpoint suppression occurs by some other mechanism(s).Genome instability is a potentially lethal event for a eukaryotic cell, and a mutational force for genetic diseases such as cancer. DNA double-strand breaks (DSBs) can drive genome instability and are sensed by the DNA damage checkpoint, a defined set of evolutionarily-conserved proteins that bind the DSB to signal a pause or arrest of the cell cycle8 and were constructed as follows: The 5’ flanking region, theH4 gene, and the 3’ flanking region of each histoneH4 gene were separately cloned into a pFA vector 5’ of the selection marker . Approximately 500 base pairs 3’ to the initial fragment was cloned 3’ of the selection marker to the appropriate vector. The inclusive distance between the last A for the histone H4 TAA stop codon and the first G in the Asc I site from the pFA6a marker was 441 bp from H4.1 to+arg3, 707 bp from H4.2 to+his3 and 464 bp from H4.3 to+ura4  or FY1646 9. Confirmation of integration was done by restriction digestion and DNA sequencing of the PCR product of the H4 gene. The strains withhhf1 (+h) andhhf3 (-h) marked and/or mutated were crossed to generate a strain in whichhhf2 is the only unmarked gene copy. The resulting+h strain was then crossed with thehhf2 (-h) marked strain to generate a strain in which all three loci of the histone H4 gene contain a selectable marker and are either wild type or mutated to arginine at lysine 20. Confirmation via digestion and DNA sequencing was performed after each cross. The H4K20R strain has been previously shown to be sensitive to DNA damaging agents8. The strains and primers used during strain construction are available upon request.Wild type (yJRE20-1) and histone H4 lysine 20 mutant (yJRE21-1) strains were previously describedet al.10 and AHRULK + G418 contains 225 mg/l adenine, histidine, arginine, uracil, leucine, lysine and 200 mg/l G418 sulfate). Mid-log cells (9–12 × 106/ml or 0.8-1.2 OD600) were cross-linked with 1% formaldehyde for 15 min at room temperature and washed twice with cold HBS buffer . Cell pellets were stored at -80°C. All subsequent steps were performed at 4°C. Cell pellets were resuspended in ChIP-lysis buffer11 and lysed using mechanical disruption by bead-beater (Bio Spec Mini-Beadbeater-16) with 0.5 mm glass beads (Biospec 11079105) using 4 cycles of 45 sec followed by 60 sec on ice. The lysate was sonicated for 10 cycles on maximum power (30 sec ON and 59 sec OFF) in a Diagenode Bioruptor XL with sample tubes soaked in an ice water bath. Solubilized chromatin protein (2–4 mg) was used for each ChIP while 5 µl was saved as Input. Antibodies (2 µg) against H4K20me2 (GeneTex GT282 [RRID: AB_2728656] Lot #41582) or total histone H4 (Abcam ab10158 [RRID: AB_296888] Lot #GR133660-1) were mixed with chromatin and incubated at 4°C while rocking for 4 h. Dynabeads Protein G  was then added and rocked overnight at 4°C. Beads were washed with ChIP lysis buffer, ChIP lysis buffer with 500 mM NaCl, Wash buffer and TE buffer  successively11. Beads were then resuspended in 145 µl of TES (1X TE with 1% SDS). Supernatant (120 µl) was recovered and incubated in a Thermomixer at 65°C, 1000 rpm (rotation per min) overnight to reverse cross-linking. For Input samples, TES buffer (115 µl) was added and incubated in the Thermomixer with the ChIP samples. Samples were treated with RNase A (2 µl of 10mg/ml added to each sample)(Roche 10109142001) for 15 min at 37°C and Proteinase K (2 µl of 20mg/ml added to each sample)(Roche 03115879001) for 30 min at 55°C, and purified by QIAgen PCR purification column (Cat.No. 28106)14. All samples from the same assay were processed for ChIP assay at the same time.The strains used are described in2O while beads-only-ChIP, H4-ChIP and H4K20me2-ChIP samples were diluted to 1/10. Template DNAs (4 µl) were added to 5 µl of Roche LightCycler 480 SYBR Green I Master (2X) and primers  for a 10 µl total reaction volume. Each sample was run in triplicate on the same 384-well PCR plate  in a Roche LightCycler 480. H4K20me2 immunoprecipitate levels were normalized to the total H4 immunoprecipitate levels at each locus17. The primers used are shown inInput samples were diluted to 1/100 with ddH7 cells) were collected and resuspended in 200 µl SDS loading buffer without dye and reducing agent . Cells were lysed using mechanical disruption by FastPrep 120 (Thermo Savant) with 0.5 mm glass beads, in cold room, using 2 cycles of 40 sec of disruption followed by 60 sec on ice. Cell lysis efficiency, monitored under microscope, always reached a minimum of 90%. The lysate was collected by poking holes on the bottom of the tubes and spinning into new tubes at 1000 rpm for 1 min at 4°C. The protein concentration was measured by BCA assay (Pierce 23225) on 96-well plate. After adding 4X SDS loading buffer, lysate of 10 µg was heated at 95°C for 5 min, spun down, and loaded into each lane on SDS-PAGE gel. The rest of the lysates were kept at -20°C.Cells of 5 OD (5 × 10Recombinant histone H4 (MLA-modified) H4K20me1 or H4K20me2 or H4K20me3  and unmodified recombinant histone H4 (Active Motif® 31223) were resuspended in PBS buffer (in HPLC grade water) and used at the working concentration of 50 ng/μl except for H4K20me3 which was at 2.5 ng/μl. After adding 4X SDS loading buffer, 200 ng of recombinant histone H4 was heated at 95°C for 5 min, spun down, and loaded into each lane on SDS-PAGE gel. The rest of the proteins were stored at -20°C.TM softwareImage Studio (v. 4.0).SDS-PAGE gels were prepared with a 15% resolving gel and a 4% stacking gel using 40% Acrylamide/Bis solution (BioRad 161-0146), Tris buffer and SDS. The gel was run in 1x SDS-Glycine-Tris running buffer with Odyssey One-Color Molecular Weight Protein Marker (Li-Cor 928-40000). The proteins were transferred onto nitrocellulose membrane (Li-Cor 926-31092) using Genie transfer system for 1 h with 1X transfer buffer with 20% methanol and 0.05% SDS. The membrane was stained with Ponceau S and the blot above the 25 kDa marker band was removed. The cut membrane was then rocked with Odyssey blocking buffer (Li-Cor 927-40000) at room temperature for 1 h, followed by incubation with anti-H4K20me2 antibodies (GeneTex GT282 [RRID: AB_2728656] Lot #41582) diluted 1:2000 in Odyssey blocking buffer with 0.2% Tween-20 at 4°C overnight. In some experiments, GT282 was replaced with Abcam ab9052 ([RRID:AB_1951942] lot #GR99672-1). Anti-H4 antibody (Abcam ab10158 [RRID: AB_296888] Lot #GR133660-1) was diluted at 1:10000 in Odyssey blocking buffer with 0.2% Tween-20. The anti-H4K20me2 blot was treated with the secondary antibody 680RD Goat anti-Mouse IgG (Li-Cor 926-68070 [RRID: AB_10956588]) in Odyssey blocking buffer with 0.2% Tween-20 at room temperature and rocked for 1 h and kept away from light during the incubation. For anti-H4 blots, the secondary antibody was Goat anti-Rabbit antibody IgG (800CW Li-Cor 926-32211 [RRID: AB_621843]). The blots were scanned by the Odyssey® CLx Imaging system to acquire Western blot signal and analyzed with theunedited blot imagesClick here for additional data file.Copyright: © 2018 Audry J et al.2018Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Original excel workbook containing the Ct values from the PCRs and the location of the primers within the genes and telomere repeat adjacent DNAClick here for additional data file.Copyright: © 2018 Audry J et al.2018Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Revised excel workbook containing the Ct values from the PCRs, the location of the primers within the genes and telomere repeat adjacent DNA and the ChIP values of total H4 normalized to input chromatinClick here for additional data file.Copyright: © 2018 Audry J et al.2018Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).taz1∆ cells, causing slower growth or cell cycle arrest. H4K20me2 constitutes ~25% of total H4 inS. pombe20, implicating a telomere-associated demethylase to deplete H4K20me2 at telomeres intaz1∆ cells to prevent checkpoint-mediated arrest. However, both a genome-wide screen of gene deletion mutants  and our screen of demethylase mutants failed to identify a mutant that causedtaz1∆ cells to arrest, to grow poorly or to recruit more Crb2. We therefore re-evaluated H4K20me2 levels by chromatin immunoprecipitation (ChIP). We first identified commercial antibodies specific for H4K20me2 by western analysis using 11 different samples. Positive controls were extracts from wild type cells, cells where the singleS. pombe H4K20 methylase geneset9 is marked and functional (set9-kan-wt) and recombinant H4 where the only modification is a chemical mimetic for K20me221. Negative controls included recombinant H4 where the only modifications were mimetics of 0, 1 or 3 methyl groups on lysine 20, and extracts from cells where all three copies of the H4 gene have lysine 20 mutated to arginine (H4K20R)8. A series ofset9 mutants that methylate H4K20 to contain 0 (set9∆), 1 , or 1 and 2 methyl groups (set9-F195Y) were also assayed12. The specific antibody identified .10.5256/f1000research.15166.d20937427Dataset 1: unedited blot images10.5256/f1000research.15166.d22080228Dataset 2: Original excel workbook containing the Ct values from the PCRs and the location of the primers within the genes and telomere repeat adjacent DNA.10.5256/f1000research.15166.d21973629Dataset 3: Revised excel workbook containing the Ct values from the PCRs, the location of the primers within the genes and telomere repeat adjacent DNA and the ChIP values of total H4 normalized to input chromatin. 1) that checkpoint activation is inhibited at telomeres as a result of exclusion of histone H4K20me2 at these loci. The prior observation was done via ChIP, using a polyclonal antibody from Abcam. As addressed in the present manuscript, the inability to identify a demethylase that is associated with this effect raised questions as to the certainty of the claim. The investigators of the current study have identified what appears to be a superior antibody to evaluate this modification. The antibody from GeneTex shows improved specificity for the dimethylated state relative to that observed for the previously utilized antibody from Abcam, and furthermore displays higher affinity for H3K20me2 relative to the currently available lot of the Abcam antibody. Audry et al. examine a previously published observation  of H4K20me2 in checkpoint inhibition, but the findings merit publication to ensure that other investigators continue to re-examine this model.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. 1) and more recently to nucleotide excision repair (NER) . H4K20me2 is read by 53BP1 or Crb1, respectivley, which bridges the interaction with other DNA repair proteins thereby playing an essential role in chromatin signaling events at the damage site. Telomeres can be considered specialized DSBs in that they suppress the DNA damage checkpoint activation. One potential mechanism to explain this phenomenon was put forward by Carneiro and colleagues3. In their research paper it was reported that 53BP1 (Crb2) and H4K20me2 do not locate to telomeres, which would partially explain why the DNA damage checkpoint activation was suppressed. However, this exciting finding was never recapitulated by other research groups. The DNA damage response involves the setting of specific chromatin marks that are important to recruit or maintain DNA repair factors at the damage site. One such mark is H4K20me2, which has been linked to doublestrand break repair (DSB repair)  and Rad9-Rad1-Hus1 (9-1-1) checkpoint sensor complexes are massively accumulated at telomeres and activated, due to the elimination of telomere protein Taz1. We showed that telomeres in taz1∆ cells block Chk1 phosphorylation by limiting accumulation of Crb2 (53BP1 ortholog) at telomeres, and found that forced targeting of Crb2 to telomeres leads to Chk1 phosphorylation and cell cycle arrest in taz1∆ cells, but not in wild-type cells. In order to explain this phenomenon, we investigated status of the known recruiters of Crb2, and found that 1- Rad4/Cut5 (TopBP1 ortholog) is present and activated, 2- phosphorylated H2A (γ-H2A) is also present, but 3- H4K20me2 is surprisingly missing at telomeres. Therefore, we proposed that shortage of H4K20me2 could contribute to telomere's ability to prevent Crb2-dependent cell cycle arrest. Inaddition, we determined that other subunits of fission yeast shelterin complexes, Pot1 and Ccq1, play critical roles in preventing Crb2 accumulation and Chk1 activation. Furthermore, we found that elimination of Set9, a sole enzyme responsible for generating H4K20me, me2 and me3, was able to partially rescue accumulation of Crb2 and strong cell cycle arrest phenotype found in taz1∆ ccq1∆ cells . Taken together, these observations suggested that the fission yeast shelterin complex contribute in attenuation of Chk1 activation via histone-dependent and -independent regulation of Crb2. It is also important to note that elimination of set9∆ or γ-H2A only partially abrogate checkpoint activation in response to DNA damages in fission yeast, due to the existence of CDK-dependent modulation of Cut5-Crb2 interaction that allow recruitment and activation of Crb2 at DNA damages even in the absence histone modifications.  We view with great interest the data presented by Audry and colleagues. Their work alerts the community that the basis of the checkpoint inhibition mechanism at telomeres is far from being fully understood. We do not view, however, that this constitutes a matter of incompatible results. We did not normalize H4K20me2 levels over H4 levels as in their Fig. 1B, instead we measured different forms of H4K20 methylation as a ratio of total input. While normalization could be useful in certain circumstances, it could also introduce unintended artefact. Thus, without evaluating their original raw data, it is nearly impossible to determine if their newly identified H4K20me2 antibody behave substantially different from antibody utilized in our previous study (Abcam ab9052). Normalization may simply reflect fewer H4 levels at telomeres and, consequently, provide an explanation to our previous finding that lessH4K20me2 and Crb2 accumulate at telomeres. We would also like to point out that the H4K20me2 antibody utilized in our study was originally validated by SL Sanders , and widely used (more than 20 publications to date). Using the same antibody as our study, others have since corroborated our results. ChIPseq experiments initially performed by the group of S Grewal, NIH alerted us to lower levels of this mark near telomeres . More recently, Svensson et al. 2015, looking at genome-wide distribution of H4K20me2 , independently observed reduced levels of this mark at pericentromere and subtelomere regions. Therefore, we are somewhat surprised and disappointed that this manuscript does not directly compare their newly identified antibody against the previously established H4K20me2 antibody. In any case, a potential explanation to our seemingly contradictory results may reside on different affinities ofthe antibodies used. It may be that this new reagent is able to pick up low amounts of H4K20me2 that we were previously unable to detect. If so, this would sustain a quantitative model whereby very low levels of this chromatin modification may be unable to provide sufficient enough Crb2 to trigger a sustained checkpoint response. Nevertheless, since experimental evidences strongly suggests that Set9-dependent H4K20 methylation alone cannot provide a full explanation to the refractory nature of telomeres to Crb2 as we outlined above, we do believe that it is very important to further investigate how telomeres prevent checkpoint activation in collaboration or in parallel to regulation of chromatin status, in particular, using independent techniques that would support each other. In fact, our results are experimentally different from the current study:  We openly divulge our identity:  Miguel Godinho Ferreira and Toru NakamuraWe have read this submission. We believe that we have an appropriate level of expertise to state that we do not consider it to be of an acceptable scientific standard, for reasons outlined above. Rebuttal to Ferreira and Nakamura:The inclusion of Drs. Ferreira and Nakamura as reviewers was a major failure of reviewer selection system for the F1000Research Preclinical Reproducibility and Robustness channel. We are directly disputing results from a paper where Drs. Ferreira and Nakamura were the senior authors.  While the editorial team tries to remove reviewers with a positive bias,they relied on software to select reviewers that included authors of the disputed publication and their former collaborators. Under this open review system, the editors and authors are supposed to agree on a set of reviewers, but reviewers were selected before we could respond.  The editorial staff did not vet these reviewers and thus allowed the inclusion of the most negatively biased reviewers possible.  As we show below, their comments distort the facts about our paper and are without merit. The point of our paper is that H4K20me2 is as clearly detectable in nucleosomes adjacent to telomere repeats as it is at internal loci, requiring re-evaluation of this mechanism for checkpoint suppression proposed in Carneiro et al. The review by Ferreira and Nakamura follows our discussions with them for more than one year in which we provide H4K20me2 localization and they turn the discussion to Pot1, Ccq1 and Crb2. Our paper is only about H4K20me2 localization.H4K20me2 exclusion from nucleosomes near telomere repeats was a fundamental mechanistic conclusion of Carneiro et al. 2010.  This paper frequently states H4K20me2 is excluded from telomeres and that this exclusion is the mechanism for severing the checkpoint response.  For example, their results contain the statement:taz1∆cells.”“In contrast, H4K20me2 was undetectable at telomere in earlier wild-type or Their final model in Figure 4g diagrams this exclusion and the legend states:53BP1association” (spelling error corrected)            “Pot1 and Ccq1, together with the Taz1 complex, define a chromatin-privileged region that excludes H4K20me2 and prevents stable Crb2            Their penultimate paragraph in the text constituting the discussion begins with: 53BP1is probably due to the lack of H4K20me2 epigenetic marks at chromosome ends.”  This paragraph ends with:              “The inability of telomeres to stably recruit Crb2            “In contrast, the absence of H4K20me2 marks regions where DNA perceived as damaged, such as chromosome ends, would not interfere with genome stability thus precluding a full checkpoint response.”It is worth noting that Ferreira and Nakamura also cite data in their rebuttal that are not in their paper (there is no figure S5D), and overstate the claims of some of the data shown . They also avoid mentioning their own contradictory data that Crb2 can be detected at telomeres by ChIP with another epitope tag (presented by Dr. Nakamura at the 2013 Cold Spring Harbor Telomeres and Telomerase meeting), so the overall picture is more complex than they present.  The focus of their summary on Pot1, Ccq1, Crb2 and phosphorylated H2A is irrelevant to our paper and distracts from the main point: H4K20me2 is present in nucleosomes adjacent to the telomere repeats at levels comparable to internal loci.We show below that the two main points raised by Ferreira and Nakamura are simply not true, misrepresent our data and contradict their own previous discussions with us.Objection 1.  (A) Normalization to H4 levels versus total input chromatin in ChIP and (B) the absence of the original data.              1. A.  The normalization to input chromatin in Carneiro et al. biases the results to lower levels of nucleosomes and H4 adjacent to telomeres, and leads to the erroneous conclusion that H4K20me2 is reduced near telomeres.  Telomere repeats are bound by specific binding proteins, so chromatin fragments that include telomere repeat chromatin will have fewer histones in total.  For example, the ~300 bp of telomere repeats in wild type cells are bound by non-nucleosomal telomere-specific binding proteins (i.e. Taz1 and Pot1).  A 500 bp chromatin fragment containing the telosome will have fewer histones than an internal fragment of the same size. Normalization to input chromatin will thus yield a reduced signal for H4 and will give an artifactual decrease in all H4 modifications. Our data supporting this point are presented in the new Dataset 3 described in 1. B below.            In contrast to normalization by input chromatin, normalization to total histone H4 at a locus allows one to assay the level of modification in the nucleosomes present, regardless of the adjacent non-nucleosomal chromatin. It allows the direct assay of the major claims in Carnerio et al. 2010 quoted above.              1. B.   We submitted the original data in a spreadsheet that was linked to our paper as Dataset 2, which had an associated doi in the pdf version of the paper that we proofed, but that link was not yet live active. This doi entry linked to an error webpage and was absent in the HTML version due to a processing mistake.  We are surprised that Ferreira and Nakamura state that the data were not provided when the defective hyperlink was obvious. The editorial staff would have provided the data if these reviewers asked for it.            Our revised version contains the original Dataset 2 and has used these data to show that normalization to input chromatin gives reduced levels of histone H4 at telomeres  and presents this analysis in a new third worksheet of Dataset 3. H4K20me2 signals near telomeres will therefore be low due to the artifact of normalizing non-nucleosomal chromatin fragments to input chromatin that is primarily nucleosomal.  Objection 2.  The H4K20me2-specific antibody we used.            We find the wording of this objection extremely odd as Dr. Toru Nakamura told us in 2012 that while the original SL Sanders antibody sold by Abcam (as ab9052) was specific and good for ChIP, this original antibody was no longer available and subsequent lots sold under the same catalog number did not work well for ChIP.  We therefore find this reversal of opinion to criticize our work puzzling, as repeating the experiment with the working anti-H4K20me2 antibody from Abcam is impossible as it no longer exists. crb2-F178Ymutant appears to be different with the new lot of antibody and the reactivity with the pure H4K20me2 mimic is reduced .  We do not know what else this antibody recognizes in the crb2-F178Ymutant.  In contrast, the GT282 antibody we used showed stronger selectivity and reactivity with H4K20me2 without the issues that accompany ab9052.            We did test the Abcam antibody sold as ab9052 in 2014 in case a newer lot showed better specificity.  However, our tests confirmed Dr. Nakamura’s statement that the newer antibody showed less reactivity with H4K20me2, and showed a different reactivity to the histones from the yeast mutants originally tested in by the Sanders lab in Greeson et al. (2008). The reactivity for the histones in the             We cannot comment on the Svenson et al. 2015 paper as we did not address their findings in our paper and do not know what lot of antibody they used for their experiments. We only test whether the nucleosomes adjacent to the telomere repeats are depleted in the H4K20me2 modification, and find that they are at the same level as two different internal loci.  Therefore, checkpoint suppression must occur by a mechanism distinct from H4K20me2 exclusion.Ferreira and Nakamura include another puzzling statement after these two points.              “It may be that this new reagent is able to pick up low amounts of H4K20me2”. Our data show that the levels at internal loci and telomeres are the same, so any quantitative models regarding Crb2 accumulation at telomere would apply to internal loci and would not explain telomere-specific checkpoint suppression. In conclusion, the nucleosomes adjacent to telomere repeats have the same levels of H4K20me2 as the internal genes act1 and hip3, so some other mechanism than H4K20me2 exclusion prevents checkpoint activation at telomeres.Kurt RungeThis rebuttal was reviewed and accepted by all authors of this paper by Audry et al.


PAX4) gene encodes a transcription factor which plays an important role in the generation, differentiation, development, and survival of insulin-producing β-cells during mammalian pancreas development. PAX4 is a key diabetes mellitus (DM) susceptibility gene, which is associated with many different types of DM, including T1DM, T2DM, maturity onset diabetes of the young 9 (MODY9) and ketosis prone diabetes. In this study, a novel PAX4 gene knockout (KO) model was generated through co-injection of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) mRNA/sgRNA into rabbit zygotes. Typical phenotypes of growth retardation, persistent hyperglycemia, decreased number of insulin-producing β cells and increased number of glucagon-producing α cells were observed in the homozygous PAX4 KO rabbits. Furthermore, DM associated phenotypes including diabetic nephropathy, hepatopathy, myopathy and cardiomyopathy were also observed in the homozygous PAX4 KO rabbits but not in the wild type (WT) controls and the heterozygous PAX4 KO rabbits. In summary, this is the first PAX4 gene KO rabbit model generated by CRISPR/Cas9 system. This novel rabbit model may provide a new platform for function study of PAX4 gene in rabbit and gene therapy of human DM in clinical trails.Paired-homeodomain transcription factor 4 ( PAX4 (paired-homeodomain transcription factor 4) gene belongs to the Pax gene families, encodes a transcription factor which plays major roles in the generation, differentiation, development, and survival of insulin-producing β cells during mammalian pancreas development (PAX4 is a key diabetes mellitus (DM) susceptibility gene, for single-nucleotide polymorphisms (SNPs) and mutations in PAX4 caused maturity onset diabetes of the young 9 (MODY9) (PAX4 gene in the development of DM has been manifested in the PAX4 gene knockout (KO) mice  . All rabbit experiment protocols were conducted under the approval of the Animal Care Center and Use Committee of Jilin University.in vitro transcription have been described in detail in our previous published protocols (PAX4 gene (NC_013675.1) were designed using the online CRISPR Design Tool (http://crispr.mit.edu/) as previously described (BbsI sites of a Puc57-T7-sgRNA cloning vector (Addgene ID 51306). The amplified PCR products of Puc57-T7-sgRNA vector were in vitro transcribed using the MAXIscript T7 Kit  and purified with the miRNeasy Mini Kit  according to the manufacturers’ instruction. Meanwhile, the 3xFLAG-NLS-SpCas9-NLS vector (Addgene ID 48137) was linearized with NotI and in vitro transcribed with the mMessage mMachine SP6 Kit (Ambion) and the RNeasy Mini Kit (Qiagen).The protocols for sgRNA design, vector construction and in vitro transcribed Cas9 mRNA (200 ng/μl) and sgRNA (40 ng/μl or 20 ng/μl) was co-injected into the cytoplasm of the zygotes. Then the injected embryos were transferred and cultured in EBSS medium at 38.5°, 5% CO2 and 100% humidity conditions for 30-60 min. Finally, approximately 30-50 injected zygotes were transferred into the oviducts of surrogate rabbits.Microinjection of pronuclear-stage embryos and embryo transfer were carried out based on the protocols described in our published papers  was used to isolate the genomic DNA of the newborn pups according to the manufacturers’ instruction. PCR primers used to detect the PCR products were then gel-purified and cloned into the pGM-T vector (Tiangen), at least 10 positive plasmid clones were sequenced and the sequences were analyzed using DNAman software.PAX4 KO rabbits, potential off-target sites (POTS) of the sgRNAs were predicted with the online CRISPR Design Tool (http://crispr.mit.edu/), and the top 5 POTS for each sgRNA were selected. Then the PCR products of these POTS were subjected to T7E1 assay and Sanger sequence analysis. Primers are listed in Table S1.To test whether off-target mutations occurred in The T7E1 assay was performed as previously described (PAX4 KO rabbits using TRNzol-A+ reagent (Tiangen), then treated with DNase I (Fermentas) and reverse-transcribed into cDNA using the BioRT cDNA First-Strand Synthesis Kit . qPCR was performed using the ABI PRISM 7500 , and relative gene expression was determined using the ∆∆CT method, which was normalized to that of the GAPDH gene. Primers for qPCR are listed in Table S2.Total RNA was extracted from the pancreas of 3-day-old WT and PAX4 KO rabbits were measured everyday until the homozygous PAX4 KO rabbits died. The blood samples were collected from the ear vein of the rabbits and the blood glucose was measured with the Roche blood glucose monitor (Glucotrend 2). Urinalysis was performed with the urine eight couplet test paper . All data were expressed as mean± SEM, at least three individuals of each genotype were used in all experiments. The data were analyzed with the Student’s t-test using Graphpad Prism software 7.0. P < 0.05 was considered statistically significant.The blood glucose and body weight of the WT rabbits and the PAX4 KO rabbits were fixed with 4% paraformaldehyde, embedded in paraffin wax and then cut into 4 μm sections. For histology analysis, the sections were stained with hematoxylin and eosin (H&E). For IHC, immunohistochemical analysis of the pancreas sections were performed as previously described . Besides, due to the advantages on construction of gene knockout rabbits in our laboratory, we chose rabbit PAX4 gene as the target of gene knockout. To disrupt the function of PAX4 gene in rabbits, two sgRNAs targeting the third and the fifth exon of PAX4 gene were designed. The target sites are shown in According to the evolutionary relationship provided by NJ tree of PAX4 KO rabbits, the in vitro transcribed Cas9 mRNA and sgRNA were co-injected into rabbit zygotes, and a total of 210 injected zygotes (pronuclear stage) were transferred into the oviducts of 4 surrogate rabbits, respectively  for each sgRNA were detected by T7E1 cleavage assay and Sanger sequencing. As shown in Besides, off-target effect was detected in the PAX4 KO rabbits were successfully generated by CRISPR/Cas9 system, and off-target mutations were effectively eliminated by co-injection of Cas9 mRNA and sgRNA into rabbit zygotes.These results together showed that PAX4 KO rabbits, we first detected the PAX4 mRNA expression level of the KO rabbits by qPCR. As expected, compared with the WT rabbits, the PAX4 mRNA expression level was significantly reduced in the heterozygous PAX4 KO rabbits (PAX4+/− rabbits) and homozygous PAX4 KO rabbits (PAX4−/− rabbits) .After successfully generated the PAX4 gene mutations were examined in the KO rabbits. As shown in PAX4 KO rabbits appeared normal and can’t be distinguished from the WT rabbits. However, 2 days later, the PAX4−/− rabbits showed detectable growth retardation in comparison with the WT rabbits and the PAX4+/− rabbits . Furthermore, the PAX4−/− rabbits died soon after birth due to severe hyperglycemia, and finally all of them died within 4 days  slight alveolar expansion of the lung was observed in the PAX4−/− rabbits; (3) decreased numbers of glomerular, vacuolation of renal tubular epithelial cells, and smaller space between the glomerular and the glomerular capsule of the kidney were observed in the PAX4−/− rabbits; (4) no obvious histopathological changes were observed in the spleen of the PAX4−/− rabbits; (5) the boundary between the visual cells layer and the bipolar cell layer was not clear in the retina of the PAX4−/− rabbits, and cells in the ganglion cells layer showed characteristics of hyperplasia and vacuolation; (6) inflammatory cells infiltration, collagen fibers hyperplasia, adipocytes ectopic deposition, and uneven muscle fiber thickness were shown in the skeletal muscle of the PAX4−/− rabbits; (7) similar characteristics were also observed in the cardiac muscle of the PAX4−/− rabbits, including cardiac muscle cell steatosis and thickened myocardial fibers. Significantly, the PAX4+/− rabbits only exhibited slight hepatocyte steatosis and vacuolation of renal tubular epithelial cells. These results demonstrated that DM associated phenotypes were exhibited in the PAX4−/− rabbits by inactivation of PAX4 gene.As far as we know, persistent hyperglycemia resulted in development of diabetic nephropathy, hepatopathy, retinopathy, myopathy and cardiomyopathy in patients with DM , and all the PAX4−/− rabbits died within 4 days after birth. All these phenomenons observed in the PAX4−/− rabbits were consistent with the previous studies carried out in the PAX4 gene KO mice (+/− mice exhibit insulin-producing β cells and do not develop diabetes, indicating that a single copy of PAX4 is sufficient to promote normal β cells development and function (+/− rabbits did not exhibit any obvious abnormalities, with normal body weight and blood glucose, and survived to adulthood. However, the results of H&E staining and immunohistochemistry of pancreas sections showed that the PAX4+/− rabbits also exhibited destroyed, incomplete pancreatic islet structure, decreased numbers of insulin-producing β cells and increased numbers of glucagon-producing α cells. But these changes in the PAX4+/− rabbits were not as serious as in the PAX4−/− rabbits, with the trend of slighter changes in the PAX4+/− rabbits with smaller fragment deletions of the PAX4 gene. However, based on the clinical characteristics of human MODY9, we speculate that it may take some time for the PAX4+/− rabbits to develop DM associated phenotypes. Therefore, our next plan is to continue to feed these PAX4+/− rabbits, to observe whether the heterozygotes could develop hyperglycemia and other DM associated phenotypes, and to investigate the possibility of this rabbit model used for DM gene therapy. At the same time, we will mate the heterozygotes with normal rabbits to observe their fertility. Furthermore, mutations of PAX4 gene seem to increase DM risk, thus this model may also be used to investigate the influence of other factors on the onset of DM.As far as we know, this is the first report of a function . SimilarPAX4 gene KO model in rabbits, including characteristics of growth retardation, persistent hyperglycemia, decreased number of insulin-producing β cells, increased number of glucagon-producing α cells and typical DM associated phenotypes. This novel PAX4 KO rabbit model will be useful for validating important pathological mechanisms, identifying novel therapeutic targets and screening new drugs for human DM.In conclusion, this is the first report of a


The contribution of ER to neuronal Ca2+ homeostasis is attributed to the organelle being the largest reservoir of intracellular Ca2+ and having a high density of Ca2+ channels and transporters. As such, ER Ca2+ has incontrovertible roles in the regulation of axodendritic growth and morphology, synaptic vesicle release, and neural activity dependent gene expression, synaptic plasticity, and mitochondrial bioenergetics. Not surprisingly, many neurological diseases arise from ER Ca2+ dyshomeostasis, either directly due to alterations in ER resident proteins, or indirectly via processes that are coupled to the regulators of ER Ca2+ dynamics. In this review, we describe the mechanisms involved in the establishment of ER Ca2+ homeostasis in neurons. We elaborate upon how changes in the spatiotemporal dynamics of Ca2+ exchange between the ER and other organelles sculpt neuronal function and provide examples that demonstrate the involvement of ER Ca2+ dyshomeostasis in a range of neurological and neurodegenerative diseases.By influencing Ca On. On2+ re2+ and microtubules are also involved in axon guidance  sensor, stromal interacting molecule 1 (STIM1), couples store release with microtubule polymerization via interactions with the microtubule plus-end binding protein, EB1  also regulate the growth and morphology of dendritic spines. Neuronal activity-dependent modification of the spine structure during long-term potentiation involves ER Ca2+ store depletion, STIM1-mediated induction of store-operated Ca2+ entry, and gene expression driven by the transcription factor NFATc3 ,492+ chag [Ca2+] B  . Roles fowledged A  and presynaptic plasticity in cerebellar and hippocampal slices  and decreased SV release probability [2+] was lowered to <0.5 mM from the usual 2 mM [2+ release at the Drosophila larval NMJ [2+] can be overridden by Ca2+ influx. We envision that only during periods of diminished presynaptic Ca2+ entry would the contribution of ER Ca2+ stores to SV release become apparent. For example, during high frequency stimulation when massive Ca2+ influx into postsynaptic neurons results in dramatic reduction of [Ca2+] in the synaptic cleft [2+ release could have a significantly larger impact on determining resting [Ca2+], and thus SV exocytosis. Although several studies have shown that Ca nuances ,55,61,62l slices . A potenbability . Interesual 2 mM . These prval NMJ ,68, showic cleft ,70,71,722+ is a key intermediate in the functional relationship between neuronal activity and gene expression. Alterations in cytosolic and nuclear [Ca2+] exert differential effects on neuronal gene expression, the dynamics of which are synchronized with the spatiotemporal patterns of the Ca2+ transients. Depletion of ER Ca2+ stores by application of the SERCA inhibitor, thapsigargin, in cortical, hippocampal, and other neurons leads to the dramatic and rapid increase in the transcription of the immediate early genes encoding c-Fos and Homer-1a  arising from the passive diffusion from the cytosol through the nuclear pore or due to activation of IP3Rs embedded in the nuclear envelope promotes CREB-dependent transcription and the changes in synaptic strength and dendritic arborization that characterize plasticity  to levels high enough for mitochondrial uptake and the attendant activation of OXPHOS  and MCU-mediated Ca2+ uptake . T. T2+] ist manner ,126. Confunction . 3R activation and mitochondrial ATP production ensures a critical requirement for IP3Rs in the dynamic regulation of neuronal membrane potential and excitability. This notion is exemplified by the Na+/K+ ATPase, which is needed for the maintenance of the electrical polarity across the neuronal membrane, and for this purpose the pump consumes ~75%–90% of the ATP produced in the cell . Thus, the interplay between IPthe cell . Computarivation . In thisactivity . Thus, istronger . Along t neurons . On the tability . Togethe3R-dependent cytosolic Ca2+ elevation is usually sufficient for stimulating RyRs, IP3Rs and RyRs are thought to cooperate in the initiation and propagation of CICR [2+] owing to the presence of high Ca2+-affinity activating sites and low Ca2+-affinity inhibitory sites on the proteins [2+] is below a critical threshold, Ca2+ ions activate the channels leading to store release. Upon exceeding the threshold, Ca2+ ions start to inhibit the channels, which lead to attenuation of Ca2+ release and restoration of cytosolic [Ca2+] to resting levels. In addition to IP3Rs and RyRs, other purported triggers for CICR include Ca2+ influx via TRP channels and VGCCs, and alterations in mitochondrial Ca2+ uptake [2+ homeostasis permits the amplification of even small or transient elevations in cytosolic [Ca2+]. Since IP of CICR ,135,136.proteins ,139,140.+ uptake ,143,144.2+ release is also coupled to the mechanisms of Ca2+ removal. Whereas extrusion of Ca2+ by PMCA serves to lower cytosolic [Ca2+] and limit CICR, Ca2+ uptake into the ER promotes subsequent release [4-fold [Ca2+] gradient between the ER lumen and the cytosol, the overall Ca2+ content of the ER serves as a powerful driving force for Ca2+ release   and SV release probability  could reflect a potential mode of activation. Both Iav and TRPV1 contain Ca2+/calmodulin-binding motifs in their cytosolic domains. In case of TRPV1, occupancy of these sites by Ca2+/calmodulin leads to channel desensitization . If so, a drop in cytosolic [Ca2+] would disinhibit Iav, leading to ER Ca2+ release. We predict that this axis serves as a feedback mechanism for the maintenance of resting [Ca2+] within a narrow physiologically important level at presynaptic termini. TRPVs, TRPM1, TRPM8, and TRPP2 subtypes of the TRP superfamily of cation channels have also been shown to localize and/or function in ER Ca release ,178,179.fication ,178. Whification , the act neurons ,180. Iavbability . The locto IP3Rs ,188,189. release . Involveding Iav ,188,189.ding Iav ,190, alttization . Notablyt the ER ,192. It 2+ homeostasis occurs in many neurological diseases. Being the largest intracellular reservoir of Ca2+, ER Ca2+ dyshomeostasis is of particular relevance to these diseases. In this section, we present an overview of the relationships between ER Ca2+ and pathology in a sampling of neurological diseases. We hope to highlight the tenet that qualitatively distinct alterations in ER Ca2+ signaling (either too much or too little release or uptake) can result in pathology. In fact, ostensibly similar changes in ER Ca2+ homeostasis can induce diverse spatiotemporal outcomes, indicating that changes in ER Ca2+ dynamics accompany neuronal processes perturbed in disease. Perturbed Ca2+ alterations in its pathophysiology  elevations needed for interorganellar ion transfer  in response to IP3R activation  in neurons from the 3xTg-AD mouse model is double that in control cells  and ATP production (2+ homeostasis, a worm model of ALS exhibits ER Ca2+ dyshomeostasis that led to decline of neuronal function [2+ dyshomeostasis due to combinations of elevated IP3R activity and diminished uptake into stores [Various lines of evidence also point to ER Caneration . A familethering ,276,277.ethering . In otheoduction  [279,280function . ALS-cauo stores ,283. 2+ homeostasis in neuronal functions ranging from development and plasticity to gene expression and cell survival. We also provided examples of neurological and neurodegenerative diseases that arise when the processes regulating the movement of Ca2+ in and out of the ER go awry. It is important to note that despite being one of the most extensively studied organelles, much still remains to be known about ER Ca2+ and its relationship to neuronal function and disease. In particular, we anticipate a proliferation of studies focused on the domains that the ER forms with other organelles such as the mitochondria, endolysosomes, and the plasma membrane. These studies should go a long way to illustrate that in the ultimate analysis, intracellular organelles function in an ecosystem that requires invaluable contributions from all the players. By remaining open to these possibilities, we can hope for novel therapies for a variety of debilitating neurological diseases that arise from miscommunication between neuronal organelles. In this review, we have described the involvement of ER Ca


Dear Editor,1. The mechanisms underlying the therapeutic activity of ATRA and classic chemotherapeutic agents are different2. In fact, the retinoid is primarily a differentiating/antiproliferative agent and it is largely devoid of direct cytotoxic activity against the leukemic cell. This is at the basis of the low systemic toxicity associated with ATRA in the clinics and it justifies the interest in the use of the retinoid in oncologic contexts other than APL. As for the last aspect, in the field of onco-hematology, it would be of particular relevance to establish whether specific subgroups of acute myelogenous leukemia (AML) may benefit from ATRA-based treatments. In fact, AML is a very heterogeneous disease, which can be classified into subgroups according to the presence/absence of specific genetic alterations. The few available clinical trials on the use of ATRA in AML other than APL have provided conflicting results5.ATRA is the first example of clinically effective targeted antitumor agent and it is part of the standard protocol used in the treatment of acute promyelocytic leukemia (APL)ATRA-21), capable of correctly predicting ATRA sensitivity across a large panel of breast cancer cell lines characterized for their basal gene-expression profiles at the whole-genome level6. Despite the original development in the context of breast cancer7, application of ATRA-21 to the over 400 cell lines derived from different types of leukemia and tumors present in the GDSC (Genomics of Drug Sensitivity in Cancer) database and profiled for their in vitro sensitivity to ATRA demonstrates that the predictive capability of the model is tumor-type independent6. In addition, the model correctly predicts ATRA sensitivity in the APL patients belonging to the TCGA (The Cancer Genome Atlas) and LEUCEGENE databases6.Precision medicine requires the development of diagnostic tools capable of identifying individuals and groups of patients who may be sensitive to the therapeutic activity of specific agents. In a recent study, we developed a gene-expression model, consisting of 21 genes  criteria, the ELN (European Leukemia Network)-RISK categories, and the presence/absence of clinically relevant genetic rearrangements as well as gene mutations. We compare the average ATRA-21 predictions obtained in the various AML subgroups against the remainder of the entire AML population of the four data sets and estimate the effect size. Following clustering of the cases according to the FAB classification, the results obtained confirm that M3 (PML-RARα+) patients are endowed with significantly higher average ATRA-21 values than the remainder of the AML population. On the basis of the ATRA-21 values, AML cases belonging to the favorable ELN-RISK class are predicted to be particularly responsive to ATRA. As for the AML classification based on the presence/absence of the most prevalent genetic rearrangements, the cases showing an inversion of chromosome 16 [inv(16)] or a t chromosomal translocation are characterized by ATRA-21 predictions well above the average of the remainder AML population. With respect to this, it is interesting to notice that a case report study demonstrates that ATRA induces one partial and three complete remissions in four t AML patients initially misdiagnosed as APL cases9. As for the AML subtypes defined by recurrent gene mutations, the only cluster predicted to be responsive to ATRA belongs to the larger group of patients characterized by biallelic mutations of the CEBPA gene. Indeed, AML cases presenting with CEBPA-biallelic mutations can be further divided into two relatively homogeneous subgroups on the basis of a gene expression profile (GEP) consisting of 95 genes (CEBPAMUT/GEP+ and CEBPAMUT/GEP−)10. The CEBPAMUT/GEP+ cases, which are endowed with this specific GEP, are predicted to be ATRA-sensitive on the basis of the ATRA-21 score. In contrast, CEBPAMUT/GEP− cases are characterized by very low ATRA-21 predictions. As for the data obtained in AML patients with recurrent mutations, it is noticeable that NPM1-mutated AML do not show higher than average ATRA-21 values. Taken together, the predicted responsiveness of CEBPAMUT/GEP+ AML patients to ATRA and the lack of predicted ATRA sensitivity in NPM1-mutated AML are in accordance with the findings of the AMLSG 07-04 study in which ATRA was added to intensive chemotherapy8. As a last remark, it should be emphasized that the favorable ELN-risk category encompasses the t, inv(16), mutated-NPM1 without FLT3-ITD  and mutated-CEBPA  subgroups. Clear experimental validation of these results is required and the approach that we are currently pursuing involves exposure of primary blasts obtained from AML patients to ATRA in vitro. Nevertheless, it must be emphasized that the validity of the predictions on ATRA sensitivity based on our model is fully supported by the results obtained in the two above-mentioned clinical studies9. Finally, it should be mentioned that it is entirely possible that our analysis may overlook specific subgroups of patients or individual cases that may be sensitive to ATRA. In fact, as observed in solid tumors7, even AML types characterized by low-predicted average sensitivity to ATRA include a proportion of cases that may be sensitive to the retinoid.A recent randomized clinical study (AMLSG 07-04) demonstrates that addition of ATRA to intensive treatment exerts a beneficial effect in ELN-favorable-risk and CEBPA-biallelic mutation patientsnts Fig. . We applATRA-21 may be endowed with prognostic potential  or inv(16) genetic aberrations as covariates. Statistical significance is observed for all the analyses with the exception of the ELN-RISK covariate in the TARGET data set. The observed associations between ATRA-21 and overall survival suggest that the endogenous levels of ATRA may have an inhibitory role in the growth and progression of the subgroups of AML, which are predicted to be sensitive to the retinoid. We described similar associations in other tumor types, which are predicted to be responsive to ATRA on the basis of the ATRA-21 model6.Analysis of the overall survival data available in the TCGA and TARGET data sets is in line with the idea that ial Fig. . Indeed,ATRA-21 predictions, we trained a conditional decision model in the TCGA data set. The other three data sets were used to confirm the validity of the decision tree  and inv(16) genomic aberrations, respectively. However, it is noticeable that inv(16) patients show a large dispersion of the prediction values, which suggests that determination of ATRA-21 in this subgroup may be useful to better discriminate retinoid responsive patients. The vast majority of the remainder groups of AML cases present with low-predicted sensitivity to ATRA. However, in these groups too, measurement of ATRA-21 may be of use, as there are individual patients, possibly among the FLT3 wild-type cases, which show high values of this parameter.To define the relative importance of all the single genetic aberrations and mutations observed in the AML population for the ATRA-21 predictions, i.e., biallelic CEBPA in its typical form or the GEP+ atypical form, t and inv(16). Each marker is sufficient to define predicted sensitivity to ATRA, irrespective of other concurrent genetic alterations. The validity of the ATRA-21 model developed to predict ATRA sensitivity is confirmed by the results obtained in the clinics for AML patients. In addition, the data obtained support the idea that the ATRA-21 model may have prognostic relevance in the context of AML, given the observed significant associations between ATRA-21 values and overall survival data. In conclusion, the study represents the basis for further clinical trials aimed at evaluating the therapeutic advantage achievable with the addition of ATRA to standard therapy in the above groups of AML patients. In a precision medicine perspective, ATRA-21 is likely to represent a new tool for the selection of AML patients who may benefit from treatments based on the use of ATRA.In conclusion, our predictions support the idea that other AML subtypes, besides the expected APL group, are likely to be responsive to the antiproliferative effects of ATRA. Indeed, we identify three new classes of AML with high Supplementary InformationSupplementary Table 1


The extremely complex calcium machinery includes plasma membrane bound channels as well as intracellular receptors distributed in distinct cytosolic compartments that act jointly to maintain a physiological [Ca2+]i, which is crucial for triggering many cellular mechanisms. Here, we first survey the overall notions related to intracellular Ca2+ mobilization and later highlight the involvement of this second messenger in crucial ECs functions with the aim at stimulating further investigation that link Ca2+ mobilization to ECs in health and disease.Endothelial cells (ECs) constitute the innermost layer that lines all blood vessels from the larger arteries and veins to the smallest capillaries, including the lymphatic vessels. Despite the histological classification of endothelium of a simple epithelium and its homogeneous morphological appearance throughout the vascular system, ECs, instead, are extremely heterogeneous both structurally and functionally. The different arrangement of cell junctions between ECs and the local organization of the basal membrane generate different type of endothelium with different permeability features and functions. Continuous, fenestrated and discontinuous endothelia are distributed based on the specific function carried out by the organs. It is thought that a large number ECs functions and their responses to extracellular cues depend on changes in intracellular concentrations of calcium ion ([Ca As it occurs in many cell types most of the intracellular (Ca2+) in ECs is sequestered in the endoplasmic reticulum (ER) i i and once specific downstream events have been activated, the amount of cytosolic Ca2+ rapidly decreases, bringing back the cell at its resting state. To this regard, both plasma membrane Ca2+ ATPases and sodium calcium exchangers came into play to extrude Ca2+ back into the extracellular space i. Therefore, one may expect that any condition that interferes with the complex molecular machinery heading the series of event occurring during Ca2+ mobilization may affect normal behavior of ECs, leading to pathological conditions i . Likewisle cells . Once reproteins  that in proteins ,36. To dproteins ,38. Ca2+ganelles ,40,41. Tganelles ), have bganelles . In addiganelles  and VEGFganelles ,46, furt in mice ,48. A repatients . Whatevear space ,51. At tnditions .2+]i which generate in response to disparate extracellular cues are crucial regulators of a number of vascular mechanisms linked to regulation of the vessel tone, inflammation, coagulation and regulation of vascular permeability, just to mention a few. A variety of ion channels expressed the cell surface of ECs mediate Ca2+ influx from the extracellular milieu that could be followed by intracellular Ca2+mobilization from internal stores. These include Voltage-Dependent Calcium Channels i can be monitored in living cells. To measure in-cell changes in cytosolic [Ca2+]i or agonist-induced Ca2+ mobilization, a number of fluorescent Ca2+ indicators are commercially available. These indicators have been first developed during the early 80′s and have soon became a fundamental tool for monitoring intracellular Ca2+ changes in many cell types i i ,77,78. N [Ca2+]i  is a sol [Ca2+]i ,80,81. T [Ca2+]i , a cytosd in ECs  and mored in ECs . Despited in ECs . The welthe cGMP . In addiactivity . Among pactivity ,89, the coupling . Due to 2+ mobilization in ECs already mentioned above, it has been demonstrated that histamine through its H1 receptor and VEGF/VEGFR2 pathway play a relevant physiological role in the regulation of vWF release, capillary-like formation and in vivo angiogenesis, respectively i contributes to modulate the inflammatory response of ECs. In addition, more recent studies in humans suggested that EC functions are affected by elevated circulating calcium levels that, indeed, must be finely regulated in physiological conditions in vivo. Several studies indicate a direct correlation between blood calcium level and the onset of cardiovascular diseases and atherosclerosis [2+ level and any increase of the ion in the bloodstream will increase [Ca2+]i, which is fundamental to upregulate the expression of endothelial adhesion molecules i controls the equilibrium between pro- and antiangiogenic factors remains crucial to the comprehension of the dysfunctions of the vascular endothelium. We believe that the use of genetic targeting of critical molecules involved in Ca2+signaling and the further characterization of the mechanisms controlling Ca2+ mobilization from intracellular stores in ECs is crucial to develop clinical strategies to counteract vascular dysfunctions that may lead to severe cardiovascular diseases. Moreover, although the effects of Ca2+ antagonists in ECs remain quite controversial, deepening the molecular mechanisms activated by these drugs could improve our knowledge of how Ca2+ signals originating from ECs contribute to ameliorate vascular dysfunctions, thereby reducing the risk of severe cardiovascular diseases such as atherosclerosis, hypertension, stroke and peripheral arterial disease. Notably, the expression of low voltage activated T-type Ca2+channels and their involvement in the reduction of vascular dysfunctions [2+antagonists in ECs. However, their specific activity within the vascular bed remain still unclear and need further investigation. Though we did not address this topic in this work, it has been also reported the higher sensitivity as well as an increased intracellular Ca2+ levels in endothelial progenitor cells compared to the adult ECs [2+ channels and different angiogenic capabilities should encourage further investigation aiming at linking Ca2+ signaling to revascularization. In conclusion, the main purpose of this review is to focus the reader’s attention on the calcium machinery in EC functions and to stimulate further investigation that may contribute to develop novel clinical strategies helpful in the field of vascular diseases.Undoubtedly the growing number of studies on Caunctions ,125 raisdult ECs ,127. The


Does not Make Infections 1) and its homologs CASTOR and POLLUX, are required for the establishment of legume-microbe symbioses by generating nuclear and perinuclear Ca2+ spiking. Here we show that CASTOR from Lotus japonicus is a highly selective Ca2+ channel whose activation requires cytosolic/nucleosolic Ca2+, contrary to the previous suggestion of it being a K+ channel. Structurally, the cytosolic/nucleosolic ligand-binding soluble region of CASTOR contains two tandem RCK (Regulator of Conductance for K+) domains, and four subunits assemble into the gating ring architecture, similar to that of large conductance, Ca2+-gated K+ (BK) channels despite the lack of sequence similarity. Multiple ion binding sites are clustered at two locations within each subunit, and three of them are identified to be Ca2+ sites. Our in vitro and in vivo assays also demonstrate the importance of these gating-ring Ca2+ binding sites to the physiological function of CASTOR as well as DMI1.A family of plant nuclear ion channels, including DMI1 ( Lotus japonicus ion channel required for nuclear Ca2+ spiking and establishing rhizobial and mycorrhizal symbioses. Here, via structural and functional analysis, Kim et al. show that CASTOR is a Ca2+-selective channel activated via Ca2+ binding to a soluble gating ring consisting of tandem RCK domains.CASTOR is a  This evolutionary history explains how these two endosymbioses share the same common symbiotic signaling pathway in legumes. Genetic screenings and mutation studies have identified a set of common critical components in the symbiotic signaling pathway using the two representative legumes, Medicago truncatula and Lotus japonicus as model systems5. Among them, Does not Make Infection 1 (DMI1) from M. truncatula has been recognized as a cation channel that resides in the nuclear envelope and is directly involved in generating oscillations of the nuclear and perinuclear concentration of Ca2+ often referred to as Ca2+ spiking10. Ca2+ spiking is a key process in the early stage of symbiotic signal transduction that results in the regulation of downstream symbiosis-related gene expression. Two DMI1 homologs, named POLLUX and CASTOR, were identified in L. japonicus and both of them are required to initiate symbiosis11. L. japonicus POLLUX is a close homolog of M. truncatula DMI1 whereas L. japonicus CASTOR (LjCASTOR) is more distantly related. A closer homolog of CASTOR was identified in M. truncatula, but it does not seem to play a significant role in symbiosis12. Using mutant rescue assays, it was demonstrated that M. truncatula DMI1 is sufficient to replace both POLLUX and CASTOR in L. japonicus and that reciprocally both POLLUX and CASTOR are required to replace DMI1 in M. truncatula. This functional difference between DMI1 and CASTOR/POLLUX was attributed to a Ser-to-Ala substitution in the filter region of DMI112. Interestingly, expressing DMI1 in human embryonic kidney (HEK) 293 cells allows cytosolic oscillations of the Ca2+ concentration reminiscent of Ca2+ spiking observed in plant cells13.Symbiotic associations between plants and microbes such as arbuscular mycorrhizal (AM) fungi and nitrogen-fixing rhizobia facilitate the supply of nutrients to their host plants. The AM symbiosis probably appeared with the first land plants+ Conductance (RCK) domain14. However, the second half of the soluble domain has no sequence homology to any known proteins. The RCK domain is commonly found in a majority of prokaryotic ligand-gated K+ channels19 and eukaryotic slo family K+ channels such as large conductance, Ca2+-gated K+ (BK) channel23. Initial studies using yeast rescue assays and artificial lipid bilayers suggested that CASTOR, POLLUX, and DMI1 could be K+ channels24 with the RCK-containing MthK channel used as a homology model for a part of these channels 11. Recently, DMI1 was found to interact with Ca2+-permeable cyclic nucleotide-gated (CNGC) channels at the nuclear envelopes25. One current model of the early symbiotic signaling pathway speculates that DMI1, CASTOR, and POLLUX function as ligand-gated K+ channels that can modulate the membrane potential across the nuclear envelope and that they work together with CNGC channels to modulate nuclear Ca2+ release25.CASTOR, POLLUX, and DMI1 channels were predicted to contain four transmembrane (TM) segments followed by a sizeable soluble domain. Sequence analysis suggested that the last two TM regions form the pore and the N-terminal half of the soluble domain is distantly homologous to a Regulator of K+ channel model for CASTOR, POLLUX, and DMI1. Firstly, in artificial planar lipid bilayer assays, denatured DMI1 or CASTOR proteins were used in proteoliposome reconstitution24, raising concerns that these channels may not be properly folded and reflect function in cells; up to now, there are no direct electrophysiological measurements of these channels in a cell-based expression system. Secondly, the predicted pore of these nuclear channels lacks the TVGYG signature sequence of the selectivity filter that is highly conserved in canonical K+ channels. Thirdly, due to low sequence similarity, whether the soluble domain of these channels contains an RCK domain remains to be proven structurally. Furthermore, if the soluble domain of these channels serves as a ligand binding domain similar to other RCK-regulated channels, the functional ligand remains to be identified.However, several important issues prompted us to revisit the K2+-regulated Ca2+ channel rather than as a K+ channel as previously proposed. We also determined the high-resolution crystal structure of the ligand-binding soluble domain of LjCASTOR and reveal that the entire soluble domain consists of two tandem RCK domains that assemble into a tetrameric gating ring where Ca2+ can bind and regulate the channel. As CASTOR, POLLUX, and DMI1 are localized in both the outer and the inner membranes of the nuclear envelope, with their soluble domains facing the cytosol and nucleosol, respectively8, these channels could directly mediate Ca2+ release from the nuclear envelope using their Ca2+-binding RCK gating rings to regulate the channel activity by sensing the cytosolic/nucleosolic Ca2+ concentrations.To address these fundamental issues that define the primary function of these channels and their role in the symbiotic process, we performed functional characterization of the LjCASTOR channel and demonstrate that it functions as a CaOur initial attempt to measure the channel activity by patching the plasma membrane of HEK 293 cells expressing full-length LjCASTOR was not successful due to the lack of surface expression of the channel and the majority of the expressed proteins being localized in the endoplasmic reticulum (ER) and nuclear membranes. A series of mutant constructs with N-terminal deletion was generated and expressed in HEK cells. One of the constructs, LjCASTOR-2TM that contains the pore-forming TM domains (TM3 and 4) and the entire soluble domain, yielded a small amount of plasma membrane-targeted channels, allowing us to measure the channel activity by single channel recording.+, illustrating the cationic (Na+) currents conducted by LjCASTOR-2TM. The channel has a high single-channel conductance of 220 pS in symmetrical 150 mM Na+. In the presence of cytosolic Mg2+ , the channel conduction is slightly inwardly rectified , however, Ca2+ exerts an inhibitory effect on the open probability, resulting in a bell-shaped Ca2+ activation curve reminiscent of Ca2+ activation in RYR or IP3 receptors28.Figure ied Fig. . Furtheried Fig. . At a lo+ channel24, it lacks the highly conserved TVGYG filter sequence of canonical K+ channels, and its selectivity property remains unclear. We, therefore, measured the relative selectivity of LjCASTOR-2TM among Na+, K+, and Ca2+ by single-channel recording in the outside-out patch. As shown in Fig. + with 150 mM K+ in the bath solution yielded a reversal potential of about −20 mV, indicating that LjCASTOR-2TM is slightly selective for Na+ over K+ with a permeation ratio, PNa/PK, of about 2. Replacing bath Na+ with 15 mM Ca2+ yielded a reversal potential of about 30 mV, indicating higher Ca2+ selectivity of LjCASTOR-2TM with a permeation ratio, PCa/PNa, of about 30. Under our recording condition, LjCASTOR-2TM has a Ca2+ conductance of ~11 pS. Furthermore, La3+, a lanthanide inorganic blocker of some Ca2+-permeating channels, can also block LjCASTOR-2TM from the extracellular side  and four subunits assemble into a tetrameric gating ring, confirming that the CASTOR channel functions as a tetramer ; a C-terminal subdomain (C-lobe) that interacts with its counterpart from the neighboring RCK domain in the same subunit; and a helix-turn-helix linker between the N- and C-lobes that interlocks the two tandem RCK domains within each subunit Fig. . The tan+ channels15. The protein–protein contacts at the assembly interface are mainly hydrophobic along with a salt bridge between Arg 420 and Asp 735. In addition to the protein contact at the assembly interface, two sets of inter-subunit salt-bridge interactions are noticeable: one is the Lys368–Asp390 salt bridge between two neighboring RCK1s at the top of the gating ring near the membrane surface, and the other set of interactions are between residues from two neighboring RCK2s at the bottom of the gating ring, including His651–Asp626 and Arg650–Asp 626.Four CASTOR soluble subunits assemble into a tetrameric gating ring consisting of eight RCK domains Fig. . The Ros+, Ca2+, and Mg2+, and its high-resolution structure allowed us to identify multiple bound ions clustered in two locations within each subunit. The identity of the bound ions was determined by the chemical environment of the ions such as ligand type, coordination numbers, and ion–ligand distances, along with the anomalous scattering signal from Ca2+, which is much stronger than that of Na+ or Mg2+.The CASTOR soluble domain was crystallized in the presence of Na2+; it is coordinated by five water molecules and one carboxylate oxygen from D444 with an averaged O–Mg distance of about 2.1 Å, reminiscent of octahedral [Mg(H2O)6]2+. The other ion is defined as Ca2+ and is labeled as the Ca(I) site; it is coordinated by eight oxygen atoms including multiple negatively charged carboxylates, and it has ion–ligand distances of 2.4–2.5 Å  site mediates extensive interactions between the two N-lobes that lock them in a fixed position relative to each other. These interactions consist of a network of hydrogen bonds and salt bridges  and Ca(III) sites, respectively  site and was identified as Na+ for two reasons: first, the bound ion has no anomalous diffraction signal; second, the bound ion has a coordination number of five with ion–ligand distances of 2.3–2.4 Å, which match that of the Na+ ion. Other than one carboxylate oxygen atom from E493, which is also shared with the Ca(II) site, the rest of the Na+ ligands are all from backbone carbonyls of D485, G488, E490, and C492  site and also shares the same oxygen ligand from E493, we suspect that Na+ and K+ binding can exert a different competing effect on Ca2+ affinity and thereby have different modulation effect on Ca2+ activation.Interestingly, K0 Å Fig. . It is i+ channels such as BK and MthK, the gating rings undergo conformational changes in response to Ca2+ binding and open the pore32. To reveal if Ca2+ binding induces a similar conformational change in CASTOR, we also crystallized the apo form of CASTOR gating ring with the presence of 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). Compared to the Ca2+-bound form, CASTOR gating ring is biochemically less stable in the absence of Ca2+ as demonstrated by the broader and less symmetrical protein elution peak on a gel-filtration column. The obtained crystals diffracted to a much lower resolution and the final structure was determined to 3.3 Å.In RCK-regulated KP 21 and contains four subunits per asymmetric unit; the four subunits exhibit slight conformational heterogeneity among them and form an asymmetrical gating ring . In this assay, Ljcastor-4 mutant plants were transformed with empty vector (negative control), a vector expressing the wild-type CASTOR protein (positive control), or CASTOR mutants with point mutations at the Ca2+-binding sites. After plants established robust root systems, they were transplanted into pots, inoculated with the compatible rhizobium M. loti and grown under 16-h light/8-h dark conditions at 23 °C for 3 weeks before being harvested to count the nodules. The efficiency of symbiosis formation was determined by calculating the average number of nodules per transformed root. Mutations at two of the Ca2+-binding sites were tested: D442A and D444A at the cluster I Ca(I) site and E493A or Q at cluster II Ca(II) site. As shown in Fig. 2+ binding for the physiological function of CASTOR. Interestingly, mutation of D444, which also participates in Ca(I) coordination, did not affect the symbiosis.A main biological function of these nuclear ion channels in legumes is the generation of Ca2+ sites are highly conserved in these nuclear channels  mutant with Sinorhizobium meliloti. The result was consistent with that observed in the CASTOR mutations: plants with D470A  and E521Q  mutations failed to form root nodules, whereas plants with D472A  mutation established normal root nodules similar to those with wild-type channel  that abolish root nodulation in M. truncatula also failed to trigger Ca2+ spiking, whereas the D472A mutant that does not affect nodulation yielded similar Ca2+ spiking as the wild-type channel.In this assay, DMI1 was co-expressed with Yellow Cameleon 3.6 (YC3.6) in HEK 293 cells. YC3.6 consists of CFP (cyan) and YFP (yellow) fluorophores and yields a -dependent activation in inside-out patches. While the D444A mutant exhibits similar bell-shaped Ca2+ activation as the wild-type channel, D442A has higher basal channel activity at low Ca2+, resulting in a leaky channel even in the absence of Ca2+ GNHA that is highly conserved among these plant nuclear channels. We suspect D265 plays an essential role in Ca2+ selectivity. Neutralization of this acidic residue by D265N mutation completely abolished the channel activity as the mutant failed to elicit any current in the whole-cell recordings  function to IP3 and ryanodine receptors, two classical Ca2+ release channels in ER and sarcoplasmic reticulum (SR), respectively. Our high-resolution structure reveals multiple Ca2+-binding sites in the RCK gating ring and provides structural insights into Ca2+-regulated gating of CASTOR. It is worth noting that while the RCK-regulated MthK and BK channels are also Ca2+-gated, their Ca2+-binding sites within the RCK gating ring are completely different from those in CASTOR  site plays an inhibitory role whereas Ca2+ binding at the cluster II Ca(II) and Ca(III) sites activates the channel  site mediates inter-lobe interactions between the two N-lobes within each subunit that would prevent the RCK1 N-lobe from swinging away from the central axis and expanding the ring. Cluster II sites, on the other hand, are located at the cleft between RCK1 N-lobe and the peripheral C-terminal subdomains; Ca2+ binding tethers the two parts of the protein by moving RCK1 N-lobe away from the center and thereby promotes gating-ring expansion.In RCK-regulated K2+ as the sole ligand, CASTOR has a low single-channel activity when it is isolated from the cellular environment as demonstrated in the recording of inside-out patches. On the contrary, in the cell-attached configuration, the channel exhibits much higher activity with low endogenous Ca2+  pathway are involved in the symbiotic signaling pathway in M. truncatula13. It was demonstrated that MVA could elicit nuclear Ca2+ oscillation. Further studies will be needed to demonstrate whether MVA or its derivatives could directly bind and regulate these symbiotic channels, or whether other unidentified secondary messengers activate the channels.With Ca2+-induced cytosolic Ca2+ spiking. A similar phenomenon has also been observed when the ryanodine receptor was expressed in the same type of cells38. As the overexpressed DMI1 is predominantly localized in ER and nuclear membranes  of 0.4, and then at a lowered temperature of 20 °C to OD600 of 0.8, when protein overexpression was induced with 0.4 mM isopropyl β-d-thiogalactoside. Cells were harvested 6 h after induction. Pelleted cells were washed with ice-cold lysis buffer base containing 50 mM Tris-HCl, pH 8.0, and 250 mM NaCl, and then resuspended in the lysis buffer supplemented with 4 mM β-mercaptoethanol (BME), 2 µg mL−1 DNase A, and protease inhibitors including 0.1 µg mL−1 pepstatin A, 1 µg mL−1 leupeptin, 1 µg mL−1 aprotinin, and 1 mM phenylmethyl-sulfonyl fluoride (PMSF). Cells were homogenized by sonication with Branson SFX550 Sonifier® (Emerson). After removing the insoluble fraction from the cell lysate by centrifugation at 20,000 × g for 30 min at 4 °C, the supernatant was incubated with a Talon Co2+ affinity resin (Clontech) for 2 h on ice with gentle shaking, and the beads were collected in a column with gravity-flow. For the LjCASTOR soluble domain in a calcium-bound state, the beads were first washed with 10 column volumes of buffer A , followed by another 10 column volumes of buffer A supplemented with 5 mM imidazole. The protein was then eluted using buffer A supplemented with 300 mM imidazole. The eluate was concentrated using 100 kDa cut-off Amicon Centrifugal Filter (EMD Millipore) and further purified by a Superdex-200 (10/30 GL) size-exclusion column  equilibrated in buffer A. Peak fractions were collected and concentrated to 4–5 mg mL−1 for crystallization. For protein in the apo state, CaCl2 was excluded from all the buffers used in purification. After Co2+ affinity binding, the protein was washed with the washing buffer . The eluate was treated with 5 mM EGTA for 30 min on ice, and any precipitates formed during incubation were removed by centrifugation. The supernatant was concentrated and purified by Superdex-200 column equilibrated in buffer B .A PCR fragment encoding the soluble domain (residues 312–853) of LjCASTOR was cloned into the pQE60 expression vector containing C-terminal hexahistidine tag. After transformation of −1 of protein concentration using the sitting-drop vapor diffusion method. The protein solution was mixed with an equal volume of well solution and incubated at 20 °C. For the protein in the calcium-bound state, crystals appeared within 2 days under the condition of 21% (w/v) PEG 550 MME, 50 mM MgAc2, 100 mM Tris, pH 8.2, and grew to their full size in 2 weeks. Crystals in the sitting drops were cryoprotected by replacing the well solution with a cryosolution containing 40% (w/v) PEG 550 MME, 50 mM MgAc2, 100 mM Tris, pH 8.2, followed by overnight equilibration, and flash-frozen in liquid nitrogen. The protein purified in the presence of EGTA yielded crystals in the solutions containing 20% (w/v) PEG 3350 and 100 mM LiAc. Crystals from the calcium-free condition were first observed within 2 weeks and grew to their full size in 4 weeks. Crystals were cryoprotected by slowly replacing the crystallization drop solution with cryosolution containing 50 mM Tris-HCl, pH 8.0, 250 mM NaCl, 4 mM BME, 1 mM PMSF, 0.5 mM EGTA, 100 mM LiAc, 20% (w/v) PEG 3350, and 20% (w/v) PEG400.Crystallization of the LjCASTOR soluble domain was carried out at 4–5 mg mL2+ belongs to space group I4 with the unit cell dimensions of a = b = 125.2 Å, c = 81.5 Å, and α = β = γ = 90°, and contains a single subunit per asymmetric unit; four crystallographic symmetry-related subunits form a gating ring. The protein crystal in the apo state has P 21 space group with unit cell dimensions of a = 100.4 Å, b = 116.0 Å, c = 113.0 Å, α = γ = 90°, and β = 113.9°, and contains four subunits in an asymmetric unit that form a gating ring. Phases were obtained from a single wavelength anomalous dispersion (SAD) experiment using the soluble domain of LjCASTOR with selenomethionine (SeMet) substitution. Incorporation of SeMet was carried out by protein expression in E. coli cultured in M9 medium supplemented with threonine (100 mg L−1), lysine (100 mg L−1), phenylalanine (100 mg L−1), leucine (50 mg L−1), isoleucine (50 mg L−1), valine (50 mg L−1), and SeMet (60 mg L−1)39. The selenium-labeled protein was purified and crystallized as described above.X-ray diffraction data were collected at Advanced Photon Synchrotron  and Advanced Light Source (BL 8.2.1 and BL 8.2.2 beamlines). The crystal formed in the presence of Ca40. Heavy atom positions of SeMet-substituted crystal were determined, and initial phasing and map were calculated in Phenix Autosol41. The structural model obtained by the SAD experiment was then employed as a search model for molecular replacement (MR), which has been performed using PHASER42 for all other data sets including the high-resolution native data. All structural models were manually built in COOT43 and refined in Phenix44, and further validated using Molprobity45. The data collection statistics and refinement details are reported in Supplementary Table https://pymol.org/2/).All diffraction data were integrated and scaled using the HKL2000 packageSalI and NotI restriction sites. All single-site mutants were generated using a QuikChange Site-Directed Mutagenesis Kit (Agilent) and confirmed by DNA sequencing. About 1–2 μg of the plasmid was transfected into HEK 293 cells that were grown as a monolayer in 35-mm tissue culture dishes (to ~70% confluence) using Lipofectamine 2000 (Life Technologies). About 24–48 h after transfection, the cells were dissociated by trypsin treatment and kept in complete serum-containing medium and re-plated onto 35-mm tissue culture dishes and incubated at 37 °C until recording. Patch clamps in cell-attached, whole-cell, inside-out, and outside-out configurations were employed to measure the LjCASTOR-2TM currents in HEK 293 cells overexpressing GFP-tagged proteins and their mutants. The standard intracellular solution contained 145 mM potassium methanesulfonate (KMS) or sodium methanesulfonate (NaMS), 5 mM KCl or NaCl, and 10 mM HEPES buffered with Tris-HCl, pH 7.4. For free Ca2+ concentrations <100 μM, a mixture of 5 mM EGTA and a certain amount of CaCl2 was prepared to achieve the target free Ca2+ concentration according to MAXCHELATOR (http://maxchelator.stanford.edu). The extracellular solution contained 145 mM NaMS, 5 mM NaCl, and 10 mM HEPES buffered with Tris-HCl, pH 7.4. Na+ was used as the main monovalent in most of the recordings to eliminate the effect of endogenous K+ channels commonly seen in HEK cell recording. Furthermore, there is no report of endogenous channels from HEK cells that have similar gating and selectivity properties as those of LjCASTOR in this study.To characterize channel activity of LjCASTOR using electrophysiological studies, we generated and utilized the LjCASTOR-2TM construct where a PCR fragment spanning residues 194 and 853 was inserted into the pCGFP-Eu vector between 2+]-dependent channel activation, and the single-channel open probability was measured at 100 mV of holding potential. Outside-out patches were used to measure LjCastor-2TM ion selectivity with 10 μM of free Ca2+ in the intracellular solution for channel activation, and the single-channel currents were measured at various holding potentials from −100 mV to +100 mV to determine the reversal potential (Erev). To measure the Na+ and K+ selectivity, the intracellular solution contained 145 mM NaMS, 5 mM NaCl, 10 mM HEPES buffered with Tris, pH 7.4, and 10 µM of free Ca2+; the extracellular solution was changed to 145 mM KMS, 5 mM KCl, and 10 mM HEPES buffered with Tris-HCl, pH 7.4. To measure calcium selectivity, the extracellular solution was changed to 10 mM Ca(MS)2, 5 mM CaCl2, 130 mM NMDG-MS, and 10 mM HEPES buffered with Tris-HCl, pH 7.4. The ion permeability ratios were calculated with the following equations:Erev is the reverse potential measured with tail currents, F is Faraday’s constant, R is the gas constant, and T is the absolute temperature.The data were acquired using an AxoPatch 200B amplifier (Molecular Devices) and a low-pass analog filter set to 1 kHz. The current signal was sampled at a rate of 20 kHz using a Digidata 1322A digitizer (Molecular Devices) and further analyzed with pClamp 9 software (Molecular Devices). Patch pipettes were pulled from borosilicate glass (Harvard Apparatus) and heat polished to a resistance of 3–5 MΩ. After the patch pipette attached to the cell membrane, a gigaseal (>10 GΩ) was formed by gentle suction. The inside-out configuration was formed by pulling the pipette away from the cell, and the pipette tip was exposed into the air for a short time in some cases. The whole-cell configuration was formed by short zap or suction to rupture the patch, and pulling the pipette away from the cell after the whole-cell configuration to form the outside-out configuration. Inside-out patches were used to measure -dependent FRET between CFP and YFP33. About 36–48 h after transfection, the HEK 293 cells exhibiting a high level of YC3.6 expression were selected for FRET measurement. Before imaging, the complete growth medium was replaced with 2 mL of pre-warmed (37 °C) external bath solution containing 10 mM HEPES, pH 7.4, 130 mM NaCl, 3 mM KCl, 0.6 mM MgCl2, and 10 mM glucose. The transfected cells were stimulated with stepwise addition of CaCl2 from 0 to 2 mM and then, 2 to 4 mM with a 5–10 min interval. Cytoplasmic calcium-dependent FRET signal was collected using a Zeiss LSM 770/880 Meta confocal microscope (Carl Zeiss Inc.). After excitation with a 405-nm laser, the CFP emission (445–493 nm) and YFP emission (517–589 nm) were recorded by scanning object field every 7.5 s for a total of 120 cycles. The images were analyzed using Fiji image-processing package46. Raw data were corrected for background by performing background subtraction and then bleach correction using the exponential fit method. FRET/CFP ratio was calculated for each time point and plotted on the y-axis in a scatter diagram over time (min) on the x-axis.General procedures for FRET-based calcium spiking assay were performed as previously described47. Binary vectors were designed to co-express LjCASTOR variants using the cauliflower mosaic virus 35S promoter and ER-localized red fluorescent protein (TdTomato) for screening transgenic roots. Assembled vectors were inserted into Agrobacterium rhizogenes MSU440 and used to generate transgenic roots. MtDMI1 mutants were created in the pDONR221 vector using the QuikChange II Site-Directed Mutagenesis Kit (Agilent). Individual MtDMI1 variants were transferred via the Gateway LR recombination reaction (Invitrogen) into the binary vector pK7FWG2-RR, which allowed co-expression of mutant constructs using the cauliflower mosaic virus 35S promoter and a red fluorescent protein (DsRed) for screening transgenic roots. Assembled vectors were inserted into A. rhizogenes MSU440 and used to generate transgenic roots. Lotus japonicus cv. Ljcastor-4 and Medicago truncatula dmi1-4 (FN1) mutants were scarified by treatment with sulfuric acid for 7 min, surface-sterilized in 8% sodium hypochlorite solution for 2 min, and imbibed overnight in sterile water at 22 °C. Seeds were stratified for 4 days at 4 °C on 1.5% agar plates containing 1 μM gibberellic acid, and germinated over 5–6 days (for L. japonicus) or overnight (for M. truncatula) at 22 °C. To transform the root systems of Ljcastor-4 or dmi1-4 with single mutants, the roots were cut at the hypocotyls with a scalpel, and the wound was dipped into an A. rhizogenes MSU440 containing the binary vectors with mutant constructs. Plants were placed on 1.5% agar plates of modified Fahraeus medium supplemented with 0.5 mM NH4NO348. Plants were grown under 24-h light conditions at 24 °C for 3 (for L. japonicus) or 2 (for M. truncatula) weeks until the emergence of transformed roots. For nodulation assays, the plants with their transgenic roots were transferred to 2.5″ × 2.5″ pots containing Turface® wetted with Fahraeus medium and 0.5 mM NH4NO3. The plants were watered with the same solution at first watering and with modified Fahraeus medium without NH4NO3 subsequently. Two days after potting, transgenic roots of L. japonicus and M. truncatula were inoculated with 10 mL of M. loti MAFF303099 diluted to an OD600 of 0.05 in sterile water and with 10 mL of S. meliloti CSB357 diluted to an OD600 of 0.01, respectively. Plants were grown under 16 h light and 8 h dark conditions at 23 °C. Transgenic roots of L. japonicus were harvested after 3 weeks to count nodules, and those of M. truncatula were collected after 2 weeks. Transgenic roots were identified using the fluorescent reporter, and the number of nodules was recorded for each transformed root.For the wild-type and single mutants of LjCASTOR, coding sequences were synthesized by SynBioTech . Individual constructs were transferred via GoldenGate cloning to the binary vector pAGM4673Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Reporting SummarySource Data